CA2446391A1

CA2446391A1 - Method for inducing t cells regulator with cd47 ligands

Info

Publication number: CA2446391A1
Application number: CA002446391A
Authority: CA
Inventors: Marika Sarfati
Original assignee: Individual
Current assignee: Individual
Priority date: 2003-10-24
Filing date: 2003-10-24
Publication date: 2005-04-24

Abstract

Naturally occurring CD4+CD25+T regulatory cells (Treg) activily participate to the mechanisms of peripheral tolerance by negatively regulating autoimmunity.
However, the molecular mechanisms of in vivo induction of T reg remain largely unknown.
Regulatory T cells (Tr1) can be generated in vitro from human naive T cells by repetitive stimulation with immature or IL-10-treated DC. We previously reported that CD47 ligation by either CD47 mAb or a peptide of its natural ligand, i.c thrombospondin, induced naïve T cell anergy. Herein, we demonstrate that the CD47 mAb- induced anergic T cells were actively suppressing the proliferation and cytokine production of syngeneic effector T
cells. T anergic/suppressors cells (Tas) produced little or no cytokine. The suppressive function required physical contact with the responder T cells and was IL-10 and TGF-.beta.
independent. Tas displayed :1) increased expression of CTLA-4, OX40 and GITR;
2) decreased levels of CD40L and CD28 and 3) similar levels of FAS, FASL, CCR4 and CCR7 and CD25 compared to effector T cells. Both CD4+ and CD8+Tas suppressed effector T cells proliferation and none of them regulated DC maturation. Our findings demonstrate that ligation of an endogenous receptor, i.e CD47, on naive cells generates a distinct type of regulatory T cells (Tas) which produce no IL-10 but share phenotypic and functional features with both Treg and Tr1 described in human and mice. TAS
may be re-infused to patients suffering from a large variety of chronic inflammatory diseases, graft versus host disease, graft rejection and allergic diseases. Compounds inducing in vivo the activity of TAS may be used to treat the same patients.

