CA2202680A1 - Presentation of hydrophobic antigens to t-cells by cd1 molecules - Google Patents

Abstract

Provided are CD1 presented antigens, compositions, cells, inhibitors and methods relating to the use of lipoarabinomannan (LAM) antigen presentation by CD1 molecules, including: methods for detecting the presence of a CD1presented LAM antigen in a sample; methods for isolating such CD1-presented LAM antigens and the isolated antigens; vaccines containing CD1-presented LAM antigens and vaccination methods; methods of blocking CD1 LAM antigen presentation; methods of identifying and/or isolating CD1 blocking agents and the isolated CD1 blocking agents; methods of inducing CD1 expression; and Tcells for use in the methods disclosed herein.

Description

W O 96/12190 pcTrus95ll3274 Presentation of Hydrophobic Antigens to T-Cells by CD1 Molecules Descrip~on of t~le Backgrolmd Art The Immune Sys~em and T-cel~s Animals have a comrle~r array of m~le~ul~r and cellular ~fen~es, collectively referred to as the ;..,...-~ system, that ~cco~Le and attack S ~ot~Lially harmful foreigll or endoge~uu~s but abnn-m~l cells (respectively ~e~lf se ~çd by, e.g., pathogens such as b~ or viruses, and c~ccrous or pdlhogen-infected cells), but tolerate en~ogcm,~ls normal oells. When stim~ t~ by foreign or abnormal biQm~ , the ;~ nr system undergoes a series of activities d~signçd to n~lut~li7P and destroy the pathogens, or cancerous or pathogen-infected cells, with which the foreign or abnorrnal biomolecules are associated. These activities, collectively known as an immllnt~ respo~se, may consist of a cell-m~Ai~ted immllnr response, a humoral (antibody-meAi~teA) immlln~ rcs~o~se, or an ;I~ e response that includes elements of cell-m~Ai~teA- and humoral ~cs~onses.
Humoral immllne responses are mloAi~teA by antibodies that bind specific foreign or abnormal biomolecules. Antibodies are imml~noglobulin (Ig) molecules produced by B-cells, as Iymphocytes which originate in avian bursa or in ~ n bone marrow, but migrate to and mature in other organs, particularly the spleen. Robertson, M., ~eture 301:114 (1983). Cell-mediated illl"~ r responses are the result of activities of T~ells, lymphocytes that undergo maturation within the thymus of an animal. Tizard, I.R., Irnm~nology: An Introd~ction, 2d EJ1.~ ln~l~ ~, Phil~ rhi~ (hereafter ~Tizard"), p. 163, 1988. Both T and B-cells migrate l~t~.~n ~arious organs and/or tissues within an animal's body. Lydyard, P., and Grossi, C., Chapter ..
3 in Imrn~nology, 2d Ed., Roitt, I., et al., eds., Gower ~e~ l Publishing, T~n~on, New York, 1989.
T cells mediate at least two ge~eral types of imm-~nologic functions, effector and regulatory, r~flectin~ the fact that T-cell activities vary considerably among different subpopulations of T-cells within an a~imal.

SU~TITUTE SKEET (RJ' E 2~

Rook, G., Chapter 9 ~ cnology, 2d A., Roitt, I., et al., eds., Gower Medical Publishing, T~nrlc!n, New York, 1989. Fff~tor îV~r-t;o~c include delayed ~ ~ ;vily, allograft le~ n~ tumor ;...,..~ , and ~t-versus-host ,~ ivily. F-ff~tor r,.~.Onc reflect thc ability of some T~ells S to secrete plOtC.~S called lympho~nes, .and the ability of other T~ells("~IOlo~c~ or "Idller~ T~ells) to Idll o~er cdls. T~c r~lato~y fit~n~
of T-cells are ~ tJ,d by the abili~ of hc~ T~ells. ~Ielpcr T-cells interact with, and ~lucc bio~olr~ that ;..n~ ~ the l~v;or of, both B-cells and ~otc,~uc T-cells, in order to ~ ote and direct andbody production and crlo~uc activides, .~i.,cly. Mosier, D.E., Science 158: 1573-1575 (1967). Other classes of T-celIs, in~.lntlin~ :~U~Jy~iOl T-ceIls and memoIy T-cells, also exist. ~i~m~, F., and Melief, C.J.M., Imm~nol.
Today 6:258-259 (1983); Tizard, pp. 225-æ8.

Antigen Recogn~on In order to function ~rope.l.y, the T- and B~ells of an animal's ; I I I I I I I I n f system must accurately and reliably iden~if y the er-ol . .o. c ~r of molecular compositions derived from foreign ("non-self"), or endogenous (~self~) but ~bnorm~lly eA~ , compositions that are rn~eu~n~f,~d.
Recognition and i~le ~t;~ ;on by the i~ system occurs at the molecular level. An antigen, a moleclllar ~lpo~i~n haYiDg the ~o~ l to g~ ate an;,.. If-r~onse, isco~ osedofoneormoremoleY~ r-sizedidc.~ ~g feaLu~es known as epiLopcs. A poly~tide antigen which has an amino acid s~ue~c~ which c~ iccs~ e.g., a hu~ ,d amino acids might C~..-l..;~G
doze~s of ~;~0~5, ~Le.e~ each epitope is defined by a portion of the polypeptide c~mpri~in~ from about 3 to a~out 25 amino acids. The m-ml~er of ~ pcs derivable from polypeptides alone is ,,l;,..;~l.~l to be about ten nilli~n Tizard, p. 25.
An antigen c~counl~-~,d by a T or B-cell of an animal must be idensifie~l as either. being associated wi~ noImal endogenous (i.e., self) SllBS~i~U ~ ~ SltEET ~RU~ E 26) PCTtUS95/13274 antigens, an i.. lllne response to which would be injurious to the anirnal, or with foreign or abnormal (i.e., non-self) antigens, to which an j~.~ln~
le~yonse should be mounted. As part of the ;.~ ....r system's means of idcnliryi~ a~ldgens, individual T and B-cells yl~>du~ anffgen r~c4~to. ~ which are displayed on the T or B-cell's surface and which bind ~l~r:r;c ~nti~
I`urner, M., Chapter S in Inunccnolo~y, 2d Ed., Roitt, I., et al., eds., Gower Medical Pllbli~hing, T l~nt10n, New York, 1989. B cells ylOducc and display antigen loceptors that co~ ;ce Ig mt~lmll~s which have unique ~nti~n-binding portions due to unique amino acid ~u~s in the variable regions of each of the two antibody subunits~ known as the Ig heavy and Ig light chains. Each B-cell membrane COI;~p~S from 20,000 to 200,000 id~-ntir~l Ig molecules. Tizard, pp. 78-80 and 202.
The T-cell antigen l~ptO~ (TCRs) produc~d by and displayed on individual T-cells comprise heavy (TCRB) and Iight (TCR~) chains (polypeptide subunits) which are linked by a ~ fi~ls bond on the T-cell surface. Each TCR cY and ~B subunit has a carbo~y-tç~min~l co~t region, the amino acid sequence of which does not va~y from T~ell to T-ceII, and an amino-termin~l variable region, the amino acid S~uC~ of which does vaIy from T~ell to T-cell. When TCRa and TCR~ ~b~ csoci~te with each other, the variable regions of the TCRa and TCR~B polypeptide S~lbu~Ls combine to form the unique an~gen-binding por~on of an c~:~ TCR. A
second type of TCR heterodimer, ~y:8, ha. been ~esrrihe~ but itc filnr.tion, if any, LC unknown. Davis, M.M., and Bjorkm~n, P,J., Na~ure 334:3954~4 (1988). Although at least one mi~ced TCR heterodimer of uDknown fimetion~
~:8 TCR, has been ~le~bed, T~ells bearing :~ TCR molecules are numerically do.l.i~ n;m~1.c, Hoc1~ c~ F., and B~ ,er, M.B., Nature 340:562-565 (1989).
Although each individual T- or B-cell displays id~ntic~l- antigen receptol~, the l~ptor displayed varies from cell to cell; an animal's c~llection of different antigen [~tul~ is t~us quite diverse. The genetic basis of this div~,~sily is as follows. The variable region of a~ Ig heavy chain, TI~l5T~ SHEET ~F~ULE 26) or that of a TCE~ chain, is encoded by three gene sf.~ Pnt~ the variable (V), diversity (D) and joining (J) se~ ; The variable region of arl Ig light chain, or that of a TC~a chain, is en~ ~ by V and J gene ~c~ t~
Multiple DNA se~ucnccs cnr~~ p many di~e.~i~ V, D and J gene ~,~..r~
S are present as ~ 55~ oopics in genn~ DNA; sn analog~us ~ut different co11~ctit n of variable gcnc K~ t~ for 1~ ~
present. During development of an animal, genoe c ~ diver~e Yariable regions are ge~ tlYl in individual cells of the i.~.. ;r ~rstem by the ~ 7."
joining of V, D and J, or V and J, gene ~r-~t~ e process of DNA
rear~nge.. ~ ts that gelle.dls a r~ntlomly ~CPmb'~d variable region of an Ig heavy or TCR,B subunit is called V-D-J joining; the analogous process that generates a rearranged variable region of an Ig light or TCRa subunit is called V-J joining. Sakano, H., et al., Nature 280:288-294 (1979); Early, P., et al., Cell 19:981-992 (1980); Alt, F.W., et al., Science 238:1079-1087 (1987);
Harlow, E., and Lane, D., An~ibodies: A L~boro~ory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Har~or, pages 1~18, 1988; Davis, M.M., and Bjorl~m~n, P.J., Nature 334:39~404 (1988).
A functionally rearranged Ig or TCR subunit gene is one in which the DNA rearra~g~ cnts of V-D-J or V-J joining have not resulted in a reading frame that is p.c~ely le.~ t~ ~uSe of the i~h~ducLion of stop codons or fi~ ~hir~ g mnt~tionc. Rec~l-~ each T or B~ell of the ;.. ~, system e~.~ses genes rnr~ing their r~ antigen ~ in which a unique f~mrtion~lly l~god variable region is present, many dif~e,~,~ T
or B~ells, each producing a ~t~r that has a unique an~`igen~
region, are gcnc~aled. Hay, F., Chapter 6 in Imnuu~olo8y~ 2d Ed., Roitt, I., et al., eds., Gower Medical PUhti~h;~ r nn~on, Rew Yorl~, 1989. The total catalog of different antigen l~ displayed on the T-c-dls of an animal is referred to as the animal's TCR l~ c. Bevan, M.J., et al., Science 264:796-797 (1994).
For mature T- or B-cells, binding of antigen to a cell's antigen l~ptol activates the cell, i.e., stim~ t~ the cell to undertal~e activities related to SUBS ~ ITU I t SHEET (~IU~ ' 26~

PC~r~US95/13274 gel~e.ating a cell-mPAi~toA or hurnoral ;" n ~n~r rcs~ollse. Typically, activated mature T or B~ells proliferate in ~e~l~on~ to ~ntigen In cl~nt~ct, for ;~.,n~ T or B-cells, binding of antigen to a d~l~ TCR or B cell antigen r~to~ i.,ely, r~sults in c~ n of the cel~ by a process S called ~eg~ e sel~tlirtn or clonal delçh~ln Clonal tl~l~firm oCwn; duriDg normal development of a healthy wild~pe animal, and is a ~ a-~i- rt by vhich the ;~ nne system learDs to tolerate 2he animal's ~mal ~o~ us (self) antigens, i.e., to treat the animal's ~ as no~;~ og~ic antigens. Failure of the ;...,.n..~ system to achieve or m~int~in tol~ 4 of self antigens may result in al~o; - ,nn~ r~ ses (i.e., aU~i n ~ r r~S~OllSe to self antigens) that can c~llmin~tf in au~o;~ , disease in anim~lc inr.lll(linf~
humans. At~to;"".,l~nf~- disease can occur when an a~p~opl~e ;"."...nç
response to a non-self antigen results in the production of l,."....i~ effector biomolecules (e.g., ~lto~ntibodies) or cells that cross-react with self ~ntigf n~.
Human autoi",--,~"e lice~ses include such criMli~ con~itinnC as Multiple Sclerosis (MS) and Systemic L~lpus E~ ru~ic (SLO. Roitt, I., Chapter 23 in Imm.w~logy, 2d Ed., Roitt, I., e~ al., eds., Gower Mf~lir~ b!i~hi~, London, New York, 1989; S~ l~n~ , L., Sci. Amencan 269:107-114 (1993).

Antigen Presen~on Although the antigen ~cep~o-~ of ~cells can directly bind soluble antigen, T~ells typically respond to antigen.only when it is di~layed on specific classes of other cells known gf ~ ;r~lly as an antigen-plesenl;np cells(APCs). Feldm~nn, M., and Male, D., Chapter 8 in Imm~nolo~y, 2d Ed., Roitt, I., et al., eds., Gower M~ir~l n~ hirl~, T~n~1nn, New Yo~ 1989 2S APCs, e.g., macrophages and ~endritir cells, present Qnti,~n~ d~rived ~om polypeptides via y,l~COp,o~,-~s, lmown as MHC (major hi~loco,.,i.al;bility complex) proteins, which are displayed on the surface of APCs. Bevan, M.J., et al., Science 264:796-797 (1994). The nnm~nrlQll~re for MHC gene products varies from species to species. For example, human MHC proteins S~BSTIT~TE SHEET ~UI E 26) are also rcfel~ed to as human lymphocy~e ~nti~en~ (HLA), munne MHC
~lOtcil~s are also l~,f~led to as H-2 ~nti~ens~ and rat MHC pr~ s are also callcd RTl ~ c. Tizard, p. 181. P~Li~ular MHC ~ot~,~s bind ~ *,A
classes of ~igeLIs ~th limited ~ rlr-;t~. For ~he most part, ~hc ;,~;I';rit~, det~ c in a IY~:Ag:MHC compl are (1) ~e uniquc pol,~ptide se~luel~rfs of the variable por~on of the 1~ and O ~e unique poly~ide ue~cesof~nti~en Howcver,to~omedegrcc,MHC-y~doligo~ e ~nti~ n.~ are embed~ within an MHC l~olc~vle and TCP~ loco~lion of antigen only occus~ within the conte~t of an a~Lo~ e class of MHC
molecule. Janeway, C.A., Sci. Amen~n 269:73-79 (1993). This pheno~enon, called ~IC restr~tit n, is of f~.d~ l i~l~ce to T-cell aDtigen re~g~Lion and physiology. 7i~ eJ, R.M., and Doherty, P.C., Na~ure 248:701-702 (1974).
In MHC-m~i~ted ~rw ~1~t;on of antigens, the ~:~ T~ell antigen leceptor loeGg~es peptide antigens in co~yu~lion with ~ UCl:i of MHC
genes. In the case of soluble antigens, recogTUtion occurs in conjull~tion with Class II molet ules. Por ~riral ~nti~PnC ~co~lion is in ~ouju~cLion with Class I molecules. rulLe,lllole, large soluble antigens are ~,ocessed from polypeptides by an apploplid~e a~c~jo,y cell, such as a macrophage or ~en lritic cell.
The general s~uell~ of events involved in T-cell ~ecog~ ion of polypeptide antigens in MHC r-Pst iction is as follows. A polypeptide antigen is phagoc~Losed by an antigen~ t;.~ cell, int~r,n~li7~d plv~sS~d, and then a peptide derived from the pol~pepdde is displayed on the cell surface in 2~ co.ljuuction with Class I or Class II MHC mole~l1Ps. In order to present ~ntigen, MHC Class I mol~-les requIre an ~1~it;on~1 pmtei~ 2-micmglobulin. Tizard, pp. 181-183. A T~ell antigen l~tor a:~
heterodimer then lc~ c the peptdde antdgen plus the MHC gene product.
R~Lion of peptide antigen alone or ~IC gene product alone is not s~ iPnt to signal T-cell activation. Only the MHC:Ag cQmrlex can be a~pm~lely recogDized by a TCR molPA~lle. Steward, M., Chapter ~ in SUB~TITUTE SHEtT ~F~ULE 26) ~mmunology, 2d Ed., Roitt, I., et al., eds., Gower Medical Publishing, T 4ntlon New York, 1989.
T~e genes e ~c4 li. ~ MHC ~luLc ~s are di~ e, hu~ r, unlike Ig and TCR mo~ s~ which v~y ~om cell to oell in an i~di~id~,al animal, ~IC
~nti~n~ ~ary from i~i~ imal to i~i~ ,al animal or f~m one group of related individual ~nimal.~ to another gr~. 1~ ~ of famili~l groups, .eplese~ed in the mouse by i~bred ~ains Of mice, ~hare similar M~C
antigens with each other, but not with ,Qdi~,id~ls f~om o~her strains of _ice.
Snell, G.D., Science 213:172-178 (1981); Owen, M., Chapter 4 in nology, 2d Ed., Roitt, I., et al., eds., Gower ~iç~l Publishing, T~n~10n New York, 1989. ~ler~ e variant MHC mole~ll~-s will be capable of bindiIlg different antigens, the antigens that T-cells will be able to recogr~ize (i.e., specific~l]y bind in the MHC context) and respond to varies arnong different strains of mice. Cooke, A., Chapter 11 in Imnu~nology, 2d Ed., Roitt, I., et al., eds., Gower MÇ~ir~l Publishing, T.on~on, New York, 1989.
In hllm~n~, particular genetic alleles ç~in,~ MHC (HI,A) molec~lles are more highly ~soei~t~ with ~ oi....~ e t~ oes, ~ bly be~..se these MHC mole~ es are more col--~tent at binding (and thus pl~5~ g to T-cells) self ~ntig~n~. V~llgh~n, in Irr~r~logical Diseases, 3rd Ed., Vol. II, Samter, M., ed., pp. 1029-1037 (1978); Stf~ , L., Sci. American 269:107-114 (1993).

SUBSTI~UTE SHEE I ~F~ULc 26) T-cell Subsets Classes of T-cells are to some e~ent ~iChl~lich~ on the basis that different T-cells display dirre~ CD ~lot~s on~seir ~.. r~r~. T.. ~tl.. ~ T-cells display both CD4 and CD8 ~t~ l.e., ;-. ~ T-cells a~
CD4+8+), mature helper T-cells are CD4+8- (i.e., display CD4 protesn but not CD8 protein) and rnat~lre ~lOtOiuC T~ells are CD4-8+ (i.e., display CD8 protein but not CD4 protein). Smith, L., lYancre 326:798-800 (1987~;
Weic~ ,., I.L., and Cooper, M.D., Sci. Amencan 269:65-71 (1993).
In most cases so far r~ lin~ CD8+ T lymphocytes recogn~e MHC
class I complexes, while CD4+ cells l~cog~e MHC class II complexes on antigen prc~ g cells. The involvement of CD8 and CD4 in antigen recognition by cx:,B TCRs is signifi5~nt CD4 and CD8 molecules increase the avidity of the TCR interaction Ag:MHC comple~r~s and are som~otim~
referred to as c~rcceptol~ (Bierer, B.E., et al., Ann. Rev. Imn~atol. 7:579-599 (1989); Steward, M., Chapter 7 in Imnu~nology, 2d Ed., Roitt, I., et al., eds., Gower Medical Publishing, Tl~n~on, New Yor~, 1989). ReC~nce of the importance of CD4 and CD8 in antigen recognition in the MHC context, CD4-8- (double ~eg~Live; DN~ T-cells have cl~csi~lly been c~nci-lered to be e thymic T~ell ~ Ol~. Lydyard, L., and Grossi, C., Chapterc 2 and 14 in Itrununology, 2d Ed., Roitt, I., et al., eds., Gower ~ic~l Publishing, r~nf~on, New York, 1989; Smith, L., Nature326:798-800 (1987);
Strominger, J.L., et al., Int. J. Cancer Suppl. 4:4347 (1989); Shirai, T., et al., J. Imm~nology 144:3756-3761 (19gO); Wei<~ , I.L. and Cooper, M.D., 5ci. Americ~n 269:65-71 (19g3).
2S The DN subpopulation of T cells is ~ in regard to the TCRs that they display. The majority of human DN T cells isolated from peripheral blood express ~:y TCRs. Porcelli, S., et al., Irnmur~logic~l Reviews 120:137-183 (1991). ~ large ylopo~lion (applo~il..&tely 60%) of murine DN
~:~B TCR T~ells express V~8 gene ~ dUe~S (Fowlkes, B.J., e ~2l., Na~re 329:251-254 (1987); BL'C, M., et al., J. E~p. Med. I78:901-908 (1993)).

SUBSTI~U I E SHEET ~F~ULE 26) PCTrUS95/13274 _g_ Several an~lyses in mice point to a stri};ing lack of junctional (V-J or V-D-J) divercity and restrieted use of germline V and J gene cl~ nf.~l ;, ç~eri~lly forTC~x subunits. Kos~l~i, H., et al., Proc. ~atl. Acad. Sci. USA 87:5248-5252 (1990); Kubota, H., et al., J. Imm nol. 149:I143-1150 (19g2). ~ A~;On of fresh DN ~ B TCR T-cells In 1mm~nc revcaled a ~ FlJ~lO~ A~ of an ~t (~ o.,..~l) Va24 J~ t that lac~d Nregion a~d;tionc. Porce11i, S., etal., J. E~p. Med. 178:1-16 (1993). Taken toge~er, these obs~ tions suggest that DN ~x:,B TCR T-cells may ~
a develo~.~.P ~1ly distinct su~ lation of T lympho~ytes whose limited l~tor repertoire re~lects leco~tion of a le~lliC~ set of antigens and/or antigen-yr~se ,~ g mo1e~ es.

CD1 Proteins Polypeptide molecules encoded by the genes of the CD1 loc~s are recognized by select CD4-8- T-cell clones e~resaillg either ~ or 7~:~ TCRs (Porcelli, S., et al., Nature 341:447-450 (1989); Faure, F., e~ aI., Eur. .1.
Immun. 20:703-706 (19gO)). Rer~lse of the sl~uclul~l resemblance of CD1 molecules, enrod~d by genes on human chromssom~ 1, to MHC mQl~ll1~S, enr4d~l by genes on human chrom~some 6 (Calabi, F. and Mi1.ctrin, C., Na~ure 323:540-543 (1986); Balk, S.P., et al., Proc. Na~l. Aca~. Sci. U~A
~6:252-256 (1989)), it has been sugg~sted that CDl may l~nt a fami1y of antigen ~ r~ g mol~lll~s scp~e from ~ose ~ r~d~d by the MHC
genes. Porcelli, S., et al., Nature 341:447-450 (1989); Sl~ , J.L., Ce~l 57:895-898 (1989); Porcelli, S., et al., Imml~n. Rev. 120:137-183 (1991).
The five CD1 genes reveal exon and domain ~lluc~, (a~l, o~2, cr3) that is similar to that of MHC class I genes, yet the ~loteills are only di~lly related in se~u~ce. All CDl family m~rnbers share a conse,~d cY3 ~om~in;
how~vtr, even this domain shows only 32% homology in amino acid sequence with CQnCt~ nC residues of class I ~IC a3 dom~in~ and there is no slet~le homology with al ~lom~inc. A major dirl~l~nce ~I~.~n ~IC a~d CDl SUBSTITUTE SHEET (~ULr 26) PCTrUS95/13274 molecules is polymorphism. Human MHC genes are eA~mely polymorphic:
multiple alleles have been described at each known MHC locus. In contrast, CDl genes are a~ c.lLly nor4~olymorphic. Despite these dirrc~llces, the CDl ~ s, like MHC Class I m~ les, a~re C~ ge ~ul,~
S (heavy chains) non-covaleD~ly ~c~ r~ w~ ~B~micro~ b~ n Van AgthoYen, A., and Te~hr)r~t. C., J. ~mmunol. 128:42~432 (198V; Te~hr~st C., e~ al., Cell 23:771-780 (1981)).
Five CDl genes _ave thus f~r been id~n'ifi~l in l.. a.. ~. CDla, CDlb, CDlc, CDld and CDle. Four of the five CD1 gene ~r~duets have been defimed serologically, are referred to as CDla, CDlb, CDlc and CDld and are distinguished by uIlique heavy chains with ap~r~ te molecular weights of 49kDa, 45~a, 43kDa and 48kDa ,c~ liv.,ly (Amiot, M., e~ al., J. Imml~nol. 136:1752-1758 (1986); Porcelli, S., et al., ~nununol. Rev.
120:137-183 (1991); Bleicher, P.A., et al., Science 250:679-682 (1990)).
CDl proteins are displayed on a nurnber of APCs in~lrl~i~ Lang~"l~s cells (which are the rnajor den~tir antigen-pr~s~r~til~g cells in the skin), activat~dB-cells, dendritic cells in lymph nodes, and on a~ a~i blood monocytes (Porcelli, S., et al., Nan~re 360:593-597 (1992); Leu~ e l~ping IV, Knapp, W., ed., Oxford U~e~ily Press, Oxford, U.K., pp. 251-269, 1989; Tissue Antigens, Kissmeyer-Nielsen, F., ed., Munksgard, Copenhagen, Denm~rk, pp. 65-72, 1989.
Previous work has shown that CD1 protc~s are recognized by CD4-8-T-cell lines derived from patients with SLE. Porcelli, e al., Na~ure 341:447-450 (1989). T ~nk~ mi~ cells t~s~g Cl~L ~ s were lysed by the T-cells in~epentl~nt of MHC reshirtion, even though no foreigu (non-self) antigen was present. DN T-cells lysed le.~k~ ;r cells in a CDl-depen~nt ~a.~cl in the ~bsenu of ~ntigt~n Thus, the possibility exists that CDI
-~roteil~s play a role in aulo;~n~ r ~i~s.
The central dogma of imm~ ology has been that the ~ L.i~e system does not normally react to self. AU~O;~ ;L~ defines a state in which the natural u~ eness or tolçr~ e to self l~ h~. As a result, ~UBSTI~UTE SHEET (F~ULE 26) antibodies or cells react with self concti~lerltc, thereby cau ing diseace. There is as yet no estahlich~ unifying concept to explain the origin and pathogenesis of the various a~lto~ ..,.r disorders. The disease plOU~Si may be caused, among other things, by s~ ~c;l ,~1 T lymphocytes. These ly~h~
S tissue lesionc by poorly nn~çrstQod . . .~h~ , .. c w~ich may inYolve the releasc of dest~uctive l~---phot ;. ~s or ~hich a~act other ;..n ..~ r~,ly oeIls to thelesion. For a review of aulO~ see Theofilopoulos, A.N., Chapter 11 in Basic and Clinical Irnm~logy, 6~ Stites, D.P., et ~l., eds., Appleton a~d L~ang, I987.

