T-cell Abundance Research Articles

Donor-Derived CD4+ T Cells and Human Herpesvirus 6B Detection After Allogeneic Hematopoietic Cell Transplantation.

We sought to determine whether donor-derived human herpesvirus (HHV) 6B-specific CD4+ T-cell abundance is correlated with HHV-6B detection after allogeneic hematopoietic cell transplantation. We identified 33 patients who received HLA-matched, non-T-cell-depleted, myeloablative allogeneic hematopoietic cell transplantation and underwent weekly plasma polymerase chain reaction testing for HHV-6B for 100 days thereafter. We tested donor peripheral blood mononuclear cells for HHV-6B-specific CD4+ T cells. Patients with HHV-6B detection above the median peak viral load (200 copies/mL) received approximately 10-fold fewer donor-derived total or HHV-6B-specific CD4+ T cells than those with peak HHV-6B detection at ≤200 copies/mL or with no HHV-6B detection. These data suggest the importance of donor-derived immunity for controlling HHV-6B reactivation.

Diversity of Tumor-Infiltrating, γδ T-Cell Abundance in Solid Cancers.

γδ T-cells contribute to the immune response against many tumor types through their direct cytolytic functions and their capacity to recruit and regulate the biological functions of other immune cells. As potent effectors of the anti-tumor immune response, they are considered an attractive therapeutic target for immunotherapies, but their presence and abundance in the tumor microenvironment are not routinely assessed in patients with cancer. Here, we validated an antibody for immunohistochemistry analysis that specifically detects all γδ T-cell subpopulations in healthy tissues and in the microenvironment of different cancer types. Tissue microarray analysis of breast, colon, ovarian, and pancreatic tumors showed that γδ T-cell density varies among cancer types. Moreover, the abundance of γδ tumor-infiltrating lymphocytes was variably associated with the outcome depending on the cancer type, suggesting that γδ T-cell recruitment is influenced by the context. These findings also suggest that γδ T-cell detection and analysis might represent a new and interesting diagnostic or prognostic marker.

Tumor fitness, immune exhaustion and clinical outcomes: impact of immune checkpoint inhibitors

Recently proposed tumor fitness measures, based on profiling neoepitopes for reactive viral epitope similarity, have been proposed to predict response to immune checkpoint inhibitors in melanoma and small-cell lung cancer. Here we applied these checkpoint based fitness measures to the matched checkpoint treatment naive Cancer Genome Atlas (TCGA) samples where cytolytic activity (CYT) imparts a known survival benefit. We observed no significant survival predictive power beyond that of overall patient tumor mutation burden, and furthermore, found no association between checkpoint based fitness and tumor T-cell infiltration, cytolytic activity, and abundance (tumor infiltrating lymphocyte, TIL, burden). In addition, we investigated the key assumption of viral epitope similarity driving immune response in the hepatitis B virally infected liver cancer TCGA cohort, and uncovered suggestive evidence that tumor neoepitopes actually dominate viral epitopes in putative immunogenicity and plausibly drive immune response and recruitment.

Abstract A31: High aneuploidy levels are linked to a reduced immune-cell abundance in metastatic castrate-resistant prostate cancer

