WO2010104617A2

WO2010104617A2 - Novel methods to induce a state of immune tolerance

Info

Publication number: WO2010104617A2
Application number: PCT/US2010/021994
Authority: WO
Inventors: Salvatore Albani
Original assignee: Salvatore Albani
Priority date: 2009-01-23
Filing date: 2010-01-25
Publication date: 2010-09-16
Also published as: WO2010104617A3

Abstract

This invention relates to novel methods to induce a state of immune tolerance in situations where it is desirable for clinical purposes. More specifically, this invention relates to novel methods to induce a state of immune tolerance in immune -mediated diseases. Even more specifically, this invention relates to inflammatory autoimmune disorders, including but not limited to rheumatoid arthritis, and other human autoimmune diseases and disorders, and the prevention and treatment of transplantation rejection and the effects of the method thereof comprising the induction of immune tolerance via a novel population of T cells with regulatory/ suppressive properties.

Description

Novel Methods to Induce a State of Immune Tolerance

2.0 Detailed Description of the Invention

The present invention provides for methods of the induction of immune tolerance in situations where it is clinically desirable for the treatment of a variety of autoimmune disorders, including but not limited to rheumatoid arthritis, juvenile idiopathic arthritis, inflammatory bowel disease, Crohn's disease, multiple sclerosis, and the prevention and treatment of transplant rejection. Briefly, the induction of tTreg cells, heretofore not described in the art, may be achieved via in vivo or ex vivo methods.

All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the spirit and scope of the invention. More specifically, the described embodiments are to be considered in all respects only as illustrative and not restrictive. All similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit and scope of the invention as defined by the appended claims.

All patents, patent applications, and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of, and "consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that use of such terms and expressions imply excluding any equivalents of the features shown and described in whole or in part thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. 2.1 Field of the Invention

2.2 Background of the Invention

The following description includes information that may be useful in understanding the present invention. It is not an admission that any such information is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.

Recent therapies for treating rheumatoid arthritis and other human autoimmune diseases have been based on non-specific suppression of the immune system. Treatment regimens so based target inflammatory immune pathways, which result in having to be concerned with balancing toxicity caused by the non-specificity with the intended perceived benefits of disease remission. For example, with respect to rheumatoid arthritis, first generation biologic agents have included those that interfere with the inflammatory cascade by blocking one or another component, for example an inflammatory cytokine such as TNFα. Such direct biological interference with pathogenic pathways is being considered for an ever-increasing number of molecules, primarily cytokines, to replace generalized pharmacological immuno-suppression for a more tailored treatment route.

Given the further understanding that recognition of self is a physiologic and necessary phenomenon, and that "quality" and "intensity" of the immune responses is regulated by complex mechanisms that ensure that recognition of self does not lead to damage, and that necessary inflammatory responses, aimed at clearing perceived "dangers" such as an infection, are down regulated once "danger" is eliminated, there is a need in the art for treatment methods which take into account the complex set of complementary and interactive pathways that contribute to the qualitative and quantitative regulation of immune responses in order to prevent tissue damage. In this context affecting the immune system towards tolerance is akin to employing a dimmer switch; i.e., rather that in causing the complete on/off shifting of pathways, the reactivity is lessened. Thus, further treatment regimens focused on tolerance pathways should consider this complexity in order to not bring about toxic immune reactivity. Some tolerance pathway-focused methods, such as in induction of remission by therapies for rheumatoid arthritis, do not have a specific approach. Data suggests that blunting, or dimming immune responses in a non-specific fashion may lead to undesirable effects with regard to frequency and gravity of occurrence, which varies according to the individual therapy and regimen. Some of the key contributors in the regulation and mediation of tolerance induction are disclosed below, namely, regulatory T cell (Treg) populations, effector T cell (Teff) populations and co-stimulatory molecules.

Current art in the area of induction of Treg for treatment of autoimmunity relies eminently on ex vivo cellular therapy and also lacks specificity. This pertains both to the types of T cells developed and to their ability to opt between antigen specific and polyclonal activation, and between ex vivo and in vivo applications. This lack of flexibility and the inherent complexity of most of the protocols has hampered the application of these technologies to human diseases (examples: 1-6).