Tuberculosis Mycobacteria are a genus of aerobic intracellular bacterial or~ani~mc which upon invasion of their host, sunive within endosomal C~ Cll~ of monocytes and macrophages. H~ an ~ycobacterial diseases include tuberculosis (caused by M. tr~erculosis), leprosy (cause~l by M. ~eprae), Rairn~ale ulcers (caused by M. ulurans), and various infections caused by M. mannun, M. kans~sii, M. scrofulaceum, ~. szulgai, M. xenopi, M.
for~iPurn, M. c~elonei, M. haemop~ulum and M. intr~7cel1T~nre. Wolinsky, E., Chapter 37 in Microbiology: Including Irrun~logy and Molecular G~nencs, 3rd Ed., Harper & Row, Philadelphia, 1980, hei~Lel "Wolink~y";
Daniel, T.M., MiIler, R.A. and rl~e~n~n, S.D., Chapters 119,120and 121, ~csyec~ively~ in Ha~ison 's Principles of Internal Medicine, 11th Ed., BldUll~ d, E., e~ a~., eds., McGraw-Hill, New Yo~c, 1987.
One third of the world's population harbors M. tuberculosis (M tb.) and is at risk for developing tuberculosis (IB), which is ~pecifically 2~ res~onsible for 18.5% of deaths in adults aged I5 to 59. Bloom, B.R., and Murray, C.J.L:, Science 257:1055-1064 (1992). ReC~llse i~ylOvcd public health and antibiotic therapy have greatly reduced the OC~ lce and/or scveli~ of TB in the Unite~ States, these ~1~rrnin~ st~Si~i~ derive largely from third-world cou~ ies. Unfortunately, with the advent of A~DS, ~iJBSTl~UTc SHEE r ~,ULE 26) tuberculosis is increasing at a nearly log~ ie Iate, and multidrug ~
strains are a~ g and now dCeOUnt for one third of all cases in New York City. Bloom, B.R., and Mu~ay, C.J.L., Science 2S7:1055-1064 (1992);
U.S. Co~glcss, Officc of T~hnr~logy ~ r..~t, The ~4~.~i"r~ g ~n~ e S of Tu~erc~losis, OTA-H-574, U.S. Go~ g Of fice, W~
D.C., 1993. M~.,ol~ trains which ve~e y~ c~r~;~od to bc ~o~l-d~h~genic ~trains (e.g., M. au~n) havc now boc~r major Icillers of immllnG~ ppl~dAIDS~t;~ t~. Moseover,curceDtM~cob~
are either inade~uate, in the case of the BCG ~accine to M. tb., or, with regard to M. Ieprae, unavailable. K~.. f,.. ~nl-, S., Microbiol. Sci. 4:32~328 (1987); U.S. Congress, Office of Technology ~s~ , The C~ cing ChaUenge of TubercuIosis, pp. 62-67, OTA-H-574, U.S. Co~ .ç- .~ g Office, Washington, D.C., 1993.
The major r~o~se to mycobacteri~ invol~es cell ~ Ai~lA,d delayed hy~cl~nsilivily (Dl~ re~ionC with T cells and macr~hagcs playing major rol~s in the intr~cp~ r kiIli~g and co,.~ or walling off ~msloma forrn~tion) of the OL~ A major T-cell l~o~e involves CD4+
lym~hocytes that lOOOg~ ob~c~ ;~1 hea~ shoc~ ~o~s (such as hsp65) as immlln~o~in~n~ an~igens. ~ .f..\~ ', S.H., etal., Eur. J. Inun~nol.
17:351-357- (1987).

Leprosy Leprosy (Hansen's disease) is a chronic ~Tam~ .us fnfection of l...n.~nc which ~ttacks ~u~ r~ l tissues, ~i~lly the s~in a~ ~
nerves. Accou~ of leprosy extend back to the carliest l-.~t~ 1 records aDd do~-m~nt a stiy~ of le~rosy patien~s which ~ ~s c~
religious bo~ ;ps. Miller, R.A., Chapter 120 in IIarnson's Pnr~pks of Inten~al ~e~icine, 11th Ed., 13~du~dld, E., et al, eds., McGraw-Hill, New Yor~, 1987, l~c.~. "Miller." ~ cient ~mes leprosy wæ ~ t throughout most of the world, but for u~ ow~ reasons it died out in Europe SUBSTlTl~TE SnEt~ (RULE 26) in the sixteenth century and now occurs there only in a few isolated pockets.
Wolinsky, p. 741.
There arc probably 10 to 20 million persoDs afr~d with leprosy in the world. The disease is more c~ n n/)n in tropical ~J ~h ~5, in many of which the prevalent rate is 1 to 2 perccnt of the pop~ ticn A warm ellvil;o~e.lt is not critical for ~ nC;..;~,ion, as leprosy also oa urs in ccrtain regions with cooIer cti n~t~s, such as ~orea and c ntral Mc~co. D;.~ ll;on of illÇ~ct~ individuals within cu~ h .s is ~very -- nh~l.nog.,n~u~
in which 20 ~,c~nt of the ~ dliO~ iS aff~ct~yl caIl be fwnd. Miller, p.
633.
In the Uruted States, leprosy occurs particularly in Te~as, California, Louisiana, Florida, New York C~ity, and Hawaii, usually in pc~sons originally from Puerto Rico, the Philippinoe, Me~ico, Cuba, or Samoa. Indigenous ~l~lnc~ni~sion occurs primarily in Hawaii, the Pacific Island t~llilolies, and specifically along the Gulf coast. Several h~ed patie.,ls are cared for at the national l~ros~iulll in Casville, Lulliçi~. Wolirlsky, p. 741.
Mycobaaerium leprae, or Hansen's bacillus, is the causal agent of ieprosy. It is an acid-fast rod ~c~igT~ to ~e family M~l~'oaete~;aceae on the basis of morphologic, biock~ ~i~l, antigenic, and genetic simil~rities to other mycob~ leprae causes chronic ~rn~lo~ J~ lesions closely resembling those of tuberwlosis, with ~pithelioid and g~L ceSls, but w~
c~ceation The ol~ani~nc in the loeions are predo~ lj inter~ellular and can evidently proliferate w;ithin ~o2hages~ like tubercle bacilli. Wolincky, p. 740.
2~ Although A~. Ieprae has not been cultivated in ~ lilicial media or tissue cul~e, it can be co~ .opag~b_d in ~e foot pads of mice S~h....c infections with ma~ ;onC similar to those of hum~n disease can be in~U~ in ~rm~Aillos and m~ey ~uo~. The bacillus multiplies e~ ~in~ly slowly, ~ith an e,~ t~ ~ optimal doubling time of 11 to 13 days du~ing lo~ ir gro~th in mouse foot pads. The mouse model hac been used e~ ely for the s~dy of antileprosy dsugs, and the high bacterial SIJ~STI~UTE SHEET ~RUEE 26) yield from a~illos has been cmcial for ;~ ge~c studies. Miller. p.
633.
I~rosy is a~ e~q~ t ~ ~ when c.~u~ 5 of ~ 15 ...~ kli~nr lesions and sl~n ulcers reach s};in ~ innc; it is not highly con~Ad~ls and ~tic~ nccd not be icn1At~ yo~g rhil~ a~ to acquire thc disease on briefer contact th n adults. The i~ .A~ pcciod is oet~ ed to ~ange from a few mon~hs to 30 yea~ or more. A~ , M. ~me can lie clorm~nt in ~ssues for plulongod peri~s~ Wolinsky, p. 741. I~prosy can present at any age, ~lth~lJgh cas~ in infants less ffLan one ycar of agc are eA~e~ely rare. The~age-~f~;rle ~ rnr~ peaks during childhood in most devdoping c~ t;oe, w`ith up to 20% of cases oce~ in children under 10.
Since leprosy is most p`reYalcD~ in pooxr soc;oec~nn~ roups, this may simply reflect the age dis~ibution of the high-risk pop~ tion The se% ratio of leprosy ~ during chiklhood is ess~nti~lly 1:1, but males pre~lo.n;.~e by a 2:1 ratio in adult cases. Miller, p. 633.
Lcprosy is distinguishe~ by its chronic, slow }"og,es~ and by its mlltil~ti~ and Aicfi~lri~ loeions. These may be so ~ ;ve that the tliq~cic is appaLe,ll at a glance; or the clinical ..~ ;r ~I;nnc may be so subtle as to escape ~let~tion by any except the most e~periPn~e~l obsc.~e,~
armed with a high iIlde~ of ~ ~p~ The o~ hac. a pr~il~io~ for skin and for nene. In ~e ~ ~ ~ c form of the ~ice~ce~ large, fi~m n~nll-s (leplo~c) are ~i~l ;h~J~d widcly, and on the facc~ey creatc a ch~r~C~ ;~I;c leonine ~ ~. In the normal fo~m, se~n~ of pe~h~ rGc are involved, more or less at ra~om, leading to 1~1i7e~ patchec of ~n~l].~
The loss Of s~n~tion in fingers and tooe li~Cl~S the L~ue~y of minor t~lm~ lead~g to s~nAz~ i..r~;o~-~ and mlltil~ti~ inrl~i~ Both forms may be present in the same patien~
In either form of leprosy, three phases may be ~ Jlcl~cd- (1) In the lcp~on~o~c or pro~le~ci~., tgpe, the lesions co~tain many lepra cells:
macrophages with a ~ ct~- ~cl;~ y foamy cytop!~cm, in which acid-fast bacilli are ~b~n~l~nt When theæ lesions are pio~ , the l~lu~ test is SUBST~Tll ~ ~ SHFET (RULE 26) WO 96112190 ~CTIUS95/13274 nsually negdtiYe, p~ ably owing to dcs~ ;on by massive ~moltt tC of endogenous lepromin, and the ce~ n~iqt~3 ;.. ~ln~ ctionc to spe~ifir, and nnr~ rir s~nuli are m~ y ~;.nin.ct.~ The ~e is then in a P1~;L~ phase and ~c PLO2~SS~S is poor. (2) lnthc tUbC--~ ~10;t~ or healing phase of thc ~iice~ce~ in COI~-4~I~ thC lcsions contain few lepra cells and bacilli, fibrosis is ~o. ~ , and ~e i~ul~LiLL test is usually posiLi~. (3) In thc i~t~ pe of ~ ~, bacil}i arc ~ee~ in ar~is of ILcL~osis but are rare else~YL~.~, ~e skin t~ is ~ositi~, and the long-ran,ge outloolc is f~ir.
Shifts t~om o~e pbase to ~n~thl~r~ with - ~r"b~ siQn of the ~is~se are co no~.
~n~n's baci~ c rnay be widely ~li~ibut~ in the tissues of ~
with leprosy, inrlutli~ the liver aud spleen. Neverthelcss, no de~lLuc~ e lesions or di~l ance of fim~ on are obsen~ed in these organs. Most deaths in leprous patients are due not to leprosy per se but to i~telc~nt infections with other Irucroor~nicmc--often tuberculosis. Leprosy itseLf often causes death through the comrlir~tion of amyloidocic, which L chzr~c~ri7ed by rnassive wasy deposits, co~ f~ ab~n~i~nt p~ s of fra~ nt.c of in~ oglobulin light chain in kidneys, li~er, spleen, a~d other organs.
Wolinsky, pp. 740 741.
l~eriologic Ai~nsic of lepro.cy is accomplished by d~Tnnn~ting acid-fast bacilli in s~ al. ~s from ~lcerat~ lesions, or in fluid e A~.esscd from sl~erfirizl ilr;c4,-c oYer non-l~l~t~ le~ionc~ No useful serologic test is available, but lJdtie~lS with leprosy frequently have a false-posiliv~ serologictest for syphilis. ALo usefill in the ~berculoid phase i the s~ test with lc~o~, an antigenic bacillary m~t~i~l pl~p~ ~xl by boi~ human c~. ztc~s tissue or inf~d ~r~ni~lill~ t~ssues, which ~s ~pic~lly s~ndaldi~d to con~ain 160 x 106 acid-fast bacillilml. Wolin~, pp. 74~741.
Therapy wit~ ~lzrsQn~- (4,4 ~i~min~;l)h~ fQne) or related compou~ds usually produces a gradual improvement over several years, and is contimle~ for a prolonged period a~ter a~)pale~ clinical remission.
However, re~Cict~nr~ to sulfonic dmgs, with a conromitt~nt relapse, may be SUBS ~ I~U I E SHEET ~RULE 26) W O 96/12190 PCTrUS95/13274 noted after years of a~p~c.ltly ~ ~r~ 2ir~.. ,l.i.- and clvr~ ..;..r (B663, a phrn~ d~ ) are p~ agen~ now under invectig~tinn for ~ g leprosy. T~e~ esults may be e~aluated by co~ ti.~ the acid-fast bacilli in se~ial biopses and ~ikin c - ~r ~s wO~, S p.741.
use of the ~e~ a~re of l~prosy, and ~ e ~a~ e--r-~ of 1~r m~ol~roe a b~ ;Q "~
apps~acl~, in~ludin~ conc~ t~re ~s ~ as .~ cA~r, op~ Y olog,Y, and ~h~ sical thera$~r in t~ t;r~n to ~n; ..;~ obial c~ ..~tt.~
In any event, ho~ ,,r, ~ fr~ eurologic ;~r~ is limit~, Miller, pp. 635-636.

Summary of the ~nvenfion Thc present invention is based on the novel and ~cted obsc, ~aLion that CDl mol~ll~s function to prese~t foreign lipoarab;,..-., A,.,.An a~AM) ~ntig~.n~ to T-cells. The i~ve.l~ioll is furthcr bas~ on the obse,~Lioll that icolqt~ blood mono~;~s can be inducod to e~rss CDl, and ~,~ro,e become co~ ~nt to present LA~ ~igc"s to T-cclls, by co,~l"r.~ the onoc,y~s with c~yto~ Based on thcse two o~se.~lions, thc ~ lL
inYen~ion ~ clo~s m~th~s of i~l~ti~ CDl+ LAM antige~,~ c~ ~l;"g cells (CDl~ APCs) which are used to ide~, isolate, and purify CDl~l~.. tl,d LAM ~liE,c,~ arious .n~ll.~c for ~1~t~ in;~ wl~clLcr a sample cr..sS~in~
one or more CDl-~,ese,lt~ IAM ~gc.,s, m~-th~lS for i.col~ting arld -~lc~t~ IJAJ~ n~ r~ C~l-p~es~t~d I.Ai c icol:~tr~ by ~he ...f~ll.n~$ ~ s~ here~ and --- It-~ of ~lo~iii~g and using i~ol:~t~Y3 CDl-~ " n~ in ~aCl~-h~s~
In one emboAim~nt~ ~e p ~ t ~lion pro~ides m~oth~s for ~1~ t,~.,",;~ e w~e~ler a saunple C~t:~;A~ a CDl-~ L~d I.A;M ~ntig~n In one such m~-.th~, the ~l~ncc of a CO1-~1cs~d LAM an~igen in the sample can be dct~ d by (1) Co~t~ t_e sample with celLs which ~ave ~UBSTITUTE S~EET (RULE 26) been in~uc~ to express a CDl protein, (2) c~nt~ n~ the cells from the first step with T~ells that spcc;rr~lly l C~,J';'~ a CDl-p~ ntig~n, and (3) m~ ri~ the prOlirf~ or cytolytic ~ Qn~e of the T-cells, wl~eill increased T-cell prQlif~-~ti~n or T-cell-~ A;~d c3rtolysis of CD1 S target~ells, ~cs~i~ly, CQ~ 5 ~ith the ~"fS~-nr~ of a CDl~
LAM ~ntiE~en In a related ~rn~;..-r-lt~ the pr~sent i~ iQll proYides for mf thods of ~e~ wL~ r a sam~lcc~-~t~ c a CDl bloCl~gageD$, i.e., a co~o~ilion that inhihitc CDl-~ ;- ~ LAM a~en 1~ ~--t~ t;~ the related emwim~nt~ ~e assay for Cl~ t ~ LAM an~igen ~
above is ~l~ed in ~tplir~ ~ith a first (con~rol) a~say being pe r~. .. ~1 as above, a~d a secon~ assay ~t1itinn~11y C~ ;~, a sample ~ ~ of c~nt~inin~ a CD1 bloc~ ager~ Thc p~ e of CD1 bl~c~i~ agents in the sam~le correlates with a T-cell prnlif~'ivc or cy~olytic ~onse in the second assay that is less than that measured in the first assay.
The present invention further provides mPthy3s for ;~Je;-~ CD1 expression in cells, such as mo~o~s, in order to e~Dn~.~t~ CD1+ LAM
an~igen-~lc ;~--1;~ cells (APCs). In one n~.otho~, CD1 ~ l"~ cm is in~UG~d in i~ol~t~d blood monocytes by cnnt~rti~ the cells with one or more cytoines. The ~cfe~cd cytoincs for CD1 in~uctinn are gramllocyte-macrophage colony stim~ tin~ factor (GM-CSF), GM CSF in c~mhin~ti-m with intPrle~lkin~ ~,4), or int~r~nlkin-3 ~3). CD1+ APCs are cells tha~
express and display CD1 ~lotcilL ar~l are thus ~lly~Ct~'~d to present CD
restrir-t~ ~ntigenC to TCR+ T~ells. CD1+ APCs are usell in sc~eIal of the m~-th~lc t1icclosed hereim l~e present invention further provides m~-th~ls for i.cgl~tir~ a CD1-p~ ntig~n from a sam~le. In one such m.othotl, a sam~le cont~ini~ a CD1-plcsc~l~ LAM antige~ is first frar~ion~te~l ILCing convention~l techniques. The res~llti~ f~etionc are then tested usi~g known s, or those di~Clos~ herein, for the ~ ce of a CD1-1~lesenLcd LAM ~ntigen The fraction cont~inin~ the CD1-p~ Led LAM antigen are SUBSTlTUTt SHtET ~RlJLE 26) W O 96/12190 PCTrUS95/13274 then either used in the development of VaC~ ~ S or are ~her fr~ticm~tP~ to obtain higher levels of purity of the CDl-~l~nted I;AM ~ntig~n The present invention fu~her provides ~ h~c for ~
CDl-l"~d I,AM ~tnt.ig~n~ from a sample which rcly on the ability of a S CDl-~l~se.~t~ ntig~-n to bind eithcr j~l~t~ CDl or CD1 c~,~d on a cell sttrf~e.~P., In one ~uch m.~.thnd, a sample c~ h~ l~ a CDl~ c ~
LAM antigen is ;~Y~vb~tt~l wi~ either CDl~ s or ~ A C~Dl mol~-tll~os Therest~ltir~ complexes of LAM ~1;g, ~ CD1~ APCorLAM
an~ig~n CDl mol~lle are tllen ,~ om the sample a~d ;,ubj~d to conAitinn~ in which the CD1 m~ l~s thc bo~ CDl~ 1ed LAM antig~n. The ,deas~d CDl-~l~d LAM antigen is then purified away from either the CD1~ APC or the ~---;f;rA CD1 m~ and may be further ch~r~c~eri7~ using conyenl;on~l imm~lnological, biocl t- . ~l a~dlor genetic m~th~l5. Purified CD1-~ese~ed LAM antig~nc, or synthetic or g~tir~lly engin~red derivatiYes the~eof, are then tes~d for CD1~
LAM antigen acti~i~r using Imown ~Locodu~C, or those Aicclos~ herein, and may be used in the form~ tion of ~ r;n~5 Utilizing the above~ s for iColztin~ a CD1-p~ed LAM
antigen, the precent invention further provides i~olz~A CD1-~A LAM
~ntigenc which haYe been ~c~d by the m~-th~5 Ai.crlos~ he~ L The isolated CD1-p-~.llL~ LAM ~nti~Pn~ ~d by the Ai~,los~d l~tho lc can ~e used either in the rhz ~c~ n of the Da~re of CD1-~,~nle~ LAM
~ntig~ n~, irl t.he deve~o~m~ont or fo~ ti~n of vao~s and lhr~ ;cs.

B~ef Descnp~on of fhe Figures FIG. lA-~I show data for the c~y~ion of CDla, CDlb and CD1c by ~onocy~s c~ltus~d with GM CSF aDd ~A, and surfaoe ~cn~ ~ of CD1b-lc,l.;-~1 T cells ~ for ~obs~tmum tuberculosis; Flow JIIIC~t- iC analysis of ~ ;~hF ~l blood ~ono~ s cul~red for 60 hours in m~ii--m co..l~ GM-CSF and IIA slluwillg c~ion of CDla (FIG.

SUBSTITUTE SH~ET (RUL~ 26) lA), CDlb (FIG. lB), CDlc (FIG. lC), and HLA (FIG. lD). Cells were stained with control mon~clQn~l antibody (mAb) (dotted line) or _Abs with the ~ i~ in cash ~ ,g,~ box (solid lines). Mo~s cultured in the ~bsenre of cyto~nes or with ~t~.re.o~y did not e~prcss Si~,-,r;r~,-, levels of CDla, CDlb orCDlc(da~anot~hown). F~ow .,~--~ h j~`
analysis of T~ell line DNl sll~w~g its r~of a:~B TCRs ~E:IG. lE~, non c~ ~sion of CD4 ~;IG. lF~, and ~1 or non ~ of CD8 (FIGS. lG, 1}~. Dot~ed and solid lines ~t conlrol and ~ c mAbs as in FIGS. la-ld.
FIG. 2A-E show data for antigen ~y and self l~~ ;nn of prolir~ e responses of CD4-8- T-cell line DNl and its subclon~, DNl.C7.
FIG. 2A shows data for the prolirf" ~ , r~onses (counts per miDute (CPM)) of 3H-thymidine incorporated) of DNl to M tuberculoszs (solid s~2ares), M.
Ieprae (solid circles), Escherichi~ coli (open circles) and tetanus toxoid (opensquares). Andgen ~lC5~ cells were ~erologous GM~SF- and treatcd CDl+ monocytes. Antigen COI~C~ I;on (based on protein content) is shown on the x-axis. FIG. 2B shows data for the prol;rC ~ response of T~ell line DNl to M. tu~erculos~ g protein/ml) ~equiL~s CDl + antigen pl`CS~ g cells ( CDl+ APCs). APCs inAie~t~ by symbols as follow: ~o APCs, open ~quare; GM{~SF and IIA ~:d ~uO~ lcS (CDl+ APCs), closed ci~les; IFN~ treated ~llono~s (CDl~), opencircles; freshly i~ol~t~l monocytes (CDl+), open !-;~1''S. IllC rnlmber of APCs added to each cul~re is shovrn on the ~c-a~s. FIG. 2C shows data inrlirat~ that APC's from all donors tested auppci~t~d thc pro!;rr,. ~ ~ of T-cell line DNl to M. tuberculosis. Open bars, T~ells plus APCs ~nthout M. tuberadosis;
solid bars, T~ells plus APCs ~nth M. n~e~ ~losis (1 ~Lg protein/ml). APCs were GM CSF and IL,4 treated P~ h ~ 211 blood ~ cl~ cells ~om five unrelated donors. HLA typing ~O ~r; -Pd that allele of the HLA-A, -B, -C,-DR, -DP or -DQ loci was shar~ asnong all five donors (data not shown).
I;IGS. 2D, 2E show data in-lir~ti~ that anti~Dlb mAb s~ ly iDhibited the proliferative response of DNl (FIG. 2D) and DNl.C7 (FIG. 2E) to M.

