Intrinsic versus environmental influences on T-cell responses in aging

Summary: A decline in T‐cell responses and a switch to memory T‐cell predominance occur with aging. We have used the T‐cell receptor (TCR) transgenic mouse model to study age‐associated changes in T‐cell responses that are a consequence of shifts in subset representation versus changes intrinsic to T cells versus changes in the ‘aged’ microenvironment. We found that naive transgene‐expressing (Tg⁺) CD4⁺ T cells from aged mice respond to antigen with reduced interleukin‐2 (IL‐2) production, decreased cell expansion, and limited differentiation to effectors. Comparable to the characteristic accumulation of memory phenotype T cells in aged humans and conventional rodents, Tg⁺ CD4⁺ T cells from old OTII and 6.5 TCR transgenic mice acquire a memory phenotype without immunization and become hyporesponsive. The naive Tg⁺ CD8⁺ T cells from aged 2C mice expressed activation markers, produced IL‐2, proliferated, and differentiated into cytotoxic T lymphocytes as efficiently as their young counterparts. Responses by adoptive transferred Tg⁺ cells from young mice, immunized in young and old conventional hosts, indicated that the host age influences the onset of cell division, level of cell expansion, and number of cytokine‐producing cells. Co‐transfer of dendritic cells (DCs) from young and less so from aged conventional mice partially restored responses. Furthermore, DCs and T‐cell migration to draining lymphoid organs was reduced due to deficiencies intrinsic to aged cells and the aged environment. Thus, alterations in T‐cell responses in aging are attributable to intrinsic and environmental influences.     

Correction of antigen-specific T cell defects in aged murine gut-associated lymphoid tissues an immune intervention by combined adoptive transfer of an antigen-specific immunoregulatory CD4 T cell subset and interleukin 2 administration

Gut-associated lymphoid tissues (GALT) from aged mice enterically immunized with Mycobacterium paratuberculosis protoplasmic antigen show hyperreactive humoral immune responses; this hyperresponsiveness can be corrected to a considerable extent, but not entirely, by systemic administration of interleukin 2 (IL 2) alone. The aim of the present study was to determine further whether the hyperreactivity in the antigen-specific humoral immune responses in aged GALT could be fully restored by adoptive transfer of in vitro expanded antigen-specific IL 2-dependent helper (CD4⁺VV^?) T cells from GALT in conjunction with recombinant IL 2 administration. The results show that the age-associated hyperresponsiveness in gut mucosal antigen-specific humoral immune responses can be entirely corrected by adoptive transfer with the antigen-specific GALT T helper cells together with in vivo IL 2 adminstration. The mechanism of this restoration involves reversal of the decline in antigen-specific CD8⁺ suppressor T cell functions in aged GALT.     

Calcineurin inhibitors dampen humoral immunity by acting directly on naive B cells

Calcineurin inhibitors (CNI), used frequently in solid organ transplant patients, are known to inhibit T cell proliferation, but their effect on humoral immunity is far less studied. Total and naive B cells from healthy adult donors were cultured in immunoglobulin (Ig)A‐ or IgG/IgE‐promoting conditions with increasing doses of cyclosporin, tacrolimus, rapamycin or methylprednisolone. The effect on cell number, cell division, plasmablast differentiation and class‐switching was tested. To examine the effect on T follicular helper (Tfh) cell differentiation, naive CD4⁺ T cells were cultured with interleukin (IL)‐12 and titrated immunosuppressive drug (IS) concentrations. Total B cell function was not affected by CNI. However, naive B cell proliferation was inhibited by cyclosporin and both CNI decreased plasmablast differentiation. Both CNI suppressed IgA, whereas only cyclosporin inhibited IgE class‐switching. Rapamycin had a strong inhibitory effect on B cell function. Strikingly, methylprednisolone, increased plasmablast differentiation and IgE class‐switching from naive B cells. Differentiation of Tfh cells decreased with increasing IS doses. CNI affected humoral immunity directly by suppressing naive B cells. CNI, as well as rapamycin and methylprednisolone, inhibited the in‐vitro differentiation of Tfh from naive CD4⁺ T cells. In view of its potent suppressive effect on B cell function and Tfh cell differentiation, rapamycin might be an interesting candidate in the management of B cell mediated complications post solid organ transplantation.     