Mechanisms of T cell regulation are multiple and complex. The crucial role of regulatory T cells (Treg) is thought to be their suppression of the proliferation and cytokine production of effector T cell (Teff) populations. Thereby, Treg mediate peripheral tolerance by targeting autoreactive T cells that arise de novo or escape thymic deletion. Treg are comprised of several different subsets with overlapping but distinct actions, particularly in human disease. Treg may be identified by co-expression of CD4 and CD25, as well as functional characteristics, such as expression of the transcription factor FoxP3, production of regulatory cytokines IL-IO and TGFβ, and the ability to suppress proliferation of activated CD4+CD25- in co-cultured experiments. Purifying Treg cells has still largely relied on their CD4+CD25+ phenotype. Recent reports (7) have specifically demonstrated that the differential expression of CD 127 enabled flow-cytometry-based separation of human CD4+CD25+ Treg cells from CD 127+ non- regulatory T cells. However, it must be emphasized that particularly in humans, the Treg repertoire is dynamic and diverse. Examples include CD62L- Treg, which appear to be enhanced by Infliximab (a biologic) therapy (8). Known types of Treg include natural Treg (nTreg), TrI and Th3 cells. TrI cells resemble other Treg cells in many ways, although they do not express large amounts of CD25 on their surface. They require IL-IO for their formation and, once mature, secrete large amounts of it as well as of transforming growth factor-beta (TGBβ). TrI cells are abundant in the intestine, and their chief function may be to create a tolerance to the many antigens that are part of a diet (9). Th3 cells are also prevalent in the intestine, but unlike TrI cells, their main lymphokine is TGF-β. Also like TrI cells, they suppress immune responses to ingested antigens (10). These cells are different from the ones which we describe here. One of the most important conceptual and practical differences between tTreg and prior art is that tTreg are inducible in vivo and ex vivo and are plastic, (i.e. They share progenitors with effectors cells and can be induced from effector cells (11).

The role of co-stimulatory molecules. Mechanisms that lead to immune tolerance rely on intersecting pathways, which involve the way T cells and antigen presenting cells (APC) regulate each other through both soluble mediators and modulation exerted via complex networks. In addition to cytokines, the network of co-stimulatory molecules takes a prominent role in the mechanisms of the modulation of tolerance.

T-APC cross talk and immune tolerance. The five B7 family members, ICOS ligand, PD-Ll (B7-H1), PD-L2 (B7-DC), B7-H3, and B7-H4 (B7x/B7-Sl) are expressed on professional antigen-presenting cells as well as on cells within non lymphoid organs, providing new means for regulating T cell activation and tolerance in peripheral tissues (12-17). The discovery of new functions for the original B7 co-stimulatory molecules, together with the identification of additional B7 and CD28 family members, have revealed new ways in which this family regulates T cell activation and tolerance (18-22). B7-1/B7-2:CD28 interactions not only promote initial T cell activation, but also regulate self-tolerance by supporting CD4+CD25+ T regulatory cell homeostasis (22-24). CTLA-4 can exert its inhibitory effects on T cells in both B7-1/B7-2 dependent and independent fashions. B7-1 and B7-2 can signal bidirectionally by engaging CD28 and CTLA-4 on T cells and by delivering signals into B7-expressing cells (25,26). ICOS and PD-I are inducibly expressed on T cells, and they regulate previously activated T cells (16,21). Both the ICOS:ICOSL and the PD-1 :PD-L1/PD-L2 pathways play a critical role in regulating T cell activation and tolerance (15). CTLΛ-4 and PD-I pathways in autoimmunity and tolerance. There is consensus that both CTLA-4 and PD-I inhibit T and B cell activation and may have a crucial role in peripheral tolerance (19). Recent attention has been focused on the PD-1/PD-Ll pathway. PD-Ll, but not PD-L2, is involved in the induction and maintenance of tolerance in autoimmune diabetes by downregulating T cell responses (24,27-29). Anti-PD-Ll, but not anti-PD-L2, ameliorates colitis induced in severe combined immunodeficiency (SCID) mice (30,31). Experimentally induced autoimmune hepatitis is accelerated in PD-Ll ^7" mice (13). However, PD-L2 still seems to be involved in regulating T cell activation and may be important in the induction of immune tolerance (16,22). The conversion of naive T cells to Treg may be influenced by PD-I expression and the maturation status of dendritic cells (DC) (32). PD-I can inhibit the expression of GATA-3 and T-bet, transcription factors associated with effector T cell function (33).

Both CTLA-4 and PD-I functions have been associated with RA and other autoimmune diseases (34-38). PD-I is overexpressed on CD4⁺ T cells in the synovial fluid (SF) of RA patients (15). It has also been shown that CD4+ PD-1+, but not CD4+ PD-I-, T cells produced IL-IO (37). In the serum, auto-antibodies against PD-Ll were found in a significant portion of RA patients, and their existence correlated with active disease. Immobilized auto-antibodies to PD-Ll -stimulated CD4⁺ T cell proliferation, IL-IO production and apoptosis (35,39,40). Recently, it has been observed that several negative regulators, including CTLA-4, PD-I and PD-Ll, were overexpressed in synovial T cells and macrophages (40). Additionally, soluble forms were found at high titers in both SF and sera and correlated with rheumatoid factor levels. The level of soluble PD-I was found to correlate with TNFα in the SF.