~UBSTITI iTE SHEE~ ~RLILE 26~

tuberculosis (1 ~.~g protein/ml). APCs were GM-CSF- and IL~treated mollocyh~s. Solid bars, prol;r ..~ o~ of T-cells to APCs with M.
tuberculosis (1 ~g protein/ml); do~ed lines, lC ~yon~e tO APCs m ~c ~
of M. ~uberculosis; ~nd,~ not dr~ M~ on~l P ~1;1~;~ used werc S P3 (con~l IgG), OKT6 (an~i-CDla), WM-25 (an~-CDlb; Fa raloro, EJ., a' al.,DiseaseMarf~rs4~ 270(1986)),10~3 (an~i~Dlc),W6132(a~i-M~C
Class I), and IVA12 (an~-~IC C~ 11; Shaw, S., Hunt. Irn~ 12:191-211 (1985)).
FIG. 3 show data for a CQ..yL~-;c.~.. oft~e abili~ of a~igen ~
cell lines CRl and cy~oldne ~imnl~t~ ~o~oc~L~s to s~mlll~t~ growth of T-cell lines 2.13.DNl and G7, cloDes derived ~om T-cell line DNl. Open bars, T-cells pl~ APCs wiLllouLM~ ~ube,cklos~s; solid bars, T~ells plus APCs with M. ~uberculos~ g proteinlml).
FIG. 4A-D show data for the ~ t~lion of M. tuberculosis by CDl transfec~ants of the lymrhobl~id cell line C1R. C1R cells stably ~ r. ,.~ ~
with vector pSR~-NEO DNA (mock) or with ~o~llu.:~ of pSRtY-NEO
con~tining cDNAs enr~i~ the ;~ ~ CD1 mol~ll~ (CDla, CDlb and CDlc) were cultured for 12 hours in ~ alone (open bars) or in .~
cn..l~itl..~ M. n~bercl~losis (25 ~g proteinlml, filled bars), labeled with 51Crand use~ as target-cells for cytolytic ~say with various ~ rr ~or T~ells. The ~ rr~or T-cell to target cell ratio was 50:1. ~G. 4A. shows for M. tb.
CDlb-~,~ Ag s~fir T cell line DNl. PIG. 4B shows data for DN1 sl~bclonç DN1.C7. PIG. 4C shows data for CDla aul~ clone B1~6.
FIG. 4D shows data for CDlc aulu~ clone 3C8.
~IG. 5A-C shows data ;-~ t;-l~ that CDlb ~ " of M. tuberculosis antigen does not reqaire MEIC Class II 2egfon ~r mo~ os~ but does involve antigen ~LOcc~ . by a chlo~ ui~ sensili~.
~a~L~ay. FIG. ~A shows data for lysis of CDl 1~ Ç~ by T-cell line DNl. 1~ -ells h~ç~icd w~th vector DNA alone (mock IL~r~c~LLl) are in~ir~ by circles, and T2 cells l-~r~f~ct~ ~n~ CDlb by t~i~lPs. Open symbols~L~nttarget~ellsnotyLe: u~ ~ withM tuberculosis, andfilled ~.llBSTiTUTE S~IE~T (~ULE 26) CA 02202680 l997-04-l4 symbols ~ se.l~ target~ells p~ei.~r~ ~ for 12 hours with M. tuberculosis (10 ug protein/ml). Flow cy~m~t~i~^ analysis showed ~hat inM~bation of CDlb ~ ~ ~n~rec-~:l T2 oells with M. tuberculosis had no effect on CDlb c ~ c~
(data not shown). FIG. 5B shows data i~ti-~ti.~ that g31lt~ratl1ehyde fi-~tinn of CDlb+ APCs ~ of ~. ~be~c~sis to line DNl CDlb+ APCs (GM{~SF- and ~Sed ~ ~...~1 blood ...~ h....rl~l cells, PBMCs) wcre a~ltured for 12 hours in ~c p.~sc~ of M. I~c,w.losis (1 ~g proteinlml; ~Pulsed APCs~) or in " ~rt;~ one (~
harvested and an aliquot of each oell ~ . was fi~ed with 0.0125%
gll~t~ ehyde for 30 se~4nAC The resllti~ APC y~"p~t;~ n.C were tested for their ability to stim~ prolife~tiQn of linc DNl in the ~bs~nr~ (open bars) or y~ese~ (solid ba~) of soluble M. Iu~ osis an~igen (1 ~g pro~ein/ml). FIG. 5C shows data for inhihition of CDlb t~tion of M. tuberc7l10sis by chloroquine. CDlb+ APCs from an HLA-DR7t indi~idual were pulsed withJ~. ~berculosis antigen for 60 ~ 5 at 37C in the p~esence of the iru~ir~t~ cow~ of chlo~e, fised with glllt~ ehyde, and used as APCs inprolir~ ~lt;~e assays with line DNl (solid cir~les) or with the M. tuberu~losis srecifir, ~ILA-DR7+ ~ lhA CD4+ T-cell line DG.l (open tri~ Sj. Results are e*,i~scd as percent inhibitir~n of l~o~es co~od to fL~ed APCs pulsed with ~. I~e~w~losis in the ~hsenre of cloro~lne, and are ~pl~i.e of ~hree similar ~
PIG. 6 shows data inrt,-~l"~ the effect on ~e prQl;r~ ~;~., .~,onse of T-cell line DG. 1 to M. tubera~losis an~igen of 1 ,g~ ~l ;QI ~ of an8gen with the intlir~tç~ plOteaSC5.
FIG. 7showsdatafortheeffectonthe p~Ot;r At;~,lC~ Se, of T-cell line DNl to M. ~ubercu~osis an~gen Of ~lig~ n4f an~gen with the i,.Air~t~
~lotcases.
FIG. 8 shows d~ta for the effect on the prolir ~;1t;~ .Ce of T-cell line DN1 to M. fortl~itum antigen of rtig~stion of antigen with the in~ti~t~d p~ot~ases.

~E 5BSTI~UTE SHEET (RULE 26) FIG. 9A-C shows data in~ir?ting that a Mycobar~, ;zl ~nti~n recognized by a DN a:~B TCR+ T cell line ~ z~ cly ~ ilio~s into the organic phase after ~ I;n-~ with organic solvcn~s and is CDlb F.Ytr~ tinn with organic so~ l2 A;rr~ t~5 ~c CD~
My~iob3e~ 1 antigen f~om M~ t~ cns 1~ ~ by a co.~ 1;nn~l MHC class II ~ Y3 CD4+ :~ T~ T-ce31 linc and dle small l-Ol~plO~ M~ 1 liga~ ; ~ by DN ~ i (V^f2V~i2) TCR+ T-cells. Pfeffer, K, et al., J. I,,, v ~olegy 148:575-583 (19~2).
Total Mycob~ ;~1 son;~fs ~rere e"~-- tJ~ with c~orofo~ Ihs~--o]/~20 and the re~lt~nt three phases were assayed by cllltl~ i~ T-cells with CDlt monocytes and the intlir~tf~ ltinnc of ~e ~anous an~igen ~le~ .n~.
FIG. 9A shows data for the pr~l;rf.~ once of the CD1b~ ~ DN
T~ell line DN1 to total ~ cob~4 ;~ t~s (-, dashed line), organic phase (O, solid line), ~ OUS phase (O, solid line) or int~ r~ (--, solid liIle). Antigen cQnr~ f~l;on along the x axLs is ~epirS~ as l/~ -tion nllnn~li7~ to the standard total sc!ni~t~ p~ A~;nn P IG. 9B shows data for the proliferative respo~se of the ~A-DR7 (MHC~ Mycob;~-. ;fl specific CD4+ T-cell li~e DG.l to Myco~e~-ri~il f~1~sir~nc after e~t~t~-tion with organic sdyenLc. FIG. 9C shows data for the prQli~ e re~sponse of the V~y2V~2 T-cell do~e D&.SF68 to Myco~t~ ;~l fT~sionc after c ~ ;on with organic solvents.
FIG. 10 shows t}}e cytolytic l~onse of the DNl line to CDl !-fr~r~ t-; of ClR cells pulsed with Mycob~ ;;l antigen ~ .4l;~tn~
CDlb or CD1c ~r~ ~Porcelli, S., et al., Non~re 341:447450 (1989)) of ClR lymphokl~tQ;d cells were us~ as targets in a st~n~ ytolytic assay pulsed either withM. tuberctdoQs antigen ~ a~ c after e~ n with organic solvents (+) or media alone (-). Recog~ilion by the T-cell line DN1 of ClR cells l.~ ~A vrith CDlb ocalrs only when pulsed with ~ntigen.
No an~gen spe~ific .~,co~lion occars for CD1c~ targets.
nG. 11 shows the ckP- ~iral ~l~uc~ of 6,~trehalose dimycolate (cord factor).

S~BSTITUTE SHEET ~RULE 26) WO 96/12190 PCT/US9~!i/13274 PIG. 12A-E shows data in-1i~ti~ that the M~cobar~~ an~gen lecog~ cd by the CDlb~ 43 T cell line DNl is mycolic acid. FIG.
12A, 12B show data ;.~ that the pr~l;r ..,I;~c ~s~ e of the CDlb-T~cell line DNl c~l.,l~s with mycolic acid p~ on reverse phasc S C18HPLC. The~llnfi~Myc~ ;al acylc~ain r. ~ c~n~ allthe CDlb-l~l.; ~ an~igcn was c~o~J~h~d us~ revcrse ph~ce HPLC and the re~ ~ fractions ass~r~ for ~e abil~ty to s~imlll~b~ a p~;r"".,;~
~onse by thc T~ell linc DNl. FIG. 12A shows dabl for di~lay of the Lqhso~L.~ I~re ~OCh ~ at 254 a~ h~ as optical densit~ ~its, OD, ~ 104) (solid line) of the cluted mqtenql and t_e ~ .~ ~;r~ methylene chlor~e con~ ;on (dot~ed line) of the elution g.~ t. The large abso~ance peak elut~g I~L~.~ 2 to 6 I";,---t~5 is free br~mo~h~
bro~ude, the de~ g agent. FIG. 12B shows data for the prOlirr~
response of the T-cell li~e DN1 to each one minute r~ n. The CDlh-l~ll jr~_ an~igen ~o,.sc is seen as a broad peak oo~lel~g with mycolic acid. FIG. 12C shows data jnAj~ti~ that ~ eA 6,~tr~halose dimycolate (cordfactor),butnotsaponifiedtreha}ose~ lp~stim~ t~sap~l;~ ;vc r~onse by the CDlb ,~ ~ T~ell line DNl. Mycolic acids were ge~-~t~d by sapo~ ;on of pl- ifi~ treh~lose dimyco~ om either M.
tuberadosis (~I37Ra) or M. k~.i. Treh~l~ce Ail~h lU~e (synthetic cord factor) was treated in an iAentic~l f~hinn ,Anti~n C- n~ l;n" i~
in ~g/ml of cord factor along the x ais. ~IG. 12D, 12E show d~ for the reversed p~se ~LC aualysis of ~- lr;~ ~O~ dilLI,y~ol~e from M.
tu~era~losis (H3 7Ra) results in the~ m~ tion of the CDlb~ A, T~ell li~e DNl by La~io~ O~g to mycolic acid peaks. The sapo~,ed trehalos~ dimycolate of M. tu~sLs was c~ , rh~l as i~l the c~C.;.n~..l shown in FIG. 12A (FIG. 12D), and r.~c~;- nc assayed for the ability to induce a prol;r~ ,al;~e le,l,or~e by the line DNl (PIG. 12E). As in FIG. 12A, bioactivity collclates with early mycolic acid peaks.
FIG. L3 shows data for t~e cytol~tic l~OI~ of the DNl T-cell lule to CD trancfe~-t~nt~ of ClR cells pulsed ~nth mycolic acid ple~ ~d from M.

~UBSTITUTE SI~EET (RIJLF 26) W O 96/12190 PCTrUS95/13274 tb. cord factor (Sigma) by saponifir~tion CDla, CDlb, CDlc or mock t~ncf~ct~ntc of ClR lg~nrhnbl~ct~id celLc were pulsed with mycolic acids d from trehalose dimycolate (+) or media alone (-) and Lced a_ targets in cytolytic assays, the results of which are giYen as % D~-;r~- ly~is.
S ~G. 14A~C shows data intljr~ti~ that mycolic acid is ~ot ~ pC.~i~C, but a s~ igen l~l. ;~ by CDlb and ,~ ; ~ by thc T-cell li~e DNl. Four T-cell lines ~ ;rc for M~ d two ~*i~l T-cell lines were t~sted for the ability tO ~ to eithcr total U. ~berallosis so~ s, mycolic acid ~ ~I;n.~c from pl-nfi~ cord factor or HPLC
0 pu;~ied mycolic acids from ei~er M. tb. s ~;r~fs or cord factor. The re~onses of three re~ e MycQ~ pecific T~cell lines are show~, DNl (--) (DN, CD1b-~ict~, ~Y:,B TCR+), DG.l (O) (CD4+, HLA-DR7 ~tr~etux~ :,B TCR+) a~d DN6 (O) (DN, CDlc-lf;~ r~l A, ~ p~
TCR+. APCs for all si~ T~ell ~lC`S tested were identir~liy GM{~SF- and IL~trea~l (CD1+) PBMCs from an ~A-DR7 Losili~., individual. FIG.
14A (upper paneV shows data for the proliferative re~onses of three Myc~bacte~ ;r;l~- T~ell lines to total s~ r~s of A~. tb. (H37Ra, Sigma). Antigen C4~ ;nn is displayed on the ~c asis as cpm~c 1~3. The three T~ell lines shown all ,es~ro~d to total Myco~.et~ sonir~t~s FIG
14B (middle panel) shows data for the prol;r~ ~ to HP$~ r!~ ;r;~d mycolic acids i~ol~t~d fromM. tb. s/)~ t s. OI~1Y the CDl~r~ d T~ell line DNl l~o~ds to pl-rifi~ mycolic acid. ~;IG. 14C (bottom paI~el) shows data for the prolir r~ , lC~l~n ~S to HPLC-purified mycolic~cids g~
from ~r!-~ ;ri~ M. tb. cord factor (Sigma). Only the CDl~~ T cell line DNl pr~l; rf., ~t~s in l~s~nsc to co~d ~actor mycolic acids. Not ~own are lhree ~d~litin~l T cell l~es ~ed in ~e same ~ , SP-P3 CE2Q ~ rlo~
M.G., et al., J. Ex~. ~ediane 16~:2139-2152 (1988 (CD4+ ~:,B T~+, DRl~l.;r~, te~ sto~oid specifi~), CP.1.15 (Mo~t~ C.T., etal., ~r. J.
Il,u,~u.ol. 21:2999-3007 (1991)) a)N, V y2V~2 TCR+, M)~ob~c~
s~e~;r.r,), BK6 (Porcelli, S., Na~ure 341:447450 (1989) (I)N, ~:,B 1~+, ~u~o~c~ , to CDla). All three did ~ot ~ d to purified mycolic acids, ~IJBSTITUTt Sl lEET ~RU~E 26) but two prolife~t~,~ in response to their s~ifir an~igen (tetanus toxoid - SP-F3, <1 kDaM. t~berculosis ~ z~ nTl - CP.l.15).- BK6~ c cytolytic activit,Y against CDla, but is unable to prQlifç1~te in ,c5~n~ to C~Dlat APCs of any type tested. Porcelli, S., Na~re 341:447 450 (1989).
FIG. lSA-B shows data; Y~ e e~fect of the intlir~
m~m~rlc~n~l ~ntiboAi~s on the prQl;r~ ~ of T cdl l~ne 2.13.DNl (PIG. LSA) and 8.23.DNl (~IG. I~B).
FIG. 16A-D shows da~a for CD3.C-~5.d,;.~ t:.l;nl~ of A~
tuberczclosis an~igen to T-cell line DN2. The results of ~ytolytic assays of CRl cells tt~n~fe~ted with vector (mock, FIG. 16A) and with DNA molecules en~oding the jn lir~t~d CDl protein (CDla, CDlb an~ CDlc) (FIGS. 16B, 16C, 16D, l~ively), wlle~e~ the I ~r.~r.~ d cells were either preincllb~ with (filled circles) or without (open circles) h~ erculosis.
FIG. 17A-D shows data for CDlc~ )t;~ n of M.
tuberculosis ~ntigen to T cell line DN6. The results of cytolytic assays of CRI cells transfected with vector (mock, FIG. 17A) and with DNA moleeules er1r~~ the intli~t~ CDl protein (CDla, CDlb and CDlc) (l;IGS. 17B, 17C, 17D, re~ectively), ~L~.ein the h~f~d cells were either prein~b~t~ with (filled circles) or without (open circles) M. tu~erculosis.
FIG. 18 shows data for t}~e 1~l;r ~ . r~onse of t~e CDlc-re~i~d cell line DN6 to M. tb. ~ntigens in Sol~ir~t S after e~-ti- n of the antigens with orga~uc solvents. Prolir~ ~I;n~ is in cpm (3H ~u~ olation) displayed on the y axis. APCs were CDl C~lCS5~ l~Ol~OC~ 5. ~ntigen~
were ti~ered oYer 6 logs and the results from a ,e~Li~. point (1:3,750 ~lilntinn of antigen) are shown. Bac~uul~d cpm (defined from a media alone CO~oV were ~ubt ~ct~ ~om all values.
FIG. L9 shows data for the prQlir~ O~ of the CDlc-n~ le~ cell line DN6 to A~ ntig~n~ in sQ.~r3~s before and after sa~nifir~tion of ~e ~nfig~n~. Prolir~ ~pO1~5c ill cpm is displayed on the y axis and the co.-~c ~ ~ior~ of antigen (shown as lI lillltion) is displayed on the x axis. The equivalent of 10 mg of M. tb- (strain H37Ra; Difco) was SJBSTITUTF SHEET (RULE 26) Wo 96/12190 PCT/US95/13274 soni-~tffl in PBS and was either used directly or first sa~onil1ed. All andgen dilutions were norrn~li7ed to the s~d initial con~f~ ;on of 200 mg yophili7~d b~et~ in 5 ml.
FIG 20A-B shows data ;~ that DN a~ T-cell lines ~om S leprous skin lesions are CDl-,~ d FIG. 20A (upper panel) shows data intlinati~ that anti~Dlc m~,b ~,~.cy~ lly ;..h;h~ the ~"Ol;r4~ cG
of T-cell line LDNl to M. I~r~e. FIG. 20B (lowcr panel) sho~s data inAiC~ti~ that an~i-CDlb mAb ~c~:r;~lly ;llhnJitl ~ theprot;r. ,~
of T-cell li~le LDN4 to M. Ieprac.
FIG. 21 shows da~a for the prolif~aL~e ~ onse of T~ell line LDN4 to the int~ t~ cellular rldc~ionc of M leprae.
FIG. 22 shows the chP ~ir~l ahu~;~uie of l~poara~ n (LAM).
Abbreviations: Many = lll~oy~l~ose; Araf = arabinofilm~ose.
FIG. 23 shows data ;~Air~ that the T~ell line LDN4 re~onds to LAM in a CDl~res~icted manner.
FIG. 24 shows data for the l~nse of T~ll line LDN4 to LAM
delivdliv~s. ~bbreviations: dLAM = da~latc~l LAM; PIM =
phosph~ti~ylin~nsitol ~ .,osi~e.
FIG. 25 shows data j~il`~ti~ that the T~ell line BDN2 .~yonds to LAM f~om M. Ieprae (L~ LA~ as well as LAM from a clinical isolate of M. tu~erculosis ~TBE LAM) and a ~rimlen~ labolat~ ain of A~.
tubercl~losis (Rv LA~.
~;IG. 26A-B shows data for the flo v ~k~ h ;C analysis of OGDl and 0AB8 T-cell lines. OGD1 T~ells were stained wit~ biotin conju~a~ anti-yô
TCR monoclon~l antibody TCR~l or anti-Vôl ~IlOI)~lO~Al an~l~d~ ~CSl (revealed with P~Llc~t~vidill), (PIG. 26A). ~AB8 T-cells were ~a~elled with anti~,B TCR mon~lon~l anhlx)dy B~A031 (followed by PE-~ll~t~vidin), anti~D~llC mC!n~lOn~ tibody 0~4, anit CD8~hain-FITC monoclonal antibody ¢)~8 or anti-CD8-~ Tl C 2ST8-SH7 (~;IG.
26B).

~UBSTl~UTE SHEET (RULE 26) FIG. 27 shows data inflir~tin~ that the TCR ~ T-cell line OGDl responds to Mycobacteri~l ~ntigpnc which pa~Lition in the organic phase of a chlo~fol~Jm~oth~nOl ~ractiQn For the organic l'"~ n, whole PBS
so~-ir~tfs of My~obn~ ~berctllosis ~DIFCO, ~etroit, ~) t200 mg S b~etPrial5 ml PBS) were ~ (4:1, v/v o~ic to ~
chlor~fo.... n,PIh~nol (2:1, v/v).- Cord factor wa ~i~ ~m SIGMA
rh~-"i~1c (St. Louis, MO). To ~ i free ,~ olic acid, cord factor wæ
saponified (25% KOH in "~lh~ol- H20 1:1, 121C, 1 hr.) and mycolic acids eYt~c~ into h.o~nP rl~a~ ~t;n~c were dried ~th ~2 and ~ c~l in media/fetal calf serum and tested at ~; r~, e- " ~ tionS in standard prolifPration assay (50,000 activated monocy~s with 50000 T~ells in a 3 day a,ssay).
FIG. 28 shows data ;~Aif~ thatthe TCR y~+ T~cell line OGD1 is restricted by CDlb, a tlemo"cl~t~l by ~ytotoxic assays of the TCR y~+ T-cell line on antigen pulsed activated monocrl~s. To prepare target cells, the monocytes were in~bat~l ovemight with no andgen (open bo~es) or the organic fraction (filled boxes). Target ceL were t~en l~bell~ with 5lCr and T-cells were added to the target cells at a ratio of 10:1 with different bloc~ing antibody (no monoclonal antibodies, anti-MHC I (BB7.7)), ni~L~ of all anti-CDl monoclonal antibodies, and~Dla (OKT6), anti~Dlb (~M25) or a~ti-CDlc (lOC3)). The mea ,ule~.lL of the ~lot~Iirity was r~k~ t~ as in standard cytotoxic assays, i.e., specific cytolysis=(~
SPO11~ US) X 100.
FIG. 29 shows data il-Air~t;~.& t~at the CD8+ c~+ T cell line 0AB8 onds to Mycob~ 1 mycolic acid. MycoIic acid was purified by HPLC
from M. tb. (~l~p~,d as ~le~~ in ~ 4) and us~ at 1 ~
Conlrol an~gens are Dirhth~-ri~ to~coit (a~ailable-~om Dr. R. }~i~c.g, Dar~a Farber Cancer In.ctit~ , Boston, used at 10 ~g/ml) and A~. tb. LAM (as in Exarnple 8; 1 ~g/ml). Fr~rfinn~ were ~ A~ in medi~/fetal calf serum and tested as describe~ in FIG. 27.
FIG. 30 shows data jn li~tin~ t_at the CD8+ ~+ T-cell line 0AB8 is res~ricted by CDlc, as demo~ t~ by proliferation assays ~c~ro~ed ac i.n SUBSTITU~E SHEET ~RULE 26) FIG. 27. Monoclon~l antibodies were added to the culture at a 1/200 final ion of ascites. ~ntigenc are as ~5l`~;1~ in FIG; 29 and antibodies are the same as in PIG. 28.

