TIM-3: a novel regulatory molecule of alloimmune activation

T cell Ig domain and mucin domain (TIM)-3 has previously been established as a central regulator of Th1 responses and immune tolerance. In this study, we examined its functions in allograft rejection in a murine model of vascularized cardiac transplantation. TIM-3 was constitutively expressed on dendritic cells and natural regulatory T cells (Tregs) but only detected on CD4(+)FoxP3(-) and CD8(+) T cells in acutely rejecting graft recipients. A blocking anti-TIM-3 mAb accelerated allograft rejection only in the presence of host CD4(+) T cells. Accelerated rejection was accompanied by increased frequencies of alloreactive IFN-γ-, IL-6-, and IL-17-producing splenocytes, enhanced CD8(+) cytotoxicity against alloantigen, increased alloantibody production, and a decline in peripheral and intragraft Treg/effector T cell ratio. Enhanced IL-6 production by CD4(+) T cells after TIM-3 blockade plays a central role in acceleration of rejection. Using an established alloreactivity TCR transgenic model, blockade of TIM-3 increased allospecific effector T cells, enhanced Th1 and Th17 polarization, and resulted in a decreased frequency of overall number of allospecific Tregs. The latter is due to inhibition in induction of adaptive Tregs rather than prevention of expansion of allospecific natural Tregs. In vitro, targeting TIM-3 did not inhibit nTreg-mediated suppression of Th1 alloreactive cells but increased IL-17 production by effector T cells. In summary, TIM-3 is a key regulatory molecule of alloimmunity through its ability to broadly modulate CD4(+) T cell differentiation, thus recalibrating the effector and regulatory arms of the alloimmune response.     

Lack of TIM-3 immunoregulation in multiple sclerosis

Multiple sclerosis (MS) is an inflammatory disease of the CNS white matter associated with T cell infiltrates and alterations of immune functions that can be measured in the peripheral immune system. TIM-3 has been identified as a central regulator of IFN-gamma-secreting type 1 Th (Th1) cells and immune tolerance. In this study, using a newly generated mAb against human TIM-3, we examined TIM-3 function on ex vivo CD4(+) T cells isolated from the circulation of healthy subjects and patients with MS. Blocking TIM-3 during T cell stimulation significantly enhanced IFN-gamma secretion in control subjects but had no effect in untreated patients with MS, demonstrating a defect in TIM-3 immunoregulation. Treatment with glatiramer acetate or IFN-beta reversed this functional defect. Reduced levels and altered kinetics of T cell TIM-3 expression, which was restored in treated patients, is one mechanism that can explain the loss of TIM-3 regulation of T cell function in untreated patients with MS. These data provide functional, mechanistic data for dysregulated TIM-3 immunoregulation in a human autoimmune disease and suggest that approved therapies for the treatment of MS may function in part by restoring TIM-3 immunoregulation of T cell function.     

T Cell Ig- and mucin-domain-containing molecule-3 (TIM-3) and TIM-1 molecules are differentially expressed on human Th1 and Th2 cells and in cerebrospinal fluid-derived mononuclear cells in multiple sclerosis

T cell Ig- and mucin-domain-containing molecules (TIMs) comprise a recently described family of molecules expressed on T cells. TIM-3 has been shown to be expressed on murine Th1 cell clones and has been implicated in the pathogenesis of Th1-driven experimental autoimmune encephalomyelitis. In contrast, association of TIM-1 polymorphisms to Th2-related airway hyperreactivity has been suggested in mice. The TIM molecules have not been investigated in human Th1- or Th2-mediated diseases. Using real-time (TaqMan) RT-PCR, we show that human Th1 lines expressed higher TIM-3 mRNA levels, while Th2 lines demonstrated a higher expression of TIM-1. Analysis of cerebrospinal fluid mononuclear cells obtained from patients with multiple sclerosis revealed significantly higher mRNA expression of TIM-1 compared with controls. Moreover, higher TIM-1 expression was associated with clinical remissions and low expression of IFN-gamma mRNA in cerebrospinal fluid mononuclear cells. In contrast, expression of TIM-3 correlated well with high expression of IFN-gamma and TNF-alpha. These data imply the differential expression of human TIM molecules by Th1 and Th2 cells and may suggest their differential involvement in different phases of a human autoimmune disease.     