Of note, current art focuses exclusively on the anergic effects of the expression of PD-I on T cells (examples: 41-48).

The above examples of the data obtained to date of how various cell populations may regulate, influence, and mediate immune tolerance fail to translate into adequate protection in patients suffering from autoimmune disorders. Thus, a need still exists in the art for a novel method to utilize these principals in the induction of immune tolerance to advance the immune treatment arts.

2.3 Summary of the Invention The invention relates to the discovery and use for therapeutic purposes of a novel subtype of T cells with regulatory function (tTreg). tTreg cells are characterized by the expression of the PD-I molecule, possibly but not necessarily in association with CD25, Foxp3 and CTL A-4. tTreg cells: a) act as suppressors for effector cells, thus favoring an immune deviation; b) can directly lyse antigen presenting cells (APC) which are contributing to inflammation; and c) produce tolerogenic cytokines such as IL-IO and TGF -b, which directly affect both circulating immune cells in a systemic fashion and the microenvironment where a specific immune reaction is occurring. tTreg cells are inducible in vivo and ex vivo by different means. The invention describes methods of inducing such a novel population of T cells for the purposes of therapy in various conditions such as the prevention or treatment of the rejection of transplant or therapy of autoimmunity.

In a first embodiment the invention comprises methods of inducing and maintaining tolerance in conditions where it is clinically desirable by exploiting mechanisms of immune tolerance via generation, augmentation or restoration of tTreg function in vivo.

In a second embodiment, the invention comprises methods of inducing and maintaining tolerance in conditions where it is clinically desirable by inducing tTreg cells ex vivo by pharmacological and immunological means. Such induction further determines the restoration of the mechanisms of immune tolerance for the treatment of inflammatory autoimmune disorders, including but not limited to rheumatoid arthritis and other human autoimmune diseases and disorders, in addition to the prevention or treatment of the rejection of transplants..

2.3a In vivo induction:

Methods of induction of tTreg in vivo include but are not limited to treatment with drugs currently used for different purposes. These drugs (collectively herein the Drugs) include, but are not limited to hydroxychloroquinine (HCQ) and its metabolites; cytokines therapies, including but not limited to interleukin 10 (IL-IO), transforming growth factor beta (TGFβ), interferon beta (IFNβ), interferon gamma (IFNg); small molecules designed to mimic the drugs used, their active metabolites and/or their receptors. These therapies can be combined with toleration to antigen specific therapies (collective herein Epitopes), such as immunologically relevant fragments of antigens which initiate, perpetuate or are targets of an undesired pathologic mechanism. In the case of autoimmune diseases, examples include peptides derived from myelin, collagen, heat shock proteins, their derivatives, including conservative and non conservative amino acid substitutions, frame shifts in the sequence, polynucleotides encoding for such peptides, mimics of the epitopic surfaces comprising the docking surface of the T cell receptor to the combined surface of the epitopic component of the peptide and the HLA molecules which present the peptide. In the case of transplant, the relevant antigens are derived from the variable and hypervariable regions of HLA molecules, their derivatives, including conservative and non conservative amino acid substitutions, frame shifts in the sequence, polynucleotides encoding for such peptides, mimics of the epitopic surfaces comprising the docking surface of the T cell receptor to the combined surface of the epitopic component of the peptide and the HLA molecules which present the peptide.

We have discovered that concomitant or antecedent treatment of patients with the Drugs, alone or in combination with the Epitopes, generates an unexpected cascade of events which can be described as follows:

1. Treatment with the Drugs, using doses and formulations known to those skilled on the art, induces a regression (or de novo formation) of dendritic cells form a pro-inflammatory to a more immature, more tolerogenic/immature phenotype (iDC), which can be defined as CD83, TLR4 ++. Particular relevance must be given to the expression by iDC of molecules which are ligands of PD-I and CTLA-4 on T cells. Examples of these molecules are B7-2, B7H1, B7H2, B7 H3, B7H-4, CD 86, PDlL-I, PD1L-2, PD1L-3.

2. iDC induce the differentiation of T cells which are characterized by the expression of the PD-I molecule, possibly but not necessarily in association with CD25, Foxp3 and CTLA-4. These T cells are highly differentiated cells, which can exert their role both in an antigen specific and non specific fashion. They can act independently from an antigen by docking their PD-I and CTLA-4 molecules to the corresponding ligands on DC.

3. Treatment with the Epitopes, using doses and formulations known to those skilled in the art, contributes to the generation of tTreg which are antigen specific. In this case, tTreg will also use the specific interaction between their T cell receptor and the antigen/HLA complexes on APC.