Abstract Single-agent immunotherapeutic approaches are promising for treating castrate-resistant prostate cancer (CRPC) patients; however, to date no significant improvement in survival has been reported. Mixed response to therapy occurs because metastatic lesions are highly heterogeneous. These lesions exhibit distinct genomic changes that are linked to a tumor microenvironment that is often poorly populated by immune cells. In other tumor types, a high percent genome altered (e.g., aneuploidy) associates with an overall reduction in immune response in the primary tumor microenvironment. However, the association between genomic changes and immune cell composition is poorly understood in metastatic prostate cancers. Thus, we quantified the abundance of 22 immune cell types in 330 tumors from two independent public domain CRPC cohorts and 491 primary prostate tumors from TCGA using the Absolute Mode from CIBERSORT. The estimation in CRPC was performed using whole-transcriptome data from West Coast Dream Team (Abida et al., 2019; n=212) and East Coast Dream Team (Robinson et al., 2015; n=117) samples. We did not identify any significant correlations between mutational load and immune cell abundance in the three investigated cohorts. In primary tumors, aneuploidy was weakly negatively correlated with CD8+ and CD4+ T-cell abundance (R=-0.14 and R=-0.09, P<0.05). Overall, metastatic lesions demonstrated a low absolute abundance of the majority of immune cell subtypes, with higher levels of CD8+ T-cells, memory CD4+ T-cells, macrophages, and plasma cells. We found that bone lesions had the lowest abundance of CD8+ T-cells (Kruskal-Wallis, p=0.05) and the highest abundance of macrophages (P<0.0001) compared to lymph node and liver samples. Overall, aneuploidy was negatively correlated with immune cell abundance in both metastatic cohorts (Spearman correlation, P<0.05). Interestingly, lymph node metastases showed the strongest negative correlation between aneuploidy and T-cell abundance in both West and East cohorts (R=-0.36 and R=-0.32, respectively, p<0.01). Our preliminary results show that the extent of chromosomal aberrations in CRPC is significantly associated with underlying immune cell infiltrates. These results have potential implications for future immunotherapy trials in prostate cancer. Citation Format: Thiago Vidotto, Daniela C. Salles, Tamara L. Lotan. High aneuploidy levels are linked to a reduced immune-cell abundance in metastatic castrate-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A31.

Abstract A101: Single-cell analysis reveals the pivotal role of the innate immune compartment in aCTLA-4 antitumor response

Abstract Immunotherapies have revolutionized and continue to revolutionize cancer patient care; however, much of our understanding of the mechanism of action of these therapies is currently limited. The accumulation of regulatory T cells (Tregs) in the tumor hampers antitumor activity and correlates with bad prognosis in several human cancers. Anti-CTLA-4 monoclonal antibodies (mAbs) have been extensively studied, and their activity depends both on the blockade of the CTLA-4 coinhibitory molecule, as well as the intratumoral depletion of Tregs, increasing effector cell abundance, and favoring tumor rejection. Fc-gamma receptor (FcgR) coengagement has been proven to be important for the action of aCTLA-4, in addition to its Treg-depleting activity via antibody-dependent cellular cytotoxicity (ADCC); however, it is unclear what are the cellular changes involved. We therefore wished to dissect the differences between aCTLA-4 mIgG1, an antibody that only blocks the CTLA-4 receptor, and aCTLA-4 mIgG2a, which has a dual activity of both blocking the receptor and depleting Tregs. To address this, we performed single-cell RNA-seq of infiltrating leukocytes from tumors in mice treated with aCTLA-4 mIgG1, mIgG2a, or left untreated (UT). This high-resolution comparison revealed unique cellular profiles generated by each treatment. While the blocking-only mIgG1 was similar to UT, mIgG2a with ADCC effector function demonstrated major changes. Tumors in mIgG2a treated mice showed an immediate decline of immune suppressive macrophages, and the emergence of proinflammatory monocytes, as well as NK cells and naive CD8 T cells. In addition, mIgG2a-treated mice displayed a gradual increase of CD4 T-cell subsets throughout treatment. Finally, a vast tissue repair signature was observed in later time points of aCTLA-4 mIgG2a treatment, comprising MRC1+ macrophages, neutrophils, and myeloid-derived suppressor cells (MDSCs), while CD8 T-cell abundance declined. In summary, our findings provide an in-depth view of the differences in mechanisms of action between an aCTLA-4 blocking mAb and an optimized blocking-depleting mAb, underscoring how FcgRs coengagement leads to enhanced antitumor response via the innate immune compartment. Citation Format: Ido Yofe, Adam Jelinski, Isabelle Solomon, Tomer Landsberger, Marc Robert de Massy, Karl Peggs, Sergio Quezada, Ido Amit. Single-cell analysis reveals the pivotal role of the innate immune compartment in aCTLA-4 antitumor response [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A101.