Assessment of alloreactive T cell subpopulations of aged mice in vivo. CD4+ but not CD8+ T cell-mediated rejection response declines with advanced age

The present investigation explored age-related alterations in T cell populations mediating allospecific responses in vivo. Healthy aged and young H-2^b and H-2^bxH-2^k mice were engrafted with major histocompatibility complex (MHC) class II-disparate bm12 skin, rejection of which requires CD4⁺ T cells, and MHC class I-disparate bm1 skin, rejection of which requires CD8⁺ T cells. Aged mice of both genders exhibited prolonged survival of bm12 skin grafts relative to their young counterparts but rejected bm1 skin grafts at a rate equivalent to that of young mice. Consistent with prolonged survival of bm12 skin grafts, markedly diminished levels of Ia^bm12 CTL activity were elicited from T cells of aged mice in vitro. However, no such decline was observed in the level of K^bm1 CTL from T cells of aged mice. The alterations in Ia^bm12 allospecific responses were not attributable to quantitative changes in CD4⁺ T cells of aged mice, and addition of soluble T cell helper factors to response cultures of aged mice did not augment Ia^bm12 cytotoxic T lymphocytes activity. These data demonstrate that aging fundamentally affects CD4⁺ T cell-mediated allospecific responses particularly in vivo, and that deficient generation of soluble T cell helper factors alone cannot explain this deficit.     

P21‐activated kinase 2 is essential in maintenance of peripheral Foxp3+ regulatory T cells

The p21‐activated kinase 2 (Pak2), an effector molecule of the Rho family GTPases Rac and Cdc42, regulates diverse functions of T cells. Previously, we showed that Pak2 is required for development and maturation of T cells in the thymus, including thymus‐derived regulatory T (Treg) cells. However, whether Pak2 is required for the functions of various subsets of peripheral T cells, such as naive CD4 and helper T‐cell subsets including Foxp3⁺ Treg cells, is unknown. To determine the role of Pak2 in CD4 T cells in the periphery, we generated inducible Pak2 knockout (KO) mice, in which Pak2 was deleted in CD4 T cells acutely by administration of tamoxifen. Temporal deletion of Pak2 greatly reduced the number of Foxp3⁺ Treg cells, while minimally affecting the homeostasis of naive CD4 T cells. Pak2 was required for proliferation and Foxp3 expression of Foxp3⁺ Treg cells upon T‐cell receptor and interleukin‐2 stimulation, differentiation of in vitro induced Treg cells, and activation of naive CD4 T cells. Together, Pak2 is essential in maintaining the peripheral Treg cell pool by providing proliferation and maintenance signals to Foxp3⁺ Treg cells.     

Intrinsic T‐ and B‐cell defects impair T‐cell‐dependent antibody responses in mice lacking the actin‐bundling protein L‐plastin

Germinal center development, critical for long‐term humoral immunity, requires the trafficking of T and B lymphocytes to defined tissues and locations after antigenic challenge. The molecular mechanisms that support lymphocyte trafficking through the linkage of extracellular chemotactic and adhesive cues to the actin cytoskeleton are not yet fully defined. We have previously identified the actin‐bundling protein L‐plastin (LPL) as a requisite intermediary in both naive B and T lymphocyte migration and in T‐cell activation. We tested the hypothesis that humoral immunity would require LPL. We show that mice lacking LPL demonstrated defective germinal center formation and reduced production of T‐cell‐dependent antibodies. T cells from LPL^?/? mice exhibited defective expansion of the follicular helper T population. Reduced expansion of LPL^?/? follicular helper T cells correlated with impaired trafficking to or retention of cells in the spleen following challenge, highlighting the importance of initial lymphocyte recruitment to the eventual success of the immune response. Furthermore, LPL^?/? B cells demonstrated cell‐intrinsic defects in population expansion and in differentiation into germinal center B cells. LPL thus modulates both T‐ and B‐cell function during the germinal center reaction and the production of T‐cell‐dependent antibody responses.     

