Grégory Verdeil

CD8 T cell help for innate antitumor immunity

Innate immunity is considered to initiate adaptive antitumor responses. We demonstrate that monoclonal CD8 T lymphocytes reactive to tumor Ag P1A on P815 mastocytoma cells provide essential “help” to NK cells for rejection of P1A-deficient tumors. RAG-deficient mice have normal NK cells but do not reject either tumor. Reconstitution of these mice with P1A-specific T cells conferred resistance to both P1A-expressing and -deficient tumor cells provided they were present at the same site. Elimination of Ag-negative tumor variants required both activated T and NK cells. Gene expression profiling of NK cells infiltrating P1A-positive tumors in mice with specific CD8 T cells demonstrated an activated effector phenotype. However, CD8 T cell help to NK cells appeared ineffective for P1A-negative variants separated from the P1A-positive tumor. Local tumor Ag-specific T cell-NK cell collaboration results in the elimination of tumor cells whether they express or not the T cell tumor Ag epitope, thus containing the emergence of tumor escape variants before metastasis.

Inhibitory Receptors Beyond T Cell Exhaustion

Inhibitory receptors (iRs) are frequently associated with “T cell exhaustion”. However, the expression of iRs is also dependent on T cell differentiation and activation. Therapeutic blockade of various iRs, also referred to as “checkpoint blockade”, is showing unprecedented results in the treatment of cancer patients. Consequently, the clinical potential in this field is broad, calling for increased research efforts and rapid refinements in the understanding of iR function. In this review, we provide an overview on the significance of iR expression for the interpretation of T cell functionality. We summarize how iRs have been strongly associated with “T cell exhaustion” and illustrate the parallel evidence on the importance of T cell differentiation and activation for the expression of iRs. The differentiation subsets of CD8 T cells (naïve, effector, and memory cells) show broad and inherent differences in iR expression, while activation leads to strong upregulation of iRs. Therefore, changes in iR expression during an immune response are often concomitant with T cell differentiation and activation. Sustained expression of iRs in chronic infection and in the tumor microenvironment likely reflects a specialized T cell differentiation. In these situations of prolonged antigen exposure and chronic inflammation, T cells are “downtuned” in order to limit tissue damage. Furthermore, we review the novel “checkpoint blockade” treatments and the potential of iRs as biomarkers. Finally, we provide recommendations for the immune monitoring of patients to interpret iR expression data combined with parameters of activation and differentiation of T cells.

The tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20) imposes a brake on antitumor activity of CD8 T cells

Significance Mechanisms controlling immune reactivity prevent excessive inflammation and autoimmunity, but generally dampen antitumor activity. The tumor necrosis factor alpha-induced protein 3 gene encoding the A20 protein, a key molecule controlling NF-κB activation, has been linked to the development of multiple inflammatory pathologies in humans, some of which are recapitulated in mice with selective deletion of A20 in myeloid, dendritic, or B cells. Here, mice with selective deletion of A20 in mature conventional T cells presented no detectable pathology. CD8 T cells from these mice showed increased antigen sensitivity with enhanced production of IL-2 and IFNγ. Importantly, A20-deleted CD8 T cells possessed heightened antitumor activity in vivo. Targeting this gene in adoptively transferred CD8 T cells could represent a promising mechanism to achieve tumor rejection. The transcription factor NF-κB is central to inflammatory signaling and activation of innate and adaptive immune responses. Activation of the NF-κB pathway is tightly controlled by several negative feedback mechanisms, including A20, an ubiquitin-modifying enzyme encoded by the tnfaip3 gene. Mice with selective deletion of A20 in myeloid, dendritic, or B cells recapitulate some human inflammatory pathology. As we observed high expression of A20 transcripts in dysfunctional CD8 T cells in an autochthonous melanoma, we analyzed the role of A20 in regulation of CD8 T-cell functions, using mice in which A20 was selectively deleted in mature conventional T cells. These mice developed lymphadenopathy and some organ infiltration by T cells but no splenomegaly and no detectable pathology. A20-deleted CD8 T cells had increased sensitivity to antigen stimulation with production of large amounts of IL-2 and IFNγ, correlated with sustained nuclear expression of NF-κB components reticuloendotheliosis oncogene c-Rel and p65. Overexpression of A20 by retroviral transduction of CD8 T cells dampened their intratumor accumulation and antitumor activity. In contrast, relief from the A20 brake in NF-κB activation in adoptively transferred antitumor CD8 T cells led to improved control of melanoma growth. Tumor-infiltrating A20-deleted CD8 T cells had enhanced production of IFNγ and TNFα and reduced expression of the inhibitory receptor programmed cell death 1. As manipulation of A20 expression in CD8 T cells did not result in pathologic manifestations in the mice, we propose it as a candidate to be targeted to increase antitumor efficiency of adoptive T-cell immunotherapy.

Molecular profiling of CD8 T cells in autochthonous melanoma identifies Maf as driver of exhaustion.

