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Supplemental Methods. Table S1. Frequency of hematopoietic subsets in PBMC at all time points for patient samples used for in vitro immunogenicity experiments Table S2: Clinical characteristics of follicular lymphoma cohorts Table S3. Amino acid changes encoded by somatic mutations identified in candidate genes in follicular lymphoma cohort Table S4. Patient HLA class I alleles for in vitro immunogenicity cohort Figure S1. The number of mutant peptides predicted to bind patient-specific HLA class I molecules in the sequencing cohort. Figure S2. Clinical timeline for 13-patient cohort selected for in vitro immunogenicity studies. Figure S3. CD8 status of mutant peptide-reactive T cells.

Purpose: Cancers accumulate mutations over time, each of which brings the potential for recognition by the immune system. We evaluated T-cell recognition of the tumor mutanome in patients with ovarian cancer undergoing standard treatment.Experimental Design: Tumor-associated T cells from 3 patients with ovarian cancer were assessed by ELISPOT for recognition of nonsynonymous mutations identified by whole exome sequencing of autologous tumor. The relative levels of mutations and responding T cells were monitored in serial tumor samples collected at primary surgery and first and second recurrence.Results: The vast majority of mutations (78/79) were not recognized by tumor-associated T cells; however, a highly specific CD8+ T-cell response to the mutation hydroxysteroid dehydrogenase–like protein 1 (HSDL1)L25V was detected in one patient. In the primary tumor, the HSDL1L25V mutation had low prevalence and expression, and a corresponding T-cell response was undetectable. At first recurrence, there was a striking increase in the abundance of the mutation and corresponding MHC class I epitope, and this was accompanied by the emergence of the HSDL1L25V-specific CD8+ T-cell response. At second recurrence, the HSDL1L25V mutation and epitope continued to be expressed; however, the corresponding T-cell response was no longer detectable.Conclusion: The immune system can respond to the evolving ovarian cancer genome. However, the T-cell response detected here was rare, was transient, and ultimately failed to prevent disease progression. These findings reveal the limitations of spontaneous tumor immunity in the setting of standard treatments and suggest a high degree of ignorance of tumor mutations that could potentially be reversed by immunotherapy. Clin Cancer Res; 20(5); 1125–34. ©2013 AACR.

PDF file - 72K, Tested minimal peptides and corresponding MHC binding scores for Patient 3.

PDF file - 248K, Patients 1 and 2 failed to respond to any of the mutant peptides at any of the time points.

Purpose: CD8+ tumor-infiltrating lymphocytes (TIL) are key mediators of antitumor immunity and are strongly associated with survival in virtually all solid tumors. However, the prognostic effect of CD8+ TIL is markedly higher in the presence of CD20+ B cells, suggesting that cooperative interactions between these lymphocyte subsets lead to more potent antitumor immunity.Experimental Design: We assessed the colocalization patterns, phenotypes, and gene expression profiles of tumor-associated T- and B-lineage cells in high-grade serous ovarian cancer (HGSC) by multicolor IHC, flow cytometry, and bioinformatic analysis of gene expression data from The Cancer Genome Atlas.Results: T cells and B cells colocalized in four types of lymphoid aggregate, ranging from small, diffuse clusters to large, well-organized tertiary lymphoid structures (TLS) resembling activated lymph nodes. TLS were frequently surrounded by dense infiltrates of plasma cells (PC), which comprised up to 90% of tumor stroma. PCs expressed mature, oligoclonal IgG transcripts, indicative of antigen-specific responses. PCs were associated with the highest levels of CD8+, CD4+, and CD20+ TIL, as well as numerous cytotoxicity-related gene products. CD8+ TIL carried prognostic benefit only in the presence of PCs and these other TIL subsets. PCs were independent of mutation load, BRCA1/2 status, and differentiation antigens but positively associated with cancer–testis antigens.Conclusions: PCs are associated with the most robust, prognostically favorable CD8+ TIL responses in HGSC. We propose that TLS facilitate coordinated antitumor responses involving the combined actions of cytolytic T cells and antibody-producing PCs. Clin Cancer Res; 22(12); 3005–15. ©2016 AACR.