IL‐7 is superior to IL‐2 for ex vivo expansion of tumour‐specific CD4+ T cells

It is well established that tumours hinder both natural and vaccine‐induced tumour‐specific CD4⁺ T‐cell responses. Adoptive T‐cell therapy has the potential to circumvent functional tolerance and enhance anti‐tumour protective responses. While protocols suitable for the expansion of cytotoxic CD8⁺ T cells are currently available, data on tumour‐specific CD4⁺ T cells remain scarce. We report here that CD4⁺ T cells sensitized to tumour‐associated Ag in vivo, proliferate in vitro in response to IL‐7 without the need for exogenous Ag stimulation and accumulate several folds while preserving a memory‐like phenotype. Both cell proliferation and survival accounts for the outgrowth of tumour‐sensitized T cells among other memory and naive lymphocytes following exposure to IL‐7. Also IL‐2, previously used to expand anti‐tumour CTL, promotes tumour‐specific CD4⁺ T‐cell accumulation. However, IL‐7 is superior to IL‐2 at preserving lymphocyte viability, in vitro and in vivo, maintaining those properties, that are required by helper CD4⁺ T cells to confer therapeutic efficacy upon transplantation in tumour‐bearing hosts. Together our data support a unique role for IL‐7 in retrieving memory‐like CD4⁺ T cells suitable for adoptive T‐cell therapy.     

Steady‐state antigen‐expressing dendritic cells terminate CD4+ memory T‐cell responses

CD4⁺ T cells are important effectors of inflammation and tissue destruction in many diseases of immune dysregulation. As memory T cells develop early during the preclinical stages of autoimmune and inflammatory diseases, immunotherapeutic approaches to treatment of these diseases, once established, must include the means to terminate memory T‐cell responses. Traditionally, it has been considered that, due to their terminally differentiated nature, memory T cells are resistant to tolerance induction, although emerging evidence indicates that some immunotherapeutic approaches can terminate memory T‐cell responses. Here, we demonstrate that CD4⁺ memory T‐cell responses can be terminated when cognate antigen is transgenically expressed in steady‐state DC. Transfer of in‐vitro‐generated CD4⁺ memory T cells establishes, in nontransgenic recipients, a stable and readily recalled memory response to cognate antigen. In contrast, upon transfer to mice expressing cognate antigen targeted to DC, memory CD4⁺ T cells undergo a phase of limited proliferation followed by substantial deletion, and recall responses are effectively silenced. This finding is important in understanding how to effectively apply immunotherapy to ongoing T‐cell‐mediated autoimmune and inflammatory diseases.     

Acute stimulation generates Tim‐3‐expressing T helper type 1 CD4 T cells that persist in vivo and show enhanced effector function

T‐cell immunoglobulin and mucin domain 3 (Tim‐3) is a surface receptor expressed by T helper type 1 (Th1) effector CD4 T cells, which are critical for defence against intracellular pathogens and have been implicated in autoimmune disease. Previous studies showed that Tim‐3 expression makes Th1 cells more susceptible to apoptosis and also marks functionally impaired T cells that arise due to chronic stimulation. However, other studies suggested that Tim‐3‐expressing Th1 cells do not always have these properties. To further define the relationship between Tim‐3 and Th1 cell function, we analysed the characteristics of Th1 cells that expressed Tim‐3 in response to brief stimulation in vitro or an acute viral infection in vivo. As expected, cultured CD4 T cells began expressing Tim‐3 during Th1 differentiation and secondary stimulation generated Tim‐3^? and Tim‐3⁺ fractions that were separated and further analysed. When injected into naive mice, Tim‐3⁺ cells down‐regulated Tim‐3 and survived equally well compared with Tim‐3^‐ cells. Further, Tim‐3^? and Tim‐3⁺ Th1 cells had similar functional responses when transferred into naive mice that were subsequently infected with lymphocytic choriomeningitis virus (LCMV). Cultured Th1 cells that expressed Tim‐3 following T‐cell receptor stimulation had a greater capacity to express signature Th1 cytokines than their Tim‐3^? counterparts and showed differential expression of genes that regulate CD4 T‐cell function. Consistent with these findings, Tim‐3⁺ Th1 cells generated in response to LCMV infection displayed augmented effector function relative to Tim‐3^? cells. These results suggest that Tim‐3 expression by Th1 cells responding to acute stimulation can mark cells that are functionally competent and have an augmented ability to produce cytokines.     