Desc,.~t~on of ~ie Prefened Embrdi~enf-~

SGlossary Ant~gen: A ~nol~lle, c..~a~d or c~ of matter ~hich (I) induces an i~."..~ response in an an~mal, alone or in c~mhin~tion with an adiuvant; and (2) ihl~.acts s~ f~ ly with one or more antigen-r~og~g co-,-pone~ of the animal's ll1111l4'~- systan.
0Foreign an~igen: An antigen which is not endoge~,ous to a normal, healthy animal Au~ou""~,e an~igen: An endogcnous mol~ d or composition of rnatter in an anima~ which colupl~es an antigen in an ~u~.n~l~lll..n li~se. This term is S,~Ou~lu~ with ~self andgen~ and 15~ lo~"~;gen.~
CD1-presen ed an~gen: An andgen which is bound by a m~mh~r of the CD1 family of p~otc~s and displayed on the surface of an G01~ APC.
CDl-pr~u~ wls vary in their s~e and c4~ ;nn ~c~ ~ on their origin a~d the ~r of CDl family that they are ~ A by. As used 20herein, the term ~ ~ antigen~ c5 those ~n~ ns jtl~ntifi~
herein and/or those. ~ C ;~ t~ using the known y~ccAI 1~5 or those i.~losed herein. This term is ~l~OUs with ~CDl-f~ ntig~o.n "
~CDl~ound an~gen~ d~Ci~n~t~C a CDl-~a antigen that is bound to its appl~liate CDl mole~ule.
25'.,~M an~igen refers to antigens co~ at least one lipoarabinol.-anr~ or a de~i~ali~_ thereof.
CDI fami~ of proteins: A co~ )n of ~O~s which have been ntifi~d by ~eir struc~re, I.,.,..l~-ologic cross-~eaCLi~L~ and/or distribution, ~UBS~ITl ITE SHEET (RUL~ 26) W O 96/12190 PCT~US95/13274 as being related to }alown CDl mol~ll~. A SpeCiflC CDl protein may be ~ef~ d to as a m-omher of the CDl family of ~,ote,ns. h~mbe~ of the CDl family of ~ ,~s in~lude.t but are not limited to, CDla, CDlb, CD~c, CDld and CDle (see, Porcelli, S., etal., n1tu~ Rev. I20.137-183 (1991)).
S CZ)I pos~vc ccll: A cell which ~s and d;~l~ one or more momhe~ of the CDl faily of ~lO~S. This ~m ~
~rCDl+ cell.~ One skilled in the art can use 2he },~ herein, or known in the art, for ~t ~-,...:. :~ ~l~c~ a a~ is ~y,~ oie or more m~ ; of the CDl faily of ~ s (see F.~mrle 1 and Porcelli, S., Inu7uul. Rev. 120:137-183 (1991)).
An~igen-p~ .hng cell ~ An APC ~ l~ any cell which displays an~igen mo~ es on its surface ~ia protein carriers and which ~e~ls antigen to T-cells. An~igen-binding protein carriers include MHC
class I molecules, MHC class II molecules and CDl mol~-l~; col~cs~onding APCs are ~c;~.~ d MHC I+ APCs, M~IC II+ APCs and CD1+ APCs.
CDl-restncted T-cell: A ma~re TCR positive CICR+) T cell which can recogri7e a CDl-bound CDl-~l~d ~nti~n CD1-r~ ~i T~ells include any subset of T cells which interact with a CDl-blound CD1-pl~sen~d antigen.
CD~ ~eU: A mature TCR+ T~ell which does not e~press CD4 and CD8. This te~n is ~llOUs with "doublc ~gaL~, T cell~ DN
T~ell. ~ Tecl~i~lu~c for ide~ir~i~g CD4~ T-cells are well known in the art and can readily be employed in t~}e present ~nliol~, for ~ .le using flow ~ ~Eh,~ as d~ ed in ~le 1, andlor in r~ h~, G., ct al., J. ~mnu~o. 147:3360-3369 (1991)). Using such ~ s, th;ree CD4-8-T~ell lines, d~i~t~ DNl, DN2 and DN6, have bee~ a~ and a~e described herein.
Adju~ r~; A mol~ll~, eo~ou~d or co~silion of matter which, when in~oduced into an animal with an antigen, enh~nr~s one or more ;~ r lO5 ~ L5es to ~hat ~nti~n SUBSTITUTE SHEET (RULE 26) W O 96/12190 PCTrUS95113274 Genetic engineering: rbis term refers to any human mqnir~llqtion ~-n~e~ to introduce genetic change.
5an~1e: Any sollltinn~ emlllcion~ J~r.~.n,~ or e~tract which can be tested using known y~O~l~s, or those ~ ~d herein. A ~le may be, S but is not limited to, a volublc e~tract or an t~ e~ E~mp~es I and 2 prwide various non~ ypcs of cqmrles dcrivcd ~omM~ob~ ~,.iu,.
tuberculosis.
C~ontn~ng: The process of ;~ bol;~ or plac~g in ~lu~ one item with another. For eYq-n-r1~, when a cell is ~o..~ with a sqmrle, the cell is intylbat~ with the c~qmple~
Fracnonanng: Subje~ g a ~le to cor~;l onc or ~lv~lul~s which sep~ate the components of the sample based on physical or ch ";~A1 plopc~Lies such as, but not limited to, size, char~e, solnkility, or co~>o~i~ion.
E~smples of fr~ction~ti~n ~lO ~ d ~ csin~ de. but are not limP,ed to, sel~i~, ylcci~ilalion~ organic ~ n, size ~ siO.~ dialysis or clllO~t~a~hy, and ion ex~r~e chromatography.
E~re~,ng: The process of produci~ a product involving ~ .tion of a DNA molecllle to gc~ ate a CCill~ ~O ~ Ig mRNA mol~ll~o that is optionally tr~n~l~t~ into a polypeptide by ribosomes and ~ t~ cellular factors.
Di~pkying: The process of loc~li7in~ a protein, or a ylo~ .~ A.n;grn c~mrl~,.to the u~ ~l surface-of a cell ~vhere the protein or ~ro~ A..li~en comrk~ is ~c~ibl~- to a second cell or to mol~lt~
displayed by a second cell. A protein, or a protein-ar~igen cQ~ -, is said to be displayed by a cell when it is pr~ t on ~e u.J~ O~l surface of the cell and is thus ~ hle to a second eell and/or to ~ d by a second eell.
~oc~.,u~g of arztigen: The proeess by which an antigen is trea~ by cellular factors in order to be rnade colu~tent for di~laying.
CDl bloG~g agent: ~ co~osi~ion or c~o- lld which is eapable of blocldng the .n~c~ion of a CDl-~ tcd antigen with CDl, orof blocking SUBSTITIJTE SHE~T (RULE 26) the inte.dc~ion bc~-.cen CDl:andgen complexes and their cognate T-cell l~ptO~. Blocking agents include (1) agents which bind to CD1, (2) agents which bind to a CD1-y~ t~d antigen, (3) agents which bind to a CDl:A.-~i~n co...~l~, (4) agents which bind to a T-ceU ~ t~l that S l~COg~S a CDl A~ n c~ lr and (S) agents which p~ ~1 the p~ c~...g of a CDl-y~ t~ Anti~n The Present Invention The present invention is based on the u~A~tcd obse. ~alion that CD1 moleculoe filnrtion to pnesent LAM zl~2;gc -~ to T-cells. The invention is further based on the observation that cells can be ;.. ~J~d to e~ress CD1, and therefore become co~ L to proee~t a.lligc"s to T~ells. Such p.~ t;on can involve cont~rtin,~ the cells vith cytokinoe such as, but not limited to, granulocytel~cl~yhage colony stinm~ p fa~;lor (GM-csp) and intrrl~llkin~
(~A).
Based on these two obse.~alioQs, the present invention cc.,.~ es.
_ m.-th~s for d~ t~ the yiese~ of a CDl-~lcse.lt~d LAM an~igen in a sample;
_ mPth~c for icot~tin~ such CD1-pl~i ~t~ens and the isolated ~ntigenc;
- ~ac~ s ~ cD~ ~d A~ cQ~ and ~ m~th~s m.oth~c of bl~t~ CDl an~igen ~lc ~
mPtho~s of ide~ir~g and/or icol~ti~ CD1 blocking agents and the icol~ted CDl blocking agents;
_ m.-thgt.c of ;".t.~ o~
- T-cells for use in the m~thr~rtc ~ O~d herein.
In one embo~ , the p~cent ~ lion pro~idPs m~th~lc for ~lc t~ ........ ....;.-i-~ whet~er a sample COI~ c a CDl-pl~t~d ~nti~en In one such mPth~, the ~.~se~ of a CDl-p~ t~ an~igen in a sample can be de te ~ ~ ~; n~ by: f~;t, col ~ the sample ~ith a CD1 ~osiliY~ cell; second, SUBSTITI iTE SHEET ~RULE 26) -c~ntarti~ the cell of the first step with a T-cell; and third, m~oaQlri~ the pro!ifer~ n of the T-cell.
Mçtho~s of chat~c~ classes of T-cells, a~d of icol~ir~
subpop~ ti~n~ of T-oells, are l~own. See, e.g., Wyso~i, L.J., and Sato, S V.L., Proc. N~l. Acad. Sa. (U5~1J 75:2844-2848 (1978); Wasik, M.A., al~d ~0~ 910, C., J. Imnu~l. 14~:3334 3340 (1990); u~,.;...~.~, G.R., ct a~, J. rmnr~ru~l 145:42062414 (1990); ~oulo~a, L., a al., J. ~I~"",~l 145:2035-2043 (1990); St~, ~, aIld Male, D., rh~r~r 25 in Imrr~nology, 2d Ed., Roit~, I., ~ al., ods., Gower M'Air~l Publishing, ~ ~n(1~n, New York, 1989.
MethrAs of ~lltllrin~ T-cells in vi~o, and of immortali7ing T-cells ~a fusion to non~rowth ~c~ ~ cells such æ mye~ , are also knowlL &e, e.g., Paul, W.E., e~ al., l'l~ture 294:697-6g9 (lg81); WilLiams, N., Nature 296:605 606 (1982).
Techniques for ldc.~lilying T-cells subsets tha~ are reac~ive with CD1 p~ ed andgens are also kno~n in the art and can readily be employed in the present invendon, for e~nlrle using flow c~ hr as described in the E~amples andlor by known ~~ s such as those deseibed in p~h~ .oorthy, G., et al., J. Imnu~. 147:33643369 (1991)). The present inven~ion ad~a~s these t~hniquoe by plC~ridi~ m~hn~s for ~nri~ hi~ T~ell pop~ Si~n~ to obtain j.col~t~d T~ell clones which are ~ . to CDl-plese,~Led?"~.g. n~ For e~mrle, a popl~ inn of T-cell;s is allowed to di~ride and a ~Ub~ tion of mi~ed T-cells is ~olated based on p~l;f~l;on in the p~ of CDlf APCs and CDl-~lese,lt~d a~igen, or on cytolytic activity against t- a- ~<ç~.t~l~ cells c~ g CDl n ol~ll~ s in the y~ c~ of a CD1-L~ 5mtiE~n US~ng such l~.oc~lu ~s, non~ e~ of such CDl ~ . T cell subsets have been j~l~.ntifi~, ;,r-l.~Ai,~ CI~4-8- T-cell lines, CD8+ TCR a~+ T-ceU lines, and TCR ~y~+ T-ceU lines.
The present invention further provides mf-th~S for inducin~ CD1 e~.ession on a cell. In one such m~th~, a cell can be in~luce~ to express CD1 by co~-~; rl; ~ the cell ~ith one or more ~lO~S, as known in the art.

SUBSTITUTE SHEtT ~RULE 26) CA 02202680 l997-04-l4 W O 96J12190 PCTrUS95/13274 The pser~ d cytokines for CDl ;n.l.~ I;on are gT~n~-locyte/"la-;lo~ge colony stim~ ting factor (GM-C~S~, GM-C~SF in CO m ~inqti~n with i ntl,l/.-k;.. 4 ~
4), or inl~ -k~in_3 ~3). F. ---,lr 1 Aicrln~es that Luonoc~L~s can be ;~A~JCQi to e~ress various ...( n~;, of the CDl family by cc. ~ the S ~on~j~ ~ith 100 units cach of GM CSF snd lIA for ~0 hours in RP~-1640 supp1~-m~nt~ with 10% fetal calf ~ rn. Using t~e . ~ C as~d m ~t~ri~ 5 p ~ereLn, one ~kiled rl the art can readUly vary thc COnt~cti~ tLme, c~4~iuc Iypc, cytokinc CQn~ ;nn~ aJId c4~t~
cQnAitiQnc to obtain favorable results so long as the ~r"~ SteP iS
sllffiri~nt to induce CDl CA~SidL1.
Several ~roc~lul~s are known in the art for ~et~ the proliferation of T~ells an~ can bc used in the above mf~tho~lc. One skilled in the art can readily adapt such pn~lu.~s for use in the present invention.
One such pro~edure, described in FY~mple 1, ~ul~s the rate of incoIporation of 3H-thymidine ~ria liquid s~intill~tiQn and by m~th~c described in Morita, C.T., e~ al., Ei~r. J. In~nol. 21:2999-3007 (lg91)).
The present invention fur~er proYides mPthr~As for isolating a CD1-~sented antigen from a sample. In one such T-~tb~ a sa~nple is first fr~etion~t~ using c~ ;on~l ~r~l"les. The f~ction~ of the sample are then tested for the ~se~x of a CD1 pl~d antigen as o~ i.~ above.
Examples 2 and 3 descr~ c f~etiQn~tinn p10~lUles USiDg organic e~trac~on ~vith chlorofo~ l and silicic acid c~oll.aL~g,~pl~ to f-~I;o~ a sample cor~ g an actract of M. tuberculosk to purify a CDl-pl~nLd ~nti~ n 2S The present invention further provides m~th~s for isolating a CD1-~ed an~igen w~ich relY on the ~ Iy ~f binding of CD1 to a CD1-ese~ed antigen. In one such m~th~d, a sample CO~t~ a CD1-~ od antigen is first cont~rt~d with either purified CD1, or a cell w_ich C ~JlU;SCS
and displays CD1 (a "CDlf cell~). The resul~ng ~ntig~n-CD1 c~mple~, or ~ntigen CDl+ cell complex, is then se~ f~i from the sample. Using such a procedure, a purified antigen:CD1 complex or antigen:CD1~ cell complex ~UBSTITUTE SHEET ~RU~E 26) WO 96tl2190 PCTIUS95/13274 is obtained. To further purify the CD1-~,ese~d ~ntig~n~ either type of c4mplex is treated under a~p~ ~ con~itinn~ such that the CD1-bound antigen will be released from the CD1 molecule.
The above nvo i~ n ,..-1l.~$ can be comhin~ by the skilled artisan to derive other .,rll,yl.c for ~ ti~ CD1~ tl ~ , In one such cnml.;n~ n, a sam~le is f~-~;o~A~ as ~rJn~ above, ~rior to ~-r~ ~ a ~ ;on method which ~elies on the b;~ of a CD1-Ld antigen to CD1.
The present u~c-l~ion fur~er pro~ t~l ~ ,c nc which are identifi~:l or isolated ILing known ~ es, or those li~los~ herein.
Unlike MHC-~r~se"lod ~ntig~nc, CDl-~l~d antigens are not limited to polypeptides. One CDl-p~ t;~le ~ntigen~ d~b~ in detail in Examples 2~, is a lipid antigen isolated from M. tu~era~losis that co~ ;~s mycolic acids. Another CDl-~ t~l antigen from M. tuberculosis, described in E~amples S and 6, is a more c~mpl~ lipid. A CDl-~d antigen from M. Ieprae is lipoara~;n~ nn~n (I,AM), described in F~mrl~C
7-9. CDl-pl~nt~d antigens are hydrophobic and have use in vaccine form~ tion and deYelopment.
The CDl-~ ntigenc of the present invention, i.e., those id~nfffi~ or icol~t~ using known ~lOC~u~S, or thosc tlicrlose~ hercin, are readily usable as v~rCin~-s. A s~lled artisan can cmploy routine formnl~tion ~r~ccduLes in order to fo mlll~t~ an isolated CDl~~ d antigen for usc as a vaccine. In ~ itit~n tO the C~1 ~lese~t~,d antigen in such ~rr~ s, the ~aeCLL~ can further CO~ ;ce at least one nd~litinn~l C~1 mol~ll~ or at least one MHC-I or MHC-I~ nti~n See, e.g., R~,~,.g~,.'s P*~rmn~rrff~7l Sciences, 18~ EJ1., GeDD~ro. A.R., ed., M~ck, F~ton 1990;
The P~Iannacologist Basis of Tf~lics, 7th Ed., t3ilm~n~ A.G., et al., eds., M~r~ n~ New York, 1985; Avery'sDn~g ~re~ Princip~es and Practice of Ciinical Pharmacolog~ and Therapeuncs, 3rd e~lifion, ADIS Press, I~d., Williams a~d Will~s, R~ltimore~ MD (1987).

SUBSTITUTE SHEET (RULE 26) The CDl~ sent~d antigens of the present invention can be purified as ~ c-lose~ herein over a wide range of purities. A sldlled artisan vvill know how to employ various l,...;fil~z~ n ~ ;~ in order to obtain a CDl-p~Led ~nti~n which has been purified to the e,~dcnt ,~u~ or an intPnrl~ use.
The v~cc~cs of the present invcn~ion can be forrn~ g a purified CDl-~ t~l an~igen or can be fo....~l~ uQng a CDl~ound ~ntigen ~CG CDl~ hA .~I;g~c are p~ ~ to T-cells ~s a c~mrlPx of ~nti~:n and CDl, the use of an ~.~I;g~n CDl c~ pk~ can, in some c~ses, provide s ~rPrior i.. ,.;,~;on pro~.lies.