Description

A METHOD FOR THE INDUCTION OF HUNAN REGULATORY T CELLS BY
A MONOCLONAL ANTIBODY TO CD4~ ANTIGEN
BACKGROUND OF THE INVENT ION
The role o.f the immune system is to maintain the homeostasis of the organism and protect against dangerous components of the environment, mainly micro-organisms. In some situations, the immune response ( IR) is undesired and harmful ; for example it may reject allograft or mediate graft versus host disease (CTVHD) after allogenic bone marrow transplantation. In other cases, the IR may be aberrant and generate chronic inflammatory diseases. The latter results from a T cell-dependent IR against :1) self antigens (occurring in conditions like autoimmune diseases, namely multiple sclerosis, type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosous) :2) innocuous and ubiquitous components of the environment such as airborne antigens or food (occurring in conditions like allergic diseases, namely asthma, allergic rhinitis, atopic eczema) :3) commensal and innocuous bacteria (occurring in conditions like chronic, inflammatory bowel diseases).
The failure of normal individuals to respond to self Ag and develop autoimmune disease results from both central tolerance (intra-thymic deletion of auto-reactive T
lymphocytes) and peripheral tolerance ( clonal deletion, anergy of auto-reactive cells or suppression of their activity by regulatory T cells (Treg). There is increasing eW dence, derived mainly from animal models, that Treg also prevent and control allergic diseases and chronic inflammatory bowel diseases. Treg are also involved in long term transplantation tolerance and prevent experimental CiVH.
Regulatory T cells, specialized in tlic suppr.-.ssiom of the imnunc response arc heterogeneous and may belong to the CD4+, CD8 '- or NKT cell lineage. Treg control the response to self antigens, tumor antigens, allogeneic cells, innocuous airborne or dietary Ags, commensal or pathogenic bacteria. The most extensively investigated Treg are the naturally occurring CD4+ CD25+ T cells and the induced Trl (Type 1 regulatory T cells).
Naturally occurring CD4* CD25k T cells are generated in the thymus where they are selected following high affinity interaction with still undefined self ligand(s). CD4+
_1_ CD25~ T cells constitute 5-10% of the normal peripheral CD4F T cell repertoia~e of mice and humans. They are long-lived and display the phenotype of previously activated T
cells; for example, human CD4+ CD25+ T cells are CD45R0+ and express increased levels of HLA-DR and CTLA-4. Gene expression analysis of mouse CD4+ CD25+ Treg confirmed that they have been recently activated and revealed that they constitutively express three members of the TNF-R superfamily (i.e.OX40, 4-1BB and CiITR) that are transiently expressed on au~tivated T cells. CD25+ CD4+ T cells are anergic, and in vitt°o, , they inhibit the response of CD25- T cells by blocking the transcription of IL-2 gene in CD4+ T cells amd the expression of IL-2 R cc chain on CD8+ T cells. he vztr~o, the suppression is contact-dependent and not inhibited by neutralizing Abs to IL-10 or TGF-(3; however, i~ vivo,the suppressive activity of CD25+ CD4+ T cells is overcome by neutralization of TCTF-(3 or IL-10. Although human and mouse CD4+ CD25~ T
cells wer a reported to express membrane bound TGF-[3 on their surface, the role of this cytokine in mediating the suppressive activity has been challenged. Naturally occurring Treg may also be CD25-; these are derived from mature naive CD4 T cells outside the thymus (without the help of CD25+ Treg) and display similar phenotypic and functional features as CD25+ Treg.
The term Trl was initially used to describe Treg generated in vitro a$er repetitive stimulation ofnaive T cells in the presence of IL-10. Tr1 cells suppressed in vitro and in viva Thl and Th2 immune responses and their suppressive activity was ascribed to their secretion of IL-10 and TGF-(3. Different methods have been developed to generate Trl cells z~ vitro including: (1) the addition of IFN-a, to T cells activated in the presence of IL-10,(2) the ligation of either CD2, 4C8 or CD46 on TCR-activated T cells in the absence of other co stimulatory signal, (3)T cell activation in the presence of 1,25(OH) ~Vit D3 and dexamethasone ,(4) repetitive stimulation of naive T cells by immature allogeneic DCs and(5) naive T cell activation by dendritic cells pre-incubated with the heavy chain of ferritin t~Uith the exception of those induced b:y naive T cell activation in the presence of immunosuppressive agents and neutralizing vAbs to IFN-y and IL-4, the Trl described in the above studies were anergic and produced variable levels of IFN-y and IL-5, in addition to IL-10 and TGF-(3. In viva, Trl cells may be induced by _2_ infectious agents such as Bordetella Pertussis, Mycobacterium tuberculosis, and Leishmania major. The lack of pro inflammatory immune response to inhaled protein antigens was ascribed to the development of Ag-specific Trl cells. The latter were generated during the interaction between Ag-specific naive T cells and airway DC by mechanisms involving ICOS/ICOSL interactions and IL-10 production by DC.
Similarly tolerance to dietary Ags and commensal intestinal bacteria is mediated by a subset of Treg producing high levels of TGF-~i known as Th3 cell.
The induction of regulatory T cells presents a clinical potential. Agonistic or antagonistic compounds for such induction ~~ill enhance or suppress the activity of Suppressor T cells.
