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Abstract Cutaneous T-cell lymphoma (CTCL) is a rare cancer of skin-homing T cells. A subgroup of patients develops large cell transformation with rapid progression to an aggressive lymphoma. Here, we investigated the transformed CTCL (tCTCL) tumor ecosystem using integrative multiomics spanning whole-exome sequencing (WES), single-cell RNA sequencing, and immune profiling in a unique cohort of 56 patients. WES of 70 skin biopsies showed high tumor mutation burden, UV signatures that are prognostic for survival, exome-based driver events, and most recurrently mutated pathways in tCTCL. Single-cell profiling of 16 tCTCL skin biopsies identified a core oncogenic program with metabolic reprogramming toward oxidative phosphorylation (OXPHOS), cellular plasticity, upregulation of MYC and E2F activities, and downregulation of MHC I suggestive of immune escape. Pharmacologic perturbation using OXPHOS and MYC inhibitors demonstrated potent antitumor activities, whereas immune profiling provided in situ evidence of intercellular communications between malignant T cells expressing macrophage migration inhibitory factor and macrophages and B cells expressing CD74. Significance: Our study contributes a key resource to the community with the largest collection of tCTCL biopsies that are difficult to obtain. The multiomics data herein provide the first comprehensive compendium of genomic alterations in tCTCL and identify potential prognostic signatures and novel therapeutic targets for an incurable T-cell lymphoma. This article is highlighted in the In This Issue feature, p. 1171

Background Emerging evidence from studies in sarcoma and melanoma immune checkpoint blockade(ICB) trials demonstrated enhanced efficacy in tumors harbouring tertiary lymphoid structures(TLS)1–3 - lymph node like aggregates in the tumor microenvironment postulated to recruit lymphocyte infiltration and coordinate tumor antigen presentation and lymphocyte priming. Previously, our group described a 12-chemokine metagene signature that reflects strong intratumoral chemotactic signalling and accurately predicts the presence of TLS in colorectal carcinoma.4 Grounded in these works, we attempted to correlate high 12CK signature with TLS formation in the context of muscle invasive bladder cancer(MIBC), and then sought to define its prognostic implications and its ability to predict response to ICB. Methods A total of 130 MIBC samples were arrayed on Affymetrix microarrays and 12CK scores were assessed. Scores were normalized using 12CK>1 to denote 12CK-High(n=24). We then investigated the the presence of TLS with the associated immune cellular infiltration evaluated by immunohistochemistry and gene signature deconvolution method (xCell).5 12CK scores were also correlated with survival in our institutional cohort and validated using data from TCGA. Finally, 12CK scores were extracted from the IMVIGOR210 study to examine its ability to predict response to ICB.6 Results Type III TLS, consisting of germinal center-like structures and discrete T-cell zones were found in 7/22 12CK-High vs. 0/21 12CK-Low tumors (p=0.009) (figure 1a). Additionally, a more robust immuno-environment was seen in 12CK-High tumors, consisting of increased infiltration of CD4+ T lymphocytes (p=0.1), CD8+ T lymphocytes (p=0.02), activated dendritic cells (p=0.047), and B lymphocytes (p=0.006) on immunohistochemistry (figure 1b). Furthermore, on xCell deconvolution, M1 macrophage, NK cells, CD8+ Tem, CD4+ Tem, and memory B cells were enriched in 12CK-High tumors, suggesting both a heightened innate and adaptive immune response (figure 1c, d). Kaplan-Meier survival analyses of our internal cohort revealed improved PFS (HR 0.25, p=0.003 ), CSS (HR 0.25, p=0.003), and OS (HR 0.55, p=0.03) amongst 12CK-High patients (figure 2a-c). From the TCGA, similar improvements were found in PFS (HR 0.55, p=0.007), CSS (HR 0.40, p=0.002), and OS (HR 0.59, p=0.01) in 12CK-High patients (figure 2d-f). From the IMVIGOR 210 study, complete responders exhibited significantly higher 12-CK scores than all other groups (figure 2g). Strikingly, the 12CK-High signature conferred a median overall survival benefit of almost 1 year in the atezolizumab-treated patients (figure 2h). Conclusions In muslce invasive bladder cancer, 12CK-High scores corresponded with formation of TLS in the TME; favourable prognosis in surgically treated MIBC patients; and CR in atezolizumab-treated patients. References Cabrita R, Lauss M, Sanna A, et al. Tertiary lymphoid structures improve immunotherapy and survival in melanoma. Nature 2020;577(7791):561–565. Helmink BA, Reddy SM, Gao J, et al. B cells and tertiary lymphoid structures promote immunotherapy response. Nature 2020;577(7791):549–555. Petitprez F, de Reynies A, Keung EZ, et al. B cells are associated with survival and immunotherapy response in sarcoma. Nature 2020;577(7791):556–560. Aran D, Hu Z, Butte AJ. xCell: digitally portraying the tissue cellular heterogeneity landscape. Genome biology 2017;18(1):220. Rosenberg JE, Hoffman-Censits J, Powles T, et al. Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: a single-arm, multicentre, phase 2 trial. Lancet (London, England). 2016;387(10031):1909–1920.