This file contains revised supplementary methods for the revised manuscript.

PDF file - 69K, Tested minimal peptides and corresponding MHC binding scores for Patient 2.

PDF file - 46K, Patient MHC class I alleles.

PDF file - 68K, Tested minimal peptides and corresponding MHC binding scores for Patient 1.

PDF file - 133K, Lymphocyte infiltration and MHC expression in HGSC.

Purpose: A fundamental challenge in the era of next-generation sequencing (NGS) is to design effective treatments tailored to the mutational profiles of tumors. Many newly discovered cancer mutations are difficult to target pharmacologically; however, T-cell–based therapies may provide a valuable alternative owing to the exquisite sensitivity and specificity of antigen recognition. To explore this concept, we assessed the immunogenicity of a panel of genes that are common sites of driver mutations in follicular lymphoma, an immunologically sensitive yet currently incurable disease.Experimental Design: Exon capture and NGS were used to interrogate tumor samples from 53 patients with follicular lymphoma for mutations in 10 frequently mutated genes. For 13 patients, predicted mutant peptides and proteins were evaluated for recognition by autologous peripheral blood T cells after in vitro priming.Results: Mutations were identified in 1–5 genes in 81% (43/53) of tumor samples. Autologous, mutation-specific CD8+ T cells were identified in 23% (3/13) of evaluated cases. T-cell responses were directed toward putative driver mutations in CREBBP and MEF2B. Responding T cells showed exquisite specificity for mutant versus wild-type proteins and recognized lymphoma cells expressing the appropriate mutations. Responding T cells appeared to be from the naïve repertoire, as they were found at low frequencies and only at single time points in each patient.Conclusions: Patients with follicular lymphoma harbor rare yet functionally competent CD8+ T cells specific for recurrent mutations. Our results support the concept of using NGS to design individualized immunotherapies targeting common driver mutations in follicular lymphoma and other malignancies. Clin Cancer Res; 22(9); 2226–36. ©2015 AACR.

Supplementary Table S1. Antibody dilutions used in single-color IHC. Supplementary Table S2. Antibody dilutions and chromogens in multi-color IHC. Supplementary Table S3. Additional patient characteristics of cohort A. Supplementary Table S4. Immune cell densities and absolute counts in matched pre- and post-NACT tumor samples with multiple comparisons (n=26).

Supplementary Fig. S1. CONSORT diagram of patient selection and cohort construction. Supplementary Fig. S2. Plasma cell infiltrates before and after NACT. The data was generated and analyzed as in Figure 1. Supplementary Fig. S3. Example of intraepithelial PD-1+ FoxP3+ TIL in a quadruple-color IHC of PD-L1 CD8 PD-1 FoxP3. Supplementary Fig. S4. Unsupervised clustering of TIL based on post-NACT data.

PDF file - 159K, Flow cytometric assessment of the prevalence of Vβ13.6 in TAL from Patient 3.

PDF file - 128K, Defining the minimal peptide containing the HSDL1L25V mutation recognized by Clone 1.

Supplementary methods. Table S1. PC-derived immunoglobulin heavy (IGH) V, D, and J region usage and consensus junction amino acid sequences for distinct VDJ families as determined by Sanger sequencing and IMGT V-QUEST alignment Table S2. Genes with greater than 10-fold differential expression between tumors with high numbers of PC ("PC", n=4) versus no B-lineage cells ("no PC/B cells" n=3), or high numbers of PC versus tumors with CD20+ TIL but without PCs ("B cells", n=2) (based on average transcript counts in NanoString analysis of FFPE tumor tissue). Table S3. Differentiation, overexpressed, and CT antigen genes analyzed for expression in TCGA microarray data. Table S4. List of genes that by hierarchical clustering were differentially expressed in tumors with a PC signature (Fig. S3). Table S5. Clinical characteristics of HGSC patients represented on the 172-case TMA used for survival analysis; P-values refer to results of univariate analyses between the indicated variable and disease-specific survival using one-way ANOVA or chi-squared tests. Table S6. Antibodies used in this study. Fig. S1. Immunoglobulin heavy chain expression in HGSC. Standardized read count (FPKM) data for 9 immunoglobulin heavy chain classes were plotted using TCGA HGSC RNA-seq data. Fig. S2. Lack of association between the PC gene signature and immunogenic mutations, TP53 mutations, and differentiation/overexpressed antigens in HGSC. Fig. S3. Heatmap of CT antigen expression.

