Predictors of responses to immune checkpoint blockade in advanced melanoma.

Immune checkpoint blockers (ICB) have become pivotal therapies in the clinical armamentarium against metastatic melanoma (MMel). Given the frequency of immune related adverse events and increasing use of ICB, predictors of response to CTLA-4 and/or PD-1 blockade represent unmet clinical needs. Using a systems biology-based approach to an assessment of 779 paired blood and tumor markers in 37 stage III MMel patients, we analyzed association between blood immune parameters and the functional immune reactivity of tumor-infiltrating cells after ex vivo exposure to ICB. Based on this assay, we retrospectively observed, in eight cohorts enrolling 190 MMel patients treated with ipilimumab, that PD-L1 expression on peripheral T cells was prognostic on overall and progression-free survival. Moreover, detectable CD137 on circulating CD8+ T cells was associated with the disease-free status of resected stage III MMel patients after adjuvant ipilimumab + nivolumab (but not nivolumab alone). These biomarkers should be validated in prospective trials in MMel.The clinical management of metastatic melanoma requires predictors of the response to checkpoint blockade. Here, the authors use immunological assays to identify potential prognostic/predictive biomarkers in circulating blood cells and in tumor-infiltrating lymphocytes from patients with resected stage III melanoma.

TIM-1 signaling is required for maintenance and induction of regulatory B cells.

Apart from their role in humoral immunity, B cells can exhibit IL-10-dependent regulatory activity (Bregs). These regulatory subpopulations have been shown to inhibit inflammation and allograft rejection. However, our understanding of Bregs has been hampered by their rarity, lack of a specific marker, and poor insight into their induction and maintenance. We previously demonstrated that T cell immunoglobulin mucin domain-1 (TIM-1) identifies over 70% of IL-10-producing B cells, irrespective of other markers. We now show that TIM-1 is the primary receptor responsible for Breg induction by apoptotic cells (ACs). However, B cells that express a mutant form of TIM-1 lacking the mucin domain (TIM-1(Δmucin) ) exhibit decreased phosphatidylserine binding and are unable to produce IL-10 in response to ACs or by specific ligation with anti-TIM-1. TIM-1(Δmucin) mice also exhibit accelerated allograft rejection, which appears to be due in part to their defect in both baseline and induced IL-10(+) Bregs, since a single transfer of WT TIM-1(+) B cells can restore long-term graft survival. These data suggest that TIM-1 signaling plays a direct role in Breg maintenance and induction both under physiological conditions (in response to ACs) and in response to therapy through TIM-1 ligation. Moreover, they directly demonstrate that the mucin domain regulates TIM-1 signaling.

Effector and regulatory T-cell subsets in autoimmunity and tissue inflammation.

Many autoimmune diseases are driven by self-reactive T helper cells. Until recently, organ-specific autoimmune diseases were primarily associated with Th1 cells but not Th2 cells. However, the discovery of a number of new effector T-cell subsets, like Th17 and Th9 cells, and regulatory T cells, like Tregs and Tr1 cells, has changed the way we view and understand autoimmunity at cellular and molecular levels. In recent years, IL-17-producing Th17 cells have emerged as major players in autoimmunity. The complicated relationship between Th1 and Th17 cells, as well as the intricate balance between Tregs and Th17 cells, provides a basis for understanding the immunological mechanisms that induce and regulate autoimmunity. Here, we give an overview of the interplay between different effector T-cell subsets and regulatory T-cell subsets, and how they contribute to the development of autoimmunity and tissue inflammation.

CD226 Gly307Ser association with multiple autoimmune diseases.

Genome-wide association studies provide insight into multigenic diseases through the identification of susceptibility genes and etiological pathways. In addition, the identification of shared variants among autoimmune disorders provides insight into common disease pathways. We previously reported an association of a nonsynonymous single nucleotide polymorphism (SNP) rs763361/Gly307Ser in the immune response gene CD226 on chromosome 18q22 with type 1 diabetes (T1D) susceptibility. Here, we report efforts toward identifying the causal variant by exonic resequencing and tag SNP mapping of the 18q22 region in both T1D and multiple sclerosis (MS). In addition to the analysis of newly available samples in T1D (2088 cases and 3289 controls) and autoimmune thyroid disease (AITD) (821 cases and 1920 controls), resulting in strong support for the Ser(307) association with T1D (P=3.46 x 10(-9)) and continued potential evidence for AITD (P=0.0345), we provide evidence for association of Gly307Ser with MS (P=4.20 x 10(-4)) and rheumatoid arthritis (RA) (P=0.017). The Ser(307) allele of rs763361 in exon 7 of CD226 predisposes to T1D, MS, and possibly AITD and RA, and based on the tag SNP analysis, could be the causal variant.