CD4+ memory T cells: functional differentiation and homeostasis

Summary: CD4⁺ T cells are central regulators of both humoral and cellular immune responses. There are many subsets of CD4⁺ T cells, the most prominent being T‐helper 1 (Th1), Th2, Th‐17, and regulatory T cells, specialized in regulating different aspects of immunity. Without participation by these CD4⁺ T‐cell subsets, B cells cannot undergo isotype switching to generate high‐affinity antibodies, the microbicidal activity of macrophages is reduced, the efficiency of CD8⁺ T‐cell responses and CD8⁺ T‐cell memory are compromised, and downregulation of effector responses is impaired. It therefore stands to reason that memory CD4⁺ T cells are likely to fulfill an important facilitator role in the maintenance and control of protective immune responses. This review discusses some issues of importance for the generation of memory CD4⁺ T cells and focuses in particular on their heterogeneity and plasticity, with respect to both phenotypic characteristics and function. Finally, we discuss a number of factors that affect long‐term maintenance of memory CD4⁺ T cells.     

T cells in aging mice: genetic, developmental, and biochemical analyses

Summary: A combination of approaches – gene mapping, biomarker analysis, and studies of signal transduction – has helped to clarify the mechanisms of age‐related change in mouse immune status and the implications of immune aging for late‐life disease. Mapping studies have documented multiple quantitative trait loci (QTL) that influence the levels of age‐sensitive T‐cell subsets. Some of these QTL have effects that are demonstrable in young‐adult mice (8 months of age) and others demonstrable only in middle‐aged mice (18 months). Biomarker studies show that T‐cell subset levels measured at 8 or 18 months are significant predictors of lifespan for mice dying of lymphoma, fibrosarcoma, mammary adenocarcinoma, or all causes combined. Mice whose immune systems resemble that of young animals, i.e. with low levels of CD4⁺ and CD8⁺ memory T cells and relatively high levels of CD4⁺ T cells, tend to outlive their siblings with the opposite subset pattern. Biochemical analyses show that T cells from aged mice show defects in the activation process within a few minutes of encountering a stimulus and that the defects precede the recognition by the T‐cell receptor of agonist peptides on the antigen‐presenting cell. Defective assembly of cytoskeletal fibers and hyperglycosylation of T‐cell surface glycoproteins contribute to the immunodeficiency state, and indeed treatment with a sialylglycoprotein endopeptidase can restore full function to CD4⁺ T cells from aged donors in vitro.     

Optimal stimulation for CD70 induction on human monocyte‐derived dendritic cells and the importance of CD70 in naive CD4+ T‐cell differentiation

Studies in mice have shown that CD70 on dendritic cells (DCs) is sufficient to convert T‐cell tolerance into immunity and hence induce anti‐tumour immune responses. Therefore, it is important to investigate (i) optimal stimuli to induce CD70 on human monocyte‐derived DCs (MoDCs), which are widely used for tumour immunotherapy, and (ii) the role of CD70 in functional differentiation of naive CD4⁺ and CD8⁺ T cells stimulated with MoDCs. We show that interferon‐α (IFN‐α) is a key cytokine to differentiate monocytes into DCs with the capacity to express CD70 upon maturation. CD70 expression on IFN‐α‐induced MoDCs was elicited by different categories of maturation‐inducing factors (Toll‐like receptor ligands, CD40 ligand and pro‐inflammatory mediators), among which prostaglandin E₂ was most effective. Naive T cells stimulated with MoDCs also expressed CD70. Stimulation with MoDCs promoted naive CD4⁺ T cells to acquire the ability to produce T helper type 1 and 2 cytokines in a CD70‐dependent manner. In contrast, the CD70–CD27 interaction diminished the production of an immunoregulatory cytokine IL‐10. The CD27 signal did not play a dominant role in the induction of effector molecules in naive CD8⁺ T cells during the stimulation with MoDCs. This study adds a novel function to the versatile cytokines, type I IFNs, that is, the induction of CD70 on MoDCs. CD70 promotes naive CD4⁺ T cells to acquire immunostimulatory activity through the DC–T‐cell and T‐cell–T‐cell interactions during the stimulation with MoDCs. Hence, the CD70–CD27 interaction may play an important role in inducing effective immune responses in DC‐based immunotherapy.     