Purpose: Some forms of chemotherapy can enhance antitumor immunity through immunogenic cell death, resulting in increased T-cell activation and tumor infiltration. Such effects could potentially sensitize tumors to immunotherapies, including checkpoint blockade. We investigated whether platinum- and taxane-based chemotherapy for ovarian cancer induces immunologic changes consistent with this possibility.Experimental Design: Matched pre- and post-neoadjuvant chemotherapy tumor samples from 26 high-grade serous carcinoma (HGSC) patients were analyzed by immunohistochemistry (IHC) for a large panel of immune cells and associated factors. The prognostic significance of post-chemotherapy TIL patterns was assessed in an expanded cohort (n = 90).Results: Neoadjuvant chemotherapy was associated with increased densities of CD3+, CD8+, CD8+ TIA-1+, PD-1+ and CD20+ TIL. Other immune subsets and factors were unchanged, including CD79a+ CD138+ plasma cells, CD68+ macrophages, and MHC class I on tumor cells. Immunosuppressive cell types were also unchanged, including FoxP3+ PD-1+ cells (putative regulatory T cells), IDO-1+ cells, and PD-L1+ cells (both macrophages and tumor cells). Hierarchical clustering revealed three response patterns: (i) TILhigh tumors showed increases in multiple immune markers after chemotherapy; (ii) TILlow tumors underwent similar increases, achieving patterns indistinguishable from the first group; and (iii) TILnegative cases generally remained negative. Despite the dramatic increases seen in the first two patterns, post-chemotherapy TIL showed limited prognostic significance.Conclusions: Chemotherapy augments pre-existing TIL responses but fails to relieve major immune-suppressive mechanisms or confer significant prognostic benefit. Our findings provide rationale for multipronged approaches to immunotherapy tailored to the baseline features of the tumor microenvironment. Clin Cancer Res; 23(4); 925–34. ©2016 AACR.

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Purpose: Some forms of chemotherapy can enhance antitumor immunity through immunogenic cell death, resulting in increased T-cell activation and tumor infiltration. Such effects could potentially sensitize tumors to immunotherapies, including checkpoint blockade. We investigated whether platinum- and taxane-based chemotherapy for ovarian cancer induces immunologic changes consistent with this possibility. Experimental Design: Matched pre- and post-neoadjuvant chemotherapy tumor samples from 26 high-grade serous carcinoma (HGSC) patients were analyzed by immunohistochemistry (IHC) for a large panel of immune cells and associated factors. The prognostic significance of post-chemotherapy TIL patterns was assessed in an expanded cohort (n = 90). Results: Neoadjuvant chemotherapy was associated with increased densities of CD3+, CD8+, CD8+ TIA-1+, PD-1+ and CD20+ TIL. Other immune subsets and factors were unchanged, including CD79a+ CD138+ plasma cells, CD68+ macrophages, and MHC class I on tumor cells. Immunosuppressive cell types were also unchanged, including FoxP3+ PD-1+ cells (putative regulatory T cells), IDO-1+ cells, and PD-L1+ cells (both macrophages and tumor cells). Hierarchical clustering revealed three response patterns: (i) TILhigh tumors showed increases in multiple immune markers after chemotherapy; (ii) TILlow tumors underwent similar increases, achieving patterns indistinguishable from the first group; and (iii) TILnegative cases generally remained negative. Despite the dramatic increases seen in the first two patterns, post-chemotherapy TIL showed limited prognostic significance. Conclusions: Chemotherapy augments pre-existing TIL responses but fails to relieve major immune-suppressive mechanisms or confer significant prognostic benefit. Our findings provide rationale for multipronged approaches to immunotherapy tailored to the baseline features of the tumor microenvironment. Clin Cancer Res; 23(4); 925–34. ©2016 AACR.

