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Gamma delta (γδ) T cells reside within human tissues including tumors, but their function in mediating antitumor responses to immune checkpoint inhibition is unknown. Here we show that kidney cancers are infiltrated by Vδ2− γδ T cells, with equivalent representation of Vδ1+ and Vδ1− cells, that are distinct from γδ T cells found in normal human tissues. These tumor-resident Vδ2− T cells can express the transcriptional program of exhausted αβ CD8+ T cells as well as canonical markers of terminal T-cell exhaustion including PD-1, TIGIT and TIM-3. Although Vδ2− γδ T cells have reduced IL-2 production, they retain expression of cytolytic effector molecules and co-stimulatory receptors such as 4-1BB. Exhausted Vδ2− γδ T cells are composed of three distinct populations that lack TCF7, are clonally expanded and express cytotoxic molecules and multiple Vδ2− T-cell receptors. Human tumor-derived Vδ2− γδ T cells maintain cytotoxic function and pro-inflammatory cytokine secretion in vitro. The transcriptional program of Vδ2− T cells in pretreatment tumor biopsies was used to predict subsequent clinical responses to PD-1 blockade in patients with cancer. Thus, Vδ2− γδ T cells within the tumor microenvironment can contribute to antitumor efficacy.

Supplementary Table S1. Patient characteristics by cohort. Supplementary Table S2. Treatment-related toxicity. Supplementary Table S3. Individual clinical response of patients treated with ipilimumab at {greater than or equal to} 3mg/kg/dose. Supplementary Table S4. Comparison of absolute counts of T cell subsets between LTS and STS.

Cytotoxic T-lymphocyte–associated antigen-4 (CTLA-4) blockade can induce tumor regression and improved survival in cancer patients. This treatment can enhance adaptive immune responses without an exogenous vaccine, but the immunologic biomarkers associated with improved clinical outcome in cancer patients are not fully established. A phase Ib trial in patients with metastatic, castration-resistant prostate cancer was performed combining ipilimumab with sargramostim (GM-CSF). In addition to evaluating ipilimumab dose, patients were followed clinically for response and overall survival, and for immunomodulation of circulating T cells. PSA declines of ≥50% and radiographic responses were observed at doses of ≥3 mg/kg/dose. Timing of clinical responses could be either immediate or delayed. Durable responses were also observed off treatment. A subset of patients experienced long-term survival with or without objective clinical responses. The relationship between T-cell phenotype in peripheral blood and overall survival was examined retrospectively. We found that the treatment induced an increase in the levels of CD4+ effector T (Teff) cells, regulatory T cells, PD-1+ CD4 Teff cells, and PD-1+ CD8 T cells. However, these increased levels were not associated with overall survival. Instead, low pretreatment baseline levels of PD-1+ CD4 Teff cells were found to correlate with longer overall survival. Furthermore, baseline levels of PD-1+ CD4 Teff cells from patients with shorter overall survival were higher than from cancer-free male control subjects. These results suggest that preexisting expression of immunologic checkpoint marker PD-1 on CD4 Teff cells may help identify patients that may benefit from ipilimumab treatment. Cancer Immunol Res; 3(9); 1008–16. ©2015 AACR.

Supplementary Figure S1. Patient baseline characteristics.

The supplementary file contains 3 supplemental figures. Supplementary Figure S1 shows the kinetics of Ki67 expression by CD4 and CD8 T cells following treatment with ipilimumab. Supplementary Figure S2 shows the change in TCR clonality over time following treatment with ipilimumab. Supplementary Figure S3 shows the changes in clonotype frequencies of sorted CD4+ and CD8+ T cells with ipilimumab.