DC‐induced CD8+ T‐cell response is inhibited by MHC class II‐dependent DX5+CD4+ Treg

CD4⁺ T cells are important for CD8⁺ T‐cell priming by providing cognate signals for DC maturation. We analyzed the capacity of CD4⁺ T cells to influence CD8⁺ T‐cell responses induced by activated DC. Surprisingly, mice depleted for CD4⁺ cells were able to generate stronger antigen‐specific CD8⁺ T‐cell responses after DC vaccination than non‐depleted mice. The same observation was made when mice were vaccinated with MHC class II^?/? DC, indicating the presence of a MHC class II‐dependent CD4⁺ T‐cell population inhibiting CD8⁺ T‐cell responses. Recently we described the expansion of DX5⁺CD4⁺ T cells, a T‐cell population displaying immune regulatory properties, upon vaccination with DC. Intriguingly, we now observe an inverse correlation between CD8⁺ T‐cell induction and expansion of DX5⁺CD4⁺ T cells as the latter cells did not expand after vaccination with MHC class II^?/? DC. In vitro, DX5⁺CD4⁺ T cells were able to limit proliferation, modulate cytokine production and induce Foxp3⁺ expression in OVA‐specific CD8⁺ T cells. Together, our data show an inhibitory role of CD4⁺ T cells on the induction of CD8⁺ T‐cell responses by activated DC and indicate the involvement of DX5⁺CD4⁺, but not CD4⁺CD25⁺, T cells in this process.     

Induced regulatory T cells are phenotypically unstable and do not protect mice from rapidly progressive glomerulonephritis

Regulatory T (Treg) cells are a suppressive CD4⁺ T‐cell subset. We generated induced Treg (iTreg) cells and explored their therapeutic potential in a murine model of rapidly progressive glomerulonephritis. Polyclonal naive CD4⁺ T cells were cultured in vitro with interleukin‐2 (IL‐2), transforming growth factor‐β1, all‐trans‐retinoic acid and monoclonal antibodies against interferon‐γ and IL‐4, generating Foxp3⁺ iTreg cells. To enhance their suppressive phenotype, iTreg cultures were modified with the addition of a monoclonal antibody against IL‐12p40 or by using RORγt^–/– CD4⁺ T cells. Induced Treg cells were transferred into models of delayed‐type hypersensitivity and experimental glomerulonephritis. The iTreg cells exhibited comparable surface receptor expression and in vitro suppressive ability to natural Treg cells, but did not regulate antigen‐specific delayed‐type hypersensitivity or systemic inflammatory immune responses, losing Foxp3 expression in vivo. In glomerulonephritis, transferred iTreg cells did not prevent renal injury or modulate systemic T helper type 1 immune responses. Induced Treg cells cultured with anti‐IL‐12p40 had an enhanced suppressive phenotype in vitro and regulated dermal delayed‐type hypersensitivity in vivo, but were not protective against renal injury, losing Foxp3 expression, especially in the transferred cells recruited to the kidney. Use of RORγt^–/– CD4⁺ T cells or iTreg cells generated from sensitized CD4⁺ Foxp3^– cells did not regulate renal or systemic inflammatory responses in vivo. In conclusion, iTreg cells suppress T‐cell proliferation in vitro, but do not regulate experimental glomerulonephritis, being unstable in this inflammatory milieu in vivo.     

Thymic function in young/old chimeras: substantial thymic T cell regenerative capacity despite irreversible age-associated thymic involution

Age-associated thymic involution results in a diminished capacity to regenerate T cell populations, although the magnitude of this effect is unknown. In this report, thymic function was studied in aged vs.young adult mice after lethal irradiation and administration of T cell-depleted bone marrow (BM) from young mice. Abnormalities observed in aged thymi (reduced thymocyte numbers, histologic abnormalities) were not reversed by administration of young BM via bone marrow transplantation (BMT), but agend thymi displayed a normal thymocyte subset distribution and appropriately deleted Mls-reactive T cells after BMT. Aged BMT recipients regenerated significantly reduced numbers of splenic T cells compared to young recipients and showed increased peripheral expansion of thymic emigrants since a higher proportion of BM-derived T cells expressed a memory phenotype in aged vs.young BMT recipients. Because peripheral expansion of thymic emigrants could substantially increase the number of thymic progeny present in the spleen, we sought to measure thymic T cell regenerative capacity after BMT in a setting devoid of peripheral expansion. To do this, TCR-transgenic (Tg⁺) T cell-depleted BM was administered to aged and young recipients lacking antigen specific for the Tg⁺ TCR. Aged recipients regenerated approximately 50 % of the TCR Tg⁺ cells regenerated in young BMT recipients, providing evidence that even very aged thymi retain the capacity to regenerate significant numbers of mature T cell progeny. Therefore, thymic function is reduced with aged but it is not lost, suggesting that therapeutic approaches to enhance thymic function may be successful even in very aged hosts.     