SUBSTITUTE SHEET (RUEE 26) The CDl-pll,sellled ~nti~enc of the present invendon can be ~An~ pled to vertebrate ~nim~ls, inrlllAir~ ----alc, The vaccines of this invendon will have both human and ve~cli~Ly app~ ;nnc as ~lo~ ic and ~ .JI;r vt rc;~cs Further, when used thr,..~ lly, these ~,~s ca2l be co~ d with chemotherapy to ~r~duce, a more effecdve h~.. f.n of ~ny ~ cr.
The a~tigenic m~t~ri~lc Can be employed in a~ ue with c~ ;r,n~1 excipients; i.e., ph~ y 7~llt~hle organic or ;n~lg,.n;r carriers ~rhich do not deleteriously react with the i~ logically-active C4...pOI~f nt~ and which are suitable for parenkl~l, mllcos~l, or even topical applir~tinnc Such a carrier incllldes but is not limited to saline, burr~l~ saline, dextrose, water, glycerol, ethanol, oils, and comhinqtion-c thereof. The carrier and compositdon can be sterile. The forrnuladon should suit the mode of ~fl...;n~ tion P~enteldl a~minictration can include the introduction of sllbsl;1nces into an organis~ by intravenous, subcut~nfous or ~tl".".~sc..l~r meanc~ inr~ ing by imr!~nt Mucosal ~minictradon inrllldes plllmon~ry~ int~n~c~l, oral, vaginal, or rectal a~minictradon.
The carrier can be added to the vaccine at any convenient dme. In the case of a lyophilized vaccine, the carrier can, for example, be added imm~AiAtPly prior to ~tlminictration. ~1le . IA~i vely, the f~al product can be mAmlf~rtllred with the carrier.
The present invention provides- a Yariety of pl.A~ A~c llirAl composidonc.
Such compositions comprise a the.dlx~llir-Ally (or prophyl~rtir-Ally) effective arnount of an antigen or a CDl:Antig~n comrl~Y, and a carrier. The composition, if desired, can also contain minor amounts of wefflDg or emulsif,ving agentc, or pH
2s b~rr~,~gagents, or~lcS~l~aLii,S. Typical p~eS~Ya~ nr1~de,~o!P~ u~--sorbate, sodium metabisulfite. methyl pQ~ben~ propyl ~ben, Ihi~lle~osal, etc.
The composition can be a liquid solution, ~u~pe~sion, ~rmllcion, tablet, pill, capsule, sllstQin~d release formlllQtion, or powder. The method of ~ministrationcan dictate how the composilion will ~e fonmllQt~ For example, the composition can be formlllQt~ as a ~up~Osiluly, with ~itir~nAl binders and SUBSTITUTE SHEET (~U~E 26) carriers such as triglycerides. Oral fonmll~tion can include plant In~t~rl~lc, Haq, T.A., et al., Science 268; 714-716 (1995), or standard carriers such as ph~nn~rentir~l grades of m~nnitol, lactose, starch, m~is~ stearate, sodium s~ccl.~.;llF, ce~ lose, m~.uc;.. carbonate, etc.
S A variety of adju~ s can also be used to ampli~ ce~l-m~i~t~ and hnmoral ~ol~ses when mi~ced with a CDl-~lesen~d an~igerL The adJuvant of choice for human ~ ;on iS an ~ -- salt such as alum, ~l~....i.......
hydro~ide or ~ .. ,. rho~h~te Other adju~s, for e~mrl~ oil-based emlllciQnc that co~ain b~ egT~ bl~ m~trri~l~, G~ be tested in co~ n with the antigen and found to be effectiYe an~l safe. Adju~d~ ~hat are oil-based emtllcionc include Syntex fonmll~tion SAF-l, Ciba-Geigy fonm~l~tions, and Ribi formul~tion. See, Rabinovich, N.R. et al, Science 265:1401-14W (1994).
Freund's inoornrlet~ or c~mplet~ adjuvants can also be effective.
Methods of a~l",i~ dlion will va~y in accordance with the type of dLsorder and microorganism sought to be controlled or era~ir~t~. The dosage of the vaccine will be dependent upon the amount of antigen, it's level of ~ntigPnirity, and the route of ~ inn A person of ordinary skill in the art can easily and readily titrate the dosage for an imm--nogenic response for each antigen and method of a~lminictrati-n For ~ale~ dl applir~tion~ particularly sl~it~ble are injectable, sterile solutions, preferably oily or aqueous sollltinncl as well as ~ ~n~io~, emnlcionc, or imrl~ntc, inr~ in~ suppositori~s. For erlteral or mllcos~l applir~tion (inrl~ ing via oral and nasal mucosa), particularly suitable are tablets, liquids, drops, suppositories or c~rs~ -s. A syrup, eli~cir or the like can be used wL~,e~ a s~ d vehicle is employed. Topical applir~ti~n caD also be used for ~Y~mrle, in intraoclll~r a~l,ni~ dlion. ~ iv~ m~th~c of ~minictration can include an stim,ll~t;n~ comrl~x (ISCOM) as desrr~ l in U.S. paterlt No. 4,900,549 (or l~op~ Patent PUb!ir~tion No. 0 604 727 Al (Publ. July 6, 1994). In ~ition, viral vectors, liposome and microspheres, and microc~rsllles are available and can be used. See, Rabinovich, supra.
For ~ e~es of the lungs, such as tuberc~llosis, F~llmn~ mini~tration may be ~erc.Led for prophylactic yul~oses or for imm~i~te and sp~ific locali~ed SU~STITUTE SHEET ~RULE 26) PCTrUS95/13274 tre~tm~nt Pu~nonary atlmini.ctration can be accomrlich~ for example, using any of various delivery devices know in the art. See, e.g., Newman, S.P. (1984) in Aerosols and theLung, Clarke and Davia (eds.), BulLtl~vu~lhs~ Ton~r~n, Fngl~n~1,pp. 197-224; PCT Publication No. WO 92/16192; PCT P~lbliration No. WO
91/08760; NIIS Patent App~ tion 7,504,047 (1990), inr~ but not limited to nebulizers, metered dose inh~kors~ and powder inh~1~rc~ Var.ious delive~r devices are co.. e,~ially available and can be employed, e.g., Ultrave~t n~b~li7~r (~11inr~rodt~ Inc., St. Louis, ~ficsolJ~i); Acor~l II nPb~lli7ler ~Marquest ~e~1ir~l Products, Englewood, CO). Such devices typically en~ail the use of fonml1~tionc lo suitable for dis~elaing from such a devioe, in which a prop~ nt m~t~ri~l may be present.
The discovery that LAM or its deLiv~Livcs elicit T-cell proliferation extends the spect~um of antigens pl~sen~ed by CDl molPc~ s and provides the oppGlLu~ily for vaccines comprising CD1-presented antigens that are err~cLive against all gram negative and most gram positive bacte~i~ (inrlu-lin~ Streptococcus sp. and Stap~ylococcus sp.), and a variety of parasitic p~oto~oa. All gram negative bacteria contain lipopolysar~h~ri~s (I~PS) which are similar in ~LI~lc~L~e to lipom~nn~nc. Most gram positive bacteri~ contain ~ dlly-related glycolipids such as lipoteichoic acids. In ad~lition, the rk~ composition of many disease-causin~ protozoa includes LAM-like glycolipids Cuch as the lipophosphoglycans ofLeishmania. Orlandi, P.A. and S.J. Turco, J. Biol. Chem. 262:10384-10391. It is likely that the cell walls and other cellular components of the fungi also con~
lipogly~ans; therefore, vaccines comrricin~ CD1-})~ed ~ntig~nc can be used to pleVel~t or treat fungal infections of ~ CS. These antigens will include LAM, its derivatives, or its fi~nction~l equivalent in rnicrobial or~nicmc, whether prokaryotic or eukaryotic in nature.
~t preserlt, vaccines agai~st p~ OLo~ ~ sn~s are ei~her n~)nryict~nt or not feasible for mass; ~ tion. See, e.g., Nusse.~ ;g, R.S. and C.A. Long, Science 265:1381-1383 (1994). Examples of ~ice~ces caused by p~oto~oa include, but are not limited to, malaria, trirhinn~cic~ fil~ri~cic, trypanosomiaciS~
schictosomiasis, toxoplasmosis and le;c~ ni~cic. ~ulO~o~l. infections can be more ~1imrl)lt to control and eradicate than baet~ l infections beca.lse compounds SUBSTITUTE Sl iEET (RULE 26) that kill a protozo~n parasite are often to~cic to the host. For eY~mple, most of the dnugs used to treat .li~P~eS caused by Trypanosoma species can cause serious side effects and even death. In ~diti< n, dmg r~ ;.nf~ of many protozoal species is bec~omi~ increasiIlgly rommon in most parts of the world The presence of glycolipids in ~ ozl)a opens the door to a whole new class of ~cCCiD.C_ bascd on the T~ell ~"ol;r~ ~1;nn ~ a CDl-~-csc.lt~d hydrophobic ~n1ig~n Vaccines ~ rt;~i/lC or ~olipid d ;~a~,S either by s or comhin~ ~nth a prote~ a~ oould pmv-c asl rrr~r~r ~JS ana cost clr~i~c h~a~.n.~ a~t ~,~ o~ asites. An adY~gc of such a lo vaccine is that to~cic m~ (A~ t~ may not ha~re to be a~ e-u~ or can be ., d in l~ducod dosages in c~ h a CDl-y~ n~en to control the infec~ion, The present invention furthcr providoe m~thr~As for assaying for inhibitors of CD1-rcsl . ic~A antigen ~ se ~tAt;~ to T-cells. i.e., CD1 bloclri~agen~s. In one such m.-thoA, CD1 an~gen ~le~ tVAI;~ is inhibit~A by using a CD1 blocldng agent to block the ability of a CD1-r~ A antigen to bind to CD1 As used herein, a CD1 bl~l'i~ agent is said to ~inhibit CD1-res~rt~ antigen ~l~S~ ,I At;~ n" when thc CD1 blocl~ng agent d~ ases (1) the binding of a CD1-yl~ ntigen to a CD1 mo~ or (V the binAing of a CDl:CDl-pres~nte~ antigen comrl~ to its cogDate T-cell xe~t~s. Some CDl blocking agents are able to block such binding to l~nAetr~t~ble levels while ather CDl blocking agents only sligh~y dec.edse such binding. CD1 blocking agents include, but are not limited to, (1) agents which bind to CDl, (2) agents which bind to the CDl-plesc~t~d an~igen, (3) age2ts which bind to the CD1:antigen complex, and (4) age~ts which bind to the T~ell .~c~
that recognize the CD1:antigen comple~ c ~ .les of bloc~i~
agents include, but are not limited to, (1) polyclonal or mon~clonal antibodies which bind to and block the portion of a CD1 mol~le that binds a CD1-~,e~.lLed antigen, (2) polyclonal or mOnt)ClOrl~l an~ ~ which bind to a~d block the portion of a CDl-~ tl~ antigen that binds CDl, (3) synthetic oligopeptides that are derived from the CD1:an~igen-bi~i~ portion of a T-cell receptor and which bind to and block the portion of the CD1:a~igen complex bou~d by intact T-cell l~ , and.(4) synthetic componnds S-uBsTlTuTE SHEET (RULE 26) comprising a CDl-p~sen~ed antigen chf ~ ly linked to a pllnfif~l CD1 molecule or a synthetic deli~aLi~e thereof.
In an ~l~f .~ . method for ;~ geD ~ir ~ ~ f CD1-rejl. ;r~A antigens, a CD1 l~l~t ;.~ agent can be cmployed which blocks t~e ;~tJv~n~l;n~oftheA~ rl~CD1~ ,lr-withthcl~moleculesontheT-cell.
By inh~ g the ~ ,t~ ,, step, the a~--lio~of ~ C f;r su~sets of T~ells can be ;~ ;
Pilot trials are .u~ underway of ~ "/~ l~ of l.. ~ .rr~
from an ~u~i.. ~.~ disease with }~ s derivrd from TCR mol~llP
Oksenberg, J.R., et al., J. Neurol. Sci. 115 (S.yppl.):S29-S37 (1993).
DNA mol~ s en~odi~ TCR polypeFti~l~s displayed by T-cells that rocog~e the CDl-~ ed ~nti,~enc of the iu~ Lion are iSol~s~d accol.iiùg to methods known in the art. O~ ,g, J.R., et al., Proc Natl. Acad. Sci.
(IISA) 86:988-992 (1989); O~n~.g, J.R., etal., ~anue 345:344 346 (1990) and ~ -, Na~we 353:94 (1991); Uematsu, Y., et a~., PrOG Natl. Acad.
Sci. (USA) 88:53~538 (1991); Pa~zara, M.A., et al., Biotechniques 12:728-735 (1992); Uem~tcl~. Y., Inun~ogenet. 34:17~178 (1991).
The DNA s~uehce is co-l~. .~d into a polypeptide s~uence, and the portion of the polypeptide se~u~n~R that co,l~o~ds to the antigen-binding variable region of a TCR polypeptide is u~ed to design ~y~ c oligopeptides that bind CDl:antigen comple~es on APCs, thereby inhibiting antigen ~lt,S~ ;on Oligopeptides are rh~ ir~lly ~ rc;~f~ aCcol~mg to staI~dard rneth~s(StewartandYoung, SolidPhasePeptideSynthesis, PierceCh Co.,R~ n~l~Tllinnic,, 1985)andpurifiedfromreaction~l-u~sby~
phase high ~ ue liquid cl~u~l~-~.pl;~ (HPLC~.
A~ ition~lly or allr ~ ly, mP-h~c for g41~f, ~;llg anti-TCR
antibotli~s and anti-TCR binding peptides are well l~own in the art with regard to MHC ~ ;oi~ and can readily be ~r~ to the herein disclosed CD1 ~ inn system. Sl~.. ;.~er, J.L., CeU 57:895-898 (1989); Davis, M.M., and BiorkTn~n~ P.J., ~ure 334:395404 (1989).

SUESTITUTE SHEET ~RULE 26) CA 02202680 l997-04-l4 WO 96/12190 PCTtUS95tl3274 A skilled artisan can readily employ ~own m~thr>dc of antibody generation, as well as ratirm~l blocking agent design in order to obtain ~e blocldng agents of the present i~ n. Harlow, E., and ~ne,-D., Antikodies: A L~bora~ory Manua/, Cold Spring Ha~or P~, Cold Spring S Harbor, 1988; ~ynthetic Peptides: ~nswc~ Gsddc, r.~ W.H., Ne~t York, 1991; K~r7~k. A.A., ~ /J~h ~ 9230-9238 (1989).
Atl~ition~lly or ~ y~ 5 of m~ rly divcrse ~ol~ s can be sc~cd for individual --f --~ r m~llrs Wllicli are CD1 ~ agents.
Effective CDl bloc~ agents a~e id, ~ ~ by lheir abili~r to inhibit CDl-m~i~t~d T~ell prolif~lali~., and/or cytolgtic ~ onses using the mqt~ri~l5 and methods t~ ribe~l herein.
The embo~lim~ntc of the invention d~se~bed above can be used for the inrli(~t~l ~ oses, alone or in coml.il~l;on with each other or other complem.ont~ry methods and/or compositions.
The ~a~l and method of c~~ g out the present invention may be more fully understood by those of skill by le~,.e~ce to the following ~Y~mrl~, which e~r~mrles are not int.~i in any matter to limit the scope of the present invention or of the claims directed thereto.

Example 1: Antigen Presen~on by CDlb Methods Flow cytometry was pc rv, ~ as described previously rhA~. .oorthy, G., et al., J. Imn~no~ogy 147.336~3369 (1991 usIng the following monoclon~l antibodies (mAbs): P3 (IgG1 cont~ol; p~"~l~al"~
- G., et al., J. Imm~logy 147:3360 3369 (lg91)), 0~6 (anti~Dla;
p~inh~ol7., E., et al., Proc. Natl. Acad. Sci. (USA) 77:1588-1592 (1980)), 4A7.6 (anti{~Dlb; Olive, D., et al., ~mm~genefics 20:253-264 (1984)), 10C3 (anti~Dlc; Martin, L.H., et al., Proc. Na~l. Acad. Sci. (~JSA) 84:9189-9193 (1987)), W6132 (anti-HLA-A,B,C; BrodsXy, F.M., and Parham, P.P., S~3BSTITU~ SHEET ~RULE 26) J. Inununology 128:129-135 (1982)), B~L~031 (an~ TCR; Lalier, L.L., et al., Ln Leukocyte ~yping III, ~r,~ir,h~l, A.J., ed., pp. 175-178, Oxford U~ .sil~ Press, 1987), o~r4 (an~CD4; ~inh~r7~ E., et al., Proc. Natl.
AGod. Sci. ~SAJ 77:1588-1592 (1980)), 0~r8 (~n~C~8cr, ~Pinh~r~, E., S et al., Proc. Natl. Acod. Sci. ~5~) 77:1588-1592 (1980)) aDd 2ST8-SII7 (an~CD8~; Sh~ue, L., et o1., J. E~p. Med. 168:19g3-2005 (1988.
Mol~oc~hs were solst~d froDl t~ U~,~/~ c~n~ s of norI~ donors by plas~c r~h,r~-nr~ (Anego~, I., e al.,- ~. Inmuaw~ogy 147;3973-3980 (1991)), and ~e~hch~ by .~ A at 37C in ph~ r.Atç bu~e~a s~line (PBS) with 0.53 rnM EDTA (PBS/EDTA). Adherent~ells were t~pically >~0% CD14+ andMHCclassII+, andncgaliveforCDla, CDlbandCDlc as ~t~...;~.~ by surface ~ "j,~ (data not shown). To induce CDl e~r~ssion, mon~s were cul~d for 60 hours in RPMI-1640 (Gibco) cont~inin~ 10% fetal calf r.erum (FCS, Hyclone) with 100 Units/ml each of GM CSF and IL~ (Genetics ~ch Tr,~ -J~). Cells were l~led using PBSIEDTA as above.
T~ell line DNl was ~st~b1ichPd from a ran~l~m normal donor's peripheral Uood. Non~ Prent mon~m~rhp~r cells were treated with mAbs OKT4 and OKT8 and rabbit complem~-nt, and the ~ g viable cells were su~e~ded in a ~ of OKT4 (a~ti~D4), 0~8 (anti-CD8~) and a~ti-TCR~l (Porcel~i, S., etal., ~ ev. 120:137-183 (lg91)) mAbs for 1 hour, washed and ir..-~b~te~ for 30 ~ s at 4C with goat anti-mouse oglobulin coupled m~ n~ti~ beads (Dynal). After m~nl~tir ~ n and remo~ral of CD4+ and/or CD8+ andlor ~TCR Gells, the l~ g CD4-8- :~ TC~+ Gells were cllltured with equal ~ ~ of autologous mo~oe~les i~l Gomplete ...~.~ (RPM-1640 with 10% FCS and ~dr1ition~1 suppl~ ~-f .~I~ as previously desc~ by Morita, C.T., et al. (Eur. J. m77um.
21:2999-3007 (1991)) with 100 UIml each of GM CSF and IL 4.
M. tubetculosis soluble e~act, produced by sonir~tion of ~;(`C~t~d bacilli (s~ain H37Ra (Difco)) in PBS followed by centrifugation at 100,000 g to clarify (i.e., to remove insoluble mqteri~l from) the sonir~tes, was added to SUBSTITUT~ SHEET (RlJLE 26) W O 96/12190 PCTrUS95/13274 a b~ctPri~l protein cQnrc~ ~ion of 10 ~g/ml. More soluble ~nti~nic (i.e., T~ell prol;rr a~ ) activity is obt~in~ from soluble .~ eQllC sonir~t~s of M.
tb. by adding det~gc~s such as CHAPS or octyl,~l~lc4si~e during the sonir~;o~l step; wiawu~ hion of de~ , 90to 95% of ~ c~ activity S is los,t duri~g post-s~ . ;fi~ h~S wese ~
10 to 14 days w,ith U. tuberculosis and L~1~OIJS CDl+ Illol~,y~s (in~l~eeA, to c~press CDl as A,c~ d above) Ll c ~ pkle .~A;...~, and were fed every three to four days w,ith fresh .. ~A;.. ~.I~1i.. ;.~ 1 nM ~c4~i~
intrrl kin-2 (IL,2).
T-cell proliferation ,c~o.,se assays were carr,ied out in triplicate with 5 x 104 each of T-cells and irr~ t~ (5,000 Rad) APCs in 200 ~1 complete m~illm in 96 well flat bottom l~iiClo~iLc~ plates (Linbro). M. Ieprae and Escherichia coli soluble e~racts were ~ ~uce~ as ~esrribe~i for M.
tuberculosis. Monoclonal antibo~es were added as pll ifi~ ;~ nglobulin to a final ~onrf n~ ;on of 25 ~g/ml. Culturcs ~ere l~d on day five (day three for mAb blocking) after a si~ hour pulse with 1 ~Ci 3H-thymIdine (6.7 Ci/mmol, New F.~]~ml Nuclear), and 3H ~co,~ion ~lot~ ....n~3 by liquid SCinti tinn Cuu~ esults are eA~,~scd as the mean counts per minute (CPM) of 3H-thymudine i~co~laLion of trirlir~te cultures. Isolated mo~oc~r~s orwhole ~;l kP,~l bloodm~n~ P~roells (PBMCs) weretreated with recombinant GM CSF and IL 4 as above, or wi~h IFN~ 100 U/ml for 60 hours prior to combini~ them with T-cells for prQt;~ ;n assays. T-cell clone DN1.C7 is ~ senlati~C of four eA~ .siirely C1~ J ~ lbclo~s derived from DNl by phytoh~-m~P~ (PHA) ~im~ tir~n in l;---~
dilution culture and propag~te~ using PHA stim~ tion and ~2 as previously ~1es~ 3 Mort~, C.T., etal., Eur. J. Imm~n. 21:2999-3007 (1991). All clones derived from line DNl had a surface phc~oty~ ;~ l~ble from that shown in Fig. lb, i.e., ~:~ TCR e~ ession, non e .~ ion of CD4, and minim~l or non~ cSsion of CD8.
T-cell cytolytic response assays were camed out as follows. Methods of ~ r~ling ClR cells and assaying specifi~ cytolyfic a~ti~ily by 51Cr SUBSTITUTE SHEET (~ULE 26) WO g6/12190 PCT/US95/13274 release have been described. BaL~c, S.P., et al., Science 253:1411-1415 (1991) and Morita, C.T., et al., Eur. J. r~ 21:2999 3007 (19913, .es~cLi~ely. BK6, an a:,~ TCR c~ uc T-ceU clone that lyses oells displaying CDla, was ico!~t~d from the blood of a patient with SLE as S prcviously ~c~ ;1~ (Porcclli, S., et al., Na~re 341.447~0 (~989, and clone 3C8, an cY:~ TCR ~Olu.uC T celi done ~at lyses ccUs d~
CDlc, was i~ t~ from the blood of a wrmal donor USiDg the samc m~tlt~x3 Transfected cells were labcled with 51Cr and used as target-cclls in cytolytic assays with an effector (T~ell) to targct (~ r,~ ) ratio of about SO:l.
Metho~s of a~ssaying 51Cr release and C~ ti~ the % spec-ific lysis have ~een l~esctiheA. Brenner, M.B., et al., Nat~re 325:689-694 (1987).
Stable transfectants of T2 cells were ~ep~cd using the method ~lesc~ for ClR cells. BaLk, S.P., et al., Science Z53:1411-1415 (1991).
APCs for glutaraldehyde fi~tion and chloroquine e~ s were GM~SF-and IL,4-treated PBMCs as d~scribeA above, and ~ t~1d~hYde fir~tion and chloroquine tre~tm~nt of APCs were performed according to p~lk!ichçd meth~lc. Ch~cm)t, R.W., et al., J. lmrnun. 129:2382-2388 (1982);
Roncarolo, M.G., et al., ~. ~mm~l. 147:781-787 (1991). CD4+ T-cell line DG.l was derived from an HLA-DR7+ rh~lm~toid arthritis patient by ~t~d stimlll~ti~n of synovial fluid lymphocytes with autologous EBV-~n~forrn.oA B-cells and M ~ercu~osis purified pluLill d~ e (PPD, Statens Serum In~titllt~; data not shown). Prolir ~ . response assays were ~c,ro.llled as above, except that 2 x 105 APCs were added per well and 3H-thymidine illco~ tion was ~e~ i after three days.