SUMMARY OF THE INV'ENTif?N
We have succeeded in developing an in vitro or ex vivo model allowing the induction of a novel type of regulatory T cells, named TAS. Unlike the naturally occurring CD25~ CD4+
T cells, TAS may be CD4+ or CD8+ and unlike the Trl cells they suppress by a contact-dependent but IL-lU and TGF-(3-independent mechanism . TAS are generated by engaging the CD47 Ag at the time of T cell activation by mitogens or pol.yclonal activators.
CD47 Ag, also known as Integrin Associated Protein (IAP), is a widely expressed multispan transmembrane protein, which is physically and functionally associated with ccv(33 integrin, the vitronectin receptor. CD47 has also been implicated in leukocyte transendothelial migration. Its recently described natural ligand, thrombospondin ('fSP), is an homotrimeric extracellular matrix protein (ECM) which is produced not only by platelets but also by monocytes and alveolar macrophages. Thrombospondin is tz~ansiently expressed at high concentration in damaged and inflamed tissues.
We recently suggested a potential role for TSP or CD47 in ianmune regulation by the finding that they regulate the in vitro production and response to IL-12. Engagement of CD47 by either mAb, TSP or 4N 1 K (a peptide of the C-terminal domain of TSP selectively binding CDA7), inhibited IL-12 release by htunan rrionocy~es. The suppression was selective for IL-12 and occurred following T cell-dependent or -independent stimulation of monocytes. We have next ohserved that CD47 engagement in primary cultures of cord blood mononuclear cells inhibits IL-12 driven Thl cell development, as revealed by the cy~tokine secretion profile at restimulation and IFN-y production at the single cell level.
Ligation of CD47 does not deviate the phenotype of IL-I2-primed cells from 'fhl to Th2, however it does not affect IL-4-induced Th2 cell development. CD47 mAb, its Fab'2 fragments or the synthetic peptide 4N1K, corresponding to the CD47-binding site of thrombospondin, all display the same activity and inhibit the production of IFN-'y and IL-2 in primary cultures as well as the expression of IL-12R(32 chaW . Inclusion of exogenous IL-2 at priming coweets IL-I2R expression at priming and at restimulation as ~~ell as the inhibition of Thl cell development.
It is an object of this invention to provide a method for interacting or interfering with the development of a disease or condition involving the activity of Suppressor T
cells, in a patient, which comprises the steps of - inducing ex vivo the number and,~or activity of human regulatory T Lens by stimulating blood lymphocyrtes in the presence of a CD47 ligand~ and - reinfusing the induced human regulatory T cells to the patient.
The CD47 ligand may be an agonistic or antagonistic thereof. More specifically, the CD47 ligand that has induced the number and activity of Suppressor T cells is a CD47 monoclonal Antibody, namely the Antibody produced by the hybridoma deposited at the ATCC under accession number B6H121Hb9771 .
This method will result in the tr eatment of diseases or conditions wherein Suppressor T
cells activity is to be enhanced or inriibited. tn trie first case, auto immune diseases, allergic diseases and inflammatory diseases would benefit i~rom such inhibition.
Enhancing the activity of Suppressor T cells would also result in recipient tolerance to a grafted tissue. In the latter case, inhibiting the activity of Supxrressor T
cells would result in greater neutralisation of infectious agents.
_q._ Another object of this invention is a method of screening a vaxiety of compounds, in order to find agonistic and antagonistic compounds capable of enhancing or suppressing the activity of Suppressor T cells. Agonistic and antagonistic compounds would represent alternatives to the monoclonal Antibody to CD47, for example.
DESCRIPTION OF THE INVENTION
This invention will be described hereinbelow, by reference to specific examples, enbodiements and figures, the propose of which is to illustrate the invention rather than to limit its scope.
BRIEF DESCRIPTION OF THE FIGURES
In fig.lA, cells were recovered after 3 days of stimulation., washed , stained with the fluorescent vital dye CFSE and cultured for another 5 days in culture medium supplemented with IL-2. Cells wore then washed and analyzed by flow cytometry . A.s seen, anti-CD47-treated cells (TAS) proliferate much less than the control cells (ThU) activated in the presence of control antibody. Tas and Th0 are comprised of >95% CD3+~
T cells, of which GU% are CD4+ and 40% CDS+.
In fig.l8 and C, TAS and Th0 were expanded in II -2-supplemented medium for 5 days, washed and restimulated with anti-CD3 mAb immobilized on L cells cotransfected vrith human CD32 B7 antigens. After 3 days of culture,the proliferative response was measured by determining the tritiated thymidine uptakes whereas the secretion of IL-10 and TCiF-(3 were measured by ELISA. As seen, TAS proliferate and secrete much less IL-lU than Th0 cells, TAS and ThU produce similar and very low levels of Tf'tF-(3.
Fig.ID, TAS were co-cultured with ThU cells at a 1/1 ratio and the mixture was stimulated for 3 days with graded doses of anti-CD3 mAb (lUU, SU and lU ng%ml) 111i111V17111GG1~ Vll CD32 L37 L 11~.11a1GC:~il.il~J. tia ~GGll, ~rLW J
gJV1.c11Lly L11111171L Lilt ~Jd VIl.rGlA.d.lVd1 and the production of IFN-y by ThU cells.

