Tania Rodriguez-Cruz

ONCOGENIC BRAF(V600E) PROMOTES STROMAL CELL-MEDIATED IMMUNOSUPPRESSION VIA INDUCTION OF INTERLEUKIN-1 IN MELANOMA

Purpose: In this study, we assessed the specific role of BRAF(V600E) signaling in modulating the expression of immune regulatory genes in melanoma, in addition to analyzing downstream induction of immune suppression by primary human melanoma tumor-associated fibroblasts (TAF). Experimental Design: Primary human melanocytes and melanoma cell lines were transduced to express WT or V600E forms of BRAF, followed by gene expression analysis. The BRAF(V600E) inhibitor vemurafenib was used to confirm targets in BRAF(V600E)-positive melanoma cell lines and in tumors from melanoma patients undergoing inhibitor treatment. TAF lines generated from melanoma patient biopsies were tested for their ability to inhibit the function of tumor antigen-specific T cells, before and following treatment with BRAF(V600E)-upregulated immune modulators. Transcriptional analysis of treated TAFs was conducted to identify potential mediators of T-cell suppression. Results: Expression of BRAF(V600E) induced transcription of interleukin 1 alpha (IL-1α) and IL-1β in melanocytes and melanoma cell lines. Further, vemurafenib reduced the expression of IL-1 protein in melanoma cell lines and most notably in human tumor biopsies from 11 of 12 melanoma patients undergoing inhibitor treatment. Treatment of melanoma-patient–derived TAFs with IL-1α/β significantly enhanced their ability to suppress the proliferation and function of melanoma-specific cytotoxic T cells, and this inhibition was partially attributable to upregulation by IL-1 of COX-2 and the PD-1 ligands PD-L1 and PD-L2 in TAFs. Conclusions: This study reveals a novel mechanism of immune suppression sensitive to BRAF(V600E) inhibition, and indicates that clinical blockade of IL-1 may benefit patients with BRAF wild-type tumors and potentially synergize with immunotherapeutic interventions. Clin Cancer Res; 18(19); 5329–40. ©2012 AACR.

BRAFV600E Co-opts a Conserved MHC Class I Internalization Pathway to Diminish Antigen Presentation and CD8+ T-cell Recognition of Melanoma.

Bradley, Chen, and colleagues show that BRAFV600E-induced internalization of MHC class I and its sequestration within endolysosomal compartments can be reversed by MAP kinase inhibitors, demonstrating a direct link between oncogenic activation of the MAPK pathway and MHC class I trafficking and localization. Oncogene activation in tumor cells induces broad and complex cellular changes that contribute significantly to disease initiation and progression. In melanoma, oncogenic BRAFV600E has been shown to drive the transcription of a specific gene signature that can promote multiple mechanisms of immune suppression within the tumor microenvironment. We show here that BRAFV600E also induces rapid internalization of MHC class I (MHC-I) from the melanoma cell surface and its intracellular sequestration within endolysosomal compartments. Importantly, MAPK inhibitor treatment quickly restored MHC-I surface expression in tumor cells, thereby enhancing melanoma antigen-specific T-cell recognition and effector function. MAPK pathway–driven relocalization of HLA-A*0201 required a highly conserved cytoplasmic serine phosphorylation site previously implicated in rapid MHC-I internalization and recycling by activated immune cells. Collectively, these data suggest that oncogenic activation of BRAF allows tumor cells to co-opt an evolutionarily conserved MHC-I trafficking pathway as a strategy to facilitate immune evasion. This link between MAPK pathway activation and the MHC-I cytoplasmic tail has direct implications for immunologic recognition of tumor cells and provides further evidence to support testing therapeutic strategies combining MAPK pathway inhibition with immunotherapies in the clinical setting. Cancer Immunol Res; 3(6); 602–9. ©2015 AACR.

The role of antigen cross-presentation from leukemia blasts on immunity to the leukemia-associated antigen PR1.