Contrasting effects of cyclosporine and rapamycin in de novo generation of alloantigen-specific regulatory T cells.

The outcome of T-cell-mediated responses, immunity or tolerance, critically depends on the balance of cytopathic versus regulatory T (T(reg)) cells. In the creation of stable tolerance to MHC incompatible allografts, reducing the unusually large mass of donor-reactive cytopathic T effector (T(eff)) cells via apoptosis is often required. Cyclosporine (CsA) blocks activation-induced cell death (AICD) of T(eff) cells, and is detrimental to tolerance induction by costimulation blockade, whereas Rapamycin (RPM) preserves AICD, and augments the potential of costimulation blockade to create tolerance. While differences between CsA and RPM in influencing apoptosis of activated graft-destructive T(eff) cells are apparent, their effects on graft-protective T(reg) cells remain enigmatic. Moreover, it is unclear whether tolerizing regimens foster conversion of naïve peripheral T cells into alloantigen-specific T(reg) cells for graft protection. Here we show, using reporter mice for T(reg) marker Foxp3, that RPM promotes de novo conversion of alloantigen-specific T(reg) cells, whereas CsA completely inhibits this process. Upon transfer, in vivo converted T(reg) cells potently suppress the rejection of donor but not third party skin grafts. Thus, the differential effects of RPM and CsA on T(eff) and T(reg) cells favor the use of RPM in shifting the balance of aggressive to protective type alloimmunity.

Structurally derived mutations define congenital heart block-related epitopes within the 200-239 amino acid stretch of the Ro52 protein.

Congenital heart block is a passively transferred autoimmune condition, which affects the children of mothers with Ro/SSA autoantibodies. During pregnancy, the antibodies are transported across the placenta and affect the fetus. We have previously demonstrated that antibodies directed to the 200-239 amino acid (aa) stretch of the Ro52 component of the Ro/SSA antigen correlate with the development of congenital heart block. In this report, we investigated the antibody-antigen interaction of this target epitope in detail at a molecular and structural level. Peptides representing aa 200-239 (p200) with structurally derived mutations were synthesized to define the epitopes recognized by two Ro52 human monoclonal antibodies, S3A8 and M4H1, isolated from patient-derived phage display libraries. Analyses by ELISA, circular dichroism and MALDI-TOF-MS demonstrate that the antibody recognition is dependent on a partly alpha-helical fold within the putative leucine zipper of the 200-239 aa stretch and that the two human anti-p200 monoclonal antibodies, M4H1 and S3A8, recognize different epitopic structures within the p200 peptide. In addition, we investigated the representation of each fine specificity within the sera of mothers with children born with congenital heart block, and in such sera, antibodies of the S3A8 idiotype were more commonly detected and at higher levels than M4H1-like antibodies.

Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family.

To simplify the analysis of asthma susceptibility genes located at human chromosome 5q23-35, we examined congenic mice that differed at the homologous chromosomal segment. We identified a Mendelian trait encoded by T cell and Airway Phenotype Regulator (Tapr). Tapr is genetically distinct from known cytokine genes and controls the development of airway hyperreactivity and T cell production of interleukin 4 (IL-4) and IL-13. Positional cloning identified a gene family that encodes T cell membrane proteins (TIMs); major sequence variants of this gene family (Tim) completely cosegregated with Tapr. The human homolog of TIM-1 is the hepatitis A virus (HAV) receptor, which may explain the inverse relationship between HAV infection and the development of atopy.

Characterization of the phenotype and function of CD8(+), alpha / beta(+) NKT cells from tumor-bearing mice that show a natural killer cell activity and lyse multiple tumor targets.