Purpose: CD8+ tumor-infiltrating lymphocytes (TIL) are key mediators of antitumor immunity and are strongly associated with survival in virtually all solid tumors. However, the prognostic effect of CD8+ TIL is markedly higher in the presence of CD20+ B cells, suggesting that cooperative interactions between these lymphocyte subsets lead to more potent antitumor immunity. Experimental Design: We assessed the colocalization patterns, phenotypes, and gene expression profiles of tumor-associated T- and B-lineage cells in high-grade serous ovarian cancer (HGSC) by multicolor IHC, flow cytometry, and bioinformatic analysis of gene expression data from The Cancer Genome Atlas. Results: T cells and B cells colocalized in four types of lymphoid aggregate, ranging from small, diffuse clusters to large, well-organized tertiary lymphoid structures (TLS) resembling activated lymph nodes. TLS were frequently surrounded by dense infiltrates of plasma cells (PC), which comprised up to 90% of tumor stroma. PCs expressed mature, oligoclonal IgG transcripts, indicative of antigen-specific responses. PCs were associated with the highest levels of CD8+, CD4+, and CD20+ TIL, as well as numerous cytotoxicity-related gene products. CD8+ TIL carried prognostic benefit only in the presence of PCs and these other TIL subsets. PCs were independent of mutation load, BRCA1/2 status, and differentiation antigens but positively associated with cancer–testis antigens. Conclusions: PCs are associated with the most robust, prognostically favorable CD8+ TIL responses in HGSC. We propose that TLS facilitate coordinated antitumor responses involving the combined actions of cytolytic T cells and antibody-producing PCs. Clin Cancer Res; 22(12); 3005–15. ©2016 AACR.

Purpose: A fundamental challenge in the era of next-generation sequencing (NGS) is to design effective treatments tailored to the mutational profiles of tumors. Many newly discovered cancer mutations are difficult to target pharmacologically; however, T-cell–based therapies may provide a valuable alternative owing to the exquisite sensitivity and specificity of antigen recognition. To explore this concept, we assessed the immunogenicity of a panel of genes that are common sites of driver mutations in follicular lymphoma, an immunologically sensitive yet currently incurable disease. Experimental Design: Exon capture and NGS were used to interrogate tumor samples from 53 patients with follicular lymphoma for mutations in 10 frequently mutated genes. For 13 patients, predicted mutant peptides and proteins were evaluated for recognition by autologous peripheral blood T cells after in vitro priming. Results: Mutations were identified in 1–5 genes in 81% (43/53) of tumor samples. Autologous, mutation-specific CD8+ T cells were identified in 23% (3/13) of evaluated cases. T-cell responses were directed toward putative driver mutations in CREBBP and MEF2B. Responding T cells showed exquisite specificity for mutant versus wild-type proteins and recognized lymphoma cells expressing the appropriate mutations. Responding T cells appeared to be from the naïve repertoire, as they were found at low frequencies and only at single time points in each patient. Conclusions: Patients with follicular lymphoma harbor rare yet functionally competent CD8+ T cells specific for recurrent mutations. Our results support the concept of using NGS to design individualized immunotherapies targeting common driver mutations in follicular lymphoma and other malignancies. Clin Cancer Res; 22(9); 2226–36. ©2015 AACR.

Objective As a negative regulator of T cells, Programmed Death Ligand 1 (PD-L1) is both an indicator and inhibitor of anti -tumor immune responses, which has led to confusion about its prognostic significance. We investigated the primary source of PD-L1 expression in epithelial ovarian cancer and its relationship to tumor-infiltrating lymphocytes (TIL) and associated gene products. Methods Tissue microarrays containing high-grade serous carcinomas (HGSC) and endometrioid, clear cell and mucinous ovarian cancers from optimally debulked patients were assessed by immunohistochemistry for expression of PD-L1 and other markers (CD68, CD3, CD8, PD-1, CD103, FoxP3 and CD25). The Cancer Genome Atlas was interrogated for associations between PD-L1 expression and immune-related transcriptional and genomic features of HGSC. Results PD-L1 was primarily expressed by tumor-associated CD68 + macrophages rather than tumor cells. PD-L1 + cells frequently co-localized with CD8, CD4 and PD-1 + TIL, CD25 + FoxP3 + Tregs, and other TIL subsets. PD-L1 + cells were prognostically favorable in HGSC. Moreover, the presence of both PD-L1 + cells and CD8 TIL was associated with better prognosis than CD8 TIL alone. PD-L1 gene expression was independent of BRCA status. At the transcriptional level, PD-L1 was associated with both cytolytic (granzyme B, T-bet and IFN-γ) and suppressive (PD-1, CTLA-4, LAG3 and IDO-1) gene products. Conclusions PD-L1 is primarily expressed by macrophages in ovarian cancer and is strongly associated with both cytolytic and regulatory TIL subsets, resulting in a net positive association with survival. Tumors containing PD-L1 + macrophages appear caught in an immunological stalemate that may require multi-pronged immunotherapy to alleviate.