T cells infiltrating neoplasms express surface molecules typical of chronically virus‐stimulated T cells, often termed “exhausted” T cells. We compared the transcriptome of “exhausted” CD8 T cells infiltrating autochthonous melanomas to those of naïve and acutely stimulated CD8 T cells. Despite strong similarities between transcriptional signatures of tumor‐ and virus‐induced exhausted CD8 T cells, notable differences appeared. Among transcriptional regulators, Nr4a2 and Maf were highly overexpressed in tumor‐exhausted T cells and significantly upregulated in CD8 T cells from human melanoma metastases. Transduction of murine tumor‐specific CD8 T cells to express Maf partially reproduced the transcriptional program associated with tumor‐induced exhaustion. Upon adoptive transfer, the transduced cells showed normal homeostasis but failed to accumulate in tumor‐bearing hosts and developed defective anti‐tumor effector responses. We further identified TGFβ and IL‐6 as main inducers of Maf expression in CD8 T cells and showed that Maf‐deleted tumor‐specific CD8 T cells were much more potent to restrain tumor growth in vivo. Therefore, the melanoma microenvironment contributes to skewing of CD8 T cell differentiation programs, in part by TGFβ/IL‐6‐mediated induction of Maf.

Active STAT5 Regulates T-bet and Eomesodermin Expression in CD8 T Cells and Imprints a T-bet–Dependent Tc1 Program with Repressed IL-6/TGF-β1 Signaling

In adoptive therapy, CD8 T cells expressing active STAT5 (STAT5CA) transcription factors were found to be superior to unmanipulated counterparts in long-term persistence, capacity to infiltrate autochthonous mouse melanomas, thrive in their microenvironment, and induce their regression. However, the molecular mechanisms sustaining these properties were undefined. In this study, we report that STAT5CA induced sustained expression of genes controlling tissue homing, cytolytic granule composition, type 1 CD8 cytotoxic T cell–associated effector molecules granzyme B+, IFN-γ+, TNF-α+, and CCL3+, but not IL-2, and transcription factors T-bet and eomesodermin (Eomes). Chromatin immunoprecipitation sequencing analyses identified the genes possessing regulatory regions to which STAT5 bound in long-term in vivo maintained STAT5CA-expressing CD8 T cells. This analysis identified 34% of the genes differentially expressed between STAT5CA-expressing and nonexpressing effector T cells as direct STAT5CA target genes, including those encoding T-bet, Eomes, and granzyme B. Additionally, genes encoding the IL-6R and TGFbRII subunits were stably repressed, resulting in dampened IL-17–producing CD8 T cell polarization in response to IL-6 and TGF-β1. The absence of T-bet did not affect STAT5CA-driven accumulation of the T cells in tissue or their granzyme B expression but restored IL-2 secretion and IL-6R and TGFbRII expression and signaling, as illustrated by IL-17 induction. Therefore, concerted STAT5/T-bet/Eomes regulation controls homing, long-term maintenance, recall responses, and resistance to polarization towards IL-17–producing CD8 T cells while maintaining expression of an efficient type 1 CD8 cytotoxic T cell program (granzyme B+, IFN-γ+).

Epithelial-Mesenchymal-Transition-Like and TGFβ Pathways Associated with Autochthonous Inflammatory Melanoma Development in Mice

We compared gene expression signatures of aggressive amelanotic (Amela) melanomas with those of slowly growing pigmented melanomas (Mela), identifying pathways potentially responsible for the aggressive Amela phenotype. Both tumors develop in mice upon conditional deletion in melanocytes of Ink4a/Arf tumor suppressor genes with concomitant expression of oncogene H-RasG12V and a known tumor antigen. We previously showed that only the aggressive Amela tumors were highly infiltrated by leukocytes concomitant with local and systemic inflammation. We report that Amela tumors present a pattern of de-differentiation with reduced expression of genes involved in pigmentation. This correlates with reduced and enhanced expression, respectively, of microphthalmia-associated (Mitf) and Pou3f2/Brn-2 transcription factors. The reduced expression of Mitf-controlled melanocyte differentiation antigens also observed in some human cutaneous melanoma has important implications for immunotherapy protocols that generally target such antigens. Induced Amela tumors also express Epithelial-Mesenchymal-Transition (EMT)-like and TGFβ-pathway signatures. These are correlated with constitutive Smad3 signaling in Amela tumors and melanoma cell lines. Signatures of infiltrating leukocytes and some chemokines such as chemotactic cytokine ligand 2 (Ccl2) that contribute to leukocyte recruitment further characterize Amela tumors. Inhibition of the mitogen-activated protein kinase (MAPK) activation pathway in Amela tumor lines leads to reduced expression of EMT hallmark genes and inhibits both proinflammatory cytokine Ccl2 gene expression and Ccl2 production by the melanoma cells. These results indicate a link between EMT-like processes and alterations of immune functions, both being controlled by the MAPK pathway. They further suggest that targeting the MAPK pathway within tumor cells will impact tumor-intrinsic oncogenic properties as well as the nature of the tumor microenvironment.

Activated STAT5 promotes long-lived cytotoxic CD8+ T cells that induce regression of autochthonous melanoma.