4. The mechanism of action of tTreg cells relies on: a) acting as suppressors for effector cells, thus favoring an immune deviation; b) directly lysing antigen presenting cells (APC) which may present peptide on their HLA and express the appropriate ligands (i.e. express PD-I ligands to bind PD-I Treg); c) produce tolerogenic cytokines IL-IO and TGF-β which directly affect both circulating immune cells in a systemic fashion and the microenvironment where a specific immune reaction is occurring. Figure 1 depicts the mechanism of action and a sequence of event in a context in which a combination of Drug and Epitope therapy is used.

In another embodiment, in vivo induction of tTreg cells is achieved by systemic and/or local administration of cytokine -based therapies. Such cytokines comprise but are not limited to IFNβ, IL-IO, IFNγ, TGF-β.

In another embodiment, in vivo induction of tTreg cells is achieved by systemic and/or local administration of biologic -based therapies. Such therapies comprise but are not limited to molecules interfering with the action of cytokine or immune receptors. Examples of the two categories include, but are not limited to, anti-TNF and anti CTLA-4.

In a further embodiment, induction of tTreg cells can be achieved by the use of small molecules designed to mimic the Drugs used, they active metabolites and/or their receptors. In another embodiment, in addition to the aforementioned methods of in vivo induction of the tTreg cells, the methods can be achieved for the induction of immune tolerance via Epitope therapy such as immunologically relevant fragments of antigens which initiate, perpetuate or are targets of an undesired pathologic mechanism. In the case of autoimmune diseases, examples include peptides derived from myelin, collagen, heat shock proteins, their derivatives, including conservative and non conservative amino acid substitutions, frame shifts in the sequence, polynucleotides encoding for such peptides, mimics of the epitopic surfaces comprising the docking surface of the T cell receptor to the combined surface of the epitopic component of the peptide and the HLA molecules which present the peptide. In the case of transplant, the relevant antigens are derived from the variable and hypervariable regions of HLA molecules, their derivatives, including conservative and non conservative amino acid substitutions, frame shifts in the sequence, polynucleotides encoding for such peptides, mimics of the epitopic surfaces comprising the docking surface of the T cell receptor to the combined surface of the epitopic component of the peptide and the HLA molecules which present the peptide.

2.3h Ex vivo induction: Induction of tTreg may be achieved ex vivo by different means, with the ultimate goal of providing, by cellular therapy, effective means for the treatment of autoimmune conditions or the treatment or prevention of transplant rejection.

In one embodiment, PBMC are obtained in sufficient quantities by methods known to those skilled in the art. PBMC are used whole or as a source of purified DC and T cells, which can be obtained by various means as known to those skilled in the art. DC from patients with inflammatory conditions, such as autoimmunity or transplant rejection, have preeminently a proinflammatory phenotype. Purified DC are stimulated with HCQ or IFNβ (HCQ optimal concentration is 5 μm. We have tested 10 μm, 20 μm, 40 μm and 100 μm. We see significant cell death starting at 10 μm. IFNβ: optimal concentration is 1000 U/ml. We tested 500 U/ml, lOOOU/ml, and 1500 U/ml) for 48 hours and matured by adding LPS to the culture the last 24 hours, after which the DC are washed and T cells are added to the culture for an additional 24 hours.

As shown in example 2, this treatment induces the development of T cells which have the phenotypical and functional characteristics of tTreg. Ttreg obtained using this procedure are reinfused in the patient and will exert in vivo their immunomodulatory effects, thus downregulating noxious inflammatory mechanisms.

The doses and means for this type of cellular therapy are similar to what is commonly used for leukapheresis and purification and re-injection of stem cells. Doses of regulatory T cells to be transferred can also be extrapolated from prior art, which includes animal models for the diseases which this technology herein addresses. These procedures are well known to those skilled in the art (49).

In another embodiment, the induction of tTreg may be achieved by adding Epitopes to the culture with either HCQ or IFNβ. Epitope comprise of immunologically relevant fragments of antigens which initiate, perpetuate or are targets of an undesired pathologic mechanism. In the case of autoimmune diseases, examples include peptides derived from myelin, collagen, heat shock proteins, their derivatives, including conservative and non conservative amino acid substitutions, frame shifts in the sequence, polynucleotides encoding for such peptides, mimics of the epitopic surfaces comprising the docking surface of the T cell receptor to the combined surface of the epitopic component of the peptide and the HLA molecules which present the peptide. In the case of transplant, the relevant antigens are derived from the variable and hypervariable regions of HLA molecules, their derivatives, including conservative and non conservative amino acid substitutions, frame shifts in the sequence, polynucleotides encoding for such peptides, mimics of the epitopic surfaces comprising the docking surface of the T cell receptor to the combined surface of the epitopic component of the peptide and the HLA molecules which present the peptide.

