IFNγ producing CD8+ T cells modified to resist major immune checkpoints induce regression of MHC class I-deficient melanomas.

Tumors with reduced expression of MHC class I (MHC-I) molecules may be unrecognized by tumor antigen-specific CD8+ T cells and thus constitute a challenge for cancer immunotherapy. Here we monitored development of autochthonous melanomas in TiRP mice that develop tumors expressing a known tumor antigen as well as a red fluorescent protein (RFP) reporter knock in gene. The latter permits non-invasive monitoring of tumor growth by biofluorescence. One developing melanoma was deficient in cell surface expression of MHC-I, but MHC-I expression could be rescued by exposure of these cells to IFNγ. We show that CD8+ T cells specific for tumor antigen/MHC-I were efficient at inducing regression of the MHC-I-deficient melanoma, provided that the T cells were endowed with properties permitting their migration into the tumor and their efficient production of IFNγ. This was the case for CD8+ T cells transfected to express an active form of STAT5 (STAT5CA). The amount of IFNγ produced ex vivo from T cells present in tumors after adoptive transfer of the CD8+ T cells was correlated with an increase in surface expression of MHC-I molecules by the tumor cells. We also show that these CD8+ T cells expressed PD-1 and upregulated its ligand PDL-1 on melanoma cells within the tumor. Despite upregulation of this immunosuppressive pathway, efficient IFNγ production in the melanoma microenvironment was found associated with resistance of STAT5CA-expressing CD8+ T cells to inhibition both by PD-1/PDL-1 engagement and by TGFß1, two main immune regulatory mechanisms hampering the efficiency of immunotherapy in patients.

Control of CD8 T cell proliferation and terminal differentiation by active STAT5 and CDKN2A/CDKN2B.

CD8 T cells used in adoptive immunotherapy may be manipulated to optimize their effector functions, tissue-migratory properties and long-term replicative potential. We reported that antigen-stimulated CD8 T cells transduced to express an active form of the transcription factor signal transducer and activator of transcription 5 (STAT5CA) maintained these properties upon adoptive transfer. We now report on the requirements of STAT5CA-expressing CD8 T cells for cell survival and proliferation in vivo. We show that STAT5CA expression allows for greater expansion of T cells in vivo, while preserving dependency on T-cell-receptor-mediated tonic stimulation for their in vivo maintenance and return to a quiescent stage. STAT5CA expression promotes the formation of a large pool of effector memory T cells that respond upon re-exposure to antigen and present an increased sensitivity to γc receptor cytokine engagement for STAT5 phosphorylation. In addition, STAT5CA expression prolongs the survival of what would otherwise be short-lived terminally differentiated KLRG1-positive effector cells with up-regulated expression of the senescence-associated p16(INK) (4A) transcripts. However, development of a KLRG1-positive CD8 T cell population was independent of either p16(INK) (4A) or p19(ARF) expression (as shown using T cells from CDKN2A(-/-) mice) but was associated with expression of transcripts encoding p15(INK) (4B) , another protein involved in senescence induction. We conclude that T-cell-receptor- and cytokine-dependent regulation of effector T cell homeostasis, as well as mechanisms leading to senescent features of a population of CD8 T cells are maintained in STAT5CA-expressing CD8 T cells, even for cells that are genetically deficient in expression of the tumour suppressors p16(INK) (4A) and p19(ARF) .

Immunosuppression in inflammatory melanoma: can it be resisted by adoptively transferred T cells?

