CA2086015A1 - Identification of cell subpopulations using modified pcr to amplify expression intermediates - Google Patents

Abstract

A modified polymerase chain reaction (PCR) which permits identification of cells characterized by expression of a particular protein, such as B-cells and T-cells is described. The modified PCR
selectively amplifies the genes encoding these proteins, which contain both a constant region and a variable region, which variable region contains a consensus sequence. The PCR employs two primers, one of which is a perfect match for the gene encoding a portion of the constant region, and the second primer is a degenerate primer designed to accomodate the gene encoding the consensus sequence. By employing these primers, most of the sequence encoding the protein containing the consensus sequence and constant region can be amplified, cloned and sequenced, thus permitting clonal identification of the cell expressing the protein. The resulting information can be used to design therapies for autoimmune diseases, lymphomas and leukemias.

Description

WO~1/19816 PCT/US91/04317 , 2 ~

IDENTIFICATION OF CELL SUBPOPULATIONS USING
MODIFIED PCR TO AMPLIFY EXPRESSION INTER~EDIATES

Technical Field The invention is directed to applying a modified form of a polymerase chain reacti~n (PC~) to the expression intermediates in cells where the nature of a cell subpopulation is characterized by these intermediates. More specifically, the invention concerns identifying subpopulations of B or T-lymphocytes by characterizing the DN~ and mRNA int~rmediates for their def ining immunoglobulin or T-cell receptor proteins.
This characterization is made possibie by amplification of these expression intermediates using a modi~ied form of PCR in which one primer is degenerate and the other is a perfect match.
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Backaround Art B-cell and T-cell subpopulations are character-ized by the nature of the immunoglobulin ~Ig) or T-cell receptor (TCR) proteins respectively produced by the ~ individual cell. These characteristic proteins are as-; sembled from germ line DNA sequences present in unrearranged form in the genomic DNA of the germ line.
Onc~ the segments encoding the complete sequences of these proteins are assembled during cellular differentiation, the lineage of the subpopulation is determined. The rearranged, assembled genes will be effectively present as an mRNA in the clones of the cell line.
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, -2-In the case of T-~ells, for example, the characteristic proteins are the TCR heterodimers. The typical TCR heterodim~r is a combination of an ~- and ~-subunit, although a small percentage of T-cells contain, instead or in addition, a similar pair designated r ~ . Both types of TCR have substantially similar features: starting from the N-terminus, the proteins are composed of a variable (V) sequence (preceded by a leader) which varies from one T-cell population to another, fQllowed by a joining (J) segment in the case of ~- and r-subunits or by both a diversity (D) and J segment in the case of ~ and ~ proteins, and then a constant region which is characteristic of each o~
the four TC~ chains and largely invariant.
Characterization of the V, D an~ J regions of these proteins would establish the identity of t~e particular subpopulation of interest. The heavy and light chains of Igs have similar structures. In various vertebrate species, multitudinous (i.e., 101-1ol3) such subpopulations have been postulated.
It is of particular interest to characteri2P
individual T-cell populations in a number of contexts.
For example, T-cells which are present in autoimmune infiltrates are thought to be those responsible for the undesirable immune response. Thus, for example, in type I diabetes, which is caused by an autoimmune response to the pan~reatic islets, infiltrates of ~hese islets should contain T-cells which are the specific su~type responsible for the disease. Identification of unique TCR expressed on these T-cells would permi~ design of therapeutic measures directed against them specifically.
Similar remarks can be made concerning T-cells which are malignant lymphomas or associated with organ graft rejection. By characterization of these subpopulations, specific immunotherapies can be devised.