In yet another preferred embodiment, induction of tTreg is achieved in vitro by incubation of T cells purified from peripheral blood with artificial antigen presenting cells (aAPC) encompassing the molecules necessary to induce tTreg. These molecules comprise but are not limited to molecules to engage and activate the T cell receptor, either specifically (Epitope and HLA complexes) or polyclonally (i.e. Anti-CD3 antibodies), in association with PD-I and CTL A-4 ligands, as commonly expressed on iDC. Adhesion molecules may also be added to enhance the binding between T cells and aAPC. There are various types of aAPC, which use either fixed or fluid supports and organize variously on their surfaces the T cell ligands. These types of aAPC are well known to those skilled in the art (50).

Purified T cells and aAPC are incubated with either HCQ, IFNβ or other immunomodulatory cytokines as described above. Ttreg are purified and re -injected for cellular therapy, as described above.

2.4a Example 1: in vivo induction

Synergy between epitope-specific therapy and concomitant Hydroxychloroquine (HCQ) use in generating tTreg. We have completed Phase I and Ha clinical trials in RA patients tolerized with a peptide, called dnaJPl to induce an immune deviation, which translated into clinical improvement. Trial results have been published. In post-hoc analyses, significant differences were found between peptide (n=45) and placebo (n=46) treated patients within the HCQ-user subgroup (Figure 2). The HCQ-peptide and HCQ-placebo groups were homogeneous in number, demographics, and clinical characteristics at baseline and concomitant therapies. Length of use and HCQ dosage were comparable between groups. AUC 112-168 for ACR20 reached a p=0-04 by CMH and 0-02 by GEE. Inclusion of the FU time point in the AUC determined a p=0-02 by CMH and p=0 002 by GEE for ACR20. When other time points were assessed, day 140 showed significant differences between groups both for ACR20 (p=0.03) and ACR50 (<0.005). Analysis of Tend also showed significant differences between groups for ACR20 (p=0.03). ACR20 and 50 for the HCQ-peptide group at day FU reached 48-9% (p=0-009 by CMH and <0 001 by GEE) and 33-3% (p=0-07 by CMH and <0 003 by GEE) respectively (Figure 2). These data show a potentially synergistic effect between peptide therapy and HCQ. There are objective reasons to support a potential synergistic effect: i) the size and homogeneity of the peptide-HCQ and placebo-HCQ groups. By chance, these groups resemble an intended randomization; and ii) the effects seen are consistent in all clinical and immunological parameters.

The potential clinical synergy observed may be the outcome of an efficient cooperation in the mechanisms of action. HCQ has direct tolerogenic effects on antigen presenting cells (APC) and blocks protein processing and endogenous peptide presentation on MHC class II . Soluble class II peptide presentation, however, is not affected. Administration of peptide, which is a high affinity HLA binder, could therefore skew the relative representation of peptides presented to T cells. These hypotheses, based on known art, however, are not sufficient to explain the synergy.In experiments we performed by exposing human PBMC to HCQ, we saw changes in the expression of PD-I ligand B7-H1 on APC and a corresponding increase of PD-I and CTL A-4 on T cells (not shown). These data suggested that concomitant or preceding treatment with HCQ may induce the immune functional phenotype necessary for response to epitope- specific immunotherapy.

A cluster of co-stimulatory molecules associated with tTreg is induced by treatment and it is necessary for susceptibility to tolerization. PBMC at baseline were obtained from 10 peptide- treated clinical responders and 10 peptide-treated non-responders. PBMC were stimulated in vitro with peptide. Gene expression was measured by TaqMan. Expression of PD-I, B7-H1, B7- DC, CTLA-4 and FoxP3 was significantly more elevated at enrollment in clinical responders than in non-responders (Figure 3).

Microarray expression analysis shows increased expression of genes associated with cytotoxic pathways, underscoring one of the mechanisms employed by tTreg in vivo. We used microarray technology to analyze gene expression unique to treatment. Four patients who improved clinically were chosen for analysis. Two patients received peptide-HCQ co-treatment and two received placebo. PBMC collected at the TO and Tend were cultured in vitro with peptide. RNA was extracted using Qiagen's RNeasy Mini Kit with on-column DNase treatment and then sent to an Agilent Certified Microarray service lab. It was hybridized to 44K Whole Human Genome Oligo slides and then scanned in Agilent's DNA microarray scanner. Data was analyzed using Agilent Feature Extraction and GeneSpring GX software. Samples from peptide-HCQ patients showed an increase (>1.3 fold) in 184 genes from TO to Tend. These same genes demonstrated either a decrease (< 1.0 fold) or a smaller increase (<1.3 fold) in expression in the placebo group. Of particular functional interest is the expression of genes related to direct lytic activity of T cells listed in Figure 4. The gene group increases significantly in the two treated patients versus the two placebo patients (p<0.001, unpaired student t-test).