Carbonic anhydrase 9 (CA9) expression in non-small-cell lung cancer: correlation with regulatory FOXP3+T-cell tumour stroma infiltration

BackgroundLow pH suppresses the proliferation and cytotoxic activity of CD8+ cytotoxic and natural killer lymphocytes. The hypoxia-regulated transmembrane protein, carbonic anhydrase CA9, converts carbon dioxide produced by the Krebs cycle to bicarbonate and protons that acidify the extracellular milieu. We examined whether CA9 is also involved in intratumoural immunosuppression pathways.MethodsA series of 98 tissue samples of primary non-small-cell lung carcinomas (NSCLC) from patients treated with surgery were analysed for the expression of CA9 and programmed-death ligand PD-L1 by cancer cells, and of FOXP3 by tumour-infiltrating lymphocytes (TILs).ResultsThere was no direct association of CA9 with PD-L1 expression or the density of TILs in the tumour stroma, but CA9 was directly related to the extent of FOXP3+ TIL density (p = 0.008). Double-stratification survival analysis showed that patients with high CA9 expression and low TIL score had significantly poorer survival compared with all other groups (p < 0.04). In a multivariate analysis stage (p < 0.0001, HR 1.95, 95% CI: 1.3–2.7), TIL score (p = 0.05, HR 0.55, 95% CI: 0.2–1.0) was an independent prognostic variable of death events. CA9 expression by cancer cells is associated significantly with FOXP3+ regulatory T-cell abundance in the tumour stroma of NSCLC.ConclusionThe study provides a basis for testing CA9 as a marker of resistance to immune-checkpoint inhibitors and as a therapeutic target to enhance the efficacy of immunotherapy.

Abstract A28: Investigation of tumor-cell-intrinsic factors regulating immune infiltration and response to immunotherapy in pancreatic cancer

Abstract Resistance to immunotherapy is one major problem of current clinical care for cancer patients. While T-cell abundance is essential for tumor responsiveness to immunotherapy, factors that dictate T-cell infiltration in tumor microenvironments are not fully understood. To understand the tumor cell-intrinsic factors underlying the heterogeneity of tumor immunity and sensitivity to immunotherapy, we established a new experimental system by generating a library of congenic pancreatic tumor cell clones from a genetic mouse model driven by mutant Kras and p53. These tumor cell clones robustly formed implanted tumors that recapitulated the T cell-inflamed and non-T cell-inflamed tumor microenvironments in human patients, associated with distinct patterns of infiltration by T cells and myeloid cells. We found that the non-T cell-inflamed phenotype was dominant over the T cell-inflamed phenotype in the local tumor microenvironment. Both quantitative and qualitative features, specifically expression of markers of prior TCR activation, of intratumoral CD8+ T cells predicted the response to immunotherapies. An integrated transcriptomic and epigenetic analysis revealed that tumor cell-intrinsic expression of the chemokine CXCL1 as a major determinant of the non-T cell-inflamed microenvironment, and ablation of tumor cell-intrinsic CXCL1 promoted T-cell infiltration and sensitivity to a combination of chemotherapies, CD40 agonist, and checkpoint blockades. Similarly, we identified tumor cell-intrinsic EPHA2 and PTGS2 as key regulators of immune infiltration and immunotherapy response in our experimental system. Ablation of tumor cell-intrinsic EPHA2 or PTGS2 enhanced T-cell infiltration and suppressed myeloid cell infiltration in implanted pancreatic tumors, and increased sensitivities of tumors to the combined immunotherapy. These results demonstrated that heterogeneity of tumor immune phenotypes is driven by tumor cell-intrinsic factors that can be manipulated to influence the outcome of immunotherapies. The observation that non-T cell-inflamed phenotype is dominant emphasized the importance of targeting mechanisms driving T-cell low phenotype for improving immunotherapy response. Citation Format: Jinyang Li, Katelyn T Byrne, Nune Markosyan, Taiji Yamazoe, Fangxue Yan, Zeyu Chen, Yu H. Sun, Jeffrey Lin, Yogev Sela, Robert J. Norgard, Salina Yuan, Allyson J. Merrell, John W. Tobias, Robert H. Vonderheide, Ben Z. Stanger. Investigation of tumor-cell-intrinsic factors regulating immune infiltration and response to immunotherapy in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A28.