Background Favorable outcomes utilizing anti–PD-1 based immune therapies for unresectable melanoma patients are hypothesized to be dependent on antigen processing and presentation mechanisms. The present study utilizes multiomics to examine the contribution of antigen presentation pathways in metastatic melanoma tumors either responsive or resistant to anti-PD-1 therapy. Methods To unveil the mechanisms that predispose unresectable, stage III/IV melanoma patients to respond to anti–PD-1-based therapies, we conducted expression proteomics as well as employed the mRNA immune oncology panel (HTG Molecular Diagnostics, Inc., Tucson, AZ). Formalin-fixed, paraffin-embedded tissues collected from 27 patients prior to anti-PD-1 therapy (previously consented and enrolled in the Moffitt Cancer Center Total Cancer Care (TCC) protocol) were utilized in this study. For the proteomics analysis, we examined 19 FFPE samples, whereas the targeted mRNA analysis utilized 25 FFPE samples with 17 samples analyzed using both omics approaches. Robust mass spectrometry analysis used a pooled sample to optimize the number of detected peptides. The melanoma patients were selected from the database based on whether they had progression free survival (PFS) greater than 1 year (n=15) or PFS less than 6 months (n=12). Results We identified more than 250 transcript/protein candidates that demonstrated differential expression between poor and good responders following anti-PD-1 therapy. Utilizing MetaCore software and subsequent downstream analyses of expression profiles for a knowledge-based curation of pathways and protein networks, we illustrated both the enrichment of Gene Ontology (GO) terms and specific antigen processing/presentation proteins. Of the top 10 GO processes, 7 were related to antigen processing/presentation and Major Histocompatibility Complex (MHC) presentation. Antigen processing/presentation and cytokine production/signaling via IFN-γ–mediated signaling through NF-κB and the JAK/STAT pathway interaction with iNOS were mechanistic candidates of response to anti-PD-1 therapy. Conclusions These comparative analyses illustrated the importance of antigen processing/presentation pathways mediated by both MHC class I and II in activating the immune system to initiate and maintain the immune-based response to anti–PD-1 therapy in metastatic melanoma patients. The current study also demonstrated the value of proteogenomics in defining mechanisms of response and resistance to anti-PD-1 therapy. Trial Registration N.A. Ethics Approval The procurement of FFPE samples was approved under IRB-approved protocol (MCC 18583, Advarra) from patients who had received a biopsy within 1 year prior to the start of anti–PD-1 therapy. Biopsy samples were obtained from the institutional biobank (Total Cancer Cancer). The radiology reads (performed by T.R.) from this research were funded by a parent research project to Moffitt Cancer Center by Navigate BioPharma to J.G. and J.M. Consent N.A.

