CD8+ T cell self-tolerance permits responsiveness but limits tissue damage.

Self-specific CD8+T cells can escape clonal deletion, but the properties and capabilities of such cells in a physiological setting are unclear. We characterized polyclonal CD8+ T cells specific for the melanocyte antigen tyrosinase-related protein 2 (Trp2) in mice expressing or lacking this enzyme (due to deficiency in Dct, which encodes Trp2). Phenotypic and gene expression profiles of pre-immune Trp2/Kb-specific cells were similar; the size of this population was only slightly reduced in wild-type (WT) compared to Dct-deficient (Dct-/-) mice. Despite comparable initial responses to Trp2 immunization, WT Trp2/Kb-specific cells showed blunted expansion and less readily differentiated into a CD25+proliferative population. Functional self-tolerance clearly emerged when assessing immunopathology: adoptively transferred WT Trp2/Kb-specific cells mediated vitiligo much less efficiently. Hence, CD8+ T cell self-specificity is poorly predicted by precursor frequency, phenotype, or even initial responsiveness, while deficient activation-induced CD25 expression and other gene expression characteristics may help to identify functionally tolerant cells.

Constitutive metanephric mesenchyme-specific expression of interferon-gamma causes renal dysplasia by regulating Sall1 expression.

Transplacental viral and parasitic infections have been shown to initiate an innate response in the mammalian embryo by increasing the expression of pro-inflammatory cytokines such as interferon-gamma (Ifng). However, the developmental consequences of an activated innate immunity and, in particular, the effects of induction of Ifng expression independent of infection have been largely overlooked. Here, we demonstrate in vivo that the conditional overexpression of Ifng in metanephric mesenchymal (MM) progenitors results in renal agenesis or hypoplasia. Cell death was observed in and around the MM region of E10.5-11.5 mutants where Ifng was constitutively expressed during early kidney development and resulted in a retardation of branching morphogenesis. Furthermore, isolated mutant or normal Ifng-treated metanephroi replicated this phenotype in culture, demonstrating the inherent nature of the aberrant morphogenesis. The expression of renal progenitor marker Sall1 was significantly decreased in the MM of mutant kidneys, suggesting that a reduction in Sall1 may be the cause of cell death in the MM during early kidney development and that, in turn, retards UB branching in the mutants. Therefore, the aberrant induction of Ifng expression, as part of an innate immune response, may contribute to renal agenesis or hypoplasia during early metanephric development by regulating the MM progenitor population.

An Immune-Inflammation Gene Expression Signature in Prostate Tumors of Smokers.

Smokers develop metastatic prostate cancer more frequently than nonsmokers, suggesting that a tobacco-derived factor is driving metastatic progression. To identify smoking-induced alterations in human prostate cancer, we analyzed gene and protein expression patterns in tumors collected from current, past, and never smokers. By this route, we elucidated a distinct pattern of molecular alterations characterized by an immune and inflammation signature in tumors from current smokers that were either attenuated or absent in past and never smokers. Specifically, this signature included elevated immunoglobulin expression by tumor-infiltrating B cells, NF-κB activation, and increased chemokine expression. In an alternate approach to characterize smoking-induced oncogenic alterations, we also explored the effects of nicotine in human prostate cancer cells and prostate cancer-prone TRAMP mice. These investigations showed that nicotine increased glutamine consumption and invasiveness of cancer cells in vitro and accelerated metastatic progression in tumor-bearing TRAMP mice. Overall, our findings suggest that nicotine is sufficient to induce a phenotype resembling the epidemiology of smoking-associated prostate cancer progression, illuminating a novel candidate driver underlying metastatic prostate cancer in current smokers.

CD4+ T Cell Help Selectively Enhances High-Avidity Tumor Antigen-Specific CD8+ T Cells.