Cross-presentation is an important mechanism by which exogenous tumor antigens are presented to elicit immunity. Because neutrophil elastase (NE) and proteinase-3 (P3) expression is increased in myeloid leukemia, we investigated whether NE and P3 are cross-presented by dendritic cells (DC) and B cells, and whether the NE and P3 source determines immune outcomes. We show that NE and P3 are elevated in leukemia patient serum and that levels correlate with remission status. We demonstrate cellular uptake of NE and P3 into lysosomes, ubiquitination, and proteasome processing for cross-presentation. Using anti-PR1/human leukocyte antigen-A2 monoclonal antibody, we provide direct evidence that B-cells cross-present soluble and leukemia-associated NE and P3, whereas DCs cross-present only leukemia-associated NE and P3. Cross-presentation occurred at early time points but was not associated with DC or B-cell activation, suggesting that NE and P3 cross-presentation may favor tolerance. Furthermore, we show aberrant subcellular localization of NE and P3 in leukemia blasts to compartments that share common elements of the classic major histocompatibility class I antigen-presenting pathway, which may facilitate cross-presentation. Our data demonstrate distinct mechanisms for cross-presentation of soluble and cell-associated NE and P3, which may be valuable in understanding immunity to PR1 in leukemia.

Natural splice variant of MHC class I cytoplasmic tail enhances dendritic cell-induced CD8+ T-cell responses and boosts anti-tumor immunity.

Dendritic cell (DC)-mediated presentation of MHC class I (MHC-I)/peptide complexes is a crucial first step in the priming of CTL responses, and the cytoplasmic tail of MHC-I plays an important role in modulating this process. Several species express a splice variant of the MHC-I tail that deletes exon 7-encoding amino acids (Δ7), including a conserved serine phosphorylation site. Previously, it has been shown that Δ7 MHC-I molecules demonstrate extended DC surface half-lives, and that mice expressing Δ7-Kb generate significantly augmented CTL responses to viral challenge. Herein, we show that Δ7-Db-expressing DCs stimulated significantly more proliferation and much higher cytokine secretion by melanoma antigen-specific (Pmel-1) T cells. Moreover, in combination with adoptive Pmel-1 T-cell transfer, Δ7-Db DCs were superior to WT-Db DCs at stimulating anti-tumor responses against established B16 melanoma tumors, significantly extending mouse survival. Human DCs engineered to express Δ7-HLA-A*0201 showed similarly enhanced CTL stimulatory capacity. Further studies demonstrated impaired lateral membrane movement and clustering of human Δ7-MHC-I/peptide complexes, resulting in significantly increased bioavailability of MHC-I/peptide complexes for specific CD8+ T cells. Collectively, these data suggest that targeting exon 7-encoded MHC-I cytoplasmic determinants in DC vaccines has the potential to increase CD8+ T-cell stimulatory capacity and substantially improve their clinical efficacy.

A Novel HLA-A*0201 Restricted Peptide Derived from Cathepsin G Is an Effective Immunotherapeutic Target in Acute Myeloid Leukemia

Purpose: Immunotherapy targeting aberrantly expressed leukemia-associated antigens has shown promise in the management of acute myeloid leukemia (AML). However, because of the heterogeneity and clonal evolution that is a feature of myeloid leukemia, targeting single peptide epitopes has had limited success, highlighting the need for novel antigen discovery. In this study, we characterize the role of the myeloid azurophil granule protease cathepsin G (CG) as a novel target for AML immunotherapy. Experimental Design: We used Immune Epitope Database and in vitro binding assays to identify immunogenic epitopes derived from CG. Flow cytometry, immunoblotting, and confocal microscopy were used to characterize the expression and processing of CG in AML patient samples, leukemia stem cells, and normal neutrophils. Cytotoxicity assays determined the susceptibility of AML to CG-specific cytotoxic T lymphocytes (CTL). Dextramer staining and cytokine flow cytometry were conducted to characterize the immune response to CG in patients. Results: CG was highly expressed and ubiquitinated in AML blasts, and was localized outside granules in compartments that facilitate antigen presentation. We identified five HLA-A*0201 binding nonameric peptides (CG1-CG5) derived from CG, and showed immunogenicity of the highest HLA-A*0201 binding peptide, CG1. We showed killing of primary AML by CG1-CTL, but not normal bone marrow. Blocking HLA-A*0201 abrogated CG1-CTL–mediated cytotoxicity, further confirming HLA-A*0201-dependent killing. Finally, we showed functional CG1-CTLs in peripheral blood from AML patients following allogeneic stem cell transplantation. Conclusion: CG is aberrantly expressed and processed in AML and is a novel immunotherapeutic target that warrants further development. Clin Cancer Res; 19(1); 247–57. ©2012 AACR.