Changes in Tumor Immune Micro-Environment in Diffuse Large B-Cell Lymphoma (DLBCL): A Comparative Study of Relapsed Versus Diagnostic DLBCL

Introduction The tumor immune microenvironment (TiME) of DLBCL at diagnosis bears many biomarkers known to predict sensitivity to checkpoint blockade therapy (CBT). Approximately 25% of DLBCL, harboring PDL1 gene alteration, exhibit an immune inflamed phenotype. However, CBT studies in relapsed DLBCL show very low response rates. The immune landscape of DLBCL in the setting of relapse has not yet been characterized. We hypothesized that the TiME can be influenced by the therapy, explaining the CBT lack of efficacy. We explored the TiME evolution by comparing the tumor infiltrating lymphocytes (TIL) and the immune evasion signature by protein and gene expression (GE) in paired biopsies of DLBCL at diagnosis and at relapse. Method This study included patients with DLBCL treated with first-line R-CHOP-like regimen, in which we had access to nodal biopsies obtained pre-treatment and at relapse. Assessment of TIL and PDL1+ cell content was carried out by CD3, CD4, CD8 immunostaining and with PDL1 (Ventana, SP263) and PAX5 double staining. These were performed on formalin-fixed, paraffin-embedded (FFPE) tissue sections cut at 4µm. The proportion of each cell-type was expressed in relation to total nucleated cells. PDL1+ cell proportion was expressed for both total cells and tumor cells. The paired non-parametric Wilcoxon test, double-sided with p=0.05, was used for comparison. GE using a panel of 760 cancer immune genes was performed with the digital hybridization NanoString platform on paired FFPE specimens. NSolver software was used for normalization on housekeeping genes, differential expression (DE) and cell-type profiling. The correlation between IHC and GE variation were computed using the Spearman test. Analysis of enriched pathways on combined patient data were performed with GSEA software, using two gene sets for T-cell activation and immune evasion validated in B-cell lymphoma, and Ingenuity Pathway Analysis (IPA) for the 339 highest changed genes. The pathway enrichment analysis was reported for false discovery rate (FDR) &lt;0.05. Results Twenty DLBCL patients with paired diagnostic and relapse biopsies were studied. IHC was performed on 19 patients (necrotic tissue for one patient) and GE by NanoString on 6 patients. The median number of treatments received prior to obtaining the relapsed biopsy was 3 (1-6). Three patients had prior exposure to CBT, none from the patients in whom GE was performed. Our population was composed of 2/3 germinal center B-cell (GCB) DLBCL. Six patients had transformed DLBCL. At relapse, CD4 and CD8 cells density was significantly reduced compared to diagnosis. The median percentages of CD3, CD4 and CD8 were 15%, 7.5% and 5% at diagnosis and 7.5%, 2.5% and 2.5% at relapse, respectively (Figure 1A). The median proportion of PDL1+ cells (n=15) did not differ at relapse compared to pre-treatment (5.25% and 5%, respectively) (Figure 1B). At diagnosis, 87% of biopsies were found to be positive for PDL1 (≥1% of positive cells) and 73% at relapse. The PDL1 positive cells were mostly immune cells; only 3 samples had tumor cells expressing PDL1 at pre-treatment and 4 at relapse with &lt;1% of positive tumor cells in all cases. The GE profile detected 63 genes that were significantly different in relapse vs pre-treatment samples (p&lt;0.005), including 8 up-regulated and 55 down-regulated. The correlation for T-cell abundance variation between IHC and GE was high (r=0.94, p=0.017). For the subset of 6 patients in whom GE was performed, the T-cells were not significantly reduced as measured by both IHC and GE. However, the GSEA for immune escape genes and T-cell activation signatures were both down-regulated (FDR=0.033 and 0.010, respectively) (Figure 2). IPA predicted decreased activation of leukocytes (z-score=-6.317) and Th2 activation pathway (z-score=-3.182). Conclusion At relapse, a proportion of DLBCL have a significant reduction in T cell infiltration. As well, they acquire a non-T cell-inflamed TiME. The T-cell inhibition seems to be independent of local T-cell immunoregulatory mechanisms as the immune escape signature is also reduced. This non immunogenic, "cold" condition could explain the lack of efficacy of CBT in the relapsed setting. Prior exposure to steroid containing therapy might be a trigger for intra-tumor T-cell desertion and downregulation. This observation must be confirmed on a larger population. Further analyses will be presented. Disclosures Johnson: BD Biosciences: Other: Provided a significant proportion of the antibodies used in this project free of cost.; Lundbeck: Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; Seattle Genetics: Honoraria; Merck: Consultancy, Honoraria; Roche: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; BMS: Consultancy, Honoraria; Abbvie: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Assouline:Janssen: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Speakers Bureau; Abbvie: Consultancy, Honoraria.