Maintaining antitumor immunity remains a persistent impediment to cancer immunotherapy. We and others have previously reported that high-avidity CD8(+) T cells are more susceptible to tolerance induction in the tumor microenvironment. In the present study, we used a novel model where T cells derived from two independent TCR transgenic mouse lines recognize the same melanoma antigenic epitope but differ in their avidity. We tested whether providing CD4(+) T cell help would improve T cell responsiveness as a function of effector T cell avidity. Interestingly, delivery of CD4(+) T cell help during in vitro priming of CD8(+) T cells improved cytokine secretion and lytic capacity of high-avidity T cells, but not low-avidity T cells. Consistent with this observation, copriming with CD4(+) T cells improved antitumor immunity mediated by higher avidity, melanoma-specific CD8(+) T cells, but not T cells with similar specificity but lower avidity. Enhanced tumor immunity was associated with improved CD8(+) T cell expansion and reduced tolerization, and it was dependent on presentation of both CD4(+) and CD8(+) T cell epitopes by the same dendritic cell population. Our findings demonstrate that CD4(+) T cell help preferentially augments high-avidity CD8(+) T cells and provide important insight for understanding the requirements to elicit and maintain durable tumor immunity.

Immunotherapy-induced CD8+ T cells instigate immune suppression in the tumor.

Despite clear evidence of immunogenicity, cancer vaccines only provide a modest clinical benefit. To evaluate the mechanisms that limit tumor regression following vaccination, we have investigated the weak efficacy of a highly immunogenic experimental vaccine using a murine melanoma model. We discovered that the tumor adapts rapidly to the immune attack instigated by tumor-specific CD8+ T cells in the first few days following vaccination, resulting in the upregulation of a complex set of biological networks, including multiple immunosuppressive processes. This rapid adaptation acts to prevent sustained local immune attack, despite continued infiltration by increasing numbers of tumor-specific T cells. Combining vaccination with adoptive transfer of tumor-specific T cells produced complete regression of the treated tumors but did not prevent the adaptive immunosuppression. In fact, the adaptive immunosuppressive pathways were more highly induced in regressing tumors, commensurate with the enhanced level of immune attack. Examination of tumor infiltrating T-cell functionality revealed that the adaptive immunosuppression leads to a progressive loss in T-cell function, even in tumors that are regressing. These novel observations that T cells produced by therapeutic intervention can instigate a rapid adaptive immunosuppressive response within the tumor have important implications for clinical implementation of immunotherapies.

T cell avidity and tumor immunity: problems and solutions.

A potent T cell response is an important component of durable anti-tumor immunity. The quality of the T cell response can, in-part, be measured by the avidity of the T cell for its tumor antigen-expressing target. While convention suggests that raising the avidity of the responding T cells may make for a more potent anti-tumor immune response, the threshold for effective tumor immunity remains unclear, as do some of the adverse effects of an inappropriately high avidity response. In this review, we discuss the relationship between T cell avidity and anti-tumor immunity, considering both experimental model systems as well as human clinical trials.

High-avidity T cells are preferentially tolerized in the tumor microenvironment.

One obstacle in eliciting potent antitumor immune responses is the induction of tolerance to tumor antigens. TCR(lo) mice bearing a TCR transgene specific for the melanoma antigen tyrosinase-related protein-2 (TRP-2, Dct) harbor T cells that maintain tumor antigen responsiveness but lack the ability to control melanoma outgrowth. We used this model to determine whether higher avidity T cells could control tumor growth without becoming tolerized. As a part of the current study, we developed a second TRP-2-specific TCR transgenic mouse line (TCR(hi)) that bears higher avidity T cells and spontaneously developed autoimmune depigmentation. In contrast to TCR(lo) T cells, which were ignorant of tumor-derived antigen, TCR(hi) T cells initially delayed subcutaneous B16 melanoma tumor growth. However, persistence in the tumor microenvironment resulted in reduced IFN-γ production and CD107a (Lamp1) mobilization, hallmarks of T-cell tolerization. IFN-γ expression by TCR(hi) T cells was critical for upregulation of MHC-I on tumor cells and control of tumor growth. Blockade of PD-1 signals prevented T-cell tolerization and restored tumor immunity. Depletion of tumor-associated dendritic cells (TADC) reduced tolerization of TCR(hi) T cells and enhanced their antitumor activity. In addition, TADCs tolerized TCR(hi) T cells but not TCR(lo) T cells in vitro. Our findings show that T-cell avidity is a critical determinant of not only tumor control but also susceptibility to tolerization in the tumor microenvironment. For this reason, care should be exercised when considering T-cell avidity in designing cancer immunotherapeutics.

FOXO3: A master switch for regulating tolerance and immunity in dendritic cells.