Background It is becoming more evident that STING activity in tumor cells can have a functional role in mediating antitumor immune responses. We have recently shown that activation of STING signaling in human melanoma cell lines enhances their antigenicity and susceptibility to lysis by human melanoma tumor infiltrating lymphocytes (TIL) through the augmentation of MHC class I molecules.1 However, the frequent impairment of this pathway through loss of cGAS and/or STING expression in melanoma cell lines limits their antigen presentation and subsequently their sensitivity to cytotoxic T cell mediated killing. In this study, we asked if this suppression is, in part, epigenetically regulated and if it is indeed a driver of melanoma resistance to T cell-based immunotherapies. Methods To determine the role of DNA methylation in melanoma STING and cGAS silencing, we performed genome-wide DNA methylation profiling across a panel of 16 human melanoma cell lines. We subjected melanoma cell lines that indicated STING and/or cGAS promoter hypermethylation to treatment with 5-aza-2’-deoxycytidine (5AZADC) and evaluated their protein expression by immunoblot. We next assessed phosphorylation of IRF3 and induction of IFN-β and CXCL10 in 5AZADC-treated melanoma cells following their stimulation with dsDNA or 2’3’-cGAMP. We also co-cultured 5AZADC-pretreated melanoma cell lines with their HLA-matched human melanoma TIL in the presence or absence of dsDNA or 2’3’-cGAMP and assessed TIL production of IFN-γ. Results Using whole genome methylation profiling, we identified a distinct correlation between promoter hypermethylation and loss of STING and cGAS expression in human melanoma cell lines. Reconstitution of STING and cGAS expression through DNA demethylation reinstated functional STING signaling in at least half of the examined cell lines as indicated by STING-dependent phosphorylation of IRF3, induction of CXCL10 (~300 pg/ml, P Conclusions We provide evidence that methylation silencing of cGAS and STING is not only a notable mechanism of STING signaling dysfunction in melanoma, but also plays a role in tumor antigen presentation and recognition by TIL. Collectively, these observations argue that targeting epigenetic loss of STING signaling in melanomas should be considered as a strategy to improve the efficacy of clinical interventions using T cell-based immunotherapies. Acknowledgements Funding: NCI P50 CA168536, Cindy and Jon Gruden Fund, Chris Sullivan Fund, V Foundation, Dr. Miriam and Sheldon G. Adelson Medical Research Foundation. Reference Falahat R, Perez-Villarroel P, Mailloux AW, Zhu G, Pilon-Thomas S, Barber GN, Mule JJ. STING signaling in melanoma cells shapes antigenicity and can promote antitumor T-cell activity. Cancer Immunology Research 2019; 7(11):1837–48.

Tumor hypoxia is an established facilitator of survival adaptation and mesenchymal transformation in glioblastoma (GBM). The underlying mechanisms that direct hypoxia-mediated survival in GBM habitats are unclear. We previously identified BIRC3 as a mediator of therapeutic resistance in GBM to standard temozolomide (TMZ) chemotherapy and radiotherapy (RT). Here we report that BIRC3 is a biomarker of the hypoxia-mediated adaptive mesenchymal phenotype of GBM. Specifically, in the TCGA dataset elevated BIRC3 gene expression was identified as a superior and selective biomarker of mesenchymal GBM versus neural, proneural and classical subtypes. Further, BIRC3 protein was highly expressed in the tumor cell niches compared to the perivascular niche across multiple regions in GBM patient tissue microarrays. Tumor hypoxia was found to mechanistically induce BIRC3 expression through HIF1-alpha signaling in GBM cells. Moreover, in human GBM xenografts robust BIRC3 expression was noted within hypoxic regions of the tumor. Importantly, selective inhibition of BIRC3 reversed therapeutic resistance of GBM cells to RT in hypoxic microenvironments through enhanced activation of caspases. Collectively, we have uncovered a novel role for BIRC3 as a targetable biomarker and mediator of hypoxia-driven habitats in GBM.

Although, the epigenetic regulation of O6-alkylguanine DNA alkyltransferase (MGMT) in GBM is an established surrogate of intrinsic resistance to temozolomide (TMZ), the evolution of GBM habitats towards adaptive resistance to TMZ relative to MGMT promoter methylation status remains unclear. We report a novel epigenetic regulation of plasticity towards adaptive resistance in GBM. Using an adaptive TMZ resistance model of MGMT-hypermethylated and MGMT-hypomethylated GBM cellular habitats, a counter-intuitive inverse correlation was noted between intrinsic MGMT-dependent TMZ resistance versus plasticity towards adaptive TMZ resistance. Upon TMZ challenge, GBM cellular habitats with lower intrinsic resistance demonstrated significant genetic perturbations and aggressive phenotypic alterations compared to GBM habitats with higher intrinsic resistance. A resulting gene signature associated with plasticity for adaptive resistance from our model significantly correlated with GBM survival in the TCGA dataset. XAF-1 emerged as a key gene whose epigenetic regulation mediated differential plasticity towards adaptive resistance to TMZ in GBM habitats. Genetic silencing of XAF-1 significantly compromised plasticity towards adaptive resistance. XAF1 expression was found to highly correlate with promoter methylation status, and negatively correlate with long-term survival in GBM patient survival. Our studies have shed some light with respect to the plasticity of GBM habitats towards adaptive resistance evolution to TMZ relative to MGMT promoter methylation status. Particularly, a novel and translational role for XAF1 in GBM has been uncovered.