PSIV-18 Effect of sub-therapeutic antibiotics and pharmacological zinc oxide on nursery pig performance and intestinal integrity and function

Abstract The objective of this study was to evaluate the impact of sub-therapeutic chlortetracycline (CTC) and pharmacological zinc oxide on nursery pig performance and intestinal integrity and function. Twenty-four newly weaned pigs (4.51 ± 0.21 kg BW) were randomly assigned to one of three dietary treatments for 21 d (n = 8/treatment): 1) Control diet (NC), 2) NC + Zinc oxide at 3000 ppm for 7 d followed by 2000 ppm for 14 d (ZnO), and 3) NC + 40 ppm CTC for 21 d (sCTC). Pig ADG, ADFI, and G:F were determined for the overall 21 d test period. At d 21, all pigs were euthanized for tissue collection. Jejunum and ileum ex vivo transepithelial resistance (TER), macromolecule (FD4) flux, and active glucose and glutamine transport were assessed in modified Ussing Chambers. Additionally, jejunum brush border enzyme and Na+/K+ ATPase activities were determined. Morphology and T-cell abundance were determined in all ileum sections by H&amp;E staining and immunohistochemistry, respectively. Compared to the NC, sCTC and ZnO increased ADG (0.14, 0.20 and 0.23 kg/d, respectively, P = 0.020). Overall ADFI was increased by 150% due to sCTC compared to the NC and ZnO pigs (P &lt; 0.05). G:F was increased due to ZnO and sCTC compared to the NC (0.82, 0.63 and 0.47, respectively, P &lt; 0.01). Jejunum and ileum ex vivo function and integrity did not differ. Ileal villus height was increased in the NC compared to sCTC treatment (P &lt; 0.01), but not different from the ZnO treatment. In the ileum, total T-cell number per unit area was significantly lower in the ZnO segments compared to sCTC and NC (0.95, 2.29 and 2.59 count per μm2, respectively, P = 0.05). These data indicate that ZnO and sCTC improved pig performance but did not directly alter small intestinal integrity and function. However, mucosal T cell abundance was altered.

Abstract LB-017: Combining tumor mutational burden, CD8+ T-cell abundance and PD1 mRNA expression accurately predicts response to anti-PD1/PDL1 therapy across cancers

Abstract Introduction Anti-PD1/PDL1 therapies provide significant survival benefits in many cancers, but the efficacy of these treatments varies considerably across different cancer types. Identifying the underlying variables associated with the cancer type specific response remains an important open research challenge. Here, we systematically evaluate a multitude of variables to determine the key factors that can accurately predict the response to anti-PD1/PDL1 therapy across different cancer types. Approach To this end we analyzed whole exome sequencing (WES) and RNA sequencing (RNAseq) of 10k patients from the Cancer Genome Atlas (TCGA) and the overall response rate (ORR) to anti-PD1/PDL1 therapy of 21 cancer types obtained from previous clinical trials. We considered more than 30 different variables of three distinct classes: those associated with (a) tumor neoantigen landscape, (b) tumor microenvironment and inflammation, and (c) the checkpoint inhibitor targets. We evaluated the performance of each of these variables and their combinations in predicting the ORR to anti-PD1/PDL1 therapy. Prediction accuracy was quantified with Spearman correlation and explained variance quantified through a standard leave-one-out cross-validation. Results Our analysis shows CD8+ T-cell abundance is the most predictive of the response to anti-PD1/PDL1 therapy across cancer types, followed by the tumor mutational burden and the fraction of samples with high PD1 gene expression in each cancer type. Notably these top three predictors cover the three distinct classes considered, and their combination is highly predictive of the ORR to anti-PD1/PDL1 therapy (Spearman R=0.9, P&amp;lt;1E-7), explaining more than 80% of the variance observed across different tumor types. Conclusions and Relevance This is the first systematic evaluation of the different variables associated with PD1/PDL1 therapy response across different tumor types. Our findings show that, overall cancer types, three key variables can explain most of the observed cross-cancer response variability, but that their relative explanatory roles may vary in specific cancer types. Citation Format: Joo Sang Lee, Eytan Ruppin. Combining tumor mutational burden, CD8+ T-cell abundance and PD1 mRNA expression accurately predicts response to anti-PD1/PDL1 therapy across cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-017.