T cell, Ig domain, mucin domain-2 gene-deficient mice reveal a novel mechanism for the regulation of Th2 immune responses and airway inflammation

The development of asthma and other atopic diseases is influenced by cytokines produced by Th2 effector T cells. How effector T cell responses are regulated once these cell populations are established remains unclear. The recently described T cell and airway phenotype regulator locus, containing the T cell, Ig domain, mucin domain (TIM) genes, is genetically associated with Th2 cytokine production and Th2-dependent immune responses. In this study, we report the phenotype of the TIM-2 gene-deficient mouse, and demonstrate exacerbated lung inflammation in an airway atopic response model. Immune responses in the TIM-2-deficient mouse reveal disregulated expression of Th2 cytokines, and adoptive transfer experiments show that the T cell compartment is responsible for the heightened inflammatory phenotype. These studies show that TIM-2 is a novel and critical regulator of effector T cell activity.     

TIM-3 Suppresses Anti-CD3/CD28-Induced TCR Activation and IL-2 Expression through the NFAT Signaling Pathway

TIM-3 (T cell immunoglobulin and mucin-domain containing protein 3) is a member of the TIM family of proteins that is preferentially expressed on Th₁ polarized CD4⁺ and CD8⁺ T cells. Recent studies indicate that TIM-3 serves as a negative regulator of T cell function (i.e. T cell dependent immune responses, proliferation, tolerance, and exhaustion). Despite having no recognizable inhibitory signaling motifs, the intracellular tail of TIM-3 is apparently indispensable for function. Specifically, the conserved residues Y265/Y272 and surrounding amino acids appear to be critical for function. Mechanistically, several studies suggest that TIM-3 can associate with interleukin inducible T cell kinase (ITK), the Src kinases Fyn and Lck, and the p85 phosphatidylinositol 3-kinase (PI3K) adaptor protein to positively or negatively regulate IL-2 production via NF-κB/NFAT signaling pathways. To begin to address this discrepancy, we examined the effect of TIM-3 in two model systems. First, we generated several Jurkat T cell lines stably expressing human TIM-3 or murine CD28-ECD/human TIM-3 intracellular tail chimeras and examined the effects that TIM-3 exerts on T cell Receptor (TCR)-mediated activation, cytokine secretion, promoter activity, and protein kinase association. In this model, our results demonstrate that TIM-3 inhibits several TCR-mediated phenotypes: i) NF-kB/NFAT activation, ii) CD69 expression, and iii) suppression of IL-2 secretion. To confirm our Jurkat cell observations we developed a primary human CD8⁺ cell system that expresses endogenous levels of TIM-3. Upon TCR ligation, we observed the loss of NFAT reporter activity and IL-2 secretion, and identified the association of Src kinase Lck, and PLC-γ with TIM-3. Taken together, our results support the conclusion that TIM-3 is a negative regulator of TCR-function by attenuating activation signals mediated by CD3/CD28 co-stimulation.     

Altered expression of T cell Immunoglobulin-Mucin (TIM) molecules in bronchoalveolar lavage CD4+ T cells in sarcoidosis

Background

Activated T helper (Th)-1 pulmonary CD4⁺ cells and their mediators are essential for the inflammation and granulomatous process in sarcoidosis. Recently, T-cell immunoglobulin and mucin domain (TIM) molecules were suggested to be important regulators of immune function. In this study, we wanted to investigate whether TIM molecules could play a role in sarcoidosis.

Methods

We used real-time polymerase chain reaction to investigate the differential gene expression of TIM-1 and TIM-3 as well as a few Th1 and Th2 cytokines (IL-2, IFN-γ, IL-4, IL-5 and IL-13) in CD4⁺ T cells isolated from bronchoalveolar lavage fluid (BALF) of patients (n = 28) and healthy controls (n = 8). Using flow cytometry, we were also able to analyse TIM-3 protein expression in 10 patients and 6 healthy controls.

Results

A decreased TIM-3 mRNA (p < 0.05) and protein (p < 0.05) expression was observed in patients, and the level of TIM-3 mRNA correlated negatively with the CD4/CD8 T cell ratio in BALF cells of patients. Compared to a distinct subgroup of patients i.e. those with Löfgren''s syndrome, BALF CD4⁺ T cells from non- Löfgren''s patients expressed decreased mRNA levels of TIM-1 (p < 0.05). mRNA expression of IL-2 was increased in patients (p < 0.01) and non-Löfgren''s patients expressed significantly higher levels of IFN-γ mRNA (p < 0.05) versus patients with Löfgren''s syndrome.