STING (stimulator of IFN genes) signaling is an innate immune pathway for induction of a spontaneous antitumor T-cell response against certain immunogenic tumors. Although antigen-presenting cells are known to be involved in this process, insight into the participation of tumor cell–intrinsic STING signaling remains weak. In this study, we find diversity in the regulation of STING signaling across a panel of human melanoma cell lines. We show that intact activation of STING signaling in a subset of human melanoma cell lines enhances both their antigenicity and susceptibility to lysis by human melanoma tumor-infiltrating lymphocytes (TIL) through the augmentation of MHC class I expression. Conversely, defects in the STING signaling pathway protect melanoma cells from increased immune recognition by TILs and limit their sensitivity to TIL lysis. Based on these findings, we propose that defects in tumor cell–intrinsic STING signaling can mediate not only tumor immune evasion but also resistance to TIL-based immunotherapies.

Tertiary lymphoid structures (TLSs) associate with better prognosis in certain cancer types, but their underlying formation and immunological benefit remain to be determined. We established a mouse model of TLSs to study their contribution to antitumor immunity. Because the stroma in lymph nodes (sLN) participates in architectural support, lymphogenesis, and lymphocyte recruitment, we hypothesized that TLSs can be created by sLN. We selected a sLN line with fibroblast morphology that expressed sLN surface markers and lymphoid chemokines. The subcutaneous injection of the sLN line successfully induced TLSs that attracted infiltration of host immune cell subsets. Injection of MC38 tumor lysate-pulsed dendritic cells activated TLS-residing lymphocytes to demonstrate specific cytotoxicity. The presence of TLSs suppressed MC38 tumor growth in vivo by improving antitumor activity of tumor-infiltrating lymphocytes with downregulated immune checkpoint proteins (PD-1 and Tim-3). Future engineering of sLN lines may allow for further enhancements of TLS functions and immune cell compositions.

The induction of ectopic lymph node structures (ELNs) holds great promise to augment immunotherapy against multiple cancers including metastatic melanoma, in which ELN formation has been associated with a unique immune-related gene expression signature composed of distinct chemokines. To investigate the therapeutic potential of ELNs induction, preclinical models of ELNs are needed for interrogation of these chemokines. Computational models provide a non-invasive, cost-effective method to investigate leukocyte trafficking in the tumor microenvironment, but parameterizing such models is difficult due to differing assay conditions and contexts among the literature. To better achieve this, we systematically performed microchemotaxis assays on purified immune subsets including human pan-T cells, CD4+ T cells, CD8+ T cells, B cells, and NK cells, with 49 recombinant chemokines using a singular technique, and standardized conditions resulting in a dataset representing 238 assays. We then outline a groundwork computational model that can simulate cellular migration in the tumor microenvironment in response to a chemoattractant gradient created from stromal, lymphoid, or antigen presenting cell interactions. The resulting model can then be parameterized with standardized data, such as the dataset presented here, and demonstrates how a computational approach can help elucidate developing ELNs and their impact on tumor progression.

Tertiary lymphoid structures (TLSs) have been identified in the parenchyma and/or in the peripheral margins of human solid tumors. Uncovering the functional nature of these structures is the subject of much intensive investigation. Studies have shown a direct correlation of the presence of human tumor-localized TLS and better patient outcome (e.g., increase in overall survival) in certain solid tumor histologies, but not all. We had identified a tumor-derived immune gene expression signature, encoding 12 distinct chemokines, which could reliably identify the presence of TLSs, of different degrees, in a variety of human solid tumors. We are focused on understanding the influence of TLSs on the tumor microenvironment and leveraging this understanding to both manipulate the anti-tumor immune response and potentially enhance immunotherapy applications. Moreover, as not all human solid tumors show the presence of these lymphoid structures, we are embarking on bioengineering approaches to design and build ‘designer’ TLSs to address, and potentially overcome, an unmet medical need in cancer patients whose tumors lack such lymphoid structures.

In the past five decades, the role for lymphocytes in host immune response to tumors has been shown, at least in some patients, to be a critical component in disease prognosis. Also, the heterogeneity of lymphocytes has been documented, including the existence of regulatory T cells that suppress the immune response. As the functions of lymphocytes have become better defined in terms of antitumor immunity, specific targets on lymphocytes have been uncovered. The appreciation of the role of immune checkpoints has also led to therapeutic approaches that illustrate the effectiveness of blocking negative regulators of the antitumor immune response. In this Masters of Immunology article, we trace the evolution of our understanding of tumor-infiltrating lymphocytes and discuss their role in melanoma prognosis from the very basic observation of their existence to the latest manipulation of their functions with the result of improvement of the host response against the tumor. Cancer Immunol Res; 3(8); 827–35. ©2015 AACR.