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- It is currently thought that foreign antigens are cleaved enzymatically and the fragments presented to T-cells in the context of a major histocompatibility complsx (MHC) encoded glycoprotein. T-cell receptors have regions which bind to both the antigen fra~ments and MHC products. Amon~ TCRs, the greatest concentration of structural variability resides in th~ J or D-J junctional reyions which seem to make the primary contacts with the specific an~igenic pep~ide fragment; the variable (V) regions, which pro~ably make con~act with the MHC
glycoprotein presenting the antigen fragment, show more limited variability. The genes for many of the variable regions present in T-cell populations for human and murine ~- and ~-su~units have been sequenced. Thus, data are available which permi~ the ascertainment of the pres-ence of consensus sequences in the Yariable regions.
~See, for example, the determinations referenced in Davis, M.M., et al., Nature (1988) 334:3950402.) Theoretically, recovered T-cells ~or B-cells) . 20 could be characterized by sequencing cDNA molecules encoding their characteristic TCR ~or Ig). However, the production of cDNA libraries necessary for applying this approach to a particular candidate TCR chain, while pos-sible, is intensely laboriou~. The present invention of-fers a way to obtain copious quantities o~ the relevant gene which can then readily be cloned and sequenced.
The invention method utilizes the recently developed polymerase chain reaction ~PCR) which involves a highly selective and highly effective amplification of ;~ 30 a desired DNA sequence at the expense of unwanted sequences. The PCR technique as oriqinally described relies on the use of completely matched (sensa and antisense) primers at the 5' and 3' ends of the sequence to be amplif ied . Polymerization in the pregence of these primers results in a million or billionfold amplification WO 91/19~16 PCI/US91/04317 3 ~

of the desired sequence in a relatively small number of polymerase rounds (Saiki, ~.K., et al., Science (1985) 230:1350~1354).
The PCR technique has been used for genomic and for cDNA cloning (Scharf, S., et al.l Science (1986) 233:1076-1078; Saiki, R.K., e~ al., Science (198~) 239:487-491). It is also known that the primers need not exactly match the gene sequence perfPctly (Lee, c.c., et al., Science (1988) 239:1288-1291). Others have also utilized restriction sites built into the primers to ~s-sist in the subsequent cloning of the amplif ied sequence.
The use of degenerate primers to amplify genes where the nucleotide sequence is predicted ~rom a known amino acid s~quence has been employed for the cloning of some genes. Generally, knowledge of an amino acid : seque~ce predicts degenerate coding sequences (see, for example, Gonzalez, G.A., et ~1., Nature ~1989) 337:749-752; Lee, C.C., et al., Scienc_ ~1988) 239:1288-1291). Others have applied the PCR reaction to the variable regions of immunoglobulins by using a 32-fold degenerate primer in conjunction with a single primer wheréin neither first or second primers was a complete match for the inherently variable regions of the protein segment sequenced. The variable region amplified contained all three of th~ Ig complementarity determining regions (CDR)~ CDR1, CDR2, and CDR3. (For the TCR, by analogy to immunoglobulins where the V-region-encoded CDRl and CDR2 regions are less variable, and the CDR3 varies the most, it is believed that the CDR1 and CDR2 regions of TCR associate with the MHC and the CDR3 region . associates with the antigenic peptide (see abova).) The PCR technique using the combination of a defined but not necessarily perfectly matched constant primer in conjunction with a degenerate primer was also applied to ganes encoding the variable heavy chain from spleen DNA

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of a mouse immun~zed with lysozyme (Ward, E.S., et al., Nature (1989) 341:544-546).
For reasons similar to those with respect to the immunoglobulin variable region, application of the classical PCR techniques to the gene encoding the T-cell-characterizing proteins is not directly possible, since, by definition, the variable region presents an unXnown sequence to which a completely matched primer cannot bs designed. The present invention overcomes this handicap by providing for the design of a consensus primer which is applicable to a m~ltitude of variable regions and by modification of the PCR protocol to accsmmodate the presumptive lac~ of total match between :~ this primer and substrate.
Disclosure of the Invention The invention provides a means for selective :: ampli~ication of the characterizing genetic material in subpopulations of B-cells and T-cells in any arbitrary candidate sample. Tha method relies on modification of the polymerase chain reaction (PCR) using a degenerate : primer for consensus portion of the variable region of genes encoding B-cell- or T-cell-characterizing protein.

Accordingly, in one aspect, the invention is directed to a method to conduct a polymerase chain re-action to amplify the codi~g region for a protein having ` a variable region and a constant region, such as a T-cell receptor subunit or an immunoglobulin light or heavy `~ 30 chain, which method comprises subjecting a cDNA encoding said subunit to a PCR using as a 3~ (antisense) primer a ~ DNA sequence which is a precise match to the gene : encoding the constant region of the subunit and as a second 5' primer a degenerate "match" to the gene encoding a consensus region in the variable region of '''' ; ~ :

, WO91/19816 PCTtUS91/04317 8~ 6-said subunit,-or; vice ver~a, a 3' (antisense) primer which is a degenerate match to the gene encoding a portion of the variable region and a 5' ~sense) primer which complements the constant region. The senses re reversed if the complement to the coding sequence is used~ In other aspects, the invention is direc~ed to methods to identify B or T-cell subpopulations in a sample by applying the modified PCR techniques of the invention to cDNA prepared from B or T-cell mRNA in said sample, cloning and sequencing the amplif ied DNA.

