Abstract Regulatory T cells (Treg) that express the transcription factor Foxp3 are enriched within a broad range of murine and human solid tumors. The ontogeny of these Foxp3 Tregs - selective accumulation or proliferation of natural thymus-derived Treg (nTreg) or induced Treg (iTreg) converted in the periphery from naïve T cells - is not known. We used several strains of mice in which Foxp3 and EGFP are coordinately expressed to address this issue. We confirmed that Foxp3-positive CD4 T cells are enriched among tumor-infiltrating lymphocytes (TIL) and splenocytes (SPL) in B16 murine melanoma-bearing C57BL/6 Foxp3EGFP mice. OT-II Foxp3EGFP mice are essentially devoid of nTreg, having transgenic CD4 T cells that recognize a class II-restricted epitope derived from ovalbumin; Foxp3 expression could not be detected in TIL or SPL in these mice when implanted with ovalbumin-transfected B16 tumor (B16-OVA). Likewise, TIL isolated from B16 tumors implanted in Pmel-1 Foxp3EGFP mice, whose CD8 T cells recognize a class I-restricted gp100 epitope, were not induced to express Foxp3. All of these T cell populations - wild-type CD4, pmel CD8 and OTII CD4 - could be induced in vitro to express Foxp3 by engagement of their T cell receptor (TCR) and exposure to transforming growth factor β (TGFβ). B16 melanoma produces TGFβ and both pmel CD8 and OTII CD4 express TCR that should be engaged within B16 and B16-OVA respectively. Thus, CD8 and CD4 transgenic T cells in these animal models failed to undergo peripheral induction of Foxp3 in a tumor microenvironment.

Abstract Tumor antigen-reactive T cells must enter into an immunosuppressive tumor microenvironment, continue to produce cytokine and deliver apoptotic death signals to affect tumor regression. Many tumors produce transforming growth factor beta (TGFβ), which inhibits T cell activation, proliferation and cytotoxicity. In a murine model of adoptive cell therapy, we demonstrate that transgenic Pmel-1 CD8 T cells, rendered insensitive to TGFβ by transduction with a TGFβ dominant negative receptor II (DN), were more effective in mediating regression of established B16 melanoma. Smaller numbers of DN Pmel-1 T cells effectively mediated tumor regression and retained the ability to produce interferon-γ in the tumor microenvironment. These results support efforts to incorporate this DN receptor in clinical trials of adoptive cell therapy for cancer.

Background

Various proinflammatory cytokines can be detected within the melanoma tumor microenvironment. Interleukin 32 (IL32) is produced by T cells, NK cells and monocytes/macrophages, but also by a subset of melanoma cells. We sought to better understand the biology of IL32 in human melanoma.

Methods

We analyzed RNA sequencing data from 53 in-house established human melanoma cell lines and 479 melanoma tumors from The Cancer Genome Atlas dataset. We evaluated global gene expression patterns associated with IL32 expression. We also evaluated the impact of proinflammatory molecules TNFα and IFNγ on IL32 expression and dedifferentiation in melanoma cell lines in vitro. In order to study the transcriptional regulation of IL32 in these cell lines, we cloned up to 10.5 kb of the 5' upstream region of the human IL32 gene into a luciferase reporter vector.

Results

A significant proportion of established human melanoma cell lines express IL32, with its expression being highly correlated with a dedifferentiation genetic signature (high AXL/low MITF). Non IL32-expressing differentiated melanoma cell lines exposed to TNFα or IFNγ can be induced to express the three predominant isoforms (α, β, γ) of IL32. Cis-acting elements within this 5' upstream region of the human IL32 gene appear to govern both induced and constitutive gene expression. In the tumor microenvironment, IL32 expression is highly correlated with genes related to T cell infiltration, and also positively correlates with high AXL/low MITF dedifferentiated gene signature.

Conclusions

Expression of IL32 in human melanoma can be induced by TNFα or IFNγ and correlates with a treatment-resistant dedifferentiated genetic signature. Constitutive and induced expression are regulated, in part, by cis-acting sequences within the 5' upstream region.