We have reported the upregulation of MARCO, a member of the class A scavenger receptor family, on the surface of murine and human dendritic cells (DCs) pulsed with tumor lysates. Exposure of murine tumor lysate-pulsed DCs to an anti-MARCO antibody led to loss of dendritic-like processes and enhanced migratory capacity. In this study, we have further examined the biological and therapeutic implications of MARCO expression by DCs. DCs generated from the bone marrow (bm) of MARCO knockout (MARCO⁻/⁻) mice were phenotypically similar to DCs generated from the bm of wild-type mice and produced normal levels of IL-12 and TNF-α when exposed to LPS. MARCO⁻/⁻ DCs demonstrated enhanced migratory capacity in response to CCL-21 in vitro. After subcutaneous injection into mice, MARCO⁻/⁻ TP-DCs migrated more efficiently to the draining lymph node leading to enhanced generation of tumor-specific IFN-γ producing T cells and improved tumor regression and survival in B16 melanoma-bearing mice. These results support targeting MARCO on the surface of DCs to improve trafficking and induction of anti-tumor immunity.

We reported that murine tumor lysate-pulsed dendritic cells (TP-DC) could elicit tumor-specific CD4(+) and CD8(+) T cells in vitro and in vivo. In some limited cases, TP-DC treatments in vivo could also result in regression of established subcutaneous tumors and lung metastases. By gene array analysis, we reported a high level of expression of a novel member of the cell surface class A scavenger receptor family, MARCO, by murine TP-DC compared to unpulsed DC. MARCO is thought to play an important role in the immune response by mediating binding and phagocytosis, but also in the formation of lamellipodia-like structures and dendritic processes. We have now examined the biologic and therapeutic implications of MARCO expressed by TP-DC. In vitro exposure of TP-DC to a monoclonal anti-MARCO antibody resulted in a morphologic change of rounding with disappearance of dendritic-like processes. TP-DC remained viable after anti-MARCO antibody treatment; had little, if any, change in production of IL-10, IL-12p70 and TNF-alpha; but demonstrated enhanced migratory capacity in a microchemotaxis assay. The use of a selective inhibitor showed MARCO expression to be linked to the p38 mitogen-activated protein kinase (MAPK) pathway. In vivo, anti-MARCO antibody treated TP-DC showed better trafficking from the skin injection site to lymph node, enhanced generation of tumor-reactive IFN-gamma producing T cells, and improved therapeutic efficacy against B16 melanoma. These results, coupled with our finding that human monocyte-derived DC also express MARCO, could have important implications to human clinical DC vaccine trials.

To be recognized by CD8+ T lymphocytes, target cells must process and present peptide antigens in the context of major histocompatibility complex (MHC) class I molecules. The nonimmunogenic, low class I-expressing, methylcholanthrene (MCA)-induced murine sarcoma cell line, MCA 101, is a poor presenter of endogenously generated viral antigens to specific CD8+ T lymphocytes and cannot be used to generate tumor infiltrating lymphocytes (TIL). Since interferon gamma (IFN-gamma) has been shown to upregulate three sets of molecules important for antigen processing and presentation, we retrovirally transduced wild-type MCA 101 (101.WT) tumor with the mIFN-gamma cDNA to create the 101.NAT cell line. Unlike 101.WT, some clones of retrovirally transduced 101.NAT tumor expressed high levels of class I, and could be used to generate CD8+ TIL. More importantly, these TIL were therapeutic in vivo against established pulmonary metastases from the wild-type tumor. Although not uniformly cytotoxic amongst several separate cultures, these TIL did specifically release cytokines (IFN-gamma and tumor necrosis factor-alpha) in response to 101.WT targets. 101.WT's antigen presentation deficit was also reversed by gene modification with mIFN-gamma cDNA. 101.NAT had a greatly improved capacity to present viral antigens to CD8+ cytotoxic T lymphocytes. These findings show that a nonimmunogenic tumor, incapable of generating a CD8+ T cell immune response, could be gene-modified to generate a therapeutically useful immune response against the wild-type tumor. This strategy may be useful in developing treatments for tumor histologies not thought to be susceptible to T cell-based immunotherapy.