Brief Descri~tion of the Drawinas Figure 1 is a diagram of the assembled genes encoding typical ~ and ~ chains of the T-cell receptor.
Figure 2 is a diagram of the assembled gPnes encoding the heavy and light chains of immunoglobulins.
Figure 3 shows the sequences of typical variable, joinin~, and constant regions of genes encoding several variants of TCR chains.
Modes of Carrvinq Out the Invention The invention methods permit the effective amplification of a DNA sequence which encodes a protein that has a predictable, constant region along with an un-known variable region wherein the variable regioncontains a framework sequence sufficient to permit the design of a degenerate consensus primer which can serve as the ~atching primer for multiple variable regions.
There are three general steps in the invention process: isolation or preparation of the B or T-lymphocytes to be characterized, extraction of mRNA and preparation of first-strand cDNA, and PCR conducted on this first-strand cDNA. This cDNA will be the antisense sequence; there is no reason its complement could not be prepared and used as the PCR substrate instead.

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WO91/198~6 PC~/U~91/04317 . ' 2~86~ 3 T and B-cells of interest are obtained from in vlvo sources such as peripheral blood, lymph nodes, spleen, and organ infil~rates caused by autoimmune re-activity, tumor reactivity, or response to tissue engraftment. Isolation of RNA from the cells of interest can be performed directly, or subsequent to functional and/or phenotypic characterization of subpop~lations, For example, the pancreatic islets of mi~e which are genetically susceptible to autoimmune diabetes (the NOD
lo strain) can be prepared from surgically removed and dis-sociated pancreata, and RNA extracted by methods generally known in the art. Since only T-cells can serve as sources of TCR RNA, removal of associated cells (such as islet cells) derived from other lineages is not necessary for this analysis. Furthermore, the T-cells, for example, in the population can be increased by stimulation with cytokines, such as IL-2, or other T-cell-specific mitogens, such as phorbol myristate acetate (PMA).
mRNA is extracted and first-strand cDNA
synthesis is conducted using standard procedures.
First-strand cDNA synthesis can also be performed on only minute quantities of extracted RNA by use o~ the PCR
technigue. For example, priming of the reverse transcription reaction can be accomplished with oligo d(T) or with antisense primers specific for a known upstream seguence. Also, for PCR reactions conducted with limiting amounts of RNA tsuch as that extraeted from 106 or fewer cells), the number of cycles can be increased to at least 60 without the requirement for further purification o~ PCR products and secondary addition of Taa polymerase (Rapopolee, D.A., et al., J
Cell ~iochem (1989) 39:1-11).
In the classical polymerase chain reaction, a DNA sequence to be amplified is provided with a sense and : . ': ': , -2 ~

antisense primer, one of which is designed as a match for the 5~ end of the sequence to be copied, and the other for the 3 ' end. As used herein, ~match~ refers either to the same sequQnce as found i~ the substrate DNA or its complement, as the case may be. By ~match~ is also meant both a pr~cise match, in which case ~his will be specified, or a match which is sufficient to permit PCR
to proceed, even if there is no primer in the degenerate mixture which in fact exactly matches the substrate DNA.
Typical degenerate primers have at least 17 b~ses in the matching portion--longer oligomers can be used. The illustrated variable TCR ~ primer has a length of 39 bases, part of which is additional sequance to provide restriction sites.
lS The method of the invention is most useful to characterize cells which are themselves characterized by the production of a particular protein. ~his situation is found in, specifically, T and B lymphocytes wherein the properties are determined by the TCR or Ig subunits produced. Because the genes encoding these protein subunits are expressed, a convenient source of the as-sembled coding sequence is the mRNA which can readily be isolated from the cells. The mRNA is reverse-transcribed to obtain a single-strand cDNA, which provides the initial template ~or the PCR amplification. The resulting cDNA will be a complement to the sense coding sequence; hence, the primer matching the sequence on the N-terminal portion should be the sense primer, and that matching the sequence close to the C-terminus should be the antisense primer.
In the case of the TCR subunits, the variable region containing the consensus sequence is near the N-terminus, as shown in Figure l. The constant region comprises the C-terminus. DNA sequences which encode the constant region and the variable regions of the TCR in : . : .: .