CD25, the alpha subunit of the IL2 receptor, is a canonical marker of regulatory T cells (Treg) and hence has been implicated in immune suppression in cancer. However, CD25 is also required for optimal expansion and activity of effector T cells in peripheral tissues. Thus, we hypothesized that CD25, in addition to demarcating Tregs, might identify effector T cells in cancer. To investigate this possibility, we used multiparameter flow cytometry and IHC to analyze tumor-infiltrating lymphocytes (TIL) in primary high-grade serous carcinomas, the most common and fatal subtype of ovarian cancer. CD25 was expressed primarily by CD4+ TIL, with negligible expression by CD8+ TIL. In addition to conventional CD25+FoxP3+ Tregs, we identified a subset of CD25+FoxP3− T cells that comprised up to 13% of CD4+ TIL. In tumors with CD8+ TIL, CD25+FoxP3− T cells showed a strong positive association with patient survival (HR, 0.56; P = 0.02), which exceeded the negative effect of Tregs (HR, 1.55; P = 0.09). Among CD4+ TIL subsets, CD25+FoxP3− cells expressed the highest levels of PD-1. Moreover, after in vitro stimulation, they failed to produce common T-helper cytokines (IFNγ, TNFα, IL2, IL4, IL10, or IL17A), suggesting that they were functionally exhausted. In contrast, the more abundant CD25−FoxP3− subset of CD4+ TIL expressed low levels of PD-1 and produced T-helper 1 cytokines, yet conferred no prognostic benefit. Thus, CD25 identifies a subset of CD4+FoxP3− TIL that, despite being exhausted at diagnosis, have a strong, positive association with patient survival and warrant consideration as effector T cells for immunotherapy. Cancer Immunol Res; 3(3); 245–53. ©2014 AACR.

SummaryHigh-grade serous ovarian cancer exhibits extensive intratumoral heterogeneity coupled with widespread intraperitoneal disease. Despite this, metastatic spread of tumor clones is non-random, implying the existence of local microenvironmental factors that shape tumor progression. We interrogated the molecular interface between tumor-infiltrating lymphocytes (TIL) and cancer cells in 143 samples from 21 patients using whole-genome sequencing, immunohistochemistry, histologic image analysis, gene expression profiling, and T- and B-cell receptor sequencing. We identify 3 immunologic response categories, which frequently co-exist within individual patients. Furthermore, epithelial CD8+ TIL were inversely associated with malignant cell diversity, evidenced by subclonal neoepitope elimination and spatial tracking between tumor and T-cell clones. Intersecting mutational signatures and immune analysis showed that foldback inversion genomic aberrations lead to worse outcomes even in the presence of cytotoxic TIL (n=433). Thus, regional variation in immune contexture mirrors the pattern of intraperitoneal malignant spread, provoking new perspectives for treatment of this challenging disease.