While immune checkpoint blockade elicits efficacious responses in many patients with cancer, it also produces a diverse and unpredictable number of immune-related adverse events (IRAE). Mechanisms driving IRAEs are generally unknown. Because CTLA-4 blockade leads to proliferation of circulating T cells, we examined in this study whether ipilimumab treatment leads to clonal expansion of tissue-reactive T cells. Rather than narrowing the T-cell repertoire to a limited number of clones, ipilimumab induced greater diversification in the T-cell repertoire in IRAE patients compared with patients without IRAEs. Specifically, ipilimumab triggered increases in the numbers of clonotypes, including newly detected clones and a decline in overall T-cell clonality. Initial broadening in the repertoire occurred within 2 weeks of treatment, preceding IRAE onset. IRAE patients exhibited greater diversity of CD4+ and CD8+ T cells, but showed no differences in regulatory T-cell numbers relative to patients without IRAEs. Prostate-specific antigen responses to ipilimumab were also associated with increased T-cell diversity. Our results show how rapid diversification in the immune repertoire immediately after checkpoint blockade can be both detrimental and beneficial for patients with cancer. Cancer Res; 77(6); 1322–30. ©2016 AACR.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease. Knowledge of circulating immune cell types and states associated with SLE remains incomplete. We profiled more than 1.2 million peripheral blood mononuclear cells (162 cases, 99 controls) with multiplexed single-cell RNA sequencing (mux-seq). Cases exhibited elevated expression of type 1 interferon–stimulated genes (ISGs) in monocytes, reduction of naïve CD4(+) T cells that correlated with monocyte ISG expression, and expansion of repertoire-restricted cytotoxic GZMH(+) CD8(+) T cells. Cell type–specific expression features predicted case-control status and stratified patients into two molecular subtypes. We integrated dense genotyping data to map cell type–specific cis–expression quantitative trait loci and to link SLE-associated variants to cell type–specific expression. These results demonstrate mux-seq as a systematic approach to characterize cellular composition, identify transcriptional signatures, and annotate genetic variants associated with SLE. INTRODUCTION: Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease with elevated prevalence in women and individuals of Asian, African, and Hispanic ancestry. Bulk transcriptomic profiling has implicated increased type 1 interferon signaling, dysregulated lymphocyte activation, and failure of apoptotic clearance as hallmarks of disease. Many genes participating in these processes are proximal to the ~100 loci associated with SLE. Despite this progress, a comprehensive census of circulating immune cells in SLE remains incomplete, and annotating the cell types and contexts that mediate genetic associations remains challenging. RATIONALE: Historically, flow cytometry and bulk transcriptomic analyses were used to profile the composition and gene expression of circulating immune cells in SLE. However, flow cytometry is biased by its use of a limited set of known markers, whereas bulk transcriptomic profiling does not have sufficient power to detect cell type–specific expression differences. Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) holds potential as a comprehensive and unbiased approach to simultaneously profile the composition and transcriptional states of circulating immune cells. However, application of scRNA-seq to population cohorts has been limited by sample throughput, cost, and susceptibility to technical variability. To overcome these limitations, we previously developed multiplexed scRNA-seq (mux-seq) to enable systematic and cost-effective scRNA-seq of population cohorts. RESULTS: We used mux-seq to profile more than 1.2 million PBMCs from 162 SLE cases and 99 healthy controls of either Asian or European ancestry. SLE cases exhibited differences in both the composition and state of PBMCs. Analysis of lymphocyte composition revealed a reduction in naïve CD4(+) T cells and an increase in repertoire-restricted GZMH(+) CD8(+) T cells. Analysis of transcriptomic profiles across eight cell types revealed that classical monocytes expressed the highest levels of both pan–cell type and myeloid-specific type 1 interferon-stimulated genes (ISGs). The expression of ISGs in monocytes was inversely correlated with naïve CD4(+) T cell abundance. Cell type–specific expression features accurately predicted case-control status and stratified patients into molecular subtypes. By integrating genotyping data and using a novel matrix decomposition method, we mapped shared and cell type–specific cis–expression quantitative trait loci (cis-eQTLs) across eight cell types. Cell type–specific cis-eQTLs were enriched for regions of open chromatin specific to the same or related cell types. Joint analysis of cis-eQTLs and genome-wide association study results enabled identification of cell types relevant to immune-mediated diseases, fine-mapping of disease-associated loci, and discovery of novel SLE associations. Interaction analysis identified variants whose effects on gene expression are further modified by interferon activation across patients. CONCLUSION: SLE remains challenging to diagnose and treat. The heterogeneity of disease manifestations and treatment response highlight the need for improved molecular characterization. In a large multiethnic cohort, we demonstrate mux-seq as a systematic approach to characterize cellular composition, identify cell type–specific transcriptomic signatures, and annotate genetic variants associated with SLE.

Background Soft-tissue sarcomas (STS) are a rare group of mesenchymal malignancies that account for approximately 1% of adult human cancer. Undifferentiated pleomorphic sarcoma (UPS) is one of the most common subtypes of adult STS. Clinical stratification of UPS patients has not evolved for decades and continues to rely on tumor-centric metrics including tumor size and depth. Our understanding of how the tumor microenvironment correlates to these clinicopathologic parameters remains limited. Methods Here, we performed single-cell flow cytometric immune-based profiling of 15 freshly resected UPS tumors and integrated this analysis with clinical, histopathologic, and outcomes data using both a prospective and retrospective cohort of UPS patients. Results We uncovered a correlation between physiologic and anatomic properties of UPS tumors and the composition of immune cells in the tumor microenvironment. Specifically, we identified an inverse correlation between tumor-infiltrating CD8 + T cells and UPS tumor size; and a positive correlation between tumor-infiltrating CD8 + T cells and overall survival. Moreover, we demonstrate an association between anatomical location (deep or superficial) and frequency of CD4 + PD1hi infiltrating T cells in UPS tumors. Conclusions Our study provides an immune-based analysis of the tumor microenvironment in UPS patients and describes the different composition of tumor infiltrating lymphocytes based on size and tumor depth.

BackgroundTherapeutic options currently available for metastatic castration-resistant prostate cancer (mCRPC) do not extend median overall survival >6 months. Therefore, the development of novel and effective therapies for mCRPC represents an urgent medical need. T cell engagers (TCEs) have emerged as a promising approach for the treatment of mCRPC due to their targeted mechanism of action. However, challenges remain in the clinic due to the limited efficacy of TCEs observed thus far in solid tumors as well as the toxicities associated with cytokine release syndrome (CRS) due to the usage of high-affinity anti-CD3 moieties such as OKT3.MethodsUsing genetically engineered transgenic rats (UniRat and OmniFlic) that express fully human IgG antibodies together with an NGS-based antibody discovery pipeline, we developed TNB-585, an anti-CD3xPSMA TCE for the treatment of mCRPC. TNB-585 pairs a tumor-targeting anti-PSMA arm together with a unique, low-affinity anti-CD3 arm in bispecific format. We tested TNB-585 in T cell-redirected cytotoxicity assays against PSMA+ tumor cells in both two-dimensional (2D) cultures and three-dimensional (3D) spheroids as well as against patient-derived prostate tumor cells. Cytokines were measured in culture supernatants to assess the ability of TNB-585 to induce tumor killing with low cytokine release. TNB-585-mediated T cell activation, proliferation, and cytotoxic granule formation were measured to investigate the mechanism of action. Additionally, TNB-585 efficacy was evaluated in vivo against C4-2 tumor-bearing NCG mice.ResultsIn vitro, TNB-585 induced activation and proliferation of human T cells resulting in the killing of PSMA+ prostate tumor cells in both 2D cultures and 3D spheroids with minimal cytokine release and reduced regulatory T cell activation compared with a positive control antibody that contains the same anti-PSMA arm but a higher affinity anti-CD3 arm (comparable with OKT3). In addition, TNB-585 demonstrated potent efficacy against patient-derived prostate tumors ex vivo and induced immune cell infiltration and dose-dependent tumor regression in vivo.ConclusionsOur data suggest that TNB-585, with its low-affinity anti-CD3, may be efficacious while inducing a lower incidence and severity of CRS in patients with prostate cancer compared with TCEs that incorporate high-affinity anti-CD3 domains.