Fis.lE shov.~s that the suppressive effect ofTAS is dose-dependent.
Fig.2 demonstrates that TAS inhibit the proliferative ( tritiated thymidine uptake) and the cytokine production by Th2 cells (IL-4) (Fig.2A). and Thl cells (Fig.2B and C). Fig.2B
and C also show that the suppressive activity of TAS is not observed if TAS
are physically separated from their target Th0 or Th1 by a semi-permeable membrane.This indicates that the suppressive activity of TAS is contact-dependant and not mediated by soluble factors. Moreover the suppressive activity of TAS is not overcome by blocking anti-IL10 and anti-TGF-(3 mAbs (not shown) Fig.3 shows the phenotype of TAS compared to that of Th0 ~a.s determined by by 2 to 4 color analysis by means of a FACSCALIBUR. TAS and Th0 cells display similar levels of CD25, CD45RO,CD3, CD4, CDR, CD28 and CD47" TAS express higher levels of OX40, GITR and CTLA-4. The proportion of cells expressing membrane bound TGF-(3 is slightly higher in TAS. About 50% of TAS co-express O~i40, GITR and CTLA-4 as compared to about 30% of Th0 cells on which these molecules are also expressed at lower levels-In the experiments summarized in figures 1 to 3, umbilical cord blood mononuclear cells were stimulated in standard conditions with a polyc,lonal activator such as Phytohemaglutinin (PHA) in the presence of a monoclonal antibody to CD47 or a control antibody.
EXAMPLES
Example 1. TAS are prepared from the peripheral blood lymphocytes of a patient with one of the above mentioned inflammatory diseases. The cells are prepared and cultured using conditions similar to those described in Avice et ai. (J.
Irnrnuno1.,2001, 167 : 2459-2468). 10 ~ to 10 g TAS are then re-infused intravenously in the patient. In this example, peripheral blood mononuclear cells are isolated from the patient by conventional fractionation method , Cells are then ac,~tivated for a tear days with a classical T cell activator, such as PHA in the presence of a CD47 ligand, such as for example anti CD47 mAb; cells are then expanded in IL-2 supplemented medium to obtain sufficient number of cells. TAS so prepared are washed and resuspended in a physiological solution before being infi~sed into the patient. The procedure may be repeated as often as necessary.
Example2. The molecular signature of TAS cells, i.e. the genes specifically expressed in these cells and induced by CD47 ligand ,are used to screen any compound with potential application in the treatment of inflammatory disease, CTVHD, graft rejection or cancer.
The genes selectively expressed in TAS cells will be identified on highly purified preparations TAS by means of conventional DNA microarrays.
If a given compound activates a signature gene of TAS, the compound will be examined and tested for its activity in in:f(ammatory diseases ( see above), CrVHD and grad rejection. Such a compound should not be used for the treatment of cancer patient as it will increase the activity of Treg inhibiting the anti-tumor immune response.
Compound inhibiting the expression of TAS signature gene may tested and used as irnmunostimulant to enhance the immune response against cancer cells or any vaccine.
The TAS may be used in assays as they are in a whole blood sample, or in a purified or semi-purified .form e.g. blood cells of a sample would be enriched in CD47 cells, by induction and~or b~~ retention on :ua affinity medium. Such techniques well lrmot~~n in the art, are easily adaptable toCD47 cells. Recombinant cells engineered to express C.D47 could also be used to screen ligands simply having affinity to CD47. A
potentially clinical compound will be developed for its capacity to be an agonistic or an antaganistic to CD47 cell activity.
The invention being hereinabove described, it will be obvious that the same be varied in many ways. Those skilled in the art recognise that other and further changes and modifications may be made thereto without departing from the spirit of the invention, and _7_ it is intended that all such changes and modifications fall within the scope of the invention, as defined in the appended claims.
_g_

Claims

1. A method for interfering with development of a disease or condition involving the activity of Suppressor T cells, in a patient, which comprises the steps of:
- inducing ex vivo the number and/or activity of human regulatory T cells by stimulating blood lymphocytes in the presence of a CD47 ligand; and - reinfusing the induced human regulatory T cells to the patient.

2. The method of claim 1, wherein the ligand is an agonist of Suppressor T
cells activity.

3. The method of claim 2, wherein the ligand is a CD47 monoclonal antibody.

4. The method of claim 3, wherein the antibody is one produced by the hybridoma deposited at the ATCC under accession number B6H12/Hb9771.