The future of cancer care and treatment lies in the concept of “personalized medicine,” a model that focuses on the individual, not just the disease. Personalized medicine in cancer care will need to use molecular signatures to match the right patients to the right drugs, first in clinical

Depleting host immune elements with nonmyeloablative regimens prior to the adoptive transfer of tumor-specific CD8(+) T cells significantly enhances tumor treatment. In the current study, superior antitumor efficacy was achieved by further increasing the intensity of lymphodepletion to a level that required HSC transplantation. Surprisingly, the HSC transplant and not the increased lymphodepletion caused a robust expansion of adoptively transferred tumor-specific CD8(+) T cells. The HSC-driven cell expansion of effector, but not of naive, CD8(+) T cells was independent of in vivo restimulation by MHC class I-expressing APCs. Simultaneously, HSCs also facilitated the reconstitution of the host lymphoid compartment, including inhibitory elements, not merely via the production of progeny cells but by enhancing the expansion of cells that had survived lymphodepletion. Profound lymphodepletion, by myeloablation or by genetic means, focused the nonspecific HSC boost preferentially toward the transferred tumor-specific T cells, leading to successful tumor treatment. These findings indicate that CD8(+) T cell-mediated tumor responses can be efficiently driven by HSCs in the myeloablative setting and have substantial implications for the design of new antitumor immunotherapies.

Dendritic cells (DCs) are highly potent antigen-presenting cells of bone marrow origin that can stimulate both primary and secondary T- and B-cell responses (1). First described by Steinman, DCs display a characteristic veiled appearance with multiple extending cellular processes. These cells possess the necessary components for potent antigen-presenting functions, including the production of a variety of important immunostimulatory cytokines and the expression of critical cell-surface molecules. Depending on their level of maturity, DCs express prominent levels of MHC class I and class II molecules, as well as costimulatory molecules such as CD40, CD80, and CD86. Animal studies show them to be responsible primarily for sensitizing naive T cells in their first exposure to antigen. Because of this unique property in inducing immunity, DCs have been termed “nature’s adjuvant” (1). Antigen distribution in the host environment often favors uptake and presentation by DCs rather than macrophages or B cells, and subsequent migration of primed DCs to lymphoid organs enhances targeted presentation of antigens to the immune system. More recently, it has also been shown that murine monocytes residing in subcutaneous tissue can become lymph-borne DCs that localize in draining lymph nodes (2). Once in the lymph nodes, these DCs can present both MHC class I– and class II–restricted antigens and can therefore stimulate both resident CD8+ and CD4+ T cells. Whether the migration to the lymph nodes is strictly required for DCs to be competent to stimulate these responses remains less certain. In this regard, Banchereau’s group has reported that in human breast carcinomas, immature DCs can reside within the tumor mass itself, whereas the mature ones are located in peritumoral areas (3). As possible evidence of an ongoing immune response in situ, some peritumoral areas of the specimens showed T cells clustered around the mature DCs, which resembled clusters often reported for secondary lymphoid organs. The maturation state of DCs appears to be important for their optimal use in immunization strategies, with more mature DCs demonstrating higher production of some key cytokines (e.g., IL-12), increased antigen presentation in vitro and in vivo, and, at least in mice, increased localization to draining lymph nodes and more potent induction of broad T-cell immunity and antitumor activity (1, 4). CD40L, LPS, monocyte-conditioned medium, and TNFα have all been used to promote DC maturation. Human DCs can arise from bone marrow–derived and cord blood–derived CD34+ hematopoietic cell progenitors and also from PBMCs and CD14+ blood monocytes. Highly enriched rodent or human DCs can now be produced in great numbers by culturing progenitor cells in the presence of cytokines, notably GM-CSF and IL-4, with or without TNFα. Virally infected human DCs can elicit potent proliferative and cytolytic T-cell reactivity in vitro (5). In animal models, immunization with antigen-presenting DCs can result in strong protective immunity to viruses (e.g., lymphocytic choriomeningitis virus, LCMV) and to tumors (4, 6, 7). With respect to the latter, tumor antigen–pulsed DCs can successfully treat established mouse tumors in vivo. Tumor-associated and model antigens, in the form of whole cell lysates, apoptotic cell bodies, peptides, proteins, RNA, and DNA have been used and may initiate tumor-specific CD4+ and CD8+ T-cell responses (7, 8). DCs have now reached a watershed — their efficacy in immunization approaches for the protection from and the treatment of human disease is finally being tested. The establishment of human DC cultures from the peripheral blood of patients has facilitated their use as immunotherapeutic agents, most notably in the treatment of infectious diseases and against a variety of human tumors. Initial clinical trials involving DC-based immunization of patients with tumors of hematologic and solid origin are promising: subjects show increased antitumor T-cell reactivity and experience partial or complete clinical responses (9–14). However, meaningful comparisons of the immunologic and clinical outcomes of these trials have been complicated by variability regarding the source of the DCs, their level of maturity, the nature of the antigen used to pretreat them, as well as the dosing regimen and route of administration used. An additional complication centers on the fact that these immunizations have been conducted in advanced cancer patients with various tumor types, at different stages of disease, and with different histories of previous therapy. The study of Dhodapkar et al. reported in this issue of the JCI represents an important step toward optimizing some of these variables (15). These same investigators reported earlier in the JCI that, remarkably, a single subcutaneous injection of fewer than 3 × 106 mature DCs could rapidly expand CD4+ and CD8+ T-cell immunity specific to several distinct antigens, including keyhole limpet hemocyanin (KLH), influenza matrix peptide (MP), and tetanus toxoid (TT) (16). Significantly, these immunizations were conducted in normal, healthy volunteers with control immunizations of antigen alone (without DCs) and DCs alone (without antigen) to determine their separate contribution, if any, to the response. The immunologic monitoring comprised state-of-the-art assays of high sensitivity and specificity. In the current report, Dhodapkar et al. follow up on a cohort of these previously immunized volunteers to examine the durability and kinetics of the immunologic responses to KLH, TT, and MP, as well as the impact of providing a booster injection of MP-primed, mature DCs. The CD8+ T-cell immune response to the MP peptide after the booster immunization was more rapid and of greater magnitude than the first immunization. These responding T cells could also recognize lower doses of the peptide. Moreover, the booster injection of the antigen-primed DCs was efficacious in the absence of any provision of additional epitopes to elicit help to the responding CD8+ T cells. Which road will human DC-based vaccines now travel? Additional key comparisons remain to be done. The present studies point to the use of mature, rather than immature, DCs and the subcutaneous, rather than intravenous or intranodal, immunization, but their level of significance can be ascertained only by comparative, randomized studies. Moreover, if mature DCs are shown to be more beneficial in immunization, additional issues remain: how best to optimize DC maturation? Which source of antigens — apoptotic cell bodies, lysates, or peptides — should be delivered to DCs to optimize the response? Does it matter whether DCs are generated from CD34+ progenitors or from monocytes? Data are eagerly anticipated from direct clinical comparisons that will address these questions.