High-grade serous ovarian cancer (HGSC) exhibits extensive malignant clonal diversity with widespread but non-random patterns of disease dissemination. We investigated whether local immune microenvironment factors shape tumor progression properties at the interface of tumor-infiltrating lymphocytes (TILs) and cancer cells. Through multi-region study of 212 samples from 38 patients with whole-genome sequencing, immunohistochemistry, histologic image analysis, gene expression profiling, and T and B cell receptor sequencing, we identified three immunologic subtypes across samples and extensive within-patient diversity. Epithelial CD8+ TILs negatively associated with malignant diversity, reflecting immunological pruning of tumor clones inferred by neoantigen depletion, HLA I loss of heterozygosity, and spatial tracking between T cell and tumor clones. In addition, combinatorial prognostic effects of mutational processes and immune properties were observed, illuminating how specific genomic aberration types associate with immune response and impact survival. We conclude that within-patient spatial immune microenvironment variation shapes intraperitoneal malignant spread, provoking new evolutionary perspectives on HGSC clonal dispersion.

Many observations bear upon the cellular and molecular requirements for CD4 T cell activation. The interaction of CD4 T cells with dendritic cells (DC), central to the induction of most immune responses, is the most studied. However, leukocytes other than DC can dramatically affect the induction and differentiation of CD4 T cells into effector cells. We recently provided indirect evidence that in vivo CD4 T cooperation facilitates the activation of CD4 T cells. Here, we demonstrate that the activation of CD4 T cells, specific for the hen egg lysozyme (HEL)(105) (-120) peptide, is optimally achieved when BALB/c mice are immunized with additional MHC class II-binding HEL peptides in incomplete Freund's adjuvant. This cooperation cannot be mimicked by the coadministration of LPS or of an agonistic antibody to CD40, at the time of immunization. In contrast, OX40-OX40L interactions are necessary for CD4 T cell cooperation in that an OX40 agonistic antibody can replace, and an OX40L-blocking antibody can abrogate, CD4 T cell cooperation in situations where such cooperation would otherwise enhance the activation of CD4 T cells.

We address here the role of CD4 T cell cooperation in the activation of CD4 T cells. Administration of aggregated hen egg lysozyme (HEL) without microbial adjuvant to BALB/c mice normally generates cytokine-producing CD4 T cells specific for the HEL major peptide, HEL(105-120), as well as CD4 T cells specific for HEL non-major peptides. The prior administration of HEL(105-120) ablates the generation of cytokine-secreting CD4 T cells specific for HEL(105-120), as well as the CD4 T cells specific for HEL non-major peptides, normally generated upon HEL challenge. Thus, the activation of HEL non-major peptide-specific CD4 T cells appears to depend upon the HEL(105-120)-specific CD4 T cell population. In contrast, when HEL(105-120) and saline-treated mice are challenged with HEL coupled to ovalbumin (OVA), CD4 T cell responses to HEL non-major peptides and to OVA are the same, whereas treated mice still do not generate cytokine-secreting cells specific for HEL(105-120). We infer that the administration of HEL(105-120) does not generate regulatory cells capable of down-regulating CD4 T cell responses to HEL and OVA peptides. OVA-specific CD4 T cells restore the generation of HEL non-major peptide-specific T cells in the absence of HEL major peptide-specific T cells. We conclude that the generation of CD4 T cells producing IL-2, IFN-gamma and IL-4 requires CD4 T cell cooperation and that this cooperation is not mediated simply by CD40-CD40L interactions. We also conclude from these observations that there is no requirement for a microbial or danger signal for CD4 T cell activation.

Peter A Bretscher Author for correspondence: Department of Microbiology & Immunology, University of Saskatchewan, A305–107 Wiggins Road, Saskatoon, Saskatchewan, S7N 5E5, Canada Tel.: +1 306 966 4322 Fax: +1 306 966 4298 peter.bretscher@usask.ca Naive CD4 T cells are activated by antigenpresenting dendritic cells (DCs) when these DCs express sufficient and appropriate costimulatory molecules. Classical studies have led to the idea that activated DCs, presenting the appropriate antigen, are sufficient for the robust activation of naive CD4 T cells. However, recent studies have demonstrated that in addition to activated DCs, B cells are also required for optimal CD4 T-cell responses. Herein, we discuss such studies and speculate on the importance of B cells in limiting the activation of autoreactive CD4 T cells.