B-Raf Regulation of Integrin α4β1-mediated Resistance to Shear Stress through Changes in Cell Spreading and Cytoskeletal Association in T Cells

Background: Contribution of MAPK members to integrin adhesion is not established. Results: Inhibition of B-Raf function or expression selectively regulates integrin α4β1 in T cells. Conclusion: B-Raf is a signaling component for integrin α4β1 cytoskeletal association, cell spreading, and adhesion. Significance: This novel association of B-Raf and integrin α4β1 suggests new therapeutic targets in T cells and indicates potential off-target effects of sorafenib. The regulation of integrin-mediated adhesion is of vital importance to adaptive and innate immunity. Integrins are versatile proteins and mediate T cell migration and trafficking by binding to extracellular matrix or other cells as well as initiating intracellular signaling cascades promoting survival or activation. The MAPK pathway is known to be downstream from integrins and to regulate survival, differentiation, and motility. However, secondary roles for canonical MAPK pathway members are being discovered. We show that chemical inhibition of RAF by sorafenib or shRNA-mediated knockdown of B-Raf reduces T cell resistance to shear stress to α4β1 integrin ligands vascular cell adhesion molecule 1 (VCAM-1) and fibronectin, whereas inhibition of MEK/ERK by U0126 had no effect. Microscopy showed that RAF inhibition leads to significant inhibition of T cell spreading on VCAM-1. The association of α4β1 integrin with the actin cytoskeleton was shown to be dependent on B-Raf activity or expression, whereas α4β1 integrin affinity for soluble VCAM-1 was not. These effects were shown to be specific for α4β1 integrin and not other integrins, such as α5β1 or LFA-1, or a variety of membrane proteins. We demonstrate a novel role for B-Raf in the selective regulation of α4β1 integrin-mediated adhesion.

Peptide/MHC tetramer-based sorting of CD8 T cells to a Leukemia antigen yields clonotypes drawn nonspecifically from an underlying restricted repertoire

Hunsucker, McGary, Vincent, and colleagues report that low-frequency, antigen-specific T-cell responses may be specifically tested using tetramer-based, single-cell sorting and sequencing of the antigen-specific TCRβ clonotypes, and then mapping them onto a patients TCRβ to quantify antigen-driven clonal expansion. Testing of T cell–based cancer therapeutics often involves measuring cancer antigen–specific T-cell populations with the assumption that they arise from in vivo clonal expansion. This analysis, using peptide/MHC tetramers, is often ambiguous. From a leukemia cell line, we identified a CDK4-derived peptide epitope, UNC-CDK4-1 (ALTPVVVTL), that bound HLA-A*02:01 with high affinity and could induce CD8+ T-cell responses in vitro. We identified UNC-CDK4-1/HLA-A*02:01 tetramer+ populations in 3 of 6 patients with acute myeloid leukemia who had undergone allogeneic stem cell transplantation. Using tetramer-based, single-cell sorting and T-cell receptor β (TCRβ) sequencing, we identified recurrent UNC-CDK4-1 tetramer–associated TCRβ clonotypes in a patient with a UNC-CDK4-1 tetramer+ population, suggesting in vivo T-cell expansion to UNC-CDK4-1. In parallel, we measured the patients TCRβ repertoire and found it to be highly restricted/oligoclonal. The UNC-CDK4-1 tetramer–associated TCRβ clonotypes represented >17% of the entire TCRβ repertoire—far in excess of the UNC-CDK4-1 tetramer+ frequency—indicating that the recurrent TCRβ clonotypes identified from UNC-CDK-4-1 tetramer+ cells were likely a consequence of the extremely constrained T-cell repertoire in the patient and not in vivo UNC-CDK4-1–driven clonal T-cell expansion. Mapping recurrent TCRβ clonotype sequences onto TCRβ repertoires can help confirm or refute antigen-specific T-cell expansion in vivo. Cancer Immunol Res; 3(3); 228–35. ©2015 AACR.