Recent findings demonstrate that dendritic cells in prostate tumors induce immune tolerance in tumor antigen-specific CD8(+) T cells. We propose that DC tolerogenicity can be regulated by expression of Foxo3; silencing Foxo3 expression enhances anti-tumor immune responses and renders FOXO3 a potential target for immunotherapy.

Isolation of immune cells from primary tumors.

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors. Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4(+) T helper cells via adoptive transfer, promotes CD8(+) T cells to maintain pro-inflammatory effector functions. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

MicroRNA-1 is a candidate tumor suppressor and prognostic marker in human prostate cancer.

We previously reported that miR-1 is among the most consistently down-regulated miRs in primary human prostate tumors. In this follow-up study, we further corroborated this finding in an independent data set and made the novel observation that miR-1 expression is further reduced in distant metastasis and is a candidate predictor of disease recurrence. Moreover, we performed in vitro experiments to explore the tumor suppressor function of miR-1. Cell-based assays showed that miR-1 is epigenetically silenced in human prostate cancer. Overexpression of miR-1 in these cells led to growth inhibition and down-regulation of genes in pathways regulating cell cycle progression, mitosis, DNA replication/repair and actin dynamics. This observation was further corroborated with protein expression analysis and 3'-UTR-based reporter assays, indicating that genes in these pathways are either direct or indirect targets of miR-1. A gene set enrichment analysis revealed that the miR-1-mediated tumor suppressor effects are globally similar to those of histone deacetylase inhibitors. Lastly, we obtained preliminary evidence that miR-1 alters the cellular organization of F-actin and inhibits tumor cell invasion and filipodia formation. In conclusion, our findings indicate that miR-1 acts as a tumor suppressor in prostate cancer by influencing multiple cancer-related processes and by inhibiting cell proliferation and motility.

Immune suppression in the tumor microenvironment: a role for dendritic cell-mediated tolerization of T cells.

Immune suppression remains a consistent obstacle to successful anti-tumor immune responses. As tumors develop, they create a microenvironment that not only supports tumor growth and metastasis but also reduces potential adaptive immunity to tumor antigens. Among the many components of this tumor microenvironment is a population of dendritic cells which exert profound immune suppressive effects on T cells. In this review, we discuss our recent findings related to these tumor-associated dendritic cells and how targeting them may serve to generate more durable anti-tumor immune responses.

FOXO3 programs tumor-associated DCs to become tolerogenic in human and murine prostate cancer.

The limited success of cancer immunotherapy is often attributed to the loss of antigen-specific T cell function in situ. However, the mechanism for this loss of function is unknown. In this study, we describe a population of tumor-associated DCs (TADCs) in both human and mouse prostate cancer that tolerizes and induces suppressive activity in tumor-specific T cells. In tumors from human prostate cancer patients and transgenic adenocarcinoma of the mouse prostate (TRAMP) mice, TADCs expressed elevated levels of FOXO3 and Foxo3, respectively, which correlated with expression of suppressive genes that negatively regulate T cell function. Silencing FOXO3 and Foxo3 with siRNAs abrogated the ability of human and mouse TADCs, respectively, to tolerize and induce suppressive activity by T cells. Silencing Foxo3 in mouse TADCs was also associated with diminished expression of tolerogenic mediators, such as indoleamine-2,3-dioxygenase, arginase, and TGF-ß, and upregulated expression of costimulatory molecules and proinflammatory cytokines. Importantly, transfer of tumor-specific CD4+ Th cells into TRAMP mice abrogated TADC tolerogenicity, which was associated with reduced Foxo3 expression. These findings demonstrate that FOXO3 may play a critical role in mediating TADC-induced immune suppression. Moreover, our results identify what we believe to be a novel target for preventing CTL tolerance and enhancing immune responses to cancer by modulating the immunosuppressive activity of TADCs found in the tumor microenvironment.

Expression of costimulatory TNFR2 induces resistance of CD4+FoxP3- conventional T cells to suppression by CD4+FoxP3+ regulatory T cells.