Summary Responses to anti-PD-1 immunotherapy occur but are infrequent in bladder cancer. The specific T cells that mediate tumor rejection are unknown. T cells from human bladder tumors and non-malignant tissue were assessed with single-cell RNA and paired T cell receptor (TCR) sequencing of 30,604 T cells from 7 patients. We find that the states and repertoires of CD8+ T cells are not distinct in tumors compared with non-malignant tissues. In contrast, single-cell analysis of CD4+ T cells demonstrates several tumor-specific states, including multiple distinct states of regulatory T cells. Surprisingly, we also find multiple cytotoxic CD4+ T cell states that are clonally expanded. These CD4+ T cells can kill autologous tumors in an MHC class II-dependent fashion and are suppressed by regulatory T cells. Further, a gene signature of cytotoxic CD4+ T cells in tumors predicts a clinical response in 244 metastatic bladder cancer patients treated with anti-PD-L1., Graphical Abstract, Highlights • Human bladder tumors contain multiple clonally expanded cytotoxic CD4+ T cell states • Cytotoxic CD4+ T cells can kill autologous tumors in an MHC class II-dependent fashion • Autologous regulatory T cells can inhibit the activity of cytotoxic CD4+ T cells • A cytotoxic CD4+ gene signature predicts response to anti-PD-L1 in bladder cancer, Single-cell RNA and paired T cell receptor sequencing highlights enrichment of cytotoxic CD4+ T cells rather than CD8+ T cells in human bladder cancer. These CD4+ T cells are capable of killing autologous tumor cells and are subjected to inhibition by Tregs.

BackgroundCTLA-4 blockade with ipilimumab is Food and Drug Administration-approved for melanoma as a monotherapy and has been shown to modulate the circulating T-cell repertoire. We have previously reported clinical trials combining CTLA-4 blockade with granulocyte-macrophage colony-stimulating factor (GM-CSF) in metastatic melanoma patients and in metastatic castration resistant prostate cancer (mCRPC) patients. Here, we investigate the effect that cancer type has on circulating T cells in metastatic melanoma and mCRPC patients, treated with ipilimumab and GM-CSF.MethodsWe used next-generation sequencing of T-cell receptors (TCR) to compare the circulating T cells of melanoma and mCRPC patients receiving the same treatment with ipilimumab and GM-CSF by Wilcoxon rank sum test. Flow cytometry was utilized to investigate specific T-cell populations. TCR sequencing results were correlated with each T-cell subpopulation by Spearman’s rank correlation coefficient. Of note, 14 metastatic melanoma patients had samples available for TCR sequencing and 21 had samples available for flow cytometry analysis; 37 mCRPC patients had samples available for sequencing of whom 22 have TCR data available at both timepoints; 20 of these patients had samples available for flow cytometry analysis and 16 had data available at both timepoints.ResultsWhile melanoma and mCRPC patients had similar pretreatment circulating T-cell counts, treatment induces greater expansion of circulating T cells in melanoma patients. Metastatic melanoma patients have a higher proportion of clones that increased more than fourfold after the treatment compared with mCRPC patients (18.9% vs 11.0%, p=0.017). Additionally, melanoma patients compared with mCRPC patients had a higher ratio of convergent frequency (1.22 vs 0.60, p=0.012). Decreases in clonality induced by treatment are associated with baseline CD8+ T-cell counts in both patient groups, but are more pronounced in the melanoma patients (r=−0.81, pTrial registration numbersNCT00064129;NCT01363206.

© Sun et al. Tumor endothelial cells (TEC) play an indispensible role in tumor growth and metastasis although much of the detailed mechanism still remains elusive. In this study we characterized and compared the global gene expression profiles of TECs and control ECs isolated from human breast cancerous tissues and reduction mammoplasty tissues respectively by single cell RNA sequencing (scRNA-seq). Based on the qualified scRNA-seq libraries that we made, we found that 1302 genes were differentially expressed between these two EC phenotypes. Both principal component analysis (PCA) and heat map-based hierarchical clustering separated the cancerous versus control ECs as two distinctive clusters, and MetaCore disease biomarker analysis indicated that these differentially expressed genes are highly correlated with breast neoplasm diseases. Gene Set Enrichment Analysis software (GSEA) enriched these genes to extracellular matrix (ECM) signal pathways and highlighted 127 ECMassociated genes. External validation verified some of these ECM-associated genes are not only generally overexpressed in various cancer tissues but also specifically overexpressed in colorectal cancer ECs and lymphoma ECs. In conclusion, our data demonstrated that ECM-associated genes play pivotal roles in breast cancer EC biology and some of them could serve as potential TEC biomarkers for various cancers.