Natural Killer (NK) T cells are a specialized T cell population that co-expresses receptors of the NK lineage with the alpha / beta TCR receptor and other T cell surface markers. Their functions, regulation and relationship to other cells in the immune system are not fully understood. This report demonstrates that tumor-bearing C57BL / 6 mice have a population of NKT cells that co-express CD8 and CD161 (NK1.1) surface markers. These cells are maintained in long-term culture with T helper 2 (Th2) cytokine interleukin-4 (IL-4), but produce large amounts of Th1 cytokine interferon-gamma (IFN-gamma) following activation. NK1.1(+)CD8(+) T cells show a potent NK-like cytotoxic activity against multiple tumor targets, and lysis is independent of major histocompatibility complex (MHC)-class I or non-classical MHC-class I molecules (Qa, TL). The NK1.1(+)CD8(+) T cells express Vbeta14 chain of the TCR. These NKT cells are not CD1d restricted, and their cytotoxic activity is CD1d independent. Therefore, they represent a unique subset of T cells with an unknown restriction element which produce large quantities of IFN-gamma following expansion with IL-4. Furthermore, their cytotoxic activity is enhanced by B7 co-stimulatory molecules present on tumor cells. CD161(+) T cells that are expanded in tumor-bearing hosts may function as a part of the innate immune system with potential role(s) in tumor surveillance.

The origin and regulation of autopathogenic T cells.

A clear understanding of the events surrounding the selection of autoreactive T cells in the thymus and their regulation in the periphery has eluded immunologists for years. However, recent work examining the expression of tissue-specific antigens in the thymus and the biochemistry of disease associated MHC alleles has provided important clues into the generation of the autoreactive T cell repertoire in the thymus. In addition, recent studies focusing on the role of immunoregulatory cytokines and cross-reactive peptide ligands has provided information regarding both the regulation and activation of autoreactive cells in the periphery. An improved understanding of the selection and regulation of autoreactive T cells will undoubtedly aid in the development of strategies for treating autoimmune disease.

Discordant effects of anti-VLA-4 treatment before and after onset of relapsing experimental autoimmune encephalomyelitis.

Initial migration of encephalitogenic T cells to the central nervous system (CNS) in relapsing experimental autoimmune encephalomyelitis (R-EAE), an animal model of multiple sclerosis (MS), depends on the interaction of the alpha4 integrin (VLA-4) expressed on activated T cells with VCAM-1 expressed on activated cerebrovascular endothelial cells. Alternate homing mechanisms may be employed by infiltrating inflammatory cells after disease onset. We thus compared the ability of anti-VLA-4 to regulate proteolipid protein (PLP) 139-151-induced R-EAE when administered either before or after disease onset. Preclinical administration of anti-VLA-4 either to naive recipients of primed encephalitogenic T cells or to mice 1 week after peptide priming, i.e., before clinical disease onset, inhibited the onset and severity of clinical disease. In contrast, Ab treatment either at the peak of acute disease or during remission exacerbated disease relapses and increased the accumulation of CD4(+) T cells in the CNS. Most significantly, anti-VLA-4 treatment either before or during ongoing R-EAE enhanced Th1 responses to both the priming peptide and endogenous myelin epitopes released secondary to acute tissue damage. Collectively, these results suggest that treatment with anti-VLA-4 Ab has multiple effects on the immune system and may be problematic in treating established autoimmune diseases such as MS.

Identification of genetic loci associated with paralysis, inflammation and weight loss in mouse experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE), a model for human multiple sclerosis, is an inducible inflammatory and demyelinating disease of the central nervous system (CNS). Susceptibility to this disease is heritable and is demonstrated by the development of an ascending paralysis accompanied by a loss in body wt 2-3 weeks following immunization with proteins derived from CNS myelin. In a previous genetic analysis of susceptibility to EAE in a cross between susceptible SJL/J mice and resistant B10.S mice, we found suggestive evidence of linkage with disease susceptibility at the telomeric end of chromosome 2 and in the central region of chromosome 3. To define these associations more precisely and to investigate the genetic factors controlling measurable phenotypes of EAE, we performed a new analysis with a larger number of mice. The results now indicate that the chromosome 2 locus significantly influences EAE-related weight loss (P = 6.7 x 10(-5)) and that the chromosome 3 locus is linked with the development of paralysis. In addition, an intriguing inheritance pattern was revealed in which female backcross mice generated from B10.S female x (B10.S x SJL/J)F(1) male parents experienced significantly more EAE-related weight loss (P = 1.2 x 10(-4)) than females generated from F1 female x B10.S male parents. After controlling for this inheritance, a new locus at the centromeric end of chromosome 8 was identified that significantly influences both the development of paralysis (P = 8.2 x 10(-6)) and the incidence of CNS inflammation (P = 7.0 x 10(-5)) in EAE.