Abstract 1493: Therapeutic efficacy and tolerability of combined immune checkpoint blockade in metastatic melanoma patients is influenced by the gut microbiome

Abstract Background: The gut microbiome is increasingly being recognized as a strong modulator of anti-PD1 based cancer immunotherapy. Compelling evidence demonstrates differential bacterial enrichment and diversity in responders (R) versus non-responders (NR), mediated by profound influences on systemic and anti-tumor immune infiltrates. However, this has not been studied in the setting of treatment with combined immune checkpoint blockade (CICB), which is associated with superior response rates, but higher rates of potentially debilitating toxicities. Methods: We assembled a cohort of patients with metastatic melanoma receiving CICB (n=54). All patients were classified as R (n=31, CR + PR) or NR (n=23, SD + PD) based on RECIST v1.1, and as having grade 3 or higher (T; n=29), or less than grade 3 (NT; n=25) immune related adverse event(s) by NCI CTCAE 4.0 criteria. Baseline stool samples were characterized by 16S rRNA sequencing. Correlative analyses of peripheral immune cell populations by flow cytometry (n=12) and circulating T cell repertoire by TCR-sequencing (n=12) were done on matched pre-treatment blood samples. Results: The overall gut microbial landscape in these patients was varied with high abundance of Bacteroidales and Clostridiales. Ordination of beta-diversity distances revealed a lack of clustering by subtype of primary tumor (uveal, mucosal, cutaneous) consistent with no significant effect of the tumor histology. While no apparent response or toxicity associations were evident based on diversity, notable compositional differences were appreciated. Comparison of relative abundances by LEfSe (LDA&amp;gt;2, p&amp;lt;0.05), and pairwise Mann-Whitney tests revealed an enrichment of Bacteroides stercoris (p=0.03), and Parabacteroides distasonis (p=0.04) in R, and Lactobacillales (p=0.005) in NR. Consistent with our prior findings, the median relative abundance of the order Clostridiales was again higher in R (0.34) versus NR (0.26). On the other hand, Bacteroides intestinalis (p=0.01) and Anaerotignum lactatifermentans (p=0.006) were enriched in T and NT, respectively. Importantly, correlative analyses with circulating immune cell subsets revealed distinct associations by differential bacterial enrichment (including positive correlations between overall CD8+ T-cell abundance and R-taxa), and a clustering effect by high or low T-cell repertoire entropy. Conclusion: These findings build on our prior work and support the notion of a close link between the gut microbiome and therapeutic outcomes to checkpoint blockade therapy. Extensive studies are underway in both matched human biospecimens and in pre-clinical models to further understand mechanisms of interactions with immune markers, and to establish causality. Taken together, these data support a critical role for the gut microbiome as both a predictive tool and therapeutic target. Citation Format: Vancheswaran Gopalakrishnan, Miles Andrews, Wei-Shen Chen, Christine Spencer, Luis Vence, Alexandre Reuben, Zachary A. Cooper, Peter A. Prieto, Michael T. Tetzlaff, MA Abdul Wadud Khan, Alexander Lazar, Courtney W. Hudgens, Lauren E. Haydu, Hussein A. Tawbi, Patrick Hwu, Wen-Jen Hwu, Rodabe N. Amaria, Elizabeth M. Burton, Scottt E. Woodman, Adi Diab, Sapna P. Patel, Isabella C. Glitza, Jianhua Zhang, Joseph Petrosino, Robert R. Jenq, Michael A. Davies, Jeffrey E. Gershenwald, Padmanee Sharma, James P. Allison, Andrew Futreal, Jennifer A. Wargo. Therapeutic efficacy and tolerability of combined immune checkpoint blockade in metastatic melanoma patients is influenced by the gut microbiome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1493.