Our previous in vivo studies show that both the amount of Ag and the number of available naive CD4 T cells affect the Th1/Th2 phenotype of the effector CD4 T cells generated. We examined how the number of OVA-specific CD4 TCR transgenic T cells affects the Th1/Th2 phenotype of anti-SRBC CD4 T cells generated in vivo upon immunization with different amounts of OVA-SRBC. Our observations show that a greater number of Ag-dependent CD4 T cell interactions are required to generate Th2 than Th1 cells. We established an in vitro system that recapitulates our main in vivo findings to more readily analyze the underlying mechanism. The in vitro generation of Th2 cells depends, as in vivo, upon both the number of responding CD4 T cells and the amount of Ag. We demonstrate, using agonostic/antagonistic Abs to various costimulatory molecules or their receptors, that the greater number of CD4 T cell interactions, required to generate Th2 over Th1 cells, does not involve CD40, OX40, or ICOS costimulation, but does involve B7/CD28 interactions. A comparison of the level of expression of B7 molecules by APC and CD4 T cells, under different conditions resulting in the substantial generation of Th1 and Th2 cells, leads us to propose that the critical CD28/B7 interactions, required to generate Th2 cells, may directly occur between CD4 T cells engaged with the same B cell acting as an APC.

Purpose: Cancers accumulate mutations over time, each of which brings the potential for recognition by the immune system. We evaluated T-cell recognition of the tumor mutanome in patients with ovarian cancer undergoing standard treatment. Experimental Design: Tumor-associated T cells from 3 patients with ovarian cancer were assessed by ELISPOT for recognition of nonsynonymous mutations identified by whole exome sequencing of autologous tumor. The relative levels of mutations and responding T cells were monitored in serial tumor samples collected at primary surgery and first and second recurrence. Results: The vast majority of mutations (78/79) were not recognized by tumor-associated T cells; however, a highly specific CD8+ T-cell response to the mutation hydroxysteroid dehydrogenase–like protein 1 (HSDL1)L25V was detected in one patient. In the primary tumor, the HSDL1L25V mutation had low prevalence and expression, and a corresponding T-cell response was undetectable. At first recurrence, there was a striking increase in the abundance of the mutation and corresponding MHC class I epitope, and this was accompanied by the emergence of the HSDL1L25V-specific CD8+ T-cell response. At second recurrence, the HSDL1L25V mutation and epitope continued to be expressed; however, the corresponding T-cell response was no longer detectable. Conclusion: The immune system can respond to the evolving ovarian cancer genome. However, the T-cell response detected here was rare, was transient, and ultimately failed to prevent disease progression. These findings reveal the limitations of spontaneous tumor immunity in the setting of standard treatments and suggest a high degree of ignorance of tumor mutations that could potentially be reversed by immunotherapy. Clin Cancer Res; 20(5); 1125–34. ©2013 AACR.

We show that the in vivo generation of cytokine-producing CD4 T cells specific for a given major histocompatibility class-II (MHCII)-binding peptide of hen egg lysozyme (HEL) is facilitated when mice are immunized with splenic antigen presenting cells (APC) pulsed with this HEL peptide and another peptide that binds a different MHCII molecule. This enhanced generation of peptide-specific effector CD4 T cells requires that the same splenic APC be pulsed with both peptides. Pulsed B cells, but not pulsed dendritic cells (DCs), can mediate CD4 T cell cooperation, which can be blocked by disrupting OX40-OX40L (CD134-CD252) interactions. In addition, the generation of HEL peptide-specific CD4 T cell memory is greater when mice are primed with B cells pulsed with the two peptides than with B cells pulsed with the HEL- peptide alone. Based on our findings, we suggest CD4 T cell cooperation is important for vaccine design, underlies the phenomenon of "epitope-spreading" seen in autoimmunity, and that the efficacy of B cell-depletion in the treatment of human cell-mediated autoimmune disease is due to the abrogation of the interactions between autoimmune CD4 T cells that facilitates their activation.