Our previous study showed that TNFR2 is preferentially expressed by CD4(+)FoxP3(+) regulatory T cells (Tregs), and expression of this receptor identified maximally suppressive Tregs. TNFR2 is also expressed by a small fraction of CD4(+)FoxP3(-) conventional T cells (Tconvs) in normal mice, and its expression is upregulated by T cell activation. This raises questions about the role of TNFR2 signaling in the function of Tconv cells. In this study, by using FoxP3/gfp knock-in mice, we showed that TNFR2 signaling did not induce FoxP3(-) CD4 cells to become suppressive. Ki-67, a marker of proliferation, was concomitantly expressed with TNFR2 by CD4 cells, independent of forkhead box P3 expression, in normal mice and Lewis lung carcinoma-bearing mice. TNFR2 is associated with greater suppressive functions when expressed by Tregs and is associated with greater resistance to suppression when expressed by Tconv cells. In mice bearing 4T1 breast tumor or Lewis lung carcinoma, intratumoral Tconv cells expressing elevated levels of TNFR2 acquired the capacity to resist suppression by lymph node-derived Tregs. However, they remained susceptible to inhibition by more suppressive tumor-infiltrating Tregs, which expressed higher levels of TNFR2. Our data indicate that TNFR2 also costimulates Tconv cells. However, intratumoral Tregs expressing more TNFR2 are able to overcome the greater resistance to suppression of intratumoral Tconv cells, resulting in a dominant immunosuppressive tumor environment.

Cutting Edge: Tumor-specific CD8+ T cells infiltrating prostatic tumors are induced to become suppressor cells.

We previously reported that naive, tumor-specific CD8(+) (TcR-I) T cells transferred into prostate tumor-bearing mice traffic to the prostate where they become tolerized. We now report that TcR-I cells suppress the proliferation of naive T cells. This suppression is mediated at least in part by secreted factors, and the suppressive activity can be blocked by Abs directed against TGF-beta. We further report that TcR-I cells must infiltrate the prostate to acquire suppressive activity. Delivery of tumor-specific CD4(+) T cells prevents the conversion of TcR-I cells into suppressor cells. Taken together, our findings may have critical implications for sustaining T cell responsiveness during immunotherapy, as the development of suppressor cells in the tumor microenvironment may eliminate the potency of T cells primed in the periphery or delivered during adoptive immunotherapy.

Immunity to murine prostatic tumors: continuous provision of T-cell help prevents CD8 T-cell tolerance and activates tumor-infiltrating dendritic cells.

We reported previously that tumor-specific CD8(+) T cells (TcR-I) become tolerant in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. In this study, we show that CD4(+) TcR transgenic (TcR-II) T cells transferred into TRAMP mice became activated in lymph nodes, trafficked to the prostate, and initially functioned as T(H)1 cells. Although a single cotransfer of TcR-II cells delayed TcR-I cell tolerization, repeated transfer of TcR-II cells was required to prevent TcR-I cell tolerization and significantly slowed progression of TRAMP prostate tumors. After transfer of TcR-II cells, dendritic cells within the tumor expressed higher levels of costimulatory molecules and displayed an enhanced ability to stimulate proliferation of naive T cells. Blockade of CD40-CD40L interactions during TcR-II transfer resulted in a profound reduction in dendritic cell stimulatory capacity and a partial loss of TcR-I effector functions and tumor immunity. These data show that sustained provision of activated tumor-specific CD4(+) T cells alters the immunosuppressive tumor microenvironment, ultimately leading to the control of tumor growth. These findings will assist in the design of more effective immunotherapeutic approaches for cancer.

Immunologic and therapeutic synergy of IL-27 and IL-2: enhancement of T cell sensitization, tumor-specific CTL reactivity and complete regression of disseminated neuroblastoma metastases in the liver and bone marrow.