Interleukin‐4‐induced loss of CD8 expression and cytolytic function in effector CD8 T cells persists long term in vivo

Activation of naive CD8⁺ T cells in the presence of interleukin‐4 modulates their CD8 co‐receptor expression and functional differentiation, resulting in the generation of CD8^low cells that produce type 2 cytokines and display poor cytolytic and anti‐tumour activity. Although this CD8^low phenotype becomes stable after about a week and can persist with further stimulation in vitro, it is not known whether it can be maintained long term in vivo. Here we report that CD8^low cells derived from oval‐bumin_257–264‐specific T‐cell receptor‐transgenic CD8⁺ T cells activated in the presence of interleukin‐4 could be detected in the spleen for at least 4 months after adoptive transfer into normal mice. A significant proportion of the long‐term surviving cells retained their CD8^low phenotype in vivo and after clonal re‐activation in vitro. Although long‐term surviving CD8^low cells lacked detectable cytolytic activity or perforin expression, they showed some anti‐tumour function in vivo. The persistence of functional cells with a CD8^low phenotype in vivo raises the possibility that such cells can contribute to effector or regulatory responses to tumours or pathogens.     

TLR5 functions as an endocytic receptor to enhance flagellin-specific adaptive immunity

Innate immune activation via TLR induces dendritic cell maturation and secretion of inflammatory mediators, generating favorable conditions for naïve T‐cell activation. Here, we demonstrate a previously unknown function for TLR5, namely that it enhances MHC class‐II presentation of flagellin epitopes to CD4⁺ T cells and is required for induction of robust flagellin‐specific adaptive immune responses. Flagellin‐specific CD4⁺ T cells expanded poorly in TLR5‐deficient mice immunized with flagellin, a deficiency that persisted even when additional TLR agonists were provided. Flagellin‐specific IgG responses were similarly depressed in the absence of TLR5. In marked contrast, TLR5‐deficient mice developed robust flagellin‐specific T‐cell responses when immunized with processed flagellin peptide. Surprisingly, the adaptor molecule Myd88 was not required for robust CD4⁺ T‐cell responses to flagellin, indicating that TLR5 enhances flagellin‐specific CD4⁺ T‐cell responses in the absence of conventional TLR signaling. A requirement for TLR5 in generating flagellin‐specific CD4⁺ T‐cell activation was also observed when using an in vitro dendritic cell culture system. Together, these data uncover an Myd88‐independent function for dendritic cell TLR5 in enhancing the presentation of peptides to flagellin‐specific CD4⁺ T cells.     

Overcoming regulatory T‐cell suppression by a lyophilized preparation of Streptococcus pyogenes

Cancer vaccines have yet to yield clinical benefit, despite the measurable induction of humoral and cellular immune responses. As immunosuppression by CD4⁺CD25⁺ regulatory T (Treg) cells has been linked to the failure of cancer immunotherapy, blocking suppression is therefore critical for successful clinical strategies. Here, we addressed whether a lyophilized preparation of Streptococcus pyogenes (OK‐432), which stimulates Toll‐like receptors, could overcome Treg‐cell suppression of CD4⁺ T‐cell responses in vitro and in vivo. OK‐432 significantly enhanced in vitro proliferation of CD4⁺ effector T cells by blocking Treg‐cell suppression and this blocking effect depended on IL‐12 derived from antigen‐presenting cells. Direct administration of OK‐432 into tumor‐associated exudate fluids resulted in a reduction of the frequency and suppressive function of CD4⁺CD25⁺Foxp3⁺ Treg cells. Furthermore, when OK‐432 was used as an adjuvant of vaccination with HER2 and NY‐ESO‐1 for esophageal cancer patients, NY‐ESO‐1–specific CD4⁺ T‐cell precursors were activated, and NY‐ESO‐1–specific CD4⁺ T cells were detected within the effector/memory T‐cell population. CD4⁺ T‐cell clones from these patients had high‐affinity TCRs and recognized naturally processed NY‐ESO‐1 protein presented by dendritic cells. OK‐432 therefore inhibits Treg‐cell function and contributes to the activation of high‐avidity tumor antigen‐specific naive T‐cell precursors.