TGF-β1 induces preferential rapid expansion and persistence of tumor antigen-specific CD8+ T cells for adoptive immunotherapy

Adoptive cell transfer of expanded, autologous tumor-infiltrating lymphocytes (TIL) into lymphodepleted melanoma patients can induce the regression of bulky, metastatic disease. To generate the large numbers of T cells needed for infusion, TIL undergo a rapid expansion protocol (REP) in vitro using anti-CD3 antibody, interleukin-2, and irradiated peripheral blood feeder cells that typically results in an approximately 1000-fold expansion over 14 days. However, we have found that the conventional REP (C-REP) often favors the expansion of CD4+ T cells at the expense of tumor antigen-specific CD8+ T cells, which are the most potent cytolytic effector cells. In this study, we demonstrate that addition of transforming growth factor (TGF)-β1 to the TIL culture at the onset of rapid expansion (T-REP ) maintained the percentage of CD8+ T cells while not inhibiting overall T-cell expansion. Of T cells expanded from different melanoma patient tumors, 13 of 15 TIL demonstrated improved yields and percentages of both CD8+ and MART-1 melanoma antigen-specific T cells after 14 days of expansion in TGF-β1 compared with the C-REP. This was associated with a marked improvement in the antitumor activity of the resulting bulk TIL culture in terms of interferon-γ production and melanoma tumor-specific cytotoxic T-lymphocyte activity. In addition, T-REP T cells demonstrated a higher potential for continued expansion in vitro for up to 3 weeks after the expansion compared with C-REP T cells, suggesting that they may also be capable of increased persistence after adoptive cell transfer. Our results suggest that TGF-β1-expanded TIL have attributes that might predict efficacy superior to that of conventional TIL.

Abstract 5396: Interferon-α receptor dependent and independent inhibition of type I interferon production in plasmacytoid dendritic cells from mice and melanoma patients

IFN-α is pleiotropic cytokine belonging to the type I Interferon (IFNs) family and can induce multiple biological effects, e.g. induction of apoptosis and inhibition of cell growth. These cytokines exhibit a long record of clinical use in patients with some types of cancer and viral diseases. Notably, certain autoimmune disorders have been postulated to be mediated by endogenous IFN-α and are often observed in some IFN-α-treated cancer patients. How IFN-α promotes the generation of antitumor T-cell immunity and its regulation mechanisms are still unclear. To address these questions, we focus on the main producer of IFNs, Plasmacytoid dendritic cells (pDCs), which reside in bone marrow and lymphoid organs and typically secrete type I IFNs on Toll-like receptor (TLR) triggering. We detected cytokines production by human pDCs from leukapheresis cells of melanoma patients who were treated with IFNs for 25 weeks. After stimulation with CpG-A, these pDCs after treatment (IFN-α-pDCs) produce 70% less IFN-α compare to before treatment, but compatible amount of other inflammatory cytokines e.g. IL-6. We observed the similar phenomena in murine models either after VSV infection or by systemic IFN-α delivery. We showed that these IFN-α-pDCs possess more active and mature morphology as DCs and they are particularly more effective in inducing T- and NK-cell immunity by increasing cell proliferation and differentiation. Of note, IFN-α-pDCs skewed the naive CD4+ T cells to a Th17 antigen-presenting subset by producing more IL-17 but less IFN-γ compare to wide type pDCs. The ensemble of these results suggests that IFN-α-pDCs could be successfully used in strategies of cancer immunotherapy, especially in melanoma patients. We also demonstrate that pDCs possess two fundamental, but separate, physiologic mechanisms for negative feedback control of IFN-α production after IFNs treatment in melanoma patients. Both signaling through the canonical TLR-MyD88 pathway and feedback inhibition from IFN-α itself through the IFN-α/βR render pDCs unable to synthesize type I interferon upon TLR triggering. Mechanistically, we found that IFN-α-pDCs show increased IRF7 mRNA and protein upon TLR stimulation, but despite efficient nuclear IRF-7 translocation, type I IFN was not produced. Interestingly, Ifn-α6 promoter activity was blocked in the IFN-α-pDCs, suggesting a block of type I IFN transcription despite nuclear IRF-7 translocation. Taken together, triggering of TLR and of IFN-α/βR act independently to tightly limit systemic IFN-α production after IFNs treatment. All this reveals the complexity of the IFN-α-pDC interactions under normal and pathological conditions and stimulates further studies for identifying optimal modalities in either using these cytokines or controlling their production in melanoma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5396. doi:1538-7445.AM2012-5396