Recent developments support the hypothesis that simian virus 40 (SV40) T antigen (Tag) is a relevant target for immunotherapy for human cancers. Contemporary assays for DNA sequences have found SV40 DNA in a majority of human osteosarcomas, bone tumors, ependymomas, choroid plexus tumors, and mesotheliomas that have been evaluated (1-5). Expression of SV40 Tag protein in mesotheliomas and brain tumors has been demonstrated, and functional studies (6,7) have shown that SV40 Tag can interfere with the functions of p53 and retinoblastoma protein in human mesotheliomas. The possible relationship between polio vaccines contaminated with SV40, which were administered to more than 98 million subjects from 1955 through early 1963, and the oncogenesis of these cancers remains controversial (8). Nonetheless, the subsequently increasing occurrence of mesothelioma, a lethal cancer that commonly expresses SV40 Tag and that has limited treatment options, is disconcerting (9). Strategies targeting SV40 Tag represent a rational avenue for developing new therapies for such SV40 Tag-expressing cancers. SV40 Tag has several attributes that make it potentially useful in recombinant vaccine strategies. First, because SV40 Tag is a genuine tumor-associated antigen that is not present in normal host cells, vaccine therapies targeting this antigen need not overcome tolerance to achieve efficacy and circumvent the potential hazard of autoimmune responses against tumor-associated antigens coexpressed by tumor and normal tissue. Second, apart from its association with certain human cancers, SV40 is also pathogenic in rodent models, providing a unique setting for optimizing potentially clinically relevant recombinant vaccines in animal models (10). Third, the immunogenicity of SV40 Tag has been studied extensively (11-13), providing insight for rational approaches to the development of new therapeutic vaccine strategies targeting this tumor-associated antigen. Despite these attributes of SV40 Tag, a cancer vaccine strategy providing safe and effective therapy for pre-established SV40 Tag-expressing tumors has previously been elusive. To determine whether recombinant poxvirus vaccines targeting SV40 Tag would provide an avenue for unprecedented therapeutic efficacy against established SV40 Tag-expressing tumors, we have made a novel recombinant vaccinia construct. This vector encodes a safety-modified SV40 Tag sequence (mTag) that excludes retinoblastoma protein binding site, p53 binding site, and the amino-terminal oncogenic CR1 and J domains to optimize potential clinical safety but that preserves immunogenic domains. After confirming expression of the expected SV40 Tag fragment by this novel recombinant vaccinia construct termed recombinant vaccinia-encoding mTag (vac-mTag), studies were undertaken to evaluate its efficacy against SV40 Tag-expressing tumors in vivo. These studies provide evidence that vac-mTag can efficiently prime the immune response to provide effective antigen-specific protection and therapy against SV40 Tag-expressing lethal tumors.