Characterization of endogenous Chinese hamster ovary cell surface molecules that mediate T cell costimulation.

Chinese hamster ovary (CHO) cells are commonly used in the generation of transfectants for use in in vitro costimulation assays. However, we have noted that nontransfected CHO cells can themselves provide a low-level B7/CD28 independent costimulatory signal for CD3-mediated murine T cell activation and IL-2 production. This study set out to identify those molecules that contribute to this CHO-dependent costimulatory activity. We describe a CHO subline capable of delivering potent CD28-independent costimulation to murine T cells and the generation of monoclonal antibodies against these CHO cells that inhibited this costimulatory activity. These blocking antibodies do not affect CHO cell-independent costimulation or bind mouse cells, suggesting an effect mediated by their target molecules on the costimulatory competent CHO cells. Immunoprecipitation and expression cloning revealed that these antibodies bound the hamster homologues of Crry (CD21/35), CD44, CD54 (ICAM-1), CD63, CD87, CD147, and an 80- to 90-kDa protein which could not be cloned. Expression of these hamster genes on COS cells demonstrated that hamster CD54 was able to costimulate both CD3-mediated IL-2 secretion and T cell proliferation by naive murine T cells independent of the other molecules identified.

Mapping and identification of autoimmunity genes.

Over the past several years, intense effort has been made to map the chromosomal locations of genes involved in the susceptibility to autoimmune diseases. The first phase of this mapping effort-performed in most cases by using microsatellite markers to scan the genome for loci that are linked with disease-has generated first-draft maps for numerous autoimmune diseases in humans, mice and rats, pointing to as many as 20 different loci in some diseases. The second phase is now beginning, with efforts focused on narrowing the loci sufficiently to allow the positional cloning of disease-associated alleles. From these mapping data it is clear that some of these loci overlap between various autoimmune diseases and preliminary results suggest that indeed there is a sharing of 'autoimmunity genes' between various autoimmune diseases.

Intercellular adhesion molecule 1 is critical for activation of CD28-deficient T cells.

Presentation of Ag to T lymphocytes in the absence of the requisite costimulatory signals leads to an Ag-specific unresponsiveness termed anergy, whereas Ag presentation in conjunction with costimulation leads to clonal expansion. B7/CD28 signaling has been shown to provide this critical costimulatory signal and blockade of this pathway may inhibit in vitro and in vivo immune responses. Although T cells from CD28-deficient mice are lacking in a variety of responses, they nonetheless are capable of various primary and secondary responses without the induction of anergy expected in the absence of costimulation. This suggests that there may be alternative costimulatory pathways that can replace CD28 signaling under certain circumstances. In this paper, we show that ICAM-1becomes a dominant costimulatory molecule for CD28-deficient T cells. ICAM-1 costimulates anti-CD3-mediated T cell proliferation and IL-2 secretion in CD28-deficient murine T cells. Furthermore, splenocytes from ICAM-1-deficient mice could not activate CD28-deficient T cells and splenocytes lacking both ICAM and CD28 fail to proliferate in response to anti-CD3-induced T cell signals. This confirms that not only can ICAM-1 act as a CD28-independent costimulator, but it is the dominant, requisite costimulatory molecule for the activation of T cells in the absence of B7/CD28 costimulation.

Mouse inducible costimulatory molecule (ICOS) expression is enhanced by CD28 costimulation and regulates differentiation of CD4+ T cells.