2S Res~lts In order to develop a system to detect antigen pl~ -l~t;on by CD1 mol~ l~les, the ability of vanous re~mbin~nt cytoldnes to induce e~lession of CDla, CDlb and CDlc on pc,;l~kf..~l blood mono-;y~s, which norm~lly do not express ci~nifi~nt levels of these mol~ll~s, was ~Cses~ Leu~ocyte SU~STlTUTE SH~ET ~RULE 26) 73~ping IV, Knapp, W., ed., Oxford University Press, Oxford, U.K., pp. 251-269, 1989. High levels of CDla, CDlb and CDlc were col-c~ ly obsel ~ on monocr~s cultured with a co . k;.,~l;o~ of ~mll~/~o~
colony stimtll~tin~ &ctor (GM CSF) and i.~f ~e.~k.. 4 (IrA) (Fig. la).
S ~ f ~ ely, GM CSF rnay be used alone, ~1thm~h ~e r~llt~nt level of CDl e~ ssion is so~ less than that rc~lltt~ ~ ~ GM CSF
and IL 4 1~ nl ~t~ .lr.~Lh~_3 (IL,-3) may also be used, alone or in combination with other cytol~nes. Mo~o~ ~s cultured in the ~bs~-nre of cytokines, or those cultured with i~.reLoll~, did not e~ress ~;~;,..ri,~..
levels of ClDa, CDlb, or CDlc (data not shown).
Because monocytes are ~r~l. ie t antigen plc5f .~il.g cells (APCs), we reasoned that CDl+ monocytes rnight stimlll;^te a CDl ~ ;rt~ T cell response to an exogenous i1ntigen. Rec~llse most CDl specific T~ells identified to date have a double negative (DN; CD4-8~ phenotype (Porcelli, S., et al., Na~ure 341:447~50 (1989); Faure, F., et al., Eur. J. ~nm~n.
20:703-706 (1990)), we focused on this subset of cells and ge~ (ift~d a T-cell line by rc~tod stimlll~tion of periph~r~l blood of :~ TCR+ CD4-8- T~ells with a soluble extract of M. tuberculosis and heterologous CDl~ monocytes (Fig. lb).
Flln^-tioni^~l studies of the resulting T-cell line (~lesign~^te :1 DNl) showed that these T ceLls gave specific prolirc~ onses to ~ntigenc derive~ from M. t~berculosis and from closely related M. leprae bacilli, but not to unrelatedb?~Ct~ri;^~l ~ntigçnc such as those from E. coli or tetanus to~coid (Pig. 2a).
These r~llses were depe~d~l on the monocylcs being ~leh~ated with GM-CSP and IL~ (Fig. 2b), and were not l~~ i by polymorphic ~IC
~etc~ ls (Fig 2c). This lack of MHC rest~tion was c~ t.~..l with antigen ~lese~lion re~trirtion by non-MHC mo~ s~ In order to ~lctr . ".;.~
if CDl molecules are ~ d for M. tuberculosis andgen ~ ;on, ~e effects of nlo~lonal andbodies (mAbs) specific for CDl or ~IC molecules on the M. tuberculosis in~ ce~3 proliferadon of T cell line DNl and a lepr~ .~t;~, subclone, DNl.C7, were ~iet~ d. Only anti-CDlb rnAb SUBSTITUTE SHEtT ~RULE 26) showed s~ blocking of the M tuberculosis in~-)c~d prolif.,~ fe rc~onse, and no ~ c;~c ~t effects were obsel~d with anti-CDla or CDlc mAbs or with rnAbs against ,~onn~no~hic d~t ....;~ c of MHC class I or class II mol~lltos (Fig. 2d).
S B ceJl ~ r~t~.,tc D crr~ targcts fora:~ TCE~CD4~-cytoly~c T~ell acti~rity. Using the B 1~ o~ J~id cell linc ClR (7~ o~, J., e~ al., J. Irnn~r~ 1~8:1941-1948 (19S 2)~, stable l...r.~r~t~..t~ w4,~si,~g aDd displaying CDla, CDIb or CDlc at c~ r le~reLs ~erc ~ ~l~tl~1 and tcsted for their ability to prescn~ M. lub~ asis in a cytolytic assay. Only ClR cells tr~n~focte~ with DNA se~luc~c~s enr~i~ CDlb and in~lb~t~ with M. tuberculosis prior to the assay were lysed by cY:~ TCR DN T-cell line DNl and its subclone DNl.C7 (Figs. 4a and b). The ~ of this CDlb restricted response was c~nr~ using two con~ol CD4-8~ B TCR+ T-cell clones, BK6 and 3C8, which were deri~ed by mitogen stimlll~tirJn without exposure to M. tuberculosis aulig.,~s. Previous studies show that BK6 a~d 3C8 lyse target-cell lines c~ Sa~g CDla and CDlc, ~ccLi~ely (data not shown). As for all other CDl .eacti~ T-cell c~ones ~escnbe~l prior to this disclosure (Porcelli, S., e~ al., Nature 341:~47-450 (1989); Paure, F., et al., Eur. J. Imm~n. 20:703-706 (1990); Balk, S.P., etal., Science253:1411-1415 (1991)), these clones appear to be autoreactiYe and ~e their nonpolymo~phic CDl ligands in the ~bscnre of exogenous antigens. As e~pected, c!ones BK6 and 3C8 lysed only ClR ll~r~ ~,A~ 5Sing CDla or CDlc .e~ ly, and lysis was not ~ l by prior inrl~b~tion of the target-cells with M. tuberculos~s OE~lgS. 4C aI~ d).
2S The lack of MHC ~ ;r~ n d~ l.i,t~A by tl~e ~,1.~;.,*
e..~ t~nlS arguedthatMHCen~o~antigen~ mo!~.les verenot involved in the CDlb r~,sl~ i'~d ~ it~;~tn of M. tuberculosis ~nti~n.c to line DNl. As a more ~ f- ~l test of this hypothesis, CDlb ~ r~.~l;.nl~i of the 1~ cell line, in which ~.~lehsi-c chromosomal del~tionc in both MHC loci r~sult in a comrlete lack of MHC class II mol~~ ssion, were produced. Salter, R.D., et al., In~u~genetics 2I:235-2A6 (1985); Er}ich, SUBSTITUTE SH~ RULE 26) H., et al., Hum. Imm~n. 16:205-219 (1986). I~e MHC linked l~anS~G~te~
genes TAP-1 and TAP-2 (~ d in Pa~am, P., Nature 357:193-194 (1992)) are also deleted from 1~, le~ g in def~ti~, e*~lession and r~ ;nn of MHC class I mn1~~ s ~n~, N.~., and Bevan, M., Saence S 248:367-370 (19gO); Wei, M., and C~sswcll, P., Na~ure 356:443 446 (1992).
Ne~ eless, ~ r~;nn of CDib i~o 12 led to c~ion of CD1b o~e cell surface at a level similar to ~hat seen on other ~ r~d B~ell lines (data not shown) and ge ~ ~d a target cen ~t ~ M. Iub~,cl~losis to line DNl (Fig. 5a).
l~e ~le~ ion of e~ogeno~s %"l-g~ to T~ells generally re~luu~s uptake and proces~ of c~mrle~ protein antigen molecules by antigen pr~5~ cells, a process which is blocked by aldehyde fir~tion of the APC
surface and by Iysoso~otropic amines such as chlo~ u~e. Ziegler, H.K., and Unanue, E.R., Proc. Na~l. Acad. Sci. VSA 79:175-179 (198V; Ch~o~mlt R.W., et al., J. Imr~n. 129:2382-2388 (1982). By these c~lt~ . CDlb restrict~l ~rese ~ ;on of M. tu~er~losis also showed a l~u~ c~,~ for a~igen uptake and ~.~s~;u~. Mild fi~ n of C~lb~ APCs ~ith gluta~ldehyde complet~ly al)r~at~ their ability to stimll1~t~ line DN1 in the ~r. senc~ of M. tuberculosis soluble a~i~e~ lthmlgh the same APCs pulsed wit_ M. tuberculosis prior to fi~tion ~ cd their ability to stimlll~te a prolir~dlive .~ponse (Fig. 5b). Fùrt~e.~or~, the ~ ;on o~
M. tuberculosis antigens to line DNl was sl~o~ly inhibited by chloro~uine with a dose depen~l~nr~ vir~ally jtl~nti-~l to that for the inhibition of MHC
class II m~li~t~d ~es~ tion of mycQba~ ~ ~1 antigens (Fig. Sc), intli~ting - 25 that ~ S~U~ of antigens for CDlb and MHC class II fe,l~icl~ responses m y inYolve similar pdl~lWd~ or or~ l~, or that the ~al~w~ share one - or more chloroquine-se~ cellular factors. I~ gly~ T2 cells have rc~ly been shown to be defective in the ~ ing of antigens yr~l~d by MHC class II molecules (Riberdy, J.M., and Cresswell, P., J. ImnuuZ.
148:258~25gO (1992)) b~ se T2 cells lack the DMA and DMB ge~es.
Morris, P., et a~., Nan~re 368:551-554 (1994); Fling, S.P., e al., Na~re SU~STITUTE SHEET (~ULE 26) W O 96/12190 PCTrUS95/13274 368:554-558 (1994). Thus, our finding that CDlb ,.~ r~ T2 cClls can present M. tuberculosis to DNl ~.~gr~lc that the antigen ~r~c~c~
l~uilc,ue,l~ for CDlb and MHC class II _Q~ s~ ~lthntlg~h simiJ;~ Wi~}2 rgard to chlor~u~c se~ili~ y, are ~ot idr~
S Several iU~.CIi~ n.;, ha~e ~ ,t ~ thatT-cells la~ng ~ .o ~ of both CD4 and CD8 molr~ es may r~r4~ cnC ~ Ld by cell surface mol~ll~s other than t_ose cnrod~c~ by rl~sie~1 ~IC class I and rl loci. Porcelli, S., e~ al., Inunun. Rev. 120:137-183 (1991); J~ ~,~.~, C.A.
Jr., et a~., Imm~n. Tod~y 6:73-76 (1988); Rl. ~)n~.~ J.A., and Ma~s, L.A., J. In~mun. 142:178~-1788 (1989). The above results show that one member of the CDl family, CDlb, can restrict the specific l~onse of MHC
unrestricted CD4-8- T-cells to an exogenous foreign antigen. Like other CDl plv~e~s, CDlb heavy chains ~ccoci~ ~oncoYalently with,~2-microglobulin (Olive, D., et al., Imm~genetics 20:253-264 (1984)) and show limited but ciy~ r~ s~ue~e homology to both MHC class I and class II m~ t~-s.
Calabi, ~., and ~ils~in~ C., Na~ure 323:54~543 (1986); Balk, S.P., et al., Proc. Natl. Acad. Sci. USA 86:252-256 (1989). These ~hu~ual fea~_s of CDlb, together with its critical role in antigen lc~g~lion (desc~ibed a~ove), support the c~nrlllsion that CDlb is a no~polyluG~Lc ~ntigen ~ g molecule çnt~d~ by a genetic locus lmlinl~ to the MHC.
These results in~ te a l)ote~lial role for CD1 re~stri~tç~l T-cells in normal host ~ef~n~e agai~st microbial ~ ~. The above results suggest a fi~n~tinn~l pa~el ~l..~n CDl and MHC cl~ss II mo]e~lles, since both ",r~ ;rln of l~ogel~ou~ ~nti~enS },loces~d through a chloroquine se~ili~e ~Jaa~way, and both can also act as ligaD~s ~or the ~s of autoreactive T-cells. Porcelli, S., a al., Na~ue 34l:~47-450 (1989);
imrh~r, I,.H., and Shevach, E.M., ~ p. Med. 156:640 645 (1982). The limited tissue dis~ibution of CDl ml~l~llPs in vivo pro~ides a further ~imil~rity with the MHC class II family since m~ml~ers of bo& f~milie5 are ~ro.. nr ~tly ~ css~d on cell types ~ol~ed in antigen ~l~5e .l~l;0n to T-cells, inr.l~ i~ T ~~e l~ 's cells, ~n~ ;c cells in lymphoid and many other SL!BS~ITlJ~E Sl iEET (RULE 26) tissues, 8 cells and possibly cytokine activated monocytes. Porcelli, S., et al., mm~n. Rev. 120:137-183 (1991). In cont~t. the lack of stnuctural polymorphism of CDl mo~ s, thcir unique cytolcine reg~ ti-~n on monG.;r~s, and the CD4-8- ~h~ c of the C~ S~ t~ T-cclls ~ n~
S herein are ~pOl~ dir~ "~ t. the CD1 and M~C an~gen p~ ~s. These l;rr~,r~s point ltO a dis~nct role for CD1 r~sl. i~-~d T-cells in cell-m~li~ted ;",~

Example 2: A Non-Pep~de An~igen is Presen~ed by CDlb Methods The CDlb-plcseu~ed arltigen is a nondialyzable ",~rlo.,.~ l1e (data not shown). More ~ntigçnic (i.e., T-cell prolirel~live) activity could be obtained from soluble aqueous so~ tes of M. tb. by adding de~ s such as CHAPS or octylglucositle during the sonication (see above). This result suggests that the antigen is hydrophobic.
In order to ch~c~-~ the C~lC ~ l rlature of the ~ntig~nc p~.~Led by CD1, mycobacteni~l antigerLC were purified from the ~.o..~ ogerlic M. tb.
strain H37Ra (Difco) and M. fornutum (a rapidly 2;10WLU~; strain that also contains antigenic activity). Bacteria were either coullue..,ially available (M.~. H37Ra, Difco) or grown and l~ d (M. furh~ sQ~;r~t~ and subjected to- se~ue,ltial fT~Ctionqtion protocols and ~nalyzed for biological activity. All fractionc ge ~ ~ were tested for their ability to stim~ t~ the - DN T-cell line DN1 in a 5 day prolifPTation assay using irr~ te~ GM~SF-and IL~treated monocytes as APCs and ~ ;"~ 3H-thymidine - incol~olalion in a 6 hour pulse (Porcelli, S., etal., ~ature 360:593-597 (1992)). Cell wall, cell LUe UIJlo~e, and cytoplasmic fractions were pl~ared from either M tb. or M. fortsutum using a method adapted from published protocols. Hunter, S.~., e al., Journal of Biological Clzemistry 265:14065-14068 (1990). Briefly, cells were lyophili7~ ded in SUBSTITUTE SHEET (RULE 26) PBS/octyl~lllcsside, soni~ted for 20 minlltes a~d subjected to differential ul~lace .l~;r~g~tion to produce cytosolic, m~mhran~ ~d cell wJI fr~ctions.
I~e cell wa~l pellets were further pllrifi~ by a ~;rr~le.~ su~se ~ k ~-l Ch~rt~ristic sllu.~ fc~ s of the three fi~^tiOnC were ~ f~d by S ncg~ ~ with clechun " ic~sco~. Themajority of tlle bio~iYi~
for the DN1 cell line was present within the cell wall f~'~-n (da~a not shown).
To directly assess whether the CDlb-r~l~ ;'`ti~1 antigen is a protein, a series of protease ~igeStionc of the an~igen were p^rform~ Using a ~ariety of endopeptidases with elther limited amino acid specificity (chymoh~sil~`
(hydrophobic residues), ~ypsin (Iys, arg), and V-8 (acidic)), or broad amino acid recognitiorl (subtilicin, pluteinase K, plO~), sonir~tes of either ~. ~b.
or M. fortuitr~m were digested and then assayed for the ability to induce T-cellprolilelaLive responses. As a control, a DR7 ~ T-cell done DG. 1, derived in this laboratory which recogr~izes a dete~nin~nt in mycobacterial PPD (purified proleiD deliv~tive) was aLco tested. Analysis by SDS-PAGE and subsequent silver stain de~nor.~ that digestion ~th V8 protease, ~roLe~ase K, pronase E or s~lbtilicin degra~lc-s the plotei~s cont~
in mycobacten~ tigen prep~ratisnc (data not shown).

Results The M. ~uberclllosis antigen l~COg~i2~ by DG.1, a ,~
CD4+ MHC Class II l~sl~ir~ l T~ell line, is rendered ;.~(rr~ , by tre~tTn~nt with V8 protease, proLe; ~ce K, or trypsin (liigure 6). As shown in Pigure 6, DG.1 cells pro~ifer~ strongIy in ~on~e to thê mock digest 2~ ûf the mycob~rt~-ri~l soniC~te, but with the e~cception of c~U~sl~r~si~ all of the other protease ~ ls c~mrlet~ly abrogated the prolir~,dli~c response.
In contrdst, the M. tuberculosis and M. for~uilu~. antige~s ylese~llsd to line DNl by CDlb is unaffected by these broadly ~acli~c p~ dses (Figures 7 and 8, lc~lively). The mycQb~c~rial antigen ~r~nled by CDlb is SUBSTITUTE SHEE ~ (RULE 26~

-WO 96tl2190 filntl~m~t~lly di~,el~ than that p~ n~ed by MHC Class I and II antigen prc3e.~ g molecvl~os~ It is well e~ct~hlicll~ that MHC mq~ es bind and present peptide ~ntig~.nc of about 8-9 amino acids for class I a~d 13-25 aniino acids for dass II. nec~ this CDlb-~ ~ antigen is a ~a~lc.~GlP~ e S which is y~asc ~ , it is unlikely to be a pept~ T~s, the CDl system is the first known antigen ~S~ t~'t;nn system which present foreign s ~ rP5 other ~lhan pepti~3~s to a:~B TCR+ T cells.

Example 3: Punficafion of a CDlb-Presen~ed An~gen from M. fuberculosis 0 Methods M fortuitum bacteria were grown in liquid culture to stationary phase and collected by centrifugation, sterilized by stream autoclaving (250C, 18 p.s.i.) and lyophilized. Desic~t~d M. tb. (strain H37Ra, Difco) or M.
fortuiturn bacteri~ were suspen~ed in phosphate buffered saline (200 mg bacteria per 5 rnl PBS), and the b~CtÇr~ ension was soni~t~i with a probe sorucator to disrupt the cells. The resll1tin~ sor~icate was extracted with organic solvents using a Folch based 2 phase e~h~rtion system (chloroformlm~oth~nol/water) which~lu~ ely ex~actsmycob~ct~-ri~1 lipids into an organic phase. Goren, M.B., and B~e~, P.J., Myco~actenal Lipids: Chemistry and BiologicActivities in Tu~erculos~s, 1979. The sonir~tç
was combined with three volumes of a chloroform ~Ptl~ (2:1 v/v) solution in a glass cont~in~r~ and the mixture was vigorously sha~en at r~om tc~pc~l~e for 24 hours. I~e phases of the ~ Lul., were s~p~ .lt~d by c~ul~irugation at 800 g, and the organic phase was co11ect~cl and ~l~Ç~
into a glass boiling flask. Each f~rtion was then dried by rotary e~a~oldLion (orgaruc phase) or lyophilized (aqueous phase and interface). After evaporation, the organic phase left a thin film of waxy m~teri~1 on the surface of the flask. In order to prepare m~teri~1 to be tested in T-cell proliferation SUBSTITUTE SHEET (RULE 26) assays, aliquots of fractions were lecoh~ ~A as li~>osol,lcs by the ~d~lition of water (20 ml per 200 mg of b~-t~ri~ in original 5O~ ) followed by sonir~tion in a water bath soni~tor. The ~ tin~ crude ~ nn was then forced le~Aly through a 0.1 r~n filter ....-...h,..~-f in order to crcatc a S li~osolllc ~ r.r.~jon of ~ irOl~ size. ~ll~.. ,.l;~,ly, T-cell media with 10%
fetal calf æmm was added to the dried r.,a~ ;- n and ul~ wiLh~uL
3dAitinn~ I ;nn For further~ t;nn, the rn~teri~l P~ omM. 12~bcr~ as described above was dissolved in hexane and applied to a c~ n of Silicic Acid. Eluting with organic solvents of ~cle~s~g polarity over silica coll~mnc achieves sep~r~tion of lipids based on their polarity. The most polar lipids such as phnspholirids bind the ~llo~ l to the silica column and e.lute last, while glycolipids g~n~rally bind less tightly and elute ea~ier. Neutral lipids such as triglycerides or sterols bind the weakest and thc~ef~le dute first.
Small open solid phase eYtr~rtinn (SPE) c~ll-mn~ (BakerBond, JT
Baker) were ~lere,lcd l~use of the ability to process many s~mrl~
simn~ r~,Jcl~. A silica based rbonded" column (covalen~ly linkod) with cyano (CN) fimr.tinnal groups was used tO fr~inn~t~ the or~anic ~ cl~; of M. tb. The organic phase of the chololorol~ nl e~act of b~ was dried and ~ 1~ in hr~an-- The e~uivalent of 5.3 mg of ~ rc~t~
bact~ria in 200 ~11 hexane was loaded onto a 0.5 gram CN SPE c~!-lmn The column w.as washed with he~ane and then with 25% (v/v) ChlON~O~m in h~Yan~. Next, the bioactive f~Cti~)n was eluted with 85% (v/v) c~lol~r~
in he~ane with over 100% l~ of bioacti~ily. Analyzin$ the active fractic)n on silica based TLC plates, accoldi~g to the m~thod of Kupke and 7P.~ r(~hrictit~:~W.~l., LpidAna~sis,p. 117,~lg~ldl~PreSS, Oxford, U.K., 1982)), only two major species of lipid were vi-c~-~1i7~cl with cupric acetate, co~ onding to free fatty acids and mycolic acids (data not shown).
This result reflects a m~rk~ 'm from the starting organic m~t~
Proliferation assays were h~ ~t~ on day 2 (DG.SF68). day 3 (DG. 1) or day S a)Nl). DG.SF68 is a Vy2V~2 T~ell clone derived in this lab SUBSTlTUTE SHEE~ (RULE Z6) (PNAS in press, CM). APCs were GM-CSF- and IIA-treated monocvtes (DN1) or PBMC (DR7+) (DG. 1), or UlllrGdted PBMC (DG.SF68). Cytolytic assays are displayed as % sper-ific Iysis and were pe~fQnn~ as ~es~
Porcelli, S., a al., Na~ure 341:447450 (1989). Data shown ~Pigure 9) are S with an effrctor to target ratio of 50:1 and with M. n(berculosis antigen at a dilution of 1:20.

Resul~s - The relevan~ antigen of M tu~erculosis is icsl~t~ from a C4~ ' lCialpl~dtion of strain H37Ra (Difco) by extraction into a ~ ~e of chlGlufol~ll and m~th~nnl dS ~srrihe~ aboYe. Although the intçrf~r~ contaills greater than 95% of the proteins, 100% of the CDlb-restrirt~ ~ntiErnir acti~ity (i.e., ability to induce an c~:,B TCR DN T-cell proli~ d~ o~
of the mycobact~ lm C~llal~l~ into the organic phase (Figure 9a). This strongly supports the ori~in~l conr.lllsion of the ~ lide nature of the relevant b~cten~l antigen. In c~ntra~ a conv~ M~C class II l~il, ;r~
antigen recognized by DN ~:~ TCR+ T-cells was located in the phase int~rface bclw~n the aqueous and organic phases (Figure 9b). In conh~
in four in~epçn~nt antigen ~ l;ons, the CDlb-~ d antigen q~ ely partitioned into the organic phase. Rwults of t~Ç~t cytqlytic assays of the phases c4 ~ .. ~ tha~ the CDl~lese.ll~d an8gen is present in the organic phase (Figure 10).
Under these con~lition~ 100% of the ~y~Cl ;~l CD1yl~se~ed antigenic activity was ~llJA.~ ti~._ly ~cco-, ~ed afterCN SPE chr~gla~hy.
tiQn, the organic phase e~acfinn served as an er.e~ pl~r;f;t~at;On step and the organic phase was used as a star~ng m~teri~l for sl~bs~ ent cl~umatography. An ~ l;ve and so~hat more general plOCCdUlc; to pur~fy the antigen for subsequent chromatography is to saponify whole or sonir~t~l bacteri~ and extract with an a~ ifi~ solution of h~Y~nes. Purther SUBSTiTUTE SHEET ~RULL 26) ~CT/US95/13274 purification of the antigen is obtained using Silicic Acid chromatography as described above.

Fxnmple 4: Mycolic Acid is a Mycobac~eri~l CDlb-Presen~ed Antigen Given the above results, and Ot~PJf~ data l>~g~ling t}lat ~e T-cell stimnl~toly activiy co cluoil~tQ~ai~h~ A on CN ln~ifi~ silica HPLC
c~ mn~ ~ith ~le~.At;ons of free fatty acid acyl chains (data not shown), it seemed plausible that the CDlb-p~nL~d antigen is a unique mycobac~
lipid, possibly a mycolic acid.
Mycobacte i~ colltain an extraordinary proportion of lipids, amoun~ng to 40% of the dry weight of bacillus and 60% of the cell waIl; the mycolic acids are ~.haps the most numerous and diverse mrm~ers of ulycobacL~
lipids. Goren, M.B., and Brenr~, P.J., A~yco~ terf41 L pids: Chemistry and Biologic Activities in T~lbercuiosis, 1979. Mycolic acids are cY-br~nrh~
,~-hydro~y fatty acids forming a u~ique set of ~LLUC~S that are found in mycobacteri~ and related b~c~ri~l species. Wolinsky, E., ~Mycob~c~
Ch~pt~r 37 in Microbiology: Incl~ng Im~nology and Molec~nr Gene~ics, 3rd Ed., Da~ris, B.H., ed., Ha~per & Row, phil~d~lphi~ 1980.
Mycolic aci~s are ~ ip~lly found in the cell wall, esterifi~ to arabino~l~c-t~n polymers linked to the core peptidogly~ (McNeil, M.R., and ~nnall, P.J., Res. Micro~iol. 142:451-563 (1991); Besra, G.S., Biochemis~ry 30:7772-7777 (1991); McNeil, M., e~ al., Journal of Biological C~lemist y 266:13217-13223 (1991)) and can be released by either ~ or acid hydrolysis (saponifi~tiQn). Minnil~in, D.~., "Mycolic acids" in CRC
H~ndbook of C~troma~ograpky~ ysis of L~pids, Mu,her~ce, R.D.. , and Weber, N., eds., CRC Press, 1993. Mycolic acids are the major component of the lipid coat ~ul~u~ in~ the or~ni~ giving the o~a~s~ its hydrophobic surface and ch~ct~ ;c acid fast stai~ing. Goren, M.B.., and SUBSTITUTE SHE~ (RULE 26) Brennan, P.J., Mycobacterial Lipids: Chemistry and Biologic Activities in Tuberculosis, 1979.
Unlike eukaryotic and b?.rteri~l fatty acids, which ~ange in size from C12~24, mycolic acids of M~c4~a~ range in size from C6~-CgO
Minnikin, D.E., "Lipids~ pl~ Lipids, t~Leir~- .ni~ , Bio~ l~aDd Roles" in The Biology of M~,~ob~r~/eri4, ~ol. ~ tl~,e, C., a~ r~3, J., ods., ~ lemir Press, T~nrlnn, 1982. Mycolic acids, in c4 ~ q to the straight chain fatty acids, ba-re a ~J~ r~ aLkyl group at tl~e c~ carl~on and a lly~yl group at the ,~ on. Goren, M.B.., and Brennan, P.J., 0 Mycobacterial Lipids: Chem~ry and Biologic Acnvines in luberculosis, 1979; ~innikin, D.E., "Lipids: r~mpl~oy Lipids, their ~kc~
Biosynthesis and Roles'r in The Biology of Myco~octena, VoI. 1, Ratledge, C., and Sanford, J., eds., A~d~mir Press, T nnt3Qn, 1982; Takaya_a, K., and Qureshi, N., rStructure and Synthesis of Lipids" in The Mycobactena: A
Sourcebook, Part A, Kll~i~, G.P., and Wayne, L.G., eds., Marcel Dekker, New York & Basel, 1984. The main long alkyl chain of the mycolic acid (the so called mero group) is hcLel~ùgenous both in leng~ and in ~tt-Arh~
functional groups. In addition to aL~ene groups (double bonds), the ~lnrtionAl groups of mycolic acids include methoxyl, keto, lone methyl bA.~nr~s, ethylenic and cyclop,upallûid groups. ~innikin, D.E., "Lipids: Complex Lipids, their Ck~ , Biosynthesis and Roles~ in 17~e Biology of Mycobactena, Vol. 1, Ratledge, C., and Sanford, J., eds., Ac~d~Tnic Press, London, 1982. The large array of fil~iontl groups available to mycolic acids, their variable chain length, and their hete.~gencily among strains, allowmycolic acids to achieve a pOk ~ ly large degree of ~tnti~Çnic variation similar to that provided by peptides with h~te,og~;ly among amino acid side chainc. Thus, these lipid mol~lles may have an imm-tn- lûgical l~,le~,~ce not previously appr~iAt*d. For each ll-jcobAr-t~ ;Al species a flictin~lichAble rllgc,~ t exists based on the ~ s of mycolic acid molecules present.
Such ~JA~ c have been ~lctr ~ ~ for individual species by thin layer chromatography (TLC). ~innikin D.E., ~Lipids: Complex Lipid_, their ~UBSTITUT~ SttE~T ~RULE 26) ChPrni.~t~y, Biosynthesis and Roles~ in The Biology of Mycobac~eria, Vol. 1, Ratledge, C., and Sanford, J., eds., Ac~mi~ Press, T nn~on 1982; Dobson, G., e~ al., Chemical Me~hods inBacterial 5~1sten7ntie~, Ara~emir Press, 1985;
Valero~uillen, P.L., et al., Joun~al of ~pli~ Bac enology 59:113-126 (1985)), gas cl~ v~ ?~ (GC) (Vale~ n~ P.L., et al., Journal of Applied Bac~eriology 59:113-126 (1985); A~lye, M., etal., Jo~rnal of Applied Ba~eriology 58:507-512 (1985); T~ in, M., etal~. Journal of Clinical Microbiology 29:12~130 (1991)) a~d by high ~ Ul~ liquid c~at~graphy (HPLC). Qureshi, N., et al., Journal of Biological Chenustry 253:5411-5417 (1978); Qures~, N., et al., Joun~al of Biological Chernistry 255:182-189 (1980); BuSler, W.R., etal., Journal of Clinical Microbiology 29:2468-2472 (1991); Butler, W.R., andKilburn, J.O., Journal of Clinical Microbiology 28:209~2098 (1990~.