Surface expression of human leukocyte antigen (HLA) class I antigens on melanoma lines was evaluated by locus-specific monoclonal antibodies (mAbs) with three different techniques: Fluorescence-activated cell sorting (FACS), immunohistochemistry with cytospin preparation (ICP), and complement-mediated cytotoxicity (CMC). Eleven HLA class I-expressing cell lines developed from metastases were used. Specific expression of HLA loci was examined under routine culture conditions and after 48-h incubation in interferon-gamma (IFN-gamma; 500 U/ml). Loss of allelic expression was seen in one line (586-MEL): Products of genes coding for HLA-A29 and -B44, in strong linkage disequilibrium, were not detectable. HLA-A antigens were consistently detected by all methodologies and minimally affected by pretreatment with IFN-gamma. HLA-B antigens were detectable in 8 of 11 lines by ICP and 3 of 11 lines by CMC. By FACS the supratypic specificity HLA-Bw6 was expressed at low levels in most lines (mean fluorescence 47.2 +/- 13.4 and rose to 259.8 +/- 45.9 after incubation with IFN-gamma; p0.001). HLA-Cw antigen detection by CMC correlated with HLA-B (p0.01), suggesting that down-regulation and sensitivity to IFN-gamma are shared by the two loci. This low expression of the HLA-B antigens may play a role in the evasion of the host immune response and its up-regulation may be useful in allowing tumor antigen recognition.

Background: Immunotherapy (IO) has improved survival for patients with advanced clear cell renal cell carcinoma (ccRCC), but resistance to therapy develops in most patients. We use cellular-resolution spatial transcriptomics in patients with IO naïve and IO exposed primary ccRCC tumors to better understand IO resistance. Spatial molecular imaging (SMI) was obtained for tumor and adjacent stroma samples. Spatial gene set enrichment analysis (GSEA) and autocorrelation (coupling with high expression) of ligand-receptor transcript pairs were assessed. Multiplex immunofluorescence (mIF) validation was used for significant autocorrelative findings and the cancer genome atlas (TCGA) and the clinical proteomic tumor analysis consortium (CPTAC) databases were queried to assess bulk RNA expression and proteomic correlates., Results: 21 patient samples underwent SMI. Viable tumors following IO harbored more stromal CD8+ T cells and neutrophils than IO naïve tumors. YES1 was significantly upregulated in IO exposed tumor cells. The epithelial-mesenchymal transition pathway was enriched on spatial GSEA and the associated transcript pair COL4A1-ITGAV had significantly higher autocorrelation in the stroma. Fibroblasts, tumor cells, and endothelium had the relative highest expression. More integrin αV+ cells were seen in IO exposed stroma on mIF validation. Compared to other cancers in TCGA, ccRCC tumors have the highest expression of both COL4A1 and ITGAV . In CPTAC, collagen IV protein was more abundant in advanced stages of disease., Conclusions: On spatial transcriptomics, COL4A1 and ITGAV were more autocorrelated in IO-exposed stroma compared to IO-naïve tumors, with high expression amongst fibroblasts, tumor cells, and endothelium. Integrin represents a potential therapeutic target in IO treated ccRCC., Competing Interests: Competing interests: The corresponding author certifies that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (ie. employment/affiliation, grants or funding, consultancies, honoraria, stock ownership or options, expert testimony, royalties, or patents filed, received, or pending), are the following: ACS, MTH, TCP, OEO, NHC, AEB, PAS, JN, CMS, NLF, PMRE, KYT, JAB, YCP, JD, LAM, WEG, and BLF have no relevant disclosures; BJM is an NCCN Kidney Cancer Panel Member and an advisor for Merck; RL received research support from Predicine, Veracyte, CG Oncology, Valar Labs, and Merck, is on the clinical trials committee for CG Oncology, is scientific advisor for Bristol Myers Squibb, Merck, Fergene, Arquer Diagnostics, Urogen Pharma, Lucence, CG Oncology, and Janssen, and has received honoraria from SAI MedPartners, Solstice Health Communications, Putnam Associates, and UroToday; JJM is Associate Center Director at Moffitt Cancer Center, has ownership interest in Aleta Biotherapeutics, CG Oncology, Turnstone Biologics, Ankyra Therapeutics, and AffyImmune Therapeutics, and is a paid consultant/paid advisory board member for ONCoPEP, CG Oncology, Turnstone Biologics, Vault Pharma, Ankyra Therapeutics, AffyImmune Therapeutics, UbiVac, Vycellix, and Aleta Biotherapeutics; NS, SK, and MG are or formerly were employees of Nanostring.