High-grade serous ovarian cancer (HGSC) is a challenging disease characterized by poor survival and relapse. Our group has revealed extensive clonal diversity of malignant cells in HGSC, which is thought to facilitate the emergence of resistance and recurrence in response to chemotherapy. Moreover, we have shown that the presence of tumor infiltrating lymphocytes (TILs) is associated with longer patient survival in HGSC, suggesting that the immune system can contend with the clonal diversity of tumors in some patients. We sought to determine how TIL abundance and repertoire diversity relates to the degree of tumor clone diversity in HGSC. We performed whole-genome sequencing, targeted amplicon deep sequencing, gene expression profiling, and T-cell receptor sequencing on 34 spatially separated HGSC tumor samples (8 patients) including ovarian masses and peritoneal foci. In addition, tissue sections were immunohistochemically stained for CD3, CD8, CD20, CD79a, and CD138 to detect major TIL subsets. Using PyClone, we decomposed each tumor sample into its constituent malignant clones and inferred clonal genotypes. We discovered profound intra- and inter-patient variation in the degree of tumor clone diversity from phylogenetic analysis. Widespread inter-patient variation was also observed in TIL counts and T-cell receptor repertoires. Intraepithelial CD3+ cell counts ranged from 2.2-173.2 per HPF and the number of unique T-cell receptor clonotypes varied from 422 to 3164 per sample. Remarkably, quantitative metrics of tumor clone diversity—as defined by phylogenetic divergence and entropy in the malignant composition of each tumor—was associated with the ‘proliferative’ molecular subtype and, accordingly, low TIL density. Tumor clone diversity showed no correlation with the number of unique T-cell receptor species. Moreover, preliminary analyses reveal no association between tumor clone diversity and the expression of several ligands and receptors involved in inhibitory immune signaling (CTLA-4, PD-1, PD-L1, PD-L2). Our results reveal an association between low TIL abundance and high diversity of malignant clones. This suggests that immune infiltration may inhibit tumor proliferation and clonal diversification, or vice versa. Collectively, our findings expose a relationship of the immune microenvironment with the clonal dynamics of malignant cells that could be exploited to overcome therapeutic resistance associated with clonal evolution in HGSC. Citation Format: Allen W. Zhang, Andrew McPherson, Andrew Roth, David R. Kroeger, Katy Milne, Wyeth W. Wasserman, Jessica N. McAlpine, Robert A. Holt, Brad H. Nelson, Sohrab P. Shah. Properties of the immune microenvironment associated with clonal diversity in high-grade serous ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4136.

The majority of in vitro studies investigating the activation of naive TCR transgenic T cells routinely employ an artificially high frequency of such cells. To assess whether employing high frequencies of TCR transgenic cells in vitro accurately reflects the in vivo activation of a normal number of T cells, we cultured between 300 and 3 × 10 6 Rag2 −/− DO11.10 T cells per well under otherwise identical conditions. We find that those T cells cultured at low frequencies proliferate more and are more potently activated, as assessed by the expression of CD44 and CD62L, each giving rise to a much larger number of cytokine producing cells, comparable to the number generated in vivo when a normal number of CD4 + T cells are activated. The effect of T cell frequency on the level of their activation was not due to differences in MHCII or CD80/86 expression by B cells, the major APC population present, nor to increased death of B cells in high frequency cultures. Taken together, our observations illustrate the necessity of culturing naive TCR transgenic CD4 + T cells at a physiological frequency if one is to more accurately recapitulate the in vivo activation of naive CD4 + T cells.

Cytotoxic T lymphocytes (CTLs), associated with Th1 responses, are the most important mediators of resistance against most tumors. We argue that most murine tumors grow progressively when a significant Th2 component to their immune response develops, which is associated with the downregulation of the CTL response. We outline recent evidence that strongly supports this Th2-skewing hypothesis as the prevalent mechanism of tumor escape in murine systems. We describe the conceptual grounds and evidence for the ‘threshold hypothesis’ that proposes how the Th1/Th2 phenotype of an immune response generated against ‘nonliving’ antigens is determined. We suggest that this threshold hypothesis also accurately describes how the Th1/Th2 phenotype of murine antitumor immune responses is determined, as this hypothesis can account for the critical and known features of these immune responses. The efficacy of several manipulations that prevent or arrest progressive tumor growth in murine models, through their effects on the antitumor immune response, can be understood within the context of the threshold hypothesis. Indirect evidence supports the view that similar relationships between cancers and the immune system are present in humans. We propose means by which these insights can be employed to optimize the harnessing of protective Th1 CTL immunity against tumors.