While immune checkpoint blockade elicits efficacious responses in many patients with cancer, it also produces a diverse and unpredictable number of immune-related adverse events (IRAE). Mechanisms driving IRAEs are generally unknown. Because CTLA-4 blockade leads to proliferation of circulating T cells, we examined in this study whether ipilimumab treatment leads to clonal expansion of tissue-reactive T cells. Rather than narrowing the T-cell repertoire to a limited number of clones, ipilimumab induced greater diversification in the T-cell repertoire in IRAE patients compared with patients without IRAEs. Specifically, ipilimumab triggered increases in the numbers of clonotypes, including newly detected clones and a decline in overall T-cell clonality. Initial broadening in the repertoire occurred within 2 weeks of treatment, preceding IRAE onset. IRAE patients exhibited greater diversity of CD4+ and CD8+ T cells, but showed no differences in regulatory T-cell numbers relative to patients without IRAEs. Prostate-specific antigen responses to ipilimumab were also associated with increased T-cell diversity. Our results show how rapid diversification in the immune repertoire immediately after checkpoint blockade can be both detrimental and beneficial for patients with cancer. Cancer Res; 77(6); 1322–30. ©2016 AACR.

Immune responses involve coordination across cell types and tissues. However, studies in cancer immunotherapy have focused heavily on local immune responses in the tumor microenvironment. To investigate immune activity more broadly, we performed an organism-wide study in genetically engineered cancer models using mass cytometry. We analyzed immune responses in several tissues after immunotherapy by developing intuitive models for visualizing single-cell data with statistical inference. Immune activation was evident in the tumor and systemically shortly after effective therapy was administered. However, during tumor rejection, only peripheral immune cells sustained their proliferation. This systemic response was coordinated across tissues and required for tumor eradication in several immunotherapy models. An emergent population of peripheral CD4 Tcells conferred protection against new tumors and was significantly expanded in patients responding to immunotherapy. These studies demonstrate the critical impact of systemic immune responses that drive tumor rejection.

Immune checkpoint blockers (ICB) have become pivotal therapies in the clinical armamentarium against metastatic melanoma (MMel). Given the frequency of immune related adverse events and increasing use of ICB, predictors of response to CTLA-4 and/or PD-1 blockade represent unmet clinical needs. Using a systems biology-based approach to an assessment of 779 paired blood and tumor markers in 37 stage III MMel patients, we analyzed association between blood immune parameters and the functional immune reactivity of tumor-infiltrating cells after ex vivo exposure to ICB. Based on this assay, we retrospectively observed, in eight cohorts enrolling 190 MMel patients treated with ipilimumab, that PD-L1 expression on peripheral T cells was prognostic on overall and progression-free survival. Moreover, detectable CD137 on circulating CD8+ T cells was associated with the disease-free status of resected stage III MMel patients after adjuvant ipilimumab + nivolumab (but not nivolumab alone). These biomarkers should be validated in prospective trials in MMel., The clinical management of metastatic melanoma requires predictors of the response to checkpoint blockade. Here, the authors use immunological assays to identify potential prognostic/predictive biomarkers in circulating blood cells and in tumor-infiltrating lymphocytes from patients with resected stage III melanoma.

Resistance to checkpoint-blockade treatments is a challenge in the clinic. We found that although treatment with combined anti-CTLA-4 and anti-PD-1 improved control of established tumors, this combination compromised anti-tumor immunity in the low tumor burden (LTB) state in pre-clinical models as well as in melanoma patients. Activated tumor-specific Tcells expressed higher amounts of interferon-γ (IFN-γ) receptor and were more susceptible to apoptosis than naive Tcells. Combination treatment induced deletion of tumor-specific Tcells and altered the Tcell repertoire landscape, skewing the distribution of Tcells toward lower-frequency clonotypes. Additionally, combination therapy induced higher IFN-γ production in the LTB state than in the high tumor burden (HTB) state on a per-cell basis, reflecting a less exhausted immune status in the LTB state. Thus, elevated IFN-γ secretion in the LTB state contributes to the development of an immune-intrinsic mechanism of resistance to combination checkpoint blockade, highlighting the importance of achieving the optimal magnitude of immune stimulation for successful combination immunotherapy strategies.

A myriad of combination immunotherapies is being developed as cancer treatments with the goal of enhancing antitumor immunity. We found that depending on the disease setting, combined immune checkpoint blockade may not always lead to synergistic antitumor effects. While combined anti-CTLA-4 and anti-PD-1 improves control of established tumors, this combination can paradoxically compromise antitumor immunity in the low tumor burden state in preclinical models as well as in melanoma patients. This paradoxical outcome results from treatment-induced apoptosis of tumor-specific T cells. These changes further alter the overall T-cell receptor repertoire. Activated tumor-specific T cells express higher levels of IFN-γ receptor and are more susceptible to apoptosis. Deficiency of IFN-γ receptor on immune cells rescues this phenotype and restores antitumor activity. Additionally, tumor-specific T cells lacking the IFN-γ receptor demonstrate a significant survival advantage compared to their wild-type counterparts in tumor-bearing mice receiving combination therapy. Finally, combination therapy induces significantly higher levels of IFN-γ in the low versus high tumor burden state on a per cell basis, reflecting their less exhausted immune status. Thus, the optimal immunotherapy strategy may depend on disease context, and may not always favor more potent combination immunotherapies. Citation Format: Chien-Chun Steven Pai, Xiaoqing Lu, Donald Simons, Michel DuPage, Kole T. Roybal, Mingyi Chen, Serena Kwek, Amy-Jo Casbon, Gillian A. Kinsbury, Lawrence Fong. Clonal deletion of tumor-specific T cells by combination checkpoint blockade compromises antitumor efficacy in low tumor burden states [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2981.