Conclusion

These findings are the first data on the expression of TIM-1 and TIM-3 molecules in sarcoidosis. The reduced TIM-3 expression in the lungs of patients may result in a defective T cell ability to control the Th1 immune response and could thus contribute to the pathogenesis of sarcoidosis. The down-regulated TIM-1 expression in non-Löfgren''spatients is in agreement with an exaggerated Th1 response in these patients.     

Epitope-dependent effect of anti-murine TIM-1 monoclonal antibodies on T cell activity and lung immune responses

The TAPR locus containing the TIM gene family is implicated in the development of atopic inflammation in mouse, and TIM-1 allelic variation has been associated with the incidence of atopy in human patient populations. In this study, we show that manipulation of the TIM-1 pathway influences airway inflammation and pathology. Anti-TIM-1 mAbs recognizing distinct epitopes differentially modulated OVA-induced lung inflammation in the mouse. The epitopes recognized by these Abs were mapped, revealing that mAbs to both the IgV and stalk domains of TIM-1 have therapeutic activity. Unexpectedly, mAbs recognizing unique epitopes spanning exon 4 of the mucin/stalk domains exacerbated immune responses. Using Ag recall response studies, we demonstrate that the TIM-1 pathway acts primarily by modulating the production of T(H)2 cytokines. Furthermore, ex vivo cellular experiments indicate that TIM-1 activity controls CD4(+) T cell activity. These studies validate the genetic hypothesis that the TIM-1 locus is linked to the development of atopic disease and suggest novel therapeutic strategies for targeting asthma and other atopic disorders.     

Memory CD8+ T cells protect dendritic cells from CTL killing

CD8(+) T cells have been shown to be capable of either suppressing or promoting immune responses. To reconcile these contrasting regulatory functions, we compared the ability of human effector and memory CD8(+) T cells to regulate survival and functions of dendritic cells (DC). We report that, in sharp contrast to the effector cells (CTLs) that kill DCs in a granzyme B- and perforin-dependent mechanism, memory CD8(+) T cells enhance the ability of DCs to produce IL-12 and to induce functional Th1 and CTL responses in naive CD4(+) and CD8(+) T cell populations. Moreover, memory CD8(+) T cells that release the DC-activating factor TNF-alpha before the release of cytotoxic granules induce DC expression of an endogenous granzyme B inhibitor PI-9 and protect DCs from CTL killing with similar efficacy as CD4(+) Th cells. The currently identified DC-protective function of memory CD8(+) T cells helps to explain the phenomenon of CD8(+) T cell memory, reduced dependence of recall responses on CD4(+) T cell help, and the importance of delayed administration of booster doses of vaccines for the optimal outcome of immunization.     

Induction of CTL and nonpolarized Th cell responses by CD8alpha(+) and CD8alpha(-) dendritic cells

Two distinct dendritic cell (DC) subpopulations have been evidenced in mice on the basis of their differential CD8alpha expression and their localization in lymphoid organs. Several reports suggest that CD8alpha(+) and CD8alpha(-) DC subsets could be functionally different. In this study, using a panel of MHC class I- and/or class II-restricted peptides, we analyzed CD4(+) and CD8(+) T cell responses obtained after i.v. injection of freshly purified peptide-pulsed DC subsets. First, we showed that both DC subsets efficiently induce specific CTL responses and Th1 cytokine production in the absence of CD4(+) T cell priming. Second, we showed that in vivo activation of CD4(+) T cells by CD8alpha(+) or CD8alpha(-) DC, injected i.v., leads to a nonpolarized Th response with production of both Th1 and Th2 cytokines. The CD8alpha(-) subset induced a higher production of Th2 cytokines such as IL-4 and IL-10 than the CD8alpha(+) subset. However, IL-5 was produced by CD4(+) T cells activated by both DC subsets. When both CD4(+) and CD8(+) T cells were primed by DC injected i.v., a similar pattern of cytokines was observed, but, under these conditions, Th1 cytokines were mainly produced by CD8(+) T cells, while Th2 cytokines were produced by CD4(+) T cells. Thus, this study clearly shows that CD4(+) T cell responses do not influence the development of specific CD8(+) T cell cytotoxic responses induced either by CD8alpha(+) or CD8alpha(-) DC subsets.     