Abstract 3415: Tumor fitness and immune exhaustion: Checkpoint vs endogenous settings

Abstract Recently proposed tumor fitness measures based on neoepitope profiling and viral epitope similarity have led to treatment efficacy and immune response prediction models in the checkpoint setting for melanoma (SKCM) and small-cell lung cancer (NSCLC). In this work we test if these checkpoint based fitness measures are associated with either tumor-infiltrating T-cell activity or abundance, and also overall patient survival, in the endogenous setting for the SKCM (n=337) and NSCLC (n = 307) TCGA cohorts. We also investigated if epitopes arising from tumor viral co-factors in Hepatitis B liver cancer (HBV+ HCC) drove T cell activity or response compared to neo-epitope burden. We find no statistically significant correlation between immune activity and response (as measured by tumor RNA-seq profiling) and the proposed neo-epitope checkpoint-based fitness measures, and similarly no significant survival effect. Further, we found firm evidence that tumor neoepitopes dominate HBV viral epitopes in putative immunogenicity and drive immune response in TCGA HCC (n = 178), and confirmed this trend in multi-regional data (12 patients, 72 samples). These results suggest that tumor-immune response and recruitment in SKCM and NSCLC, and their interplay with viral-cofactor HBV in HCC, are fundamentally driven by different factors in the endogenous setting. Finally, we propose that tumor fitness may be partially encoded by a simple T-cell exhaustion expression signature, which we demonstrate is a significant predictor of patient survival for SKCM and NSCLC and may translate more widely into other tumor types in the endogenous setting. Citation Format: Adrian Bubie, Nicholas Akers, Augusto Villanueva, Bojan Losic. Tumor fitness and immune exhaustion: Checkpoint vs endogenous settings [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3415.

CD8+ T cells in tumor parenchyma and stroma by image analysis (IA) and gene expression profiling (GEP): Potential biomarkers for immuno-oncology (I-O) therapy.

2594 Background: Distribution patterns of CD8+ T cells within the tumor microenvironment (TME) can be assessed by IA, which may reflect underlying tumor biology and serve as a potential biomarker to assess the utility of I-O therapy. These patterns are variable and may be classified as immune desert (minimal infiltrate), excluded (T cells confined to tumor stroma or to the invasive margin), or inflamed (T cells diffusely infiltrating tumor parenchyma and stroma). We hypothesized that association of a GEP signature with abundance of parenchymal and stromal T-cell infiltrates may identify biomarkers of response or resistance to I-O therapy. To test this, we applied an AI-powered IA platform to quantify CD8+ T cells by geographical location and used GEP to define both CD8 abundance and associated geographic localization to tumor parenchyma and stroma. Methods: We performed an analysis using a tumor inflammatory GEP assay and CD8 immunohistochemistry on procured specimens (335 melanoma, 391 SCCHN). Digitized slides were used to train a convolutional neural network to quantify the number of CD8+ T cells in stroma, tumor parenchyma, parenchyma-stromal interface, and invasive margin. Generalized constrained regression models were used to predict GEP signatures specifically for stromal and parenchymal CD8+ T cells. Results: Parenchymal and stromal GEP scores were highly concordant with CD8+ infiltrate geography (adj- r2: 0.67, 0.65, respectively; P ≤ 0.01). Little overlap existed between gene sets associated with parenchymal and stromal CD8 T-cell geographies. CSF1R and NECTIN2 gene expression was observed to correlate inversely with parenchymal localization and directly with stromal CD8+ T-cell abundance. Conclusions: GEP signatures can be identified that are concordant with various CD8+ T-cell localization patterns in melanoma and SCCHN, demonstrating that GEP-IA can be developed to identify the immune status of interest in the TME. The specific genes identified have potential to elucidate mechanisms of resistance and/or inform I-O targets that can be further evaluated in relation to clinical significance in future studies.

PTEN-deficient prostate cancer is associated with an immunosuppressive tumor microenvironment mediated by increased expression of IDO1 and infiltrating FoxP3+ T regulatory cells.