Bladder cancer can be responsive to immunotherapies such as anti-PD-1 and anti-PD-L1 checkpoint inhibitors, but overall response rates are low. Tumor-resident T cells demonstrate considerable heterogeneity as far as antigenic repertoire and phenotype. Whether the bladder tumor environment is enriched for specific types of T cells with a particular functional profile and antigenic specificity, and whether these tumor-specific populations are associated with treatment or response to immunotherapy, remains unclear. We performed single-cell RNA sequencing of CD4+ and CD8+ T cells from localized human bladder tumors and paired adjacent non-malignant bladder. We also assessed resected bladder tumors treated with neoadjuvant chemotherapy or atezolizumab (anti-PD-L1) as part of an ongoing phase II trial. We find both known and unexpected CD4+ T cell functional populations that are specific to the tumor microenvironment, including regulatory T cells and a novel population of cytotoxic CD4+ T cells. Furthermore, by combining whole-transcriptome data with paired T cell receptor identification on single cells, we find that tumor-specific CD4+ populations are clonally expanded and utilize an antigenic repertoire which is distinct from uninvolved bladder. These findings provide evidence for specialization of both phenotype and antigenic specificity of CD4+ T cells in the bladder tumor environment, and indicate that a limited number of tumor-specific antigens may drive in situ expansion and differentiation of specialized CD4+ subsets whose function may be critical to tumor control. Citation Format: David Yoonsuk Oh, Serena S. Kwek, Serghei Mangul, Siddharth S. Raju, Sasha Targ, Arun Burra, Eric Chow, Dvir Aran, Sima Porten, Maxwell V. Meng, Terence W. Friedlander, Chun Jimmie Ye, Lawrence Fong. Single-cell discrimination of altered human T cell states in the bladder tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4688.

CTL-associated antigen 4 (CTLA4) is a costimulatory molecule expressed on activated T cells that delivers an inhibitory signal to these T cells. CTLA4 blockade with antibody treatment has been shown to augment antitumor immunity in animal models and is being developed as a treatment for cancer patients. As has been seen in preclinical models, combining CTLA4 blockade and granulocyte macrophage colony-stimulating factor (GM-CSF)–based immunotherapies can enhance the antitumor efficacy of this approach. We therefore examined whether CTLA4 blockade could be combined with GM-CSF administration. We treated 24 patients with metastatic, castration-resistant prostate cancer in a phase I trial where sequential cohorts were treated with increasing doses of ipilimumab, a fully human anti-CTLA4 antibody. Study subjects also received s.c. injections of GM-CSF at a fixed dose. Of the six patients treated at the highest dose level, three had confirmed PSA declines of >50%, including one patient that had a partial response in visceral metastases. Expansion of activated, circulating CD25+ CD69+ CD8+ T cells occurred more frequently at higher doses of treatment and was greater in magnitude than was seen in patients who received the same doses of either ipilimumab or GM-CSF alone. By screening sera with protein arrays, we showed that our treatment can induce antibody responses to NY-ESO-1. These results show that this combination immunotherapy can induce the expansion not only of activated effector CD8 T cells in vivo but also of T cells that are specific for known tumor-associated antigens from the endogenous immune repertoire. [Cancer Res 2009;69(2):609–15]

Cytotoxic T-lymphocyte–associated antigen-4 (CTLA-4) blockade can induce tumor regression and improved survival in cancer patients. This treatment can enhance adaptive immune responses without an exogenous vaccine, but the immunologic biomarkers associated with improved clinical outcome in cancer patients are not fully established. A phase Ib trial in patients with metastatic, castration-resistant prostate cancer was performed combining ipilimumab with sargramostim (GM-CSF). In addition to evaluating ipilimumab dose, patients were followed clinically for response and overall survival, and for immunomodulation of circulating T cells. PSA declines of ≥50% and radiographic responses were observed at doses of ≥3 mg/kg/dose. Timing of clinical responses could be either immediate or delayed. Durable responses were also observed off treatment. A subset of patients experienced long-term survival with or without objective clinical responses. The relationship between T-cell phenotype in peripheral blood and overall survival was examined retrospectively. We found that the treatment induced an increase in the levels of CD4+ effector T (Teff) cells, regulatory T cells, PD-1+ CD4 Teff cells, and PD-1+ CD8 T cells. However, these increased levels were not associated with overall survival. Instead, low pretreatment baseline levels of PD-1+ CD4 Teff cells were found to correlate with longer overall survival. Furthermore, baseline levels of PD-1+ CD4 Teff cells from patients with shorter overall survival were higher than from cancer-free male control subjects. These results suggest that preexisting expression of immunologic checkpoint marker PD-1 on CD4 Teff cells may help identify patients that may benefit from ipilimumab treatment. Cancer Immunol Res; 3(9); 1008–16. ©2015 AACR.