IL-27 exerts antitumor activity in murine orthotopic neuroblastoma, but only partial antitumor effect in disseminated disease. This study demonstrates that combined treatment with IL-2 and IL-27 induces potent antitumor activity in disseminated neuroblastoma metastasis. Complete durable tumor regression was achieved in 90% of mice bearing metastatic TBJ-IL-27 tumors treated with IL-2 compared with only 40% of mice bearing TBJ-IL-27 tumors alone and 0% of mice bearing TBJ-FLAG tumors with or without IL-2 treatment. Comparable antitumor effects were achieved by IL-27 protein produced upon hydrodynamic IL-27 plasmid DNA delivery when combined with IL-2. Although delivery of IL-27 alone, or in combination with IL-2, mediated pronounced regression of neuroblastoma metastases in the liver, combined delivery of IL-27 and IL-2 was far more effective than IL-27 alone against bone marrow metastases. Combined exposure to IL-27 produced by tumor and IL-2 synergistically enhances the generation of tumor-specific CTL reactivity. Potentiation of CTL reactivity by IL-27 occurs via mechanisms that appear to be engaged during both the initial sensitization and effector phase. Potent immunologic memory responses are generated in mice cured of their disseminated disease by combined delivery of IL-27 and IL-2, and depletion of CD8(+) ablates the antitumor efficacy of this combination. Moreover, IL-27 delivery can inhibit the expansion of CD4(+)CD25(+)Foxp3(+) regulatory and IL-17-expressing CD4(+) cells that are otherwise observed among tumor-infiltrating lymphocytes from mice treated with IL-2. These studies demonstrate that IL-27 and IL-2 synergistically induce complete tumor regression and long-term survival in mice bearing widely metastatic neuroblastoma tumors.

Melanoma progression despite infiltration by in vivo-primed TRP-2-specific T cells.

Many antigens recognized by tumor-reactive cytotoxic CD8+ T cells are self-antigens. Tyrosinase-related protein 2 (TRP-2) is a melanogenic enzyme expressed by both melanocytes and melanomas that is reported to be a candidate melanoma rejection antigen. To study the role of self-reactive CD8+ T cells in tumor immunity and autoimmunity, we generated mice that bear a T-cell receptor transgene (TCR Tg) specific for the TRP-2(180-188) epitope. TRP-2 TCR Tg mice did not spontaneously develop depigmentation despite systemic expression of TRP-2 in the skin. Peripheral T cells from these TCR Tg mice exhibited a naive phenotype and proliferated in response to TRP-2 in vitro. In addition, transfer of in vitro-activated Tg T cells reduced B16 pulmonary tumor burden, but not subcutaneous tumors. We next sought to determine the in vivo responses of the Tg T cells to endogenous and tumor-derived TRP-2. Adoptive transfer of naive TCR Tg T cells into wild-type C57BL/6 mice, in combination with a TRP-2-pulsed dendritic cell vaccine, induced proliferation of the Tg T cells and resulted in migration of the Tg T cells into a subcutaneous B16 melanoma tumor. Although these tumor-infiltrating Tg T cells remained reactive against TRP-2, they did not reduce growth of the primary subcutaneous tumor; similarly, these in vivo-primed effector cells had no significant effect on the growth of pulmonary nodules. These data demonstrate that despite in vivo priming, tumor-infiltrating T cells may fail to reduce tumor burden. Determining the basis for the inability of the tumor microenvironment to sustain effective antitumor responses will be critical for designing newer, more potent antitumor immunotherapies.

The influence of IL-2 family cytokines on activation and function of naturally occurring regulatory T cells.

IL-2 is essential for CD4+CD25+forkhead box P3+ (FoxP3+) naturally occurring regulatory T cell (Treg) homeostasis and activation. Binding of IL-2 to its receptor leads to phosphorylation of STAT5, and binding of phosphorylated STAT5 to the foxp3 promoter increases foxp3 transcription, resulting in elevated levels of FoxP3 protein in Tregs. Transcriptional regulation by the elevated levels of FoxP3 is thought to be essential for the strong suppressor function seen in activated Tregs. IL-2 belongs to a family cytokines, which all depend on the common gamma-receptor chain (gammac). Given the well-documented effects of IL-2 on Treg function, the effect of other IL-2 family cytokines (IL-7, -15, and -21) on Tregs was examined. We observed that IL-7 and IL-15 induce STAT5 phosphorylation and up-regulation of FoxP3 in Tregs. STAT5 activation correlated with enhanced viability. However, only in the presence of IL-2 did Tregs acquire potent suppressor function. This finding is surprising, as IL-15 as well as IL-2 use the same IL-2R betac and gammac for signaling. In contrast, IL-21 activated STAT3 but did not activate STAT5 and had no effect on Treg viability, activation, or function. We therefore conclude that phosphorylation of STAT5, mediated through the IL-2Rgamma, promotes Treg survival in a resting and activated state. However, activation of STAT5 alone in conjunction with TCR signaling is not sufficient for the induction of potent suppressor function in Tregs, as IL-7 and IL-15 are not capable of inducing potent Treg suppressor function.