The inducible costimulatory (ICOS) molecule is expressed by activated T cells and has homology to CD28 and CD152. ICOS binds B7h, a molecule expressed by APC with homology to CD80 and CD86. To investigate regulation of ICOS expression and its role in Th responses we developed anti-mouse ICOS mAbs and ICOS-Ig fusion protein. Little ICOS is expressed by freshly isolated mouse T cells, but ICOS is rapidly up-regulated on most CD4(+) and CD8(+) T cells following stimulation of the TCR. Strikingly, ICOS up-regulation is significantly reduced in the absence of CD80 and CD86 and can be restored by CD28 stimulation, suggesting that CD28-CD80/CD86 interactions may optimize ICOS expression. Interestingly, TCR-transgenic T cells differentiated into Th2 expressed significantly more ICOS than cells differentiated into Th1. We used two methods to investigate the role of ICOS in activation of CD4(+) T cells. First, CD4(+) cells were stimulated with beads coated with anti-CD3 and either B7h-Ig fusion protein or control Ig fusion protein. ICOS stimulation enhanced proliferation of CD4(+) cells and production of IFN-gamma, IL-4, and IL-10, but not IL-2. Second, TCR-transgenic CD4(+) T cells were stimulated with peptide and APC in the presence of ICOS-Ig or control Ig. When the ICOS:B7h interaction was blocked by ICOS-Ig, CD4(+) T cells produced more IFN-gamma and less IL-4 and IL-10 than CD4(+) cells differentiated with control Ig. These results demonstrate that ICOS stimulation is important in T cell activation and that ICOS may have a particularly important role in development of Th2 cells.

Autoantigen-responsive T cell clones demonstrate unfocused TCR cross-reactivity toward multiple related ligands: implications for autoimmunity.

It has been suggested that the cross-reaction of a single T cell receptor with multiple different peptide ligands is a mechanism for maintaining a diverse yet compact immune repertoire. In the context of autoimmune disease it is important to understand how this property is balanced against the maintenance of self-tolerance. Specifically, whether the cross-reactivity inherent in the immune system is focused or unfocused will have important consequences for the development of autoimmune disease. If cross-reactivity is "focused," then in an immune response to a foreign antigen all T cell receptors that recognize the foreign antigen will cross-react with a specific autoantigenic peptide. However, if cross-reactivity is "unfocused," an immune response to a foreign antigen will result in the activation of a small number of self-reactive cells within a larger pool of cells specific for the foreign antigen. We have tested whether cross-reactivity is focused or unfocused by generating a panel of T cell clones that respond to two closely related ligands. W144 is an autoantigenic peptide of myelin proteolipid protein, PLP 139-151 (HSLGKWLGHPDKF), and Q144 is an altered peptide of PLP 139-151 bearing a glutamine for tryptophan substitution at position 144. The Q144-responsive clones have a broad degree of cross-reactivity with other position 144 substituted peptides. We find that despite their characteristic responses to Q144 and W144, the patterns of responses of these clones to other structurally related ligands are random, demonstrating that cross-reactivity is unfocused in the absence of selection. Maintaining a diverse range of cross-reactive interactions may limit nonspecific responses to autoantigens.

Fulminant spontaneous autoimmunity of the central nervous system in mice transgenic for the myelin proteolipid protein-specific T cell receptor.

Proteolipid protein (PLP)-139-151 is the dominant encephalitogenic peptide that induces experimental autoimmune encephalomyelitis (EAE) in SJL (H-2(s)) mice. To examine the contribution of T cell receptor (TCR) specificity in the induction of EAE, we generated transgenic mice expressing the rearranged TCR genes from an encephalitogenic or a nonencephalitogenic PLP-139-151/I-A(s)-specific T cell clone. Both types of transgenic lines developed spontaneous EAE, but, remarkably, the lines expressing the TCR from the nonencephalitogenic clone showed increasingly higher frequencies of disease (60-83%) in progressive SJL backcrosses and could not be propagated on the susceptible background. The T cells from the transgenic mice were not tolerized, because they responded vigorously to the antigen in vitro and mediated EAE when the mice were immunized with antigen. Besides being the only description of a TCR transgenic mice for the PLP-139-151/I-A(s) epitope, the results demonstrate that the TCR from a nonencephalitogenic PLP-specific T cell clone can induce autoimmune disease when expressed appropriately in vivo.

High frequency of autoreactive myelin proteolipid protein-specific T cells in the periphery of naive mice: mechanisms of selection of the self-reactive repertoire.