The human prostatic carcinoma cell line LNCaP is sensitive to TNF-alpha treatment and expresses wild-type p53. To analyse the possible role of p53 in TNF-alpha-mediated apoptosis, we generated a derivative of LNCaP, LN-56, expressing a dominant-negative element of p53, GSE56. P53 inactivation in LN-56 was associated with an increased resistance to apoptosis induced by TNF-alpha. Surface expression of TNF-alpha receptors was unchanged in LN-56 compared to LNCaP. TNF-alpha treatment resulted in accumulation of p53 in LNCaP and upregulation of p21/WAF1. Activation of caspase-7 and PARP proteolysis were delayed in LN-56 under TNF-alpha treatment. TNF-alpha-induced apoptosis in LNCaP cells was accompanied by caspase-dependent proteolysis of p21/WAF1 and Rb, which was significantly attenuated in LN-56. Cytochrome c release was induced by TNF-alpha treatment in both cell lines, but caspase-9 was not activated. LNCaP and LN-56 were injected s.c. in nude mice and tumors were identified in all LN-56, but not LNCaP, bearing mice indicating that p53 plays an important role in growth control of prostatic neoplasms. Interestingly, accumulation of p53 in TNF-alpha-treated LNCaP cells was decreased in the presence of the caspase inhibitor Z-VAD-FMK, suggesting a new role of activated caspases in acceleration of p53 response. In summary, these results indicate that p53 is involved in TNF-alpha-mediated apoptosis in LNCaP.

The human ING1 gene encodes nuclear protein p33ING1, previously shown to cooperate with p53 in cell growth control (Garkavtsev, I., Grigorian, I. A., Ossovskaya, V. S., Chernov, M. V., Chumakov, P. M., and Gudkov, A. V. (1998) Nature 391, 295–298). p33ING1belongs to a small family of proteins from human, mouse, and yeast of approximately the same size that show significant similarity to one another within the C-terminal PHD finger domain and also contain an additional N-terminal region with subtle but reliably detectable sequence conservation. Mouse ing1 is transcribed from three differently regulated promoters localized within a 4-kilobase pair region of genomic DNA. The resulting transcripts share a long common region encoded by a common exon and differ in their 5′-exon sequences. Two transcripts are translated into the same protein of 185 amino acids, the mouse equivalent of the human p33ING1, while the third transcript encodes a longer protein that has 94 additional N-terminal amino acids. Overexpression of the longer protein interferes with the accumulation of p53 protein and activation of p53-responsive promoters after DNA damage. Between the two products ofing1, only the longer one forms a complex with p53 detectable by immunoprecipitation. These results indicate that a single gene, ing1, encodes both p53-suppressing and p53-activating proteins that are regulated by alternative promoters.

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a surface receptor on activated T cells that delivers an inhibitory signal, serving as an immune checkpoint. Treatment with anti-CTLA-4 antibodies can induce clinical responses to different malignancies, but the nature of the induced antigen-specific recognition is largely unknown. Using microarrays spotted with over 8000 human proteins, we assessed the diversity of antibody responses modulated by treatment with CTLA-4-blockade and granulocyte macrophage colony-stimulating factor (GM-CSF). We find that advanced prostate cancer patients who clinically respond to treatment also develop enhanced antibody responses to a higher number of antigens than non-responders. These induced antibody responses targeted antigens to which preexisting antibodies are more likely to be present in the clinical responders compared to non-responders. The majority of antibody responses are patient-specific, but immune responses against antigens shared among clinical responders are also detected. One of these shared antigens is p21-activated kinase 6 (Pak6), which is expressed in prostate cancer and to which CD4+ T cell responses were also induced. Moreover, immunization with Pak6 can be both immunogenic and protective in mouse tumor models. These results demonstrate that immune checkpoint blockade modulates antigen-specific responses to both individualized and shared antigens, some of which can mediate anti-tumor responses.

Although cancer cells can be immunogenic, tumour progression is associated with the evasion of immunosurveillance, the promotion of tumour tolerance and even the production of pro-tumorigenic factors by immune cells. Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) represents a crucial immune checkpoint, the blockade of which can potentiate anti-tumour immunity. CTLA4-blocking antibodies are now an established therapeutic approach for malignant melanoma, and clinical trials with CTLA4-specific antibodies in prostate cancer have also shown clinical activity. This treatment may provide insights into the targets that the immune system recognizes to drive tumour regression, and could potentially improve both outcome and toxicity for patients with prostate cancer.

Chronic prostatitis is a common disease of unclear etiology and has no specific treatment. Mice deficient in the expression of the autoimmune regulator (Aire) gene, which are defective in thymic expression of self antigens and central tolerance, develop spontaneous prostatitis. In this study, we found that Aire-deficient mice developed spontaneous B and T cell immune responses to a prostate autoantigen, seminal vesicle secretory protein 2 (SVS2), which we believe to be novel. We show that thymic expression of this self antigen was Aire dependent. Moreover, prostatitis was induced in WT mice through immunization with SVS2, demonstrating that immunity to SVS2 was sufficient to induce prostatitis. The clinical relevance of this antigen was highlighted by our observation that patients with chronic prostatitis possessed specific autoantibodies against the human SVS2-like seminal vesicle protein semenogelin. These results provide direct evidence that spontaneous chronic prostatitis is an autoimmune disease and is regulated by both central and peripheral tolerance. Moreover, SVS2 and semenogelin are among the relevant autoantigens in mice and humans, respectively.