Control of NKT cell differentiation by tissue-specific microenvironments

CD1d-restricted Valpha14 NKT cells play an important role in both Th1- and Th2-type immune responses. To determine whether NKT cells develop two functionally distinct subsets that provoke different types of responses, we examined the phenotypes and cellular functions of NK1.1(+) and DX5(+) T cells. We found that both NK1.1(+) and DX5(+) T cells are CD1d-restricted Valpha14 T cells with identical Ag specificities, phenotypes, tissue locations, and functions. Similar to the NK1.1 marker, the DX5 marker (CD49b) is expressed on mature NKT cells in both NK1.1 allele-positive and allele-negative strains. However, when NK1.1(+) and DX5(+) NKT cells isolated from different tissues were compared, we found that thymic and splenic NKT cells differed not only in their cytokine profiles, but also in their phenotype and requirements for costimulatory signals. Thymic NKT cells displayed the phenotype of activated T cells and could be fully activated by TCR ligation. In contrast, splenic NKT cells displayed the phenotype of memory T cells and required a costimulatory signal for activation. Furthermore, the function and phenotype of thymic and splenic NKT cells were modulated by APCs from various tissues that expressed different levels of costimulatory molecules. Modulation of NKT cell function and differentiation may be mediated by synergic effects of costimulatory molecules on the surface of APCs. The results of the present study suggest that the costimulatory signals of tissue-specific APCs are key factors for NKT cell differentiation, and these signals cannot be replaced by anti-CD28 or anti-CD40 ligand Abs.     

TLR2 stimulation drives human naive and effector regulatory T cells into a Th17-like phenotype with reduced suppressive function

Naturally occurring CD4(+)CD25(+)FOXP3(+) regulatory T cells suppress the activity of pathogenic T cells and prevent development of autoimmune responses. There is growing evidence that TLRs are involved in modulating regulatory T cell (Treg) functions both directly and indirectly. Specifically, TLR2 stimulation has been shown to reduce the suppressive function of Tregs by mechanisms that are incompletely understood. The developmental pathways of Tregs and Th17 cells are considered divergent and mutually inhibitory, and IL-17 secretion has been reported to be associated with reduced Treg function. We hypothesized that TLR2 stimulation may reduce the suppressive function of Tregs by regulating the balance between Treg and Th17 phenotype and function. We examined the effect of different TLR2 ligands on the suppressive functions of Tregs and found that activation of TLR1/2 heterodimers reduces the suppressive activity of CD4(+)CD25(hi)FOXP3(low)CD45RA(+) (naive) and CD4(+)CD25(hi)FOXP3(hi)CD45RA(-) (memory or effector) Treg subpopulations on CD4(+)CD25(-)FOXP3(-)CD45RA(+) responder T cell proliferation while at the same time enhancing the secretion of IL-6 and IL-17, increasing RORC, and decreasing FOXP3 expression. Neutralization of IL-6 or IL-17 abrogated Pam3Cys-mediated reduction of Treg suppressive function. We also found that, in agreement with recent observations in mouse T cells, TLR2 stimulation can promote Th17 differentiation of human T helper precursors. We conclude that TLR2 stimulation, in combination with TCR activation and costimulation, promotes the differentiation of distinct subsets of human naive and memory/effector Tregs into a Th17-like phenotype and their expansion. Such TLR-induced mechanism of regulation of Treg function could enhance microbial clearance and increase the risk of autoimmune reactions.     

TIM-1 signaling in B cells regulates antibody production

Members of the T cell Ig and mucin (TIM) family have recently been implicated in the control of T cell-mediated immune responses. In this study, we found TIM-1 expression on anti-IgM- or anti-CD40-stimulated splenic B cells, which was further up-regulated by the combination of anti-IgM and anti-CD40 Abs. On the other hand, TIM-1 ligand was constitutively expressed on B cells and inducible on anti-CD3⁺ anti-CD28-stimulated CD4⁺ T cells. In vitro stimulation of activated B cells by anti-TIM-1 mAb enhanced proliferation and expression of a plasma cell marker syndecan-1 (CD138). We further examined the effect of TIM-1 signaling on antibody production in vitro and in vivo. Higher levels of IgG2b and IgG3 secretion were detected in the culture supernatants of the anti-TIM-1-stimulated B cells as compared with the control IgG-stimulated B cells. When immunized with T-independent antigen TNP-Ficoll, TNP-specific IgG1, IgG2b, and IgG3 Abs were slightly increased in the anti-TIM-1-treated mice. When immunized with T-dependent antigen OVA, serum levels of OVA-specific IgG2b, IgG3, and IgE Abs were significantly increased in the anti-TIM-1-treated mice as compared with the control IgG-treated mice. These results suggest that TIM-1 signaling in B cells augments antibody production by enhancing B cell proliferation and differentiation.     