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_g_ human, murine, and ra~ TCR subunits are compiled in the GenBank, ~MBL, VecBase, and NBRF data bases. Reference may be made to these compilations to ascertain the ap-propriate regions for primer ~onstruction.
As shown in Figure 2, the organization of immunoglobulin subunit heavy and light chains is similar.

DNA sequences encoding variable, diversity, joining and constant regions of various immunoglobulins are also found in the GenBank, EMBL, VecBase and NBRF data bases.
In the method of the invention, one of the primers in the modif ied PCR is preferably a precise match to the part of the gene encoding the constant sequence in the protein. However, because the conditions are adjusted to account for lack of an exact match in the lS variable region, it is not required that this be the case. Mismatches comparable to those to which ~he ; hybridization conditions are adjusted can be employed as well. In the case of TCR or Ig, the sense primer will be constructed to the downstream constant region, or a por-tion thereof when first-strand cDNA is employed as a template. Use of the co~plement to the first-strand cDNA
would require the converse. The degenerate primer, which is a "match" for a consensus region, will ~e constructed on the basis of genes encoding the variable regions in the case of TCR and Ig. Examination of the published gene sequences for the variable region will permit the ascertainment of the appropriate consensus regions and the design of appropriate sequences.
In the case o~ the T-cell receptor protein ~
and ~ subunits, a consensus region is found between amino acids 30 and 40, just downstream of what is presumed to be the "CDRl"-encoding region of the gene. A consensus region is found in a similar position in the TCR
~ubunit.

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The polymerase ~hain reaction is conducted using these primers with a modification to the protocol to account for the mismatch of the degenerate primer. In general, this involves lowerrng the temperature in the initial cycles so as to provide lower hybridization stringency. This temperature is raised in the later repeated cycles as the subsequent strands synthesized will incorp~rate the conse~sus primer and perfectly match the subs~rate.
Application of the modified PCR of the invention to the identified identification o~ T-cells is especially useful. Amplification and sequencing of the TCR ~xpressed in T-cells inYolved in autoimmune responses, graft rejection, or lymphomas will make possible the design of peptides which can be used as vaccines or monoclonal antibodies directed against the TCR for use in the treatment of these conditions. See, for example, Vandenbark, A.A~, et al., Nature (1989) 24l:541-554; Acha-Orbea, H., et al., Cell (1988) 54:263-273.
The following Examples are intended to il-lustrate, but not to limit, the invention.

ExamDle l PreParation of T-Cell Samples To obtain T-cell clones ~or analysis, DBA/Z
mice were immunized at the base of the tail with 100-200 ug sperm whale myoglobin in 50% complete Freund's adjuvant, and the draining lymph nodes were removed 8 days later. The lymph node cells were cultured with irradiated syngeneic spleen cells as antigen-presenting cells ~APC) and the sperm whale myoglobin for-12 days.
The cells were restimulated 3 times with APC and the 56-131 cyanogen bromide fragment of the sperm whale ,- :
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myoglobin at 12-13 day intervals, and the bulk cultures were cloned and subcloned by limiting dilutions.

ExamE~e 2 PreParation of cDNA
Res~ing T-cells prepared as in Example 1 (m~re than 10 days after last antigen stimulation) were Ficoll purified and resuspended at 1.5-1 x 106/ml in medla containing 10 ng/ml PMA, 250 ng/ml ionophore and 10-25 U/ml human recombinant IL-~. After 24 hours, the cultures were diluted 1:2-1:4 into media containing rIL-2 alone. Optimal RNA levels were shown after 3-5 days.
Total RNA was prepared by homogenization of 5-lo x lo6 cells in guanidine thiocyanate and centrifugation through cesium chloride, according to the method of Chirgwin, J.M., et al., Biochemistry (1979) 18:5294_5299.
A first-strand cDNA synthesis was performed on 10 mg of total RNA using oligo-dT priming with reverse transcriptase, according to the method of Gubler, U., et al., Genq (1983) 25:263-269. Ten to fifty percent of the transcription reaction was used as a template for PCR
amplification.

~xam~l~ 3 Conduct of ~CR
A. The oligonucleotide primers for the constant and consensus regions were determined from published sequences by Chien, Y., et al., Nature (1984) 312:31-35; Saito, H., et al., ibid, 36-42; Arden, B., et al., Nature (198S) 316:783-787; Becker, D.M., et al., Nature (1985) 317:430-434; McElliot, D.L., et al., J
Immunol (1988) 140:4123-4131; Yague, J., et al., Nucleic Acids Res (1988) 11355-11i63. The constant region primer for the ~ chain had the sequence 5'-TCAACTGGACCACAGCCTCAG-3'. The 216-fold degenerate : ~, : ....