Co-amplification at chromosomes 8p11-8p12 and 11q12-11q14 occurs often in breast tumors, suggesting possible cooperation between genes in these regions in oncogenesis. We used high-resolution array comparative genomic hybridization (array CGH) to map the minimal amplified regions. The 8p and 11q amplicons are complex and consist of at least four amplicon cores at each site. Candidate oncogenes mapping to these regions were identified by combining copy number and RNA and protein expression analyses. These studies also suggested that CCND1 at 11q13 induced expression of ZNF703 mapping at 8p12, which was subsequently shown to be mediated by the Rb/E2F pathway. Nine candidate oncogenes from 8p12 and four from 11q13 were further evaluated for oncogenic function. None of the genes individually promoted colony formation in soft agar or collaborated with each other functionally. On the other hand, FGFR1 and DDHD2 at 8p12 cooperated functionally with MYC, whereas CCND1 and ZNF703 cooperated with a dominant negative form of TP53. These observations highlight the complexity and functional consequences of the genomic rearrangements that occur in these breast cancer amplicons, including transcriptional cross-talk between genes in the 8p and 11q amplicons, as well as their cooperation with major pathways of tumorigenesis.

We conducted a phase II clinical trial of anti-CTLA-4 antibody (ipilimumab) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in 22 patients with metastatic melanoma and determined clinical outcomes and immunologic responses. The treatment consisted of a 3-mo induction with ipilimumab at 10mg/kg administered every 3weeks for four doses in combination with GM-CSF at 125µg/m2 for 14d beginning on the day of the ipilimumab infusion and then GM-CSF for 3mo on the same schedule without ipilimumab. This was followed by maintenance therapy with the combination every 3mo for up to 2y or until disease progression or unacceptable toxicity. Blood samples for determination of immune subsets were obtained before treatment, at week 3 (end of cycle 1) and at week 6 (end of cycle 2). Blood samples were also obtained from seven subjects who were cancer-free. The immune response disease control (irDC) rate at 24weeks was 41% and the overall response rate (ORR) was 32%. The median progression free-survival (PFS) was 3.5mo and the median overall survival (OS) was 21.1mo. 41% of the patients experienced Grade 3 to 4 adverse events. We conclude that this combination is safe and the results suggest the combination may be more effective than ipilimumab monotherapy. Further, the results suggest that lower levels of CD4+ effector T cells but higher levels of CD8+ T cells expressing PD-1 at pre-treatment could be a potential biomarker for disease control in patients who receive immunotherapy with ipilimumab and GM-CSF. Further trials of this combination are warranted.

Background: Granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim) is a bone marrow growth factor that is often used as an adjuvant in cancer vaccine clinical trials and has the potential to enhance antitumor efficacy in combination with immune checkpoint blockade in preclinical studies and clinical trials. The effect of GM-CSF remains controversial, however. In this study, we sought to characterize the systemic and tissue-specific effects of GM-CSF in prostate cancer patients in the preoperative setting. Methods: Patients with localized prostate cancer were treated on an open-label phase I/II study of neoadjuvant GM-CSF prior to planned radical prostatectomy (NCT00305669). Patients received GM-CSF 250mcg/m2 subcutaneously on days 1-14 (cohort 1), days1-21 (cohort 2), or days 1-28 (cohort 3), and underwent radical prostatectomy (RP) within 5 days after the last dose of GM-CSF. Blood collected at baseline, day 14, pre-RP, and 6 weeks post-RP were analyzed for immune cell subsets by flow cytometry. Immune infiltration in RP tissue were evaluated by immunohistochemistry and quantified by digital image analysis with comparison to RP tissue from untreated prostate cancer patients as controls. Results: 18 patients were evaluated with 6 patients at each dose level. Treatment with GM-CSF was associated with an increase in the number of myeloid cells including myeloid dendritic cells. Treatment was also associated with increases in circulating conventional CD4 and CD8 T cells and NK cells. The number of FoxP3+ regulatory CD4+ T cells was also increased, but to a much lower magnitude. At the time of radical prostatectomy, treatment was associated with a recruitment of CD3+ T cells to the tumor rim, and with cohort 3, at the centers of tumor. These T cells were predominantly CD8+ T cells, although conventional CD4+ T cells were also increased. No increase in monocytes or dendritic cells was seen in the tissue. Conclusion: Daily injection of systemic GM-CSF induces an increase in circulating antigen presenting cells as well as T and NK cells. While this treatment does not enhance the frequency of antigen presenting cells in the tumor microenvironment, this treatment recruits effector CD8+ T cells into the tumor rims. Citation Format: Xiao X. Wei, Stephen Chan, Jera Lewis, Serena Kwek, Vinh Dao, Lawrence Fong. Recruitment of effector T cells into the tumor rim and center with neoadjuvant systemic GM-CSF in patients with localized prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 276. doi:10.1158/1538-7445.AM2015-276

Inactivation of p53 and expression of Bcl-2, frequently occurring during tumor progression, have different prognostic value: while inactivation of p53 is generally associated with unfavorable prognosis, expression of Bcl-2 often correlates with better clinical outcome and delays selection of metastatic variants of experimental tumors. To analyze the mechanisms underlying the "anti-progression" function of Bcl-2, we engineered tumor cell variants differing in their p53 status and Bcl-2 expression and compared their expansion in experimental tumors. Although neither p53 suppression nor Bcl-2-expression altered cell growth properties in vitro, both variants showed rapid accumulation in growing tumors in vivo, presumably due to their resistance to hypoxia. However, no expansion of p53-deficient variants occurred in the tumors formed by Bcl-2-overexpressing cells, indicating that p53 deficiency has no selective advantages in the Bcl-2-expressing environment. Importantly, expression of Bcl-2, unlike p53 suppression, did not lead to genomic instability as judged by the frequencies of gene amplification. Thus, acquisition of Bcl-2 expression is as advantageous for tumor cell growth in vivo as is p53 inactivation but does not affect genomic stability and creates the environment restrictive for the expansion of genetically unstable and potentially malignant p53-deficient cells, causing a delay in tumor progression and explaining the different prognostic value of Bcl-2 and p53.