The autoreactive T cells that escape central tolerance and form the peripheral self-reactive repertoire determine both susceptibility to autoimmune disease and the epitope dominance of a specific autoantigen. SJL (H-2(s)) mice are highly susceptible to the induction of experimental autoimmune encephalomyelitis (EAE) with myelin proteolipid protein (PLP). The two major encephalitogenic epitopes of PLP (PLP 139-151 and PLP 178-191) bind to IA(s) with similar affinity; however, the immune response to the PLP 139-151 epitope is always dominant. The immunodominance of the PLP 139-151 epitope in SJL mice appears to be due to the presence of expanded numbers of T cells (frequency of 1/20,000 CD4(+) cells) reactive to PLP 139-151 in the peripheral repertoire of naive mice. Neither the PLP autoantigen nor infectious environmental agents appear to be responsible for this expanded repertoire, as endogenous PLP 139-151 reactivity is found in both PLP-deficient and germ-free mice. The high frequency of PLP 139-151-reactive T cells in SJL mice is partly due to lack of thymic deletion to PLP 139-151, as the DM20 isoform of PLP (which lacks residues 116-150) is more abundantly expressed in the thymus than full-length PLP. Reexpression of PLP 139-151 in the embryonic thymus results in a significant reduction of PLP 139-151-reactive precursors in naive mice. Thus, escape from central tolerance, combined with peripheral expansion by cross-reactive antigen(s), appears to be responsible for the high frequency of PLP 139-151-reactive T cells.

IL-4, IL-10, IL-13, and TGF-beta from an altered peptide ligand-specific Th2 cell clone down-regulate adoptive transfer of experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is induced in the SJL/J mouse by adoptive transfer of activated proteolipid protein peptide (PLP) 139-151-specific Th1 cells. T cells responding to altered peptide ligands (APL) of PLP, previously shown to induce Th2 differentiation and regulate disease in PLP-immunized mice, do not transfer EAE. However, the exact mechanism of disease regulation by APL-specific T cells has not been elucidated. In this report, we show that 1F1, a Th2 clone specific for an APL of PLP139-151 can prevent adoptive transfer of EAE when cocultured with PLP-encephalitogenic spleen cells (PLP-spleen). Cytokines from activated 1F1 cells were detected by hybridization of mRNA to oligonucleotide arrays (DNA chip) and by ELISA. The Th2 cytokines found to be present at the highest protein and mRNA levels were evaluated for their role in suppression of adoptive transfer of EAE from PLP-activated spleen cell cultures. Abs to individual cytokines in 1F1 PLP-spleen cocultures suggested that IL-4, IL-13, and TGF-beta played a significant role in suppressing EAE. Abs to the combination of IL-4, IL-10, IL-13, and TGF-beta completely neutralized the protective effect of 1F1. Addition of Th2 cytokines to PLP-spleen cultures showed that IL-13 and TGF-beta were each individually effective and low levels of IL-4 synergized with IL-13 to inhibit disease transfer. IL-5, IL-9, and IL-10 had little or no effect whereas GM-CSF slightly enhanced EAE. Our results demonstrate that Th2 cytokines derived from APL-specific Th2 cells can effectively down-regulate the encephalitogenic potential of PLP-spleen cells if present during their reactivation in culture.

Tuning T cell activation threshold and effector function with cross-reactive peptide ligands.

We have generated a panel of cross-reactive T cells by immunizing SJL mice (I-A(s)) with Q144 peptide, an analog of an autoantigenic peptide (W144) of myelin proteolipid protein (PLP) 139-151 (HSLGKWLGHPDKF) in which W was replaced by Q at position 144. Following immunization with Q144, T cells were expanded in vitro with W144, which is a cross-reactive, suboptimal ligand, for Q144-specific T cells. The T cell clones responded to both ligands and grew normally on the peptide W144, but were hyperstimulated when activated by Q144 in vitro. This hyperstimulation results in a heteroclitic proliferative response with secretion of additional cytokines not induced by W144. Thus expansion of T cells by a suboptimal cross-reactive ligand effectively lowers the activation threshold so that the immunizing antigen becomes a hyperstimulating ligand for the clones. Surprisingly, when the T cell clones are grown on the hyperstimulating ligand Q144, some adapt by increasing their activation threshold. This desensitization results in a loss of response to a number of cross-reactive ligands and the appearance of a more specific T cell response. Long-term culture with the hyperstimulating ligand is sometimes associated with down-regulation of CD4 expression. These results provide an explanation for the common finding of T cell heteroclicity, and suggest that although the specificity and hierarchy of the response of T cells to peptides is determined by the TCR, activation threshold and effector functions are modified by exposure to cross-reactive ligands. This observation has implications for the development and regulation of autoimmune disease.