, . , 2 ~ 1 r i oligom~ric primer for the consensus variable region purchased from Operon Technologies, San Pablo, Californi~, had the sequence 5~-TAAGCGGCCGCTGGTACZLMCAGC
ATCCXGGMGAAGGCC-3'. In this primer, Z represents 40%A/40%~/l5%C/5%T;
; L represents AjG/T;

M represents 45%A/50%G/5%C; and X represents C/T.
The degenerate primer was llsed at 1 uM final concentration in a 100 ul PCR reaction with 1 uM constant reg on 3' primer.
Th temperature of annealing segmen~s was 2 cycles at 37C, 1 cycle at 42C, and 27 cycles at 55C.
The amplified product was purified and cloned by digesting the PCR-amplified material with NotI, providing a cloning site at the 5' consensus oligo available for ligation into a polylinXer derivati~e of M13 bearing an NotI site. The general purification process and ligation into M13 vectors, with the above exception for NotI restriction site, was described by ; Acha-Orbea, H., et al., Cell ~1988) 54:267-273.
The results of the amplified sequences of six recovered TCR ~ chains were sequenced using standard techniques. The results o~ this sequencing are shown in Figure 3. The results showed that clone 8.2 is a member o~ the V-~-4 family and clones 10.3, 12.2, 14.12, and 14.16 are members of the V-~-1 family. Clone 9.4 ap-pears to define a new-family pro~isionally designated V-~-15. The clone designations in Figure 3 are on the left.
A similar protocol and the same consensus primer is also useful to amplify rat and human TCR
chains and murine TCR B chains.
B. In a manner analogous to that set forth in paragraph A of this example, PCR amplification of TCR

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ch~ins from in vlvo and in vitro cell sources is conducted. The consensus primer is designed to a con~erved region in TC~ V8 sequence similarly located to that of the ~ sequence. The ~onsensus primer is us~d in combination with a variety of TCR CB or JB
oligonucleotides to permit amplification of ~CRB chains which utilize members of the murine VB f~milies 2,5,6,8,lO,12 and 15. The sequence is 5' TAA GCG GCC GCA
; TGS LYT GGT AYW XXC AG 3' where S=G/T, L=A/G/T, Y=C/T, :~: 10 W=A/C AND X=A/G and contains a NotI restriction site near the 5' end. The PCR cycling conditions used with this primer are identical to those used with the TCR V~
consensus primer. This primer can also be used for amplification of rat TCR ~ chains.

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Claims (11)

Claims

1. A method to amplify a substrate DNA
sequence encoding a protein with a constant region and a variable region wherein said variable region contains a consensus sequence, which method comprises subjecting said substrate DNA sequence to a modified polymerase chain reaction (PCR) utilizing two primers, wherein one primer is a match for a portion of the substrate DNA
encoding said constant region and the other primer is a degenerate primer which is a match for at least a portion of the DNA encoding said consensus sequence.

2. The method of claim 1 wherein said protein is a subunit of a T-cell receptor protein.

3. The method of claim 2 wherein said TCR
subunit is the .alpha.-subunit.

4. The method of claim 3 wherein said consensus sequence comprises amino acids 30-40 of the variable region.

5. The method of claim 2 wherein said TCR
subunit is the .beta. subunit.

6. The method of claim 5 wherein the consensus sequence comprises amino acids 30-40 of the variable region.

7. A method to characterize a T-cell population which method comprises amplifying a single-strand cDNA prepared from said population wherein said cDNA contains a substrate DNA sequence encoding a TCR subunit, wherein said amplification comprises subjecting said cDNA to a modified PCR which utilizes two primers, wherein one primer is a match primer for DNA encoding the constant region of the TCR
receptor subunit and the other primer is a degenerate primer which matches the DNA encoding a consensus sequence in the variable region of said TCR receptor subunit.

8. The method of claim 7 wherein the TCR
subunit is the .alpha.-subunit.

9. The method of claim 7 wherein the T-cell population is derived from an autoimmune infiltrate, or localized site of an immune response to an exogenous antigen.

10. The method of claim 7 wherein the T-cell population is derived from a transplanted tissue or organ.

11. The method of claim 7 wherein the T-cell population is derived from a lymphoma population.