PD-I expression on Tregs is correlated with clinical response. Regulatory T cells were FACS sorted from treatment clinical responders and placebo non-clinical responders according to PD-I expression. The following cell populations were sorted by FACS; CD4+CD25+CD127- (nTreg),

CD4+CD25+CD127-PD 1+ (PD l+Treg), CD4+CD25+CD 127-PD1 - (PD l-Treg) and

CD4+CD25-CD127-PD1+ (CD4PD1+). Cells were initially negatively MACSsorted on CD4+ and CD8+ cells before FACSsorted to increase purity of lymphocyte population. Pl was gated on the lymphocyte population based on FSC-SSC characteristics. CD4+ Lymphocytes were selected by negatively selecting CD8+ cells. The remaining CD4+ lymphocytes were separated by CD 127 expression. Effector T cells were designated as CD4+CD127+ cells. CD 127- expressing were FACSsorted depending on there CD25 and PD-I expression.

The % expression of PDl+ Treg and CD4PD1+ (p<0.05) was increased in treatment clinical responders at both TO en Tend compared to placebo clinical non-responders (figure IA-B).

PDl+ Treg % expression even increased during the trial, which might support the involvement of PDl expression on Tregs in tolerance induction to dnaJPl instead of only a marker of treatment susceptibility (Figure 5).

PD-I expressing Tregs are not anergic. PD-I expression has been previously documented on T- cells as a marker of anergy and exhaustion in for instance HIV and hepatitis. A suppression

CFSE-assay showed that PDl expressing Treg cells could suppress effector T cell proliferation, which is contrary to the exhaustive and anergic state documented.

Further supporting that PDl expression on Tregs is not associated with exhaustion or anergy is the expression of FoxP3, CTLA-4 and IL-IO measured by TaqMan in both non-PDl and PDl- expressing Tregs (Figure 6).

Both functional (suppression assays, Figure 8) and expression data (Figure 7) showed that tTreg are part of fundamental mechanisms which can downregulate and modulate effector T cell responses.

Several populations of T cells with regulatory capability arise with tolerization: a role for

PD-I in active regulation. These data relate to the hypothesis that a population of PD-1/CD25⁺ ⁺ T cells is induced by APC expressing B7-H1 and has strong suppressor/regulatory capability on Teff. It is evident from our data (Table 1) that immune tolerization induces not only a restoration of the suppressor ability for "conventional" CD4/CD25⁺⁺ T cells, but also that Treg co-expressing PD-I may play a pivotal role.

These findings were further corroborated when the various populations of Treg were sorted and gene expression was studied by TaqMan (Figure 6). These data show a functional reliance of PD-1/CD25++ Treg on the production of TGFβ and IL-IO and on the expression of CTLA-4 and underscore differences in mechanism of action among the various populations studied.

CΩ4 /CD25t.ΦDl-'CD127- L37 1.62

1 SS

CD4 - ΦDΪ - CB25 *'CB J27- 61.22 5^.09

Table 1: Percent of suppression at TO and Tend of Teff proliferation induced by various categories of Treg (left column). PBMC were incubated in culture for 48 hours with anti-CD3/CD28 or 24 hours with peptide (not shown here). Cells were harvested and non-adherent cells were purified for T cells by MACS sort and followed by FACS sort for the specific T cell groups. APC (adherent cells) were then cultured with the FACS sorted Teff and Treg for 5 days. Cells were subjected to FACS analysis and the resulting data was analyzed using ModFit LT software.

TGF-b plays an important role in PDl expressing regulatory T cells. Subtractive hybridisation was performed on PBMCs from placebo non-clinical responders and dnaJPl clinical responder at Tend. Upregulation of Carboxypepsidase D precursor, a TGF-beta regulated gene, was seen in dnaJPl clinical responders. In an agilent array upregulation of latent TGF-b binding protein 4 isoform b was seen in PBMCs of dnaJPl responders. To specifically address the question if TGF-b was correlated with treatment response, PBMCs were stimulated with dnaJPl in vitro. A higher expression of TGF-β was noticed in Tregs expressing PD-I compared to PDl- Tregs (Figure 9).

2.4h Example 2: ex vivo induction

In vitro culture with HCQ induces ΪDC phenotype. Healthy PBMCs were isolated via Ficoll gradient separation. CD 14+ cells (monocytes) were purified via negative CD 14 MACS selection. The CD 14+ cells were cultured at 5%CO2 and 37C, and IL-4 and GM-CSF was added to the culture at day 0 and 4. On day 6 of culture, HCQ (5uM) or Media was added to the culture. On day 7 LPS was added, to either the HCQ or media treated group, to mature the DCs. After 24 hours of culture, the DCs were washed and T-cells were added and recultured in media for an additional 24 hours. T-cells were selected and purified by negative pan T-cell MACS selection (CD4/CD8). On day 9, T-cells and DCs were harvested via difference in plate adherence and used for FACS.