Human TIM-1 associates with the TCR complex and up-regulates T cell activation signals

The T cell, Ig domain, and mucin domain-1 (TIM-1) gene is associated with Th2 T cell responses and human atopic diseases. The mechanism by which TIM-1 influences T cell responses remains unknown. We demonstrate that TIM-1 is recruited to the TCR-signaling complex via association with CD3. TIM-1 up-regulates TCR-associated signaling events, including phosphorylation of Zap70 and IL-2-inducible T cell kinase. This activity requires TIM-1 tyrosine phosphorylation. TIM-1 expression induces formation of a novel complex that includes PI3K and ITK. Finally, the consequences of TIM-1 activation include increased expression of effector cytokines. These results demonstrate that TIM-1 is a critical component of the human T cell response and provide a mechanistic hypothesis for the role of TIM-1 in disease.     

Th cells act via two synergistic pathways to promote antiviral CD8+ T cell responses

The mechanisms of how Th cells promote CD8(+) T cell responses during viral infections are largely unknown. In this study, we unraveled the mechanisms of T cell help for CD8(+) T cell responses during vaccinia virus infection. Our results demonstrate that Th cells promote vaccinia virus-specific CD8(+) T cell responses via two interconnected synergistic pathways: First, CD40L expressed by activated CD4(+) T cells instructs dendritic cells to produce bioactive IL-12p70, which is directly sensed by Ag-specific CD8(+) T cells, resulting in increased IL-2Rα expression. Second, Th cells provide CD8(+) T cells with IL-2, thereby enhancing their survival. Thus, Th cells are at the center of an important communication loop with a central role for IL-2/IL-2R and bioactive IL-12.     

Selective expression rather than specific function of Txk and Itk regulate Th1 and Th2 responses

Itk and Txk/Rlk are Tec family kinases expressed in T cells. Itk is expressed in both Th1 and Th2 cells. By contrast, Txk is preferentially expressed in Th1 cells. Although Itk is required for Th2 responses in vivo and Txk is suggested to regulate IFN-gamma expression and Th1 responses, it remains unclear whether these kinases have distinct roles in Th cell differentiation/function. We demonstrate here that Txk-null CD4(+) T cells are capable of producing both Th1 and Th2 cytokines similar to those produced by wild-type CD4(+) T cells. To further examine whether Itk and Txk play distinct roles in Th cell differentiation and function, we examined Itk-null mice carrying a transgene that expresses Txk at levels similar to the expression of Itk in Th2 cells. Using two Th2 model systems, allergic asthma and schistosome egg-induced lung granulomas, we found that the Txk transgene rescued Th2 cytokine production and all Th2 symptoms without notable enhancement of IFN-gamma expression. These results suggest that Txk is not a specific regulator of Th1 responses. Importantly, they suggest that Itk and Txk exert their effects on Th cell differentiation/function at the level of expression.     

Ox40 costimulation enhances the development of T cell responses induced by dendritic cells in vivo.

Dendritic cells (DCs) are bone marrow-derived APCs that display unique properties aimed at stimulating naive T cells. Several members of the TNF/TNFR families have been implicated in T cell functions. In this study, we examined the role that Ox40 costimulation might play on the ability of DCs to regulate CD4(+) and CD8(+) T cell responses in vivo. Administration of anti-mouse Ox40 mAb enhanced the Th response induced by immunization with Ag-pulsed DCs, and introduced a bias toward a Th1 immune response. However, anti-Ox40 treatment enhanced the production of Th2 cytokines in IFN-gamma(-/-) mice after immunization with Ag-pulsed DCs, suggesting that the production of IFN-gamma during the immune response could interfere with the development of Th2 lymphocytes induced by DCs. Coadministration of anti-Ox40 with DCs during Ag rechallenge enhanced both Th1 and Th2 responses induced during a primary immunization with DCs, and did not reverse an existing Th2 response. This suggests that Ox40 costimulation amplifies an ongoing immune response, regardless of Th differentiation potential. In an OVA-TCR class II-restricted adoptive transfer system, anti-Ox40 treatment greatly enhanced the level of cytokine secretion per Ag-specific CD4(+) T cell induced by immunization with DCs. In an OVA-TCR class I-restricted adoptive transfer system, administration of anti-Ox40 strongly enhanced expansion, IFN-gamma secretion, and cytotoxic activity of Ag-specific CD8(+) T cells induced by immunization with DCs. Thus, by enhancing immune responses induced by DCs in vivo, the Ox40 pathway might be a target for immune intervention in therapeutic settings that use DCs as Ag-delivery vehicles.     