AIethods In order to ~et~ f- if the CD1-p~csc~d antigen ~lescrihe~l above is a mycolic acid, an HPLC method that ~p~ tes mycolic acids on C18 reverse phase column chromatography was used to prepare mycolic acids. Butler, W.R., e~ al., Journal of Clinical Microbiology 23:182-I85 (1986); Butler, W.R., et al., Joumal of C.lini~n~ Micro~iology 26:50-53 (1988); ~loyd, M.M., etal., Journal of ~ l Microbiology 30:1327-1330 (1992).
Reversed ph~se ch~o~ato~,lap~ ~ s acyl chains pIlm~nly on the basis of the length of the acyl chain or ~carbon number,~ thus it is relatively easy to achieve good sep~hdtion ~l~n free fatty acids and mycolic aciLs which are much larger.
This ~LC method l~uues an inidal ~ o~ dlion of the sample, followe~ by de~ dtion of the fatty acids or mycolic acids with the ultrdviolet (OD254) absorbing co~poulld p-bromophen~acylbromide, which ~tt~ChPs to the ~IJG~1 t~----;--,-s of an acyl chain. In pr~limin~ry e~.;,.,~nts, we ~e~ ~ ~ that the process of de~iv~ ulg the b~cteri~l SUBSTITUTE SHEET (RULE 26) fraction with p-phenacylbromide d~sllu~ bioaclivily. However, the CDlb-c~ n~igerliC actiYily could then be ~c~u~c.~ by S~ ;rc~ with h~ KOH, a p~c~s which free~ the c~l end of thc acyl chain by cle~vi lg off the l.hcDa~ll.~o~dc gr~ (as assayed by OD254 on HPLC~.
S This result ;~ t~ that the acyl ~hain must be cleaved in order to achievc a form which is ~"CS~..t~hl~ by CDl ~ , APCs and/or~hata fiee C~1JOA~
group i;s critical for ~)L~ of the CDlb~ ~I Q~n 'Ihis is ~d~litiQn~1 c~idc~ that thc antigen c4 .l . ~ s an a~yl chain The SPE CN colllmn ~ d ~ ..~lr 3) W~lS used as a sta~tng m~t~ l for the C18 ~ ~rl~S were S~l~r;r;~ with ~ ~ ~r lha ~ ir KoH and d~"iv~li~d ~yith the w absoLl~g group bromûph~na bromide. The active f.~l;ol- wa ntn on a C18 column (Alltech Nueleocil C18 5 ~lm, 25 cm x 4.6 mm) u ing a linear ~rliPnt of 30 to 90% methylene chloride in ...~-lh~..ol oYer SO ...;. ~ at 1 ml/~un. Using as a l~,fe,~,~ a C90 int~m~l standard (Ribi), ~e ~ t~ c~.~ (Figure 12, panel a, upper portion) has a pattern c~ bl~ to y~ llP~ results. Floyd, M.M., e~ al., Joumal of (~ ol Microbiology 30:1327-1330 (19~ nc were r.,~n~:l in corr~rl~te media with 109~ FCS and tested at a 1:17 r tion relative to the or~ n~l SQ~ volumc.

~esu~s ~

I~ioac~ , ass~d by the T-cell prol;r ~ w~e assay, was found to cQmi~t~ with early peaks in the region of the ~lic acids (Fig.
12a). To C4l~ that mycolic acid is l.l~d by CDlb, an ~
source of mycolic acids, ~-- ;r~ cord factor (trehalosc di~olatt,, Fig. 11) was tes~d. Upon sa~ ", ~ -- ;r~i trehaloæ dimycolate from eitherM.
tb. (from Sigma) or M. k ~ ; (gift of Patrick n~ "~ timnl~t~A the prQl;r. ".linn of the T~ell line DN1 (Fig. 12b). However, no ~im~ til~n was acL~ d by -~ ;al ~ d from sa~Q~ r ~ehalose ~ ha ~ale a ~ ct;c d~ aLi~ of cord factor which co~ two C22 fatty acid chains, ~UBSTITUTE SHEET (RUL~ 261 WO g6/12190 rather than mycolic acids, that are liberated by saponification. This strongly argues that mycolic acid, not trehalose (which is present in cach of the samples) nor fatty acids, is the antigen ~lesc.l~l by CDlb to the double slegati~c ~:~B TCR T-ccll line DN1. A HPLC ~n~lysis of the ~ PA ~igma S cord factor was then ~c~rolLuPd, and again the bioactivil~ was locatod in f~rfionc c~ o~ding to the early mycolic acid pea~s (Fig. 12c). Togcthcr, the above data ~p~n~ te that the CDlb ~ 4bact~.al antigen recogniæ~ by the T~ell lille DN1 is a species of mycolic acid.
Mycobac~.;~ are known to be hig~ly tf~ adju~ . Aldovini, A., and Young, R.A., Nature 351:479482 (1991). One sour~e of the CDlb-presented ~nti~en, mycolic acid, used herein is trehalose disnycolate (i.e.
Mycob~ct~ri~l cord factor), which has been show~l to rnh~ c antibody fo~ ion (Re~ilor~lnc~ A., et al., J. Bacteriol. 100:9S-102 (1969);
Be~ rh-nct, A., et al., Infection and lmm~uty 4:245-255 (1971);
B~ rhlnct, A., et al., Infecnon and Imm~ 4:256-263 (1971)) and to stimnl~te l~ons~ci~lc immllnity to bacterial islfectiosLc (Parant, M., et al., Infect. Imm~. 20:12-19 (1978)) and turnorc (R~rhlnCt, A., et al., Infection and Irnrm~nity 10:1044-1050 (1974).
In order to ensure that the purified antigenic activity cont~inC a boDa fide antigen and not a n~-"~l.c~ ~ilogen~ we looked at the speeifioity of the T-cell prolif,or~tive response to crude M. tb. ~lep~.~tionC and HPLC-purified mycolic acid from M. tb. and sapo~ ed cord factor. Total SQI~If~ S of M.
tb. (Fig. 14, upper panel) contain antigen recogni~ed by the MHC class 11 restri~t~ CD4+ a:,B TCR+ T-cell line DG.1, as vell as an antigen re~ognized by the CDlb-restri~ d T-cell line DN1 and the CDlc-restrir~
T-cell line DN6 (see FY~mpl~ ~, below). Ho~vever, HPLC-p~lrifiç~ mycolic acid from either M. tb. (Fig. 14, middle panel) or from sapo~ ed cord factor (Fig. 14, lower panel) cont~in~ antigens recognized only by CDlb-r~llicled DNl T-cells. This specificity is also demonstrated in the ~r~ a~ cytolytic assay (Fig. 13). The CDlb-~ ,led response of DN1 was blocked by anti-~UBSTITUTE SHEET (RULE 26) CDlb antibodies, but was not affected by antibodies to MHC class I or II
(Figure 15, upper panel).

Example 5~ gen Present~on by CDIc In ~ldhion to the ple~ ~t~t;nn of ~ ~;g~n~ by CDlb Aicr1~sed in Example 1, CDlc mo~ es alsopresenta~tig~n~. A se,~ t~!, C04~ B
TCR+ T-cell line, derived by repeated stimlll~tit~n with .~lonc~s treated with GM-CSF and ~4 (to induce CDl c,.~l~sion) and M. uberculosis antigens was isolate,d and named DN2 (also l~f.,,l~d to as 8.23.DNl).
Proliferation of DN2 is completely inhibited by the addition of mAbs to CD1c, but is not ~ffe~t~d by mAbs to CDlb (Figure 15, lower panel). A
cytolytic assay COli~ lS this result: ClR cells transfected with vector oDly or with vector encoding CDla, CDlb or CD1c were preinçl~bat~d vrith or without M. tuberculosis sonicate and used as targets in a cytolytic assay. Only CD1c+ ClR cells are reco~lii~d (Fig. 16); recognition is ~nh~nre~l by preincubation with the sonicate. Thc.~forc, CDlc molecules present M.
tu~erCuiosis antigens to DN2 T~ells.
A second CDlc~ ;ct~ CD4-8~ TCR+ T~ell liIle, derived by repeated stim~ tion with ~lono~.ylcs treated with GM CSF and IIA (to induce CDl ~ C~;on) and M. tuberculosis ~nti~en~ was isolated and named DN6.
~ytolytic assays (Fig. 17) intli~t~ that DN6 Iyses CD1c+ cells in the plcsence of M. tb. antigen.

Ex~mple 6: Charactenzafzon of a CDlc-prese~2fed An*gen from M. tuberculos~s The antigen ~lcsenl~ to DN6 T-cells by CDlc is not mycolic acid (Fig. 14, middle panel). However, ch~tnic~l ch~c~i~lion of the antigen recog~ized by DN6 in~ir~t~s that the antigen is a cornplex lipid. When M.
tb. sonicates are e:AllaCl~d with chloroforrn meth~nol (as describ~d in SUBSTlTllTE SHEET ~RULE 26) Exarnple 3), the antigenic activity is found ~u~s~n~ ly in both the phase interface and the organic phase (Fig. 18). Antigen i~cove.~d from the organic phase can be bound to CN SPE coll-mnc and eluted (as ~ ;lr~ in P~mple 3). The_e studies ~l~nl-n~ ~ that the an~gen L h~ol~h~ic and has Ihe c~o~ato,~a~hic ~u~c"ie_ of a lipid.
However, unlike the CDlb~ t ,d ~ ,.~ba. ~ ;al ~nti~n, results Of ~dllition~ .lc in~lir~te ll~t ff~e DN6~ t ~ CDlc~I
antigen is a co~rle~ lipid and not a ~ee a~yl c~a~ S~r~ a~;nn of M. tb soni~tf~ el;...;nAt~s the prot;r .a~ onse of DN6 (Figure 20). n~-~ce sapol-.~cation b~ ester linkages that conn~t acyl chains to c~bo~le back~ones, this result suggests that the antigen lo~o~iLo~ by DN6 T-cells is a, or conl~l~s an ~d~iitinn~l~ moiety other than a free acyl chain.
Saponifir~tion ~l~ably destroys or .e.llo~s the a~tlition~l moiety which may be, e.g., a polysaccharide backbone or branch point. Thus, in c~nt~ct to the recognition of free mycolic acid by the DN1 T-cell line, the DN6 T-cell line re~ogDizes a more complex lipid Shu~
It is no~,l~ that the CDl-~l~d antigens rccog.,i~d by T-cell lines DN1 and DN6 are not unique to M. tuberu~loszs. Rather, the DN1- and DN6-re~ognized CDl-~ ed ~nti~en~ are cross-l~acli~c to ~ntig~onc found in many Mycob~ l species tested to date (M. f~,hu~u,., M. a~ium, M.
bovis (BCG) and M. kprae). In the case of CDlc- ~ t~d recognition by DN6 T~cells, an ~nti~n iS ro~;~d in Co)yneba~ (data not sho~rn), a sep~te but related genera of bact~i~ which produce mycolic acids.
Takayarna, K., and Qureshi, N., ~Slluct~ and Synthesis of Lipids" in 17le Mycobactena: A Sourcebook. Part A, K ~bi~ G.P., and Wayne, L.G., eds., Marcel De~er, New York & Basel, 1984. Thus, CDl~ d antigens include at least several bar,t~n~l lipid ~nti~n~; in the case of aulO~ y, CD1-~lesen~ed antigens include endogenous lipid antigens.

~UBSTITUTE SHE~T (RULE 26) Example 7: CDl-Presented Antzgens from M. Ieprae In order to derive DN ~:,B TCR+ T-cell lines from leprous lesions, T-celIs from such lesions or PBMC were stimnl~t~d by culture with CDl+
APCs and M. Ieprae. T.\~., ...,~o. -~f~n~tir dcplction ~ cd CD4f, CD8f and S y:~-TCR+ T-cells. The ability of ,~ CD4~ a:~ TCR+ T cells to respond to M. Ieprae ~ntigen~ was ~c~ ~ by 3H-ll~idinc ~l~G~..liOn using allogenic CDl + monoc~s as APCs.
Four of si~ DN cY:,~ TCR~ T~ell lines isolated in the abo~e rlla~er proliferated strongly in the yr~sence of M. Ieprae and allogenic CDl + APCs, whereas little or no stimtll~tion of growth was det~ted in the presence of the allogenic CDl+ APCs alone, and no proliferation in reponse to M. Ieprae was detected in the ylese~ce of APCs not e~yless~g CDl proteins. Thus, the four DN ~:,B TCR+ T-celI lines respo~d to, and proliferate in the presence of, CDl-presel,~ed antigen from M. Ieprae.
Two DN c~:,B TCR+ T-cell lines, isolated from leprous lesions and d~signqt~d LDNl and LDN4, were subjected to further analyses. To deterrnine which CDl molecules (i.e., CDla, CDlb, Cdlc, etc.) were specifically responsible for A~. leprae antigen y~ ;on~ two sets of e~en~s were yerformed. First, proliferation studies were yelrolllled in which CDl + APCs were in~lbat~ with andbodies to CDla, CDIb or CD lc.
Second, ClR cells were Ll~srol~ed with genes encoding different CDl molecules and used as target cells in cytolytic assays.
M. Ieprae-in~u~d proliferation of the LDNl T~elI line was inhibited by greater than 63 % by anti-CDlc, but anti-CDla, anti-CDlb and an isot~
t~ nOn~Dl-~acLi~ antibody had no effect on the pr~lif~
to M. leprae (Pigure 20, upper panel). Similarly, LNDI cells lysed CDlc-transformed ClR cells in an M. leprae antigen specific manner, but did not lyse cells transfected with CDla or CDlb or mock transfected ClR cells.
A second M. leprae antigen reactive DN ~ TCR+ T~ell line derived from a leprous lesion, LND4, was found to be Cdlb ~sllicLed. The ability SUBSTITU~_ al~E.~ ~RUI ~ 2&) WO 96/12190 PCTtUS9~tl3274 of this T-cell line to proliferate in ~e prese~ce of M. Ieprae was complet~ly inhibited by the addition of anti-CDlb monoclonal antibodies (MAbs) (Figure 20, lower panel). Similarly, LND4 lysed CDlb-ll~Çccled ClR cells in an antigen ~cil~r l,~a~,r, but did not lyse CDla or CDlc ~ r~s .,t~;.
S I~ nre~ the ability of LND4 to Iyse an~ige~plllsed CDlb targets was blocked by anti-CDlb MAbs. In all DN cY:~ TCR~ T-cell line_ tested, antibodies to MHC class I and cl -cs II molecules had no effect o~ prQl;r ~I;nn in ,~nse to M. Ieprae.
The plese~ce of CDl-bearing cells inlepro~ lesio~Lc wac el-~n~.n~d by immllnnhictological e~ ir~t;on of tissue sectinn~ ucing MAbs to CDl (data not shown). These analyses reveal that CDla~, CDlb+ and CDlc~ cells occur in in tuberculoid gr~n~ m~c to a greater e~ent th~n in le~o...~tc,..c granulo_as. Although CDla was eA~l~ssed in the epideImis as well as the derrnal granulomas of tuberculoid leprous lesions, CDlb and CDlc were only ~*~lcs5cd in dermal ~mltnm~c Fursherrnore, Il-10, which is sllo~ly e~ ed in leprom~tollc lesions (Y~ , M., etal., Science 2~4:277-279 (1991)) could inhibit S~e .,~lcssion of CDl on ~o~oc.r~c (data not shown).
The correlation of CDl e~lession with le~ in leprosy suggests a role for CDl-m~ t-oA rcsl- ,r-l;o-- in S~e gcne.d~ion of cell-m~ s~ ;~ lll;ly.
The f~lnrtion~l role of DN cY:,B TCR+ T-cells was ~ .. ;.. ~ by studying their cytokine secretion pdll~ . Anti-CD3 stimlll~sion of DN ~.,B
TCR+ T~eL resulted in the releace of .~telÇc.~n~ ;N~) (m~i~n = 647 pg/rnl) in four of the five DN cr:~ TCR~ T cell lines, but ~loluc~ le or no IL 4 (median < 20 pg)ml), ~lSh~v~h one T-cell line produced IL~ (99 pg/ml) but no ~ t~ble IFN-r (<20 pg/ml). IL~5, II~6 and ~10 were not ~et~t~ iII the ~u~ c of anti~D3-s~im~ t~ DN T cells. These data suggest that the majori~,r of M. /eprae-l~cli~e DN ~:~ TCR+ T-cells secrete the type 1 cytokine pattern.

SU~S I I~UT~ SH~E I (~UL~- 2~) PCTrUS95/13274 Example 8: L~M is a CDlb-Presented Antigen from M.
Ieprae In order to el~r;~l~ the bio~ Al nature of the ~nti~n~ leco~
by CD1~ . ;CL~ T-cells, p-- ifi~ cell wall c4nc~ e ~s of M. leprae were 5~ ed and assayed for biological activity. LDN4, in the pl~CC of CD1 ~gantigen~ cells~lol;r-~4~ poncetolipid~ tA;~l~n fractions derived from ~he cell ~all (So~PCW), ~it not to protein & ~.;rl,~
fractions from the membrane and cytosol dlat have been ~e~lete~ of lipids (SP-) (Figure 21). No response was observed to incoluble fractions of the cell 10wall (CWC) or to mycolic acid-a~bino~l~n-peptidoglycan (mAGP).
These data intlic~te that DN ,B T-cells recognize non-peptide ~nti~enc of M.
Ieprae in a CDl-restricted manner.
The reactivities of LDN4 correlated with the presence of lipoarabino.l.~nn~.. (I,AM; Figure 22), and we subsequently found that this 15line proliferated to purified LAM. The ability of LDN4 to respond to LAM
was blocked by anti-CDlb antibody (Figure 23). BDN2, an :,B double negative T~ell line which responds to M. Ieprae in a CDlc restricted manner, also responded to LAM. The ability of BDN2 to respond to LAM was blocked by anti-CDlc arltibody (data not shown).

20Example 9: Der7va~ives andAnalogs of I~A~, and CDl-Dependen~ Uses Thereof LAM is a nonprotein molecule, one of a class known as "~mphirhil~J ~
poss~ssin~ both hydrophobic and hydrophilic components (Figure 22).
Hunter, S.W., etal., J. Biol. Chem. 261:12345-12531 (1986); Hunter, S.W., 2~et al., J. Biol. Chem. 265:9272-9279 (1990). The hydrophobic domain is a phosph~tif~yl inositol linked to a hydrophilic heteropolysaccharide composed of a ,..~nn~l~ core and bl~nch~ arabinan side chains with short m~nnnse oligos~e~h~ride l'caps." ChaU~ljec, D., etal., J. Biol. Chem. 267:6228-6233 SVBSTIT~T~ SH~t~ (RUL~ 26) WO g6/12190 PCT/US95/13274 (1992); Chatte~ee, D., et al., J. Biol. Chem. 267:6234~239 (1992).
Protocols for the pl~rific~tion of LAM have been ~lesc~ Hunter, S.W., et al., J. Biol. Ch~m. 261:12345-12531 (1986). F7st Atom Romh~rdment-Mass S~ o~etry analysis inrlir~t~s the vir~ally c~omrl~t~ purity of the M.
leprae LAM (>99.9%).
Prenously, M. lep~ae and M. nlbera~losis LAM have been shown to have distinct ~L~u~s whicll r~sult in dilr~ ~s Prinzis, S., etal., J. Gen. ~icrobiol. I39:2649-2658 (1993). Th,_,.,rol," it is co~cc;vdble that LAM from different l..yeob~ pecies may possess dis~nct T cell epitopes. It is also llOt~WULlh~ that gram positive bact~ri~ contain structuallyrelatçd glycolipids such as lipoteichoic acids, sû that recognition of these molecules by T~ells may be a part of the ;~ r response di~ generally to bacterial pathogens.
To de~rmin~ the dom~in(s) of LAM l~o~sible for stim~ ing DN
cY:~B T~ells, cll~rni~l derivatives of LAM were te~ted for their ability to stim~ e DN cr:~ T-cells. Mild ~ lin~- hydrolysis releases the fatty acid moieties of LAM, while leaving the carbohydrate intac~ This deacylated L.A~ (dLA~ loses the ability to stiml~l~te LDN4 at concenLlatio~s where the native LAM caused 5triki~ T-cell proliferation (Figure 24). In contr~st to deacy~ted LAM, rh~ph~ ylin- sitol .. ,~ nside (PIM) stim~ t~ LDN4 ten-fold more than media alone (Figure 24). These data suggest that the lipid ~om~in of LAM is required for T-cell s~im~ tion~ but that the lepca n~ arabinan bac~.on~ of LAM may not be ~
Finally, the cross-~Lion of LAMs from other mycob~ .ia was 2~ invectigated with regard to two DN <Y:~ T-cell lines (Figure 25). The leprosy-derived T-celI line LDN4 was stimlll~t~ by M. Ieprae LAM ~p LA~, but not by LAM from a clinical isolate of M tu~erculosis (I~E-LAM) or LAM
frûm a virulent laborator)~ strain of M. tuberculosis ~v J_A~. These results demo~Lctrate a species specificity of LDN4 for M. Ieprae L~/l. In con~ast, T-cell line BDN2 responded to M. tuberculosis LAM (~E LAM and Rv LAM) as well as to M. leprae LAM (Lep LAM). Taken together, these SUBSTITU I E S~ET ~RUL~ 2~) -results ~lemonct ate that both species-specific and cross-reactive detf ~ A .t~ of LAM are recogr~ized by DN cY:~ T-cells.

- Example 10: Vaccines Compnsing CDI-PresentedAn~'gens Prior to the present tlicr IQsllre, lipid-c or glycoliri~c were not known to possess pOtf-nt~ ypOterlt :~rcr;ric T_cell-mPAi~ted ;~ r~g~n~ei~y. Thc CDl-~ lipid and glycolipid ~nti~f ns or their filnrtinn~l equivalents tlesr~ihed herein can be used as ~ssç,.
componentC of compositionc deciEn~ to filnrtinn as vacci~e~s to b~ 1, fungal or protozoan pathogenc. Vaccines compri-cing CDl-p1~ed andgerL can ~e ~rlminictereddirectly by parenteral, m~lcQs~l or topical means. For e~Ample, due to the p1~sence of CDl 0 on cells found in the gastrointçstinAl epith~lillm (Bleicher, P.A., et al., Science 250:679-682 (1990)), oral a~lminictTation of vauines co...~ g CDl-p~ sen~d antigerL can introduce such vauines into an ~Aninial in need of a vauine comprising a CD1-presented antigen.
CDl-presen~ed arltiger~c can be combined with other antigeus, either another CDl-presented antigen or an MHC Class I or MHC Class II-presented antigen, to produce a more effective prophylactic or th~,d~,~lir, vaccine.
Vaccines compricin~ the CD1-~l~sell~ed antigeus of the present invention are formnl~ted according to known m~thr,(lc. Ren~ngton's Pharm~cer~nca1 Sciences, 18th Ed., Gennaro, A.R., ed., Mack, Easton, 1990; The Pharmacologist Basis of Therapeutics, 7th Ed., Gilman, A.G., et al., eds., ~Ae~~ An, New York, 1985. phAnnAr~lltir~lly 20 acceptable lipid-stabilizing and -so~ h~ ing agents kno vn to those of sldll in the art ca~ be- -added to vaccines comprising CD1-~l~sented antigens to enhAnr~ their effectiveness. Teng, N., et al., PCT published patent application WO 91/01750 (February 21, 1991); Nunberg, J.H., U.S. Patent 4,789,702 (De~mher 6, 1988).