Immunotherapy based on adoptive transfer of tumor antigen-specific CD8(+) T cell (TC) is generally limited by poor in vivo expansion and tumor infiltration. In this study, we report that activated STAT5 transcription factors (STAT5CA) confer high efficiency on CD8(+) effector T cells (eTC) for host colonization after adoptive transfer. Engineered expression of STAT5CA in antigen-experienced TCs with poor replicative potential was also sufficient to convert them into long-lived antigen-responsive eTCs. In transplanted mastocytoma- or melanoma-bearing hosts, STAT5CA greatly enhanced the ability of eTCs to accumulate in tumors, become activated by tumor antigens, and to express the cytolytic factor granzyme B. Taken together, these properties contributed to an increase in tumor regression by STAT5CA-transduced, as compared with untransduced, TCs including when the latter control cells were combined with infusion of interleukin (IL)-2/anti-IL-2 complexes. In tumors arising in the autochthonous TiRP transgenic model of melanoma associated with systemic chronic inflammation, endogenous CD8(+) TCs were nonfunctional. In this setting, adoptive transfer of STAT5CA-transduced TCs produced superior antitumor effects compared with nontransduced TCs. Our findings imply that STAT5CA expression can render TCs resistant to the immunosuppressive environment of melanoma tumors, enhancing their ability to home to tumors and to maintain high granzyme B expression, as well as their capacity to stimulate granzyme B expression in endogenous TCs.

Very Late Antigen-1 Marks Functional Tumor-Resident CD8 T Cells and Correlates with Survival of Melanoma Patients

A major limiting factor in the success of immunotherapy is tumor infiltration by CD8+ T cells, a process that remains poorly understood. In the present study, we characterized homing receptors expressed by human melanoma-specific CD8+ T cells. Our data reveal that P-selectin binding and expression of the retention integrin, very late antigen (VLA)-1, by vaccine-induced T cells correlate with longer patient survival. Furthermore, we demonstrate that CD8+VLA-1+ tumor-infiltrating lymphocytes (TILs) are highly enriched in melanoma metastases in diverse tissues. VLA-1-expressing TIL frequently co-express CD69 and CD103, indicating tissue-resident memory T cells (TRM) differentiation. We employed a mouse model of melanoma to further characterize VLA-1-expressing TIL. Our data show that VLA-1+ TRM develop in murine tumors within 2 weeks, where they exhibit increased activation status, as well as superior effector functions. In addition, in vivo blockade of either VLA-1 or CD103 significantly impaired control of subcutaneous tumors. Together, our data indicate that VLA-1+ TRM develop in tumors and play an important role in tumor immunity, presenting novel targets for the optimization of cancer immunotherapy.

29th Annual meeting of the Society for Immunotherapy of Cancer (SITC)

The 29th annual meeting of the Society for Immunotherapy of Cancer (SITC) was held November 7-9, 2014 in National Harbor, MD and was organized by Dr. Arthur A. Hurwitz (National Cancer Institute), Dr. Kim A. Margolin (Stanford University), Dr. Daniel E. Speiser (Ludwig Center for Cancer Research, University of Lausanne) and Dr. Walter J. Urba (Earle A. Chiles Research Institute, Providence Cancer Center). This meeting included over 1,600 registered participants from 28 separate countries, making it the largest SITC meeting held to date. It highlighted significant worldwide progress in the development and application of cancer immunology to the practice of clinical oncology, including advances in diagnosis, prognosis and therapy, utilizing several immunological pathways and mechanisms for a variety of oncologic conditions. Presentations and posters demonstrated that many concepts that had been pursued preclinically in the past are now being translated into clinical practice, with clear benefits for patients.

Distinct patterns of cytolytic T-cell activation by different tumour cells revealed by Ca2+ signalling and granule mobilization

Cancer‐germline genes in both humans and mice have been shown to encode antigens susceptible to targeting by cytotoxic CD8 T effector cells (CTL). We analysed the ability of CTL to kill different tumour cell lines expressing the same cancer‐germline gene P1A (Trap1a). We previously demonstrated that CTL expressing a T‐cell receptor specific for the P1A35–43 peptide associated with H‐2Ld, although able to induce regression of P1A‐expressing P815 mastocytoma cells, were much less effective against P1A‐expressing melanoma cells. Here, we analysed parameters of the in vitro interaction between P1A‐specific CTL and mastocytoma or melanoma cells expressing similar levels of the P1A gene and of surface H‐2Ld. The mastocytoma cells were more sensitive to cytolysis than the melanoma cells in vitro. Analysis by video‐microscopy of early events required for target cell killing showed that similar patterns of increase in cytoplasmic Ca2+ concentration ([Ca2+]i) were induced by both types of P1A‐expressing tumour cells. However, the use of CTL expressing a fluorescent granzyme B (GZMB‐Tom) showed a delay in the migration of cytotoxic granules to the tumour interaction site, as well as a partially deficient GZMB‐Tom exocytosis in response to the melanoma cells. Among surface molecules possibly affecting tumour–CTL interactions, the mastocytoma cells were found to express intercellular adhesion molecule‐1, the ligand for LFA‐1, which was not detected on the melanoma cells.