When HCQ is added to both iDC and mDC, PD-Ll expression on CD83- cells is increased. Meaning HCQ deverts to a more immature DC phenotype while maintaining or upregulating their PD-Ll expression (Figure 10).

PD-I expression on T-cells is induced in vitro by hydroxychloroquine treated dendritic cells. As described, DCs gain an immature phenotype when treated with HCQ. When HCQ treated DCs are co-cultured with T cells, they induce PD-I surface expression on T-cells. Immature DCs are capable of inducing PD-I expression on T-cells and when treated with HCQ become even brighter in PD-I expression. Mature DCs are however not capable to induce PD-I expression on T-cells but obtain that function when they are pre-treated with HCQ (Figure 11).

5.0 Figures Figure Legends

Figure 1: diagram of the mechanism of induction in vivo

Figure 2: Clinical outcome by ACR scores in all patients versus HCQ users. % of ACR responders of HCQ users in peptide (n=45) and placebo (n=46) treatment groups on different visit days throughout the study, p-values are marked for the test they are derived with: Φ = CMH test, ♦ = Adjusted GEE.

Figure 3: Co-expression of PD-I, CTLA-4, FoxP3, B7-H1 and B 7 -DC is a requirement for susceptibility to epitope-specific immunotherapy. PBMC from TO and Tend were obtained from patients treated with peptide and discordant for clinical response at Tend (non-responder: n=10; and responder: n=10). PBMC were incubated in vitro with peptide for 48 hrs, the cell pellets lysedfor mRNA isolation and cDNA synthesis. cDNA was preamplified for the genes of interest with the Applied Biosy stems TaqMan. The results were analyzed as a percentage of GAPDH [2^Λ- (CTgoi-CTgapdh)] . Paired t-test was used for statistical analysis.

Figure 4: The expression of all genes shown increases significantly more in the two treated patients versus the two placebo patients (p<0.0001, t-test).

Figure 5: CD4+CD25-CD127-PD1+ (CD4PD1,) and CD4+CD25+CD127-PD1+ (PDl + Treg) were increased at TO and Tend in dnaJPl treatment responders compared to placebo non- responders (CD4PD1 p<0.05, PDl+Treg). An additional increase of PDl+ Treg was seen at Tend. PDl- Treg were not increased at TO, but did increase at Tend, supporting PDl as susceptibility marker measured by TaqMan.

Figure 6: FoxP3, IL-IO and CTLA-4 was elevated at Tend in PDl+ Treg and CD4PD1 cells, CTLA-4 and IL-IO only in PDl- Treg supporting the plasticity of these cells. PBMC (n=3, each group) were cultured for 48 hours with plate bound anti-CD3CD28. Cells were harvested and non-adherent T cells were enriched by pan-T cell MACS and stained for FACS analysis and sorting. Different Treg groups, as depicted in table 4, were FACS sorted and used for real-time polymerase chain reaction (PCR). The PCR reactions were carried out using TaqMan universal PCR master mix (PE Applied Biosystems) with 900 nM oligonucleotide primers (IDT Inc., Coralville, IA), 200 nM fluorogenic probe (IDT Inc.), and 2 ul ofcDNA (100 or 200ng). For the final detection, the ABI Prism 7000 sequence detector (Applied Biosystems, Foster City, CA) and Step One Plus (Applied Biosystems, Foster City, CA) was programmed to an initial step of 2 minutes at 50⁰C and 10 minutes at 95°C, followed by 45 thermal cycles of 15 seconds at 95°C and 1 minute at 60⁰C. Each measurement was carried out in duplicate. Calculations were done by the relative standard curve method, and results are expressed as an induction index (2(-dCT) x 100, in arbitrary units), using GAPDH as a reference.

Figure 7: PBMC were cultured for 48 hours with peptide or with plate bound anti-CD3/CD28.

Cells were harvested and T cells were enriched by MACS. T cells populations were sorted by

FACS based on phenotype. RNA was obtained and message for targets genes shown here. A-G were measured by TaqMan as described in the legend to figure 6.

Figure 8: Suppression assay on treatment and placebo treated patines at Tend

Figure 9: TGF-β measured by TaqMan was highly expressed in PDl + Treg and CD4PD1 cells compared to PDl-Treg in dnaJPl responders at both TO and Tend.