SVBSTITU I L S~t-E ~ (RLII ~' 2~j PCTrUS95/13274 Example 11: Means and Methods for Inhibiting CDl-Restncted Antigen Present~ion The r'iceloselre of CDl antigen p,es~ system allows for various means and m~.tho~c of ;..l.;~ CDl~ ;r~d antigen E,Lr~ t~ Any S c~ -o~;lion which inhibits the ~ of CDl-~ ~nti~onc1 which i~.Lles with the b~diL~ of aDtigen to a CDl ~ > or whi~h i~L,r~
with the binding of the CDl ~l~ti~cn ~nmrlp~ to a l~molecule, will inhibit CDl~ ,iek~ ples~ ;nn of ~nti~en Such ~~ , called CDl biocking agents~ are useful for controlling undesired T-cell-m~i~t~ ;~ y which occurs, e.g., in autoi.~ ç 'i~zces. Oksenberg, J.R., et al., J.
Neurol. Sci. 115 (Suppl.):S29-S37 (1993).
CDl blocking agents include, but are not limited to, (1) a purified CDl molecule, or a synthetic de~iv~ e thereof, capable of binding a CDl-p~csented antigen and y~vc~ing the antigen's ;~ [~ on with CDl displayed on APCs; (2) a purified TCR polypeptide, or a synthetic de~ivaLiv~ thereof, capable of binding to a CD1-antigen cQmrle~ on a CD1+ APC and y,e~ g the complex's interaction with T~ll ~ tO,s; (3) an antigen antagonist comprising a ch~mi~lly-modified CD1-p~se~led antigen, or a sy~thetic derivative of a CDl-yl~ed antigen; (4) a p~ fi~l CDl-antigen comrl~, or a synthetic derivative thereof, capable of bindi~g to a T-cell ~ r that rccognizes a CD1-antigen c~mr!~r on a CD1 + APC and ~lc~ i~ the T~cell r~Lol's interaction with CD1-antigen complexes; (5) an antibody which binds a CD1 molecule and thus yre~ L~ the ;--~ n of the CD1 mol~lç
and a CDl-ylesen~ed antigen; (6) a polyclonal or mon~clon~ ibody which binds a CD1:antigell comrle~ and t~ "e~ the int.o~artion of the CD1:antigen complex and its cognate TCR; (7) a po~clonal or ~ocloa~l antibody which binds a TCR that necogn~es a CDl-yl~L~d antigen and thus ple~rcllLs the interaction of the TCR with its cognate CDl:antigen complex;
arld (8) a composition which blocks an ~cser~ l step in the yalhway by which CDl-presented antigens are process~d prior to being displayed.

~UBST~TUT~ SHE~ U` E 26!

The preceding Exarnples contain exemplars of CD1 blocking agents as follows.
CD1 blocl~g agents of type (5) are le~l~,.t~d by mnn~)cl~n~l antibody WM25 to CDlb, which ~ lly inhibits CDlb~ A antigen E~ ;o~ ;ig. 15, upper panel) al~d ~cl~n~ lOC3 to CD1c, which ~ r~lly inhibits CDlc-~h; ~ ~gen yl~ (Fig. 15, lower panel). A sl~lled ar~san can used ~e ~ ~s ~es~be~ herein to isolate antibodies which act as CDl bl~l'i~ age~s of t~pe (6) or ~7), and those of skill in the art know how to f~m~ t.~ ~nti~i~s for thr ~l ~..lir, pu~poses. A Critical Ana~sis of Antibo~y Therapy in Transpl~nta~ion, Burlington, W.J., e~., CRC Press, Boca Ra~on, 1992.
CD1 blocking agents of type (8) are r~le3~2ed by chloroquine (Fig.
5c), which inhibits a step in the ~ ng of CDlb-restrirt~ antigens.
Methods of form~ ting and ~minictering chloroq~ine are known to those of skill in the art. Webster, L.T., ~Drugs Used in the Chemotherapy of Protozoal Infections,r Chapters 41 and 42 in Goodman and G~lman's The Pharmacological Basis of 17~erapeuncs, 8th Ed., Gilm~n, A.G., e al., eds., Pergamon Press, New York, l9gO. ~lthough chloro~uine also inhibits MHC-restricted antigen present~titm, those skilled in the art can, using the methods~licrlo5ed herein, identify and/or isolate compositions which spe~ific~lly inhibit processing of CD1-ressrir,~ ~ntigenc Blocking agents of type (3), i.e., antigen antagonists, may be derived from CD1~ ic~d antigen~C icol~t~ by the methods of ~}e inYen~on.
Variants of MHC-l~ d peptide ~ ip,f n~, binding with weaker ~r~";,;~S
~han the origin~l peptide antigen, act as an~agon~sts for mature T~ells in MHC-restricted antigen ~l~sç ~ ;on- Wraith, D.C., et al., CeU 59:247 255 (1989); ~mile~, D.E., et al., Proc. Na~l. Acad. Sci. (~ISA) ~8:9633-9637 (1991). In like f~chi~:)n, CDl-~ led antigens are isolated by the meth~l5 of the invention and are ch~mir~lly modified, accoldi~g to standard techniquec, in order to ge~ le non-antigenic or wealdy ~nti~ ni~ CD1-sellLed antigen de.i~tiv~s. For e~ample, mycolic acid d~iv~Li~cd with p-SlJBSTiT~i I t ~Ht~ LlLt 26) bromophenacylbromide is non-antigenic (Example 4). Antigen antagonists are idPntified as CDl-presented antigen de~iY.sli~es which i~hibit or ~ ,nt the T-cell proli~,atiYe or CDl ~ r~ n~ cytolytic lC~0~5 which oc~r when only the origin~l, unmo~iified CDl~ ~ aD~igen is prcsent (F~ntrl~ 1).
S Blocking agents of ~pe ~2), i.c., TCR de~ cs which block the interaction of the antigen:CDl c~mrl~ with the TCR mo~ s on T-cells, may be yle~ar~ from ~e ~ re by ~ose skilled in the art~ DNA
molecules ~onr~lir~ TCR poly~ s di~l~ by a T-ccll line ~at l~,~S a CD1-~u~d antigen of the im~ention arc icol~t~l ~ç~ n~ to rn~thoAc known in the art. Os~n~.g, J.R, et al., Proc. Natl. Acad. Sci.
(USAJ 86:988-992 (1989); Okse.l~r~,, J.R., etal., Nature 345:34~346 (1990) .
and eildt~lul, Na~ure 353:94 (1991); Uematsu, Y., et al., Proc. Natl. Acad.
Sci. (USAJ 8~:53~538 (1991); Panzara, M.A., et al., Biotechniq~es 12:728-735 (1992); Ue~n~tcll~ Y., Irnmunogenet. 34:174-178 (1991). The DNA
s~u, uce is couvel~d into a polypeptide sequence, and the portion of the polypeptide seque~ce that corresponds to the antigen-binding vanable region of a TCR polypeptide is used to design synthetic oligopeptides that bind CD1:antigen complexes on APCs, thereby inhibidng andgen ~seul~tion.
Oligopeptides are ç~ mir~lly synth~ci7~1 according to standard mçtho~c - 20 (Stewart and Young, Solid Phase Peptide Syn~fzes~s, Pierce ~h~nir~l CO., ~orkl~nr1~ Illinois, 1985) and purified ~om reaction ~ Cs by reversed ph~se high ~res~u~e liquid ch~ou~kJ ~ph~ (~IPLC). Pilot trials of tre~tm~nt of hl-m~nc surçtlu~g from an ~ulo; .~ disease with peptides derived from MHC-r~ . ie~ :,B TCR mol~.~lles is ~ e.ltly underway. Oksenberg, J.R., et al., J. Neurol. Sci. 115 (Suppl.J:S29-S37 (1993). TCR-derived peptides ~hich ~lnrtjon as CD1 blocking agents are idr ~I;f.~ as TCR-derived peptides which inhibit or plevcùl the T-cell prol;r"~ , or CDl ~ r~ ~olytic which occur in the plesellce of CD1-~1~sellhd an~ge~ (~y~mp1e 1~
The assays for CD1-~rcse~led antigens describe~ herei~ may be used to screen molecular libraries for CD1 blocking agents. Libraries of molecularly diverse compositions are prepared by chemir~l, biochrmical TlJ ~ ~ ~HEE f (RULE 26) and/or biotechnological means. Such lil~r~ries include combinatorial libraries of synthetic peptides (Houghten, R.A., et al., BioTechnigues 13:412-421 (1992)) and fusion protein librari~s yl~p~od by lccuulbi~L DNA terhn~logy, e.g., phage display libra i~s K~ivu~, E., et al., J. Biol. ~ ~ )0~
20210 (1993). The lihr~ri~s a~e sc~ Ld for the ~ ce of .. P -~ ~ which inhibit or ~le~,lL the DN T~ell prol;~-~ c a~dlor Cl)l cy~olytic lcs~
~escrih~1 herein. CDl blorl~i~ m~mhers are i~l~t~d ~om the libray accol~ing to techniques ~own in thc ar~ and applo~,~Le for the t~pe of libraryemploye~. Lowman,H.B., etal., Rio~ ny30:10832-10838 (1991); Felicia, F., et al., J. Mol. Bio~. 22:301-310 (1991); D~nf~ , T., et al., Neurochem. Int. 7:247-253 (1985); Owens, R.A., et al., Bioplys. Res.
Commun. 181:402 408 (1991).
In order to detect a CD1 blocking agent in a sample, assays for CD1-presented antigen are perforrned in duplicate. The first (control) assay is a T-cell proliferative or cytolytic assay pe~form~d ecsenti~tly as described in Example 1. The second assay is sllhst~nti~lly the sarne as the first assay except that the second assay ~d~ition~lly c4.-~;nc a sample ~ ~d of cont~ining a CDl bloc~ng agent. The l).csence of CD1 blocking agents in the sarnple correlates with a T-cell proliferative or cytolytic response in the second assay that is sig~ Arllly less than that measured in the first assay.

Example 12: T-Cell Reactivi~ to CD1-Presented Antigens As described in Exarnple 1, T-cell reacdvity to CD1-r~sl. ;rt~ andgens was initially found among a subset of T~ells, i.e., CD4-8- TCR ~+ T-cells.
However, additional examples of CD1-res~ict~foreign antigen locog~lion of rnicrobial antigens is also found in CD8+ TCR a~+ T-cells, as well as among TCR y~+ T-cells.

~JBSTITU ~ ~ S.~t~ LJLE 26!

W O 96/12190 PCT~US9S/13274 CDI Restncts the Response of TCR y~ T-cells to an Antigen Human pc~irh~l mono".Jrl~r cells were obtaill~d from a 1~ lk p~l~
After ~letion of the mo,~ te population by plastic ~tlh~r~P~ R ~ T-cells were isol~t~d using ~ ...t;r cell SO~g (M~CS) as ~)lt~iuu~
S ~esçril~ (Srhittr~, B. and Raje~ , K., Na~ue 346:749-751 (1990.
Briefly, PBMC were l~h~ll~ with biotinylated anti~ô I~ nl~ocll~nal antibody (TCRôl), followed by ~s..nl.~,~ with FlTC-A~idin (CALTAG) and biotinylated beads (Miltenyi Biotec, S~ V~IC~ CA). Cells were then positively so~ ted in a strong ...zg..et;c field through a high capacity (3xlO' ceUs) column (Miltenyi Biotec, Sunnyvale, CA). The purity of the ceUs was ~sGcse~ by FACS imm~ tely after ~ ;on Starting from 2x109 PBMC, this proce~ure typically allows one to obtain 2~107 TCR y~+ T-cells with >98~; purity.
After three days in vitro without specific stim~ tion, the purified TCR
y~+ T~ells were cultured with autologous GM-CSF- and IL~- treated CDl +
monocytes at a 1:1 ratio in 24 well plates (Costar, C~mhri~lge, MA) (plating 2X106 T~ells per well). The antigen used to s~mlll~t~ the cells was an organic extract of Mycobacterium tuberculosis (as described in Examples 2 and 3) at a 1/3,750 t1illltion Briefly, a chlolofol~ eth~ l e~etion based on Folch's method, which paLliLions most lipids into the organic phase and protein into the aqueous phase and ~ f-r~Ce, was used. Recombinant rnterlenkin-2 (rIL,2) was added (0.3 nM) after 4 days of culture and the cells were restim~ te~ with antigen every 12 days.
Three rounds of stim~ tjo~ led to the icol~tion of the CD1b-restricted TCR ~+ T~ell lu~e called OGD1. Although the ~u.;tled TCR ~ô+ T cell population from which this T-cell line was isolated pr~o.,.;lu,~ly eA~lcssed the V~2 gene product (90%), FACS analysis of OGD1 with ~e mol~ocl~nal antibody TCR ~ TCR),- ~TCSl (V~l gene product) and BB3 (V~2 gene product) (available f,rom T Cell Di~ sli~, Cambridge, MA) demo~;L~ated that the isolated T~ells c~sod predo~ ly the V~l gene product (Figure 26A). This result suggests that this CD1 m~i~ted ssim~ tion is -~UBSTlTlJ ~; ~HFE~ (~U!~ 26) W O 96/12190 PCT~US95/13274 different than all previous demon.~ t~ examples of stimulation of TCR.~+
T cells by bacterial antigens, which invariably involve eyr~n~ion of V~y2V~2 T-cells.
The lc,l.iel;on of the OGDl cell line by CDlb was cQ~Ir~ p~i using anti-CDlb m~nnrlnn~ ibody which bloc~ ~tl~t(~ of antige~ pulsed CD1+ mono~;y~s (Figure 27) and also blor~d ~ific p~lif~aticm of the OGDl celI line to ,~ ob~ct~ nti~nc pIes~t in the organic phase of chloroforrn~ nnlt~ C~AmyCo~aetf ;~igure28). Fultherand1ysisof the fine s~ f;ir-l~ of this cell line demnn~fe~ that OGDl r~ ~
saponified cord factor (SIGMA, St. Louis, MO) (i.e., mycolic acid, a lipid component of the mycob~cte-ri~l cell wall) (Figure 28).

CD8 f TCR a,~+ T-cells Recogrsize Antigens Presented ~y the CDl Molecule In order to determine if the CD1 molecule could serve as an antigen plesenti--~ molecule for CD8+ TCR ~+ bearing T-cells, ~A~C~ s similar to those described above were ~ro~ cd u~sing pnrified peripheral blood CD8+ T-cells instead of TCR ~y~+ T-cells. T-cells were purified with m ~gnlotic beads conjugated with anti-CD8 monoclsn~l antibody (Miltenyi Biotec, SuDnyvale, CA) and purity was ~sse~sed by FACS after subsequent st~inin~ with FITC con3ugated monoclonal antibody OKT8 (anti-CD8a). The culture protocol was similar to that for TCR y~+ T~ells. After three stimul~tions of these CD8+ T-cells with CDl+ monocytes pulsed with the organic extract of Mycobactenum tu~erculosis, a cell line was obtained which was named OAB8. FACS analysis with FITC~onjugated anti-CD8 ~x-chain (OKT8) and anti~D8 ,B~hain (2ST8-5H7) monoclonal aIItibodies showed that the cell line was homogeneously posidve for e~ression of the CD8 a~
heterodimer. Staining with TCR c~-specific monoclonal antibody BMA031 showed that cell line OAB8 was homogenously positive for TCR c~.
Proliferation assays confilllled the specificity of the OAB8 cell line to the organic extract of mycobacteria and showed that OAB8 recognized purified SJBSTiTUT~ SHE.T ~RULF 26~

mycobacterial mycolic acid (Figure 29). Moreover, antibody blocking e~e,i llen~ ~rnor,~lra~ed that the 0AB8 cell line was resLric~ed by the CD1c molecule (Figure 30), as anti-CDlc mnnnclon~l andbody bloe~r~ its lcs~ se W11CLeaS antibodies to CDla, CDlb and MHC class I ms~ rs had no effect.
These data intlir~te that in s~tlitinn to the ~Clivily of CD4~ TCR
~+ bearing T-cclls to CDl~ ,t~, li.;lo~ t-~f ~C, othcr major T-ccll pop~ ti~nc are CD1 ~ r*A l~,uce CD8+ TCR ~+ l~hlg T-cclls ~ccollnt for a~plo~....~t~ly 35% of all ~ ti~ T ceLls and TCR ~ bearing T-cells ~ccollnt for ano~r 5~ of all T-cells, the ~!ot~nti y...h-~e of the CDl-restricted ;.. ~ e response is likely to be sig.. l~.. l in a variety of immnnological response and syndromes. Moreover, since these T~ell populations are known to secrete an array of cyto~es and t,o m~i~t~
cytolytic activity, the err~or capabilities of CDl-restTicted CD8+ TCR a~+
and TCR~y~+ T-cells specific for nonpeptide antigens may play a critical role in host defense.

Incorpora~on by Reference The entire text of all public~tion~ cited herein are entirely incorporated herein by reference.

~UB~TI~llT~ S~EET ~RULE 26)

Claims (22)

What Is Claimed Is:

1. A method for producing a vaccine containing a CD1-presented lipoarabinomannan or structurally-related glycolipid antigen comprising the steps of:
a) incubating a sample containing a CD1-presented lipoarabinomannan or structurally-related glycolipid antigen with CD1-positive cells;
b) separating said CD1-positive cells displaying CD1-bound lipoarabinomannan or structurally-related glycolipid antigen from said sample;
c) separating the CD1-presented lipoarabinomannan or structurally-related glycolipid antigen from said CD1-positive cells displaying said antigen; and d) formulating said separated CD1-represented lipoarabinomannan or structurally-related glycolipid antigen so as to form a vaccine.

2. A method of producing a vaccine containing a CD1-presented lipoarabinomannan or structurally-related glycolipid antigen comprising the steps of:
(a) fractionating a sample containing a CD1-presented lipoarabinomannan or structurally-related glycolipid antigen into two or more fractions;
(b) testing said fractions for the presence of a CD1-presented antigen; and (c) formulating one or more fractions which contains said CD1-presented lipoarabinomannan or stlucturally-related glycolipid antigen so as to form a vaccine.

3. The method of Claim 1 or Claim 2 wherein:
(a) said CD1-presented lipoarabinomannan or structurally-related glycolipid antigen is presented by a CD1 molecule selected from the group consisting of CD1a, CD1b, CD1c, CD1d and CD1e; or (b) said CD1-presented lipoarabinomannan or structurally-related glycolipid antigen is isolated from a Mycobacterial species selected from the group consisting of M. tuberculosis, M. bovis, M. leprae, M. fortuitum and M.
avium.

4. A vaccine containing a CD1-presented lipoarabinomannan or structurally-related glycolipid antigen producible by a method according to any one of Claims 1 to 3.

5. Use of a CD1-presented lipoarabinomannan or structurally-related glycolipid antigen for the manufacture of a medicament which induces a specific T cell response, said medicament for use in vaccination.

6. A vaccine which induces a specific T cell response in a vertebrate upon administration to said vertebrate, the vaccine comprising:
(a) an effective specific T cell-inducing amount of a CD1-presented lipoarabinomannan or structurally-related glycolipid antigen or a functional fragment or analog thereof and a pharmaceutically acceptable carrier; or (b) an effective specific T cell-inducing amount of a CD1-presented lipoarabinomannan or structurally-related glycolipid antigen or a functional fragment or analog thereof, complexed with a CD1+ molecule, and a pharmaceutically acceptable carrier

7. A vaccine according to Claim 6 further comprising:
(a) one or more cytokines or other molecules which induce CD1 expression on antigen-presenting cells; or (b) one or more different antigens; or (c) a CD1 molecule selected from the group consisting of CD1a, CD1b, CD1c, CD1d and CD1e, wherein said vaccine comprises an antigen:CD1 complex.

8. The vaccine of Claim 7, wherein at least one of the different antigens is a non-CD1 presented antigen which is selected from an MHC-I presented antigen or an MHC-IIpresented antigen.

9. A method for vaccination, comprising the step of administering a vaccine according to Claim 6, 7 or 8 in an amount effective to provide an immune response to said lipoarabinomannan or structurally-related glycolipid antigen in a bird or mammal.

10. A method according to claim 9, wherein said vaccine is administered orally or parenterally.

11. Use of a CD1-presented lipoarabinomannan or structurally-related glycolipid antigen (or its functional equivalent) for the manufacture of a medicament for enhancing the immunity of a vertebrate by inducing a specific T cell response.

12. Use of (a) a CD1-presented lipoarabinomannan or structurally-related glycolipid antigen (or its functional equivalent) and (b) one or more additional non-specific response inducing components for the manufacture of a medicament for enhancing the immunity of a vertebrate by inducing a specific T cell response and said non-specific response.

13. A vaccine according to Claim 6, wherein the lipoarabinomannan or structurally-related glycolipid antigen is:
(a) a cellular component of an organism; or (b) derived from a bacterial, fungal, or protozoan cell.

14. A method for enhancing or accelerating the immunity of a vertebrate which comprises administering to the vertebrate an effective immunizing amount of a vaccine comprising a CD1-presented hydrophobic antigen or its functional equivalent.

15. A CD1-blocking agent for use in therapy which inhibits CD1-restricted lipoarabinomannan or structurally-related glycolipid antigen presentation selected from the group consisting of an antibody, a synthetic peptide, an inhibitor of CD1-restricted lipoarabinomannan or structurally-related glycolipid antigen presentation, and an lipoarabinomannan or structurally-related glycolipid antigen antagonist derived from a CD1-presented antigen.

16. A method for inhibiting CD1-restricted lipoarabinomannan or structurally-related glycolipid antigen presentation by CD1-positive cells comprising the step of contacting cells displaying a CD1 molecule with the CD1 blocking agent of Claim 15.

17. A method for detecting a CD1-presented lipoarabinomannan or structurally-related glycolipid antigen in a sample comprising the steps of:
(a) contacting said sample with CD1 positive cells;
(b) contacting said CD1 positive cells with T cells; and (c) measuring the proliferative or cytolytic response of said T cells.

18. A method for isolating a CD1-presented lipoarabinomannan or structurally-related glycolipid antigen from a sample comprising the steps of:
(a) incubating said sample with CD1 positive cells which bind said CD1-presentedlipoarabinomannan or structurally-related glycolipid antigen, in order to generate CD1 positive cells displaying CD1-bound lipoarabinomannan or structurally-related glycolipid antigen;
(b) separating said CD1 positive cells displaying CD1-bound lipoarabinomannan or structurally-related glycolipid antigen from said sample; and (c) separating the CD1-presented lipoarabinomannan or structurally-related glycolipid antigen from said CD1-positive cells displaying said lipoarabinomannan or structurally-related glycolipid antigen.

19. An isolated CD1-presented lipoarabinomannan or structurally-related glycolipid antigen producible by a method comprising the steps of:
(a) incubating a sample with CD1 positive cells which bind said CD1-presented lipoarabinomannan or structurally-related glycolipid antigen, in order to generate CD1-positive cells displaying CD1-bound lipoarabinomannan or structurally-related glycolipid antigen;
(b) separating said CD1 positive cells displaying CD1-bound lipoarabinomannan or structurally-related glycolipid antigen from said sample; and (c) separating the CD1-presented lipoarabinomannan or structurally-related glycolipid antigen from said CD1 positive cells displaying said antigen.

20. An isolated T-cell that recognizes a CD1-presented lipoarabinomannan or structurally-related glycolipid antigen.

21. A T-cell according to claim 20, wherein said CD1-presented lipoarabinomannan or structurally-related glycolipid antigen is:
(a) presented by a CD1 molecule selected from the group consisting of CD1a, CD1b, CD1c, CD1d and CD1e;
(b) is isolated from a Mycobacterial species selected from the group consisting of M. tuberculosis, M bovis, M. leprae, M. fortuitum and M. avium.

22. A method for providing a T-cell according to claim 20, comprising the steps of:
(a) purifying T-cells which are capable of binding a CD1-presented lipoarabinomannan or structurally-related glycolipid antigen from a sample containing T-cells;
(b) incubating a mycobacterium with CD1+ cells which bind said CD1-presented lipoarabinomannan or structurally-related glycolipid antigen to generate CD1+
cells displaying said CD1-presented lipoarabinomannan or structurally-related glycolipid antigen;
(c) contacting the CD1+ cells displaying said CD1-presented lipoarabinomannan or structurally-related glycolipid antigen of (b) with the T-cells of (a); and (d) isolating T-cells that recognize an isolated CD1-presented lipoarabinomannanor structurally-related glycolipid antigen and give a proliferative response during step (c), wherein said proliferative response does not occur in the presence of antibodies to CD1.