p53 tumor suppressor is a subject of several post-translational modifications, including phosphorylation, ubiquitination and acetylation, which regulate p53 function. A new covalent modification of p53 at lysine 386 by SUMO-1 was recently identified. To elucidate the function of sumoylated p53, we compared the properties of wild type p53 and sumoylation-deficient p53 mutant, K386R. No differences were found between wild type p53 and K386R mutant of p53 in transactivation or growth suppression assays. Moreover, overexpression of SUMO-1 has no effect on p53-regulated transcription. Biochemical fractionation showed that sumoylated p53 is localized in the nucleus and is tightly bound to chromatin structures. p53 and SUMO-1 co-localized in PML nuclear bodies in 293 cells and the nucleoli in MCF7 and HT1080 cells. However, sumoylation-deficient p53 mutant showed a similar pattern of intranuclear localization, suggesting that SUMO-1 does not target p53 to subnuclear structures. These data indicate that SUMO-1 modification of p53 at lysine 386 may not be essential for p53's cellular localization, transcriptional activation, or growth regulation.

Mutations in the p53 tumor suppressor gene are frequently associated with the metastatic stage of tumor progression. Inactivation of p53 was shown to promote metastasis under experimental conditions. To determine the p53 functions that are involved in the control of tumor metastasis, we compared properties of three types of transformed mouse fibroblasts: with intact p53, with p53-mediated apoptosis suppressed by bcl-2 and with p53 inactivated by dominant negative mutants. Although expression of bcl-2 blocked apoptosis in detached cells and increased tumor cell survival in the blood circulation, it was insufficient to affect the ability of p53 to cause cell cycle arrest in detached cells and suppress experimental metastasis. For the suppression of metastasis complete inactivation of p53 was required. We conclude that the apoptotic function of p53 is dispensable for the p53-dependent suppression of experimental metastasis that is presumably achieved by controlling anchorage dependence. These data provide a possible explanation to dramatic differences in values of bcl-2 and mutant p53 as prognostic markers in human cancer.

2578 Background: CTL-associated antigen 4 (CTLA4) is a costimulatory molecule expressed on activated T cells that delivers an inhibitory signal to these T cells thereby serving as an immune checkpo...

CTLA-4 is a costimulatory molecule expressed on activated T cells that delivers an inhibitory signal to these T cells. CTLA4 blockade with antibody treatment has been shown to augment T cell responses and anti-tumor immunity in animal models and is being developed as an immunotherapy for cancer patients. We performed a phase I trial in patients with metastatic, hormone refractory prostate cancer (HRPC) where sequential cohorts of 3-6 patients were treated with escalating doses (0.5, 1.5 or 3 mg/kg) of ipilimumab, a fully human anti-CTLA-4 antibody (Medarex/BMS), given IV on day 1 of each 28-day cycle x 4 cycles. Patients also received GM-CSF (sargramostim, Berlex) 250 mg/m2/d SC on days 1-14 of the 28-day cycles. Patients were monitored for toxicity as well as for T cell activation. PSA and radiographic tests were performed at baseline and through therapy to evaluate for clinical response. 24 patients have been treated in the initial phase of this study. Of 6 patients treated on the highest dose level (3.0 mg/kg x 4), 3 (50%) had confirmed PSA declines of >50%, and one of these patients had a partial response in hepatic metastases. Immune-related adverse events associated with ipilimumab treatment were also seen more frequently at higher doses of treatment. Expansion of CD25+CD69+ CD8 T cells was also seem primarily at the highest dose level. The treatment also induced an antibody and CD8 T cell immune response to NY-ESO-1. These results demonstrate that CTLA-4 blockade induces not only the expansion of activated effector CD8 T cells in vivo in cancer patients, but also can induce from the endogenous T cell repertoire of these patients T cells that are specific for tumor-associated antigens. CD8 T cell activation, toxicity, and clinical responses also appear to be dose-dependent. Supported by NIH P50 CA89520.

Genomic analyses of human cells expressing dihydrofolate reductase provide insight into the effects of genome position on the propensity for a drug-resistance gene to amplify in human cells., Background Amplifications, regions of focal high-level copy number change, lead to overexpression of oncogenes or drug resistance genes in tumors. Their presence is often associated with poor prognosis; however, the use of amplification as a mechanism for overexpression of a particular gene in tumors varies. To investigate the influence of genome position on propensity to amplify, we integrated a mutant form of the gene encoding dihydrofolate reductase into different positions in the human genome, challenged cells with methotrexate and then studied the genomic alterations arising in drug resistant cells. Results We observed site-specific differences in methotrexate sensitivity, amplicon organization and amplification frequency. One site was uniquely associated with a significantly enhanced propensity to amplify and recurrent amplicon boundaries, possibly implicating a rare folate-sensitive fragile site in initiating amplification. Hierarchical clustering of gene expression patterns and subsequent gene enrichment analysis revealed two clusters differing significantly in expression of MYC target genes independent of integration site. Conclusion These studies suggest that genome context together with the particular challenges to genome stability experienced during the progression to cancer contribute to the propensity to amplify a specific oncogene or drug resistance gene, whereas the overall functional response to drug (or other) challenge may be independent of the genomic location of an oncogene.