Figure 10: Expression of PD-Ll and CD83 by mDC and iDC before and after culture with HCQ

Figure 11: Expression of PD-I on T cells after incubation with iDC and mDC treated or not with HCQ

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Claims

3.0 Claims

What is claimed is:

The identification and use for therapeutic purposes of a novel subtype of T cells with regulatory function (tTreg). tTreg cells are characterized by the expression of the PD-I molecule, possibly but not necessarily in association with CD25, Foxp3 and CTLA-4. tTreg cells: a) act as suppressors for effector cells, thus favoring an immune deviation; b) can directly lyse antigen presenting cells (APC) which may present peptide on their HLA and express the appropriate ligands (i.e. express PD-I ligands to bind PD-I Treg); c) produce tolerogenic cytokines such as IL-IO and TGF-β which directly affect both circulating immune cells in a systemic fashion and the microenvironment where a specific immune reaction is occurring. tTreg cells are inducible by different means, such as via a method of in vivo or ex vivo induction of immune tolerance based on induction, restoration or enhancement of tTreg cell function. Such methods by which the induction of immune tolerance is used for the therapy and prevention of immune mediated diseases.

1. Methods by which the induction of immune tolerance is achieved by treatment in vivo: a) by administration of drugs such as, but not limited to, hydroxychloroquinine, its active metabolites, and/or small molecules designed to mimic the drugs used, they active metabolites and/or their receptors; b) by administration of cytokine -based therapies. Such cytokines comprise but are not limited to IFNβ, IL-IO, IFNγ, TGF-β and/or small molecules designed to mimic the drugs used, they active metabolites and/or their receptors; c) by co induction of tTreg cells in association with therapy of autoimmune diseases, Examples comprise but are not limited to rheumatoid arthritis, juvenile idiopathic arthritis, inflammatory bowl disease, Crohn's disease, multiple sclerosis, psoriasis, SLE, JIA, uveitis, dermatomyositis, scleroderma, with IFN β; d) by co-induction of tTreg cells in the induction of immune tolerance in association with antigen-specific therapy and/or the mimetics thereof, such as immunologically relevant fragments of antigens which initiate, perpetuate or are targets of an undesired pathologic mechanism. In the case of autoimmune diseases, examples include peptides derived from myelin, collagen, heat shock proteins, their derivatives, including conservative and non conservative amino acid substitutions, frame shifts in the sequence, polynucleotides encoding for such peptides, mimics of the epitopic surfaces comprising the docking surface of the T cell receptor to the combined surface of the epitopic component of the peptide and the HLA molecules which present the peptide. In the case of transplant, the relevant antigens are derived from the variable and hypervariable regions of HLA molecules, their derivatives, including conservative and non conservative amino acid substitutions, frame shifts in the sequence, polynucleotides encoding for such peptides, mimics of the epitopic surfaces comprising the docking surface of the T cell receptor to the combined surface of the epitopic component of the peptide and the HLA molecules which present the peptide; e) by co induction of tTreg cells in association with antigen-specific therapy and/or the mimetics thereof in the induction of immune tolerance, when such antigens are derived from proteins involved in autoimmune inflammation and their epitopic and/or epitopic motifs are designed in silica using tools available to those skilled in the art; f) in vivo induction of tTreg cells is achieved by systemic and/or local administration of biologic -based therapies. Such therapies comprise but are not limited to molecules interfering with the action of cytokine or immune receptors. Examples of the two categories include, but are not limited to, anti-TNF and anti CTLA-4; g) by co induction of tTreg cells in association with antigen-specific therapy and/or the mimetics thereof in the induction of immune tolerance, when such antigens are derived from the hypervariable regions of HLA molecules whose alleles are discordant between donor and recipient in transplant procedures and their epitopic and/or epitopic motifs are designed in silica using tools available to those skilled in the art.

2. Methods of ex vivo induction of immune tolerance: a) wherein white blood cells are obtained from the patient and processed to induce desired changes in the antigen presenting cell (APC) population; wherein said APC population is comprised of dendritic cells where methods induce an immature DC phenotype and function, which is associated with tolerance. Expression of an immature phenotype may be characterized by the post- manipulation, decreased expression of co-stimulatory molecules such as CD80/CD86 and CD40. Expression on such APC of PD-I ligands leads in to the generation of tTreg cells; b) create artificial APC which would contribute to induction of tTreg cells from white blood cells obtained form the patient; c) in which either patient-derived APC or artificial APC would be incubated with patient-derived withe cells in combination with soluble mediators, including but not limited to HCQ, its metabolites, cytokines, such as IFNβ, IL-IO, IFNγ, TGF-β and by the use of small molecules designed to mimic the mediators listed above, they active metabolites and/or their receptors A method by which the induction of immune tolerance employing tTreg is used for the therapy and prevention of immune mediated diseases which are autoimmune in nature as well as the induction of immune tolerance for the therapy and prevention of transplant rejection. Examples comprise but are not limited to rheumatoid arthritis, juvenile idiopathic arthritis, inflammatory bowl disease, Crohn's disease, multiple sclerosis, psoriasis, SLE, JIA, uveitis, dermatomyositis, scleroderma. Examples of rejection of transplant include but are not limited to solid organs, stem cells and bone marrow derived cells.