A promising arsenal of targeted and immunotherapy treatments for metastatic melanoma has emerged over the last decade. With these therapies, we now face new mechanisms of tumor-acquired resistance. We report here a patient whose metastatic melanoma underwent dedifferentiation as a resistance mechanism to adoptive T-cell transfer therapy (ACT) to the MART1 antigen, a phenomenon that had been observed only in mouse studies to date. After an initial period of tumor regression, the patient presented in relapse with tumors lacking melanocytic antigens (MART1, gp100) and expressing an inflammation-induced neural crest marker (NGFR). We demonstrate using human melanoma cell lines that this resistance phenotype can be induced in vitro by treatment with MART1 T cell receptor-expressing T cells or with TNFα, and that the phenotype is reversible with withdrawal of inflammatory stimuli. This supports the hypothesis that acquired resistance to cancer immunotherapy can be mediated by inflammation-induced cancer dedifferentiation.Significance: We report a patient whose metastatic melanoma underwent inflammation-induced dedifferentiation as a resistance mechanism to ACT to the MART1 antigen. Our results suggest that future melanoma ACT protocols may benefit from the simultaneous targeting of multiple tumor antigens, modulating the inflammatory response, and inhibition of inflammatory dedifferentiation-inducing signals. Cancer Discov; 8(8); 935-43. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 899.

Background

CTLA4-blocking antibodies induce tumor regression in a subset of patients with melanoma. Analysis of immune parameters in peripheral blood may help define how responses are mediated.

Methods

Peripheral blood from HLA-A*0201-positive patients with advanced melanoma receiving tremelimumab (formerly CP-675,206) at 10 mg/kg monthly was repeatedly sampled during the first 4 cycles. Samples were analyzed by 1) tetramer and ELISPOT assays for reactivity to CMV, EBV, MART1, gp100, and tyrosinase; 2) activation HLA-DR and memory CD45RO markers on CD4+/CD8+ cells; and 3) real-time quantitative PCR of mRNA for FoxP3 transcription factor, preferentially expressed by T regulatory cells. The primary endpoint was difference in MART1-specific T cells by tetramer assay. Immunological data were explored for significant trends using clustering analysis.

Results

Three of 12 patients eligible for immune monitoring had tumor regression lasting > 2 years without relapse. There was no significant change in percent of MART1-specific T cells by tetramer assay. Additionally, there was no generalized trend toward postdosing changes in other antigen-specific CD8+ cell populations, FoxP3 transcripts, or overall changes in surface expression of T-cell activation or memory markers. Unsupervised hierarchical clustering based on immune monitoring data segregated patients randomly. However, clustering according to T-cell activation or memory markers separated patients with clinical response and most patients with inflammatory toxicity into a common subgroup.

Conclusion

Administration of CTLA4-blocking antibody tremelimumab to patients with advanced melanoma results in a subset of patients with long-lived tumor responses. T-cell activation and memory markers served as the only readout of the pharmacodynamic effects of this antibody in peripheral blood.

Clinical trial registration number

NCT00086489.

Adoptive cell therapy (ACT) consisting of genetically engineered T cells expressing tumor antigen-specific T-cell receptors displays robust initial antitumor activity, followed by loss of T-cell activity/persistence and frequent disease relapse. We characterized baseline and longitudinal T-cell phenotype variations resulting from different manufacturing and administration protocols in patients who received ACT. Patients with melanoma who enrolled in the F5-MART-1 clinical trial (NCT00910650) received infusions of MART-1 T-cell receptors transgenic T cells with MART-1 peptide-pulsed dendritic cell vaccination. Patients were divided into cohorts based on several manufacturing changes in the generation and administration of the transgenic T cells: decreasing ex vivo stimulation/expansion time, increased cell dose, and receiving fresh instead of cryopreserved cells. T-cell phenotypes were analyzed by flow cytometry at baseline and longitudinally in peripheral blood. Transgenic T cells with shorter ex vivo culture/expansion periods displayed significantly increased expression of markers associated with less differentiated naive/memory populations, as well as significantly decreased expression of the inhibitory receptor programmed death 1 (PD1). Patients receiving fresh infusions of transgenic cells demonstrated expansion of central memory T cells and delayed acquisition of PD1 expression compared with patients who received cryopreserved products. Freshly infused transgenic T cells showed persistence and expansion of naive and memory T-cell populations and delayed acquisition of PD1 expression, which correlated with this cohort's superior persistence of transgenic cells and response to dendritic cell vaccines. These results may be useful in designing future ACT protocols.