Proteome profiling of interleukin-12 treated human T helper cells

Selective activation of T helper subsets 1 (Th1) and 2 (Th2) plays a crucial role in different pathological conditions. Th1 cell response is involved in pathogenesis of autoimmune diseases, such as type II diabetes and multiple sclerosis, and Th2 cell response in pathogenesis of allergy and asthma. Cytokine interleukin-12 (IL-12) is one of the key factors in the differentiation of na?ve CD4(+) T cells into Th1 cells. In this study we used 2-DE and MS to find and identify IL-12 regulated proteins in human CD4(+) T cells. In total, 42 protein spots were found to be differentially expressed following IL-12 stimulation, of which 22 were up- and 20 down-regulated. Among the upregulated proteins there are a multifunctional cytokine macrophage migration inhibitory factor and a known IL-12 target gene Programmed cell death 4. Downregulated proteins include p21-activated kinase 2 and its upstream GTPase Cdc42. Compared to previous reports our analysis provides a new view on the IL-12 induced changes on CD4(+) T cells underscoring the importance of creating and combining the data generated at various levels to build a comprehensive view of a given biological process of the cell.     

TIM-3 Does Not Act as a Receptor for Galectin-9

T cell immunoglobulin and mucin protein 3 (TIM-3) is a type I cell surface protein that was originally identified as a marker for murine T helper type 1 cells. TIM-3 was found to negatively regulate murine T cell responses and galectin-9 was described as a binding partner that mediates T cell inhibitory effects of TIM-3. Moreover, it was reported that like PD-1 the classical exhaustion marker, TIM-3 is up-regulated in exhausted murine and human T cells and TIM-3 blockade was described to restore the function of these T cells. Here we show that the activation of human T cells is not affected by the presence of galectin-9 or antibodies to TIM-3. Furthermore, extensive studies on the interaction of galectin-9 with human and murine TIM-3 did not yield evidence for specific binding between these molecules. Moreover, profound differences were observed when analysing the expression of TIM-3 and PD-1 on T cells of HIV-1-infected individuals: TIM-3 was expressed on fewer cells and also at much lower levels. Furthermore, whereas PD-1 was preferentially expressed on CD45RA⁻CD8 T cells, the majority of TIM-3-expressing CD8 T cells were CD45RA⁺. Importantly, we found that TIM-3 antibodies were ineffective in increasing anti-HIV-1 T cell responses in vitro, whereas PD-L antibodies potently reverted the dysfunctional state of exhausted CD8 T cells. Taken together, our results are not in support of an interaction between TIM-3 and galectin-9 and yield no evidence for a functional role of TIM-3 in human T cell activation. Moreover, our data indicate that PD-1, but not TIM-3, is a promising target to ameliorate T cell exhaustion.     

Human CD4(+) T cells recognize an epitope within alpha-fetoprotein sequence and develop into TGF-beta-producing CD4(+) T cells

There is limited information on the influence of tumor growth on the expansion of tumor-specific TGF-beta-producing CD4(+) T cells in humans. alpha-Fetoprotein (AFP) is an oncofetal Ag and has intrinsic immunoregulatory properties. In this study, we report the identification and characterization of subsets of CD4(+) T cells that recognize an epitope within the AFP sequence (AFP(46-55)) and develop into TGF-beta-producing CD4(+) T cells. In a peptide-specific and dose-dependent manner, AFP(46-55) CD4(+) T cells produce TGF-beta, GM-CSF, and IL-2 but not Th1-, Th2-, Th17-, or Tr1-type cytokines. These cells express CTLA-4 and glucocorticoid-induced TNR receptor and inhibit T cell proliferation in a contact-dependent manner. In this study, we show that the frequency of AFP(46-55) CD4(+) T cells is significantly higher (p = 001) in patients with hepatocellular carcinoma than in healthy donors, suggesting that these cells are expanded in response to tumor Ag. In contrast, tumor necrosis-inducing treatments that are shown to improve survival rate can shift the Th1/TGF-beta-producing CD4(+) T cell balance in favor of Th1 responses. Our data demonstrate that tumor Ags may contain epitopes which activate the expansion of inducible regulatory T cells, leading to evasion of tumor control.