Discovery of tumor antigen (TA) recognized by autologous T cells (TCs) in patients with melanoma has led to clinical protocols using either vaccination or adoptive transfer of TA-specific TCs. However, efficacy of these treatments has been hampered by inhibitory effects exerted on tumor-infiltrating TCs by tumor-intrinsic mediators or by recruitment of immunosuppressive cells. A mouse model of autochthonous melanoma recapitulates some aspects of inflammatory melanoma development in patients. These include a systemic Th2-/Th17-oriented chronic inflammation, recruitment of immunosuppressive myeloid cells and acquisition by tumor-infiltrating TCs of an 'exhausted' phenotype characterized by expression of multiple inhibitory receptors including programmed death-1, also expressed on patients' melanoma-infiltrating TCs. Rather than using extracellular blocking reagents to inhibitory surface molecules on TCs, we sought to dampen negative signaling exerted on them. Adoptively transferred TCs presenting increased cytokine receptor signaling due to expression of an active Stat5 transcription factor were efficient at inducing melanoma regression in the preclinical melanoma model. These transferred TCs thrived and retained expression of effector molecules in the melanoma microenvironment, defining a protocol endowing TCs with the ability to resist melanoma-induced immunosuppression.

Epithelial-mesenchymal-transition-like and TGFß 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-Ras(G12V) 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 TGFß-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.

Disrupted lymph node and splenic stroma in mice with induced inflammatory melanomas is associated with impaired recruitment of T and dendritic cells.

Migration of dendritic cells (DC) from the tumor environment to the T cell cortex in tumor-draining lymph nodes (TDLN) is essential for priming naïve T lymphocytes (TL) to tumor antigen (Ag). We used a mouse model of induced melanoma in which similar oncogenic events generate two phenotypically distinct melanomas to study the influence of tumor-associated inflammation on secondary lymphoid organ (SLO) organization. One tumor promotes inflammatory cytokines, leading to mobilization of immature myeloid cells (iMC) to the tumor and SLO; the other does not. We report that inflammatory tumors induced alterations of the stromal cell network of SLO, profoundly altering the distribution of TL and the capacity of skin-derived DC and TL to migrate or home to TDLN. These defects, which did not require tumor invasion, correlated with loss of fibroblastic reticular cells in T cell zones and in impaired production of CCL21. Infiltrating iMC accumulated in the TDLN medulla and the splenic red pulp. We propose that impaired function of the stromal cell network during chronic inflammation induced by some tumors renders spleens non-receptive to TL and TDLN non-receptive to TL and migratory DC, while the entry of iMC into these perturbed SLO is enhanced. This could constitute a mechanism by which inflammatory tumors escape immune control. If our results apply to inflammatory tumors in general, the demonstration that SLO are poorly receptive to CCR7-dependent migration of skin-derived DC and naïve TL may constitute an obstacle for proposed vaccination or adoptive TL therapies of their hosts.

Tumor-initiated inflammation overrides protective adaptive immunity in an induced melanoma model in mice.

We studied the effect of the immune system on two differentially aggressive melanomas developing in mice on conditional deletion of the INK4A/ARF tumor suppressor gene, with concomitant expression of oncogene H-Ras(G12V) and a natural cancer-germline tumor antigen (TA). "Slow progressor" melanomas contained no activated T lymphocytes (TL). In contrast, "aggressive" melanomas were infiltrated by activated TLs lacking effector molecules and expressing high levels of PD-1, indicating an exhausted phenotype. Aggressive melanomas were also infiltrated by immature myeloid cells (IMC). Infiltration was associated with local inflammation and systemic Th2/Th17-oriented chronic inflammation that seemed to impair further activation of TLs, as tumor-specific T cells adoptively transferred into mice bearing aggressive melanomas were poorly activated and failed to infiltrate the melanoma. This immunosuppression also led to the incapacity of these mice to reject inoculated TA-positive tumors, in contrast to slow-progressing melanoma-bearing mice, which were responsive. To test the role of adaptive immunity in tumor progression, we induced melanomas in immunodeficient RagKO compound mice. These mice developed aggressive but not slow-progressing melanomas at a higher frequency and with a shorter latency than immunocompetent mice. Immunodeficient mice also developed abnormal inflammation and infiltration of IMCs in a manner similar to immunocompetent mice, indicating that this phenotype was not dependent on adaptive immunity. Therefore, tumor-intrinsic factors distinguishing the two melanoma types control the initiation of inflammation, which was independent of adaptive immunity. The latter delayed development of aggressive melanomas but was overridden by inflammation.