Imiquimod is a synthetic Toll-like receptor 7 (TLR7) agonist approved for the topical treatment of actinic keratoses, superficial basal cell carcinoma, and genital warts. Imiquimod leads to an 80-100% cure rate of lentigo maligna; however, studies of invasive melanoma are lacking. We conducted a pilot study to characterize the local, regional, and systemic immune responses induced by imiquimod in patients with high-risk melanoma. After treatment of the primary melanoma biopsy site with placebo or imiquimod cream, we measured immune responses in the treated skin, sentinel lymph nodes (SLNs), and peripheral blood. Treatment of primary melanomas with 5% imiquimod cream was associated with an increase in both CD4+ and CD8+ T cells in the skin, and CD4+ T cells in the SLN. Most of the CD8+ T cells in the skin were CD25 negative. We could not detect any increases in CD8+ T cells specifically recognizing HLA-A(*)0201-restricted melanoma epitopes in the peripheral blood. The findings from this small pilot study demonstrate that topical imiquimod treatment results in enhanced local and regional T-cell numbers in both the skin and SLN. Further research into TLR7 immunomodulating pathways as a basis for effective immunotherapy against melanoma in conjunction with surgery is warranted.

Purpose

It has been demonstrated that large numbers of tumor-specific T cells for adoptive cell transfer (ACT) can be manufactured by retroviral genetic engineering of autologous peripheral blood lymphocytes and expanding them over several weeks. In mouse models, this therapy is optimized when administered with dendritic cell (DC) vaccination. We developed a short 1-week manufacture protocol to determine the feasibility, safety, and antitumor efficacy of this double cell therapy.

Experimental design

A clinical trial (NCT00910650) adoptively transferring MART-1 T-cell receptor (TCR) transgenic lymphocytes together with MART-1 peptide-pulsed DC vaccination in HLA-A2.1 patients with metastatic melanoma. Autologous TCR transgenic cells were manufactured in 6 to 7 days using retroviral vector gene transfer, and reinfused with (n = 10) or without (n = 3) prior cryopreservation.

Results

A total of 14 patients with metastatic melanoma were enrolled and 9 of 13 treated patients (69%) showed evidence of tumor regression. Peripheral blood reconstitution with MART-1-specific T cells peaked within 2 weeks of ACT, indicating rapid in vivo expansion. Administration of freshly manufactured TCR transgenic T cells resulted in a higher persistence of MART-1-specific T cells in the blood as compared with cryopreserved. Evidence that DC vaccination could cause further in vivo expansion was only observed with ACT using noncryopreserved T cells.

Conclusion

Double cell therapy with ACT of TCR-engineered T cells with a very short ex vivo manipulation and DC vaccines is feasible and results in antitumor activity, but improvements are needed to maintain tumor responses.

Purpose

It has been demonstrated that large numbers of tumor-specific T cells for adoptive cell transfer (ACT) can be manufactured by retroviral genetic engineering of autologous peripheral blood lymphocytes and expanding them over several weeks. In mouse models, this therapy is optimized when administered with dendritic cell (DC) vaccination. We developed a short 1-week manufacture protocol to determine the feasibility, safety, and antitumor efficacy of this double cell therapy.

Experimental design

A clinical trial (NCT00910650) adoptively transferring MART-1 T-cell receptor (TCR) transgenic lymphocytes together with MART-1 peptide-pulsed DC vaccination in HLA-A2.1 patients with metastatic melanoma. Autologous TCR transgenic cells were manufactured in 6 to 7 days using retroviral vector gene transfer, and reinfused with (n = 10) or without (n = 3) prior cryopreservation.

Results

A total of 14 patients with metastatic melanoma were enrolled and 9 of 13 treated patients (69%) showed evidence of tumor regression. Peripheral blood reconstitution with MART-1-specific T cells peaked within 2 weeks of ACT, indicating rapid in vivo expansion. Administration of freshly manufactured TCR transgenic T cells resulted in a higher persistence of MART-1-specific T cells in the blood as compared with cryopreserved. Evidence that DC vaccination could cause further in vivo expansion was only observed with ACT using noncryopreserved T cells.

Conclusion

Double cell therapy with ACT of TCR-engineered T cells with a very short ex vivo manipulation and DC vaccines is feasible and results in antitumor activity, but improvements are needed to maintain tumor responses.