Accumulating evidence shows that tumor cell-specific genomic changes can influence the cross talk between cancer cells and the surrounding tumor microenvironment (TME). Loss of the PTEN tumor suppressor gene is observed in 20% to 30% of prostate cancers (PCa) when first detected and the rate increases with PCa progression and advanced disease. Recent findings implicate a role for PTEN in cellular type I interferon response and immunosuppression in PCa. However, the way that PTEN inactivation alters antitumor immune response in PCa is poorly understood. To investigate the changes associated with PTEN loss and an immunosuppressive TME in PCa, we used CIBERSORT to estimate the relative abundance of 22 immune-cell types from 741 primary and 96 metastatic tumors. Our in silico findings were then validated by immunohistochemical analysis of immune cells and IDO1 and PDL1 checkpoint proteins in a cohort of 94 radical prostatectomy specimens. FoxP3+ T regulatory cells (Tregs) were significantly increased in PTEN-deficient PCa in all three public domain cohorts. Loss of PTEN in bone metastases was associated with lower CD8+ T-cell abundance, but in liver metastasis, FoxP3+ Tregs were present at higher levels. PTEN-deficient lymph node metastasis had a distinct profile, with high levels of CD8+ T cells. Moreover, we found that metastatic PCa presents higher abundance of FoxP3+ Treg when compared to primary lesions. Since PTEN-deficient tumors are likely to be immunosuppressed as a consequence of increased FoxP3+ Tregs, we then evaluated the localization and expression of IDO1, PDL1 immune checkpoints, and the corresponding density of FoxP3+ Treg and CD8+ T cells using our validation cohort (n = 94). We found that IDO1 protein expression and FoxP3+ Treg density were higher in neoplastic glands compared with benign adjacent tissue. Moreover, higher densities of FoxP3+ Treg cells in both stromal (P = 0.04) and tumor (P = 0.006) compartments were observed in PTEN-deficient tumors compared to tumors that retained PTEN activity. Similarly, IDO1 protein expression was significantly increased in the tumor glands of PTEN-deficient PCa (P < 0.0001). Spearman correlation analysis showed that IDO1 expression was significantly associated with FoxP3+ Treg and CD8+ T-cell density (P < 0.01). Our findings imply that PTEN deficiency is linked to an immunosuppressive state in PCa with distinct changes in the frequency of immune cell types in tumors from different metastatic sites. Our data suggest that determining PTEN status may also help guide the selection of patients for future immunotherapy trials in localized and metastatic PCa.

Abstract A209: Targeting immune cell-specific sphingosine-1-phosphate receptor 4 to restore antitumor immunity resulting in improved therapy response

Abstract The inflammatory tumor microenvironment is a critical element of tumor development and progression, as well as a parameter predicting the response to cancer treatment. An increased abundance of suppressive immune cells such as tumor-associated macrophages (TAMs) and regulatory T-cells (Tregs) appears thereby as a limiting factor for a successful treatment due to the inhibition of tumoricidal lymphocytes such as cytotoxic CD8+ T-cells. The bioactive lipid sphingosine-1-phosphate (S1P), which signals mainly through its five G-protein-coupled receptors (S1PR1-5), emerges as an important regulator of carcinogenesis that promotes tumor growth, angiogenesis and metastasis, among others, by affecting tumor immunity. We investigated S1PR4, which is exclusively expressed by immune cells, as a potential regulator of antitumor immunity. Using a transgenic mouse model of spontaneous invasive ductal mammary carcinoma (PyMT mice) and the AOM/DSS model of colitis-associated cancer, we show that S1PR4 deficiency (S1PR4-/-) leads to a significant decrease in lung metastasis and a delay of tumor growth in case of the PyMT breast cancer model and reduced colon tumor incidence and size in S1PR4-/- mice subjected to the AOM/DSS model. S1PR4-/- PyMT tumors contained macrophages with an altered activation profile, characterized by reduced IL-10, IL-1β and IL-6 production. Likely as a consequence, S1PR4-/- mice showed a less immunosuppressive lymphocyte infiltrate composed of reduced Tregs and Th17 cells, but increased cytotoxic CD8+ T-cell numbers. Interestingly, AOM/DSS-treated colons of S1PR4-/- mice also displayed a marked increase in CD8+ intestinal epithelial lymphocytes, underlining the negative impact of S1PR4 signaling on CD8+ T-cell abundance during tumor development. Next, we asked whether increased CD8+ T-cell numbers in S1PR4-/- tumors would increase the efficacy of chemotherapy or immunotherapy. Indeed, S1PR4-/- PyMT mice exhibited a significantly improved response to treatment with doxorubicin (DXR), characterized by the absence of relapse, whereas WT PyMT tumors relapsed after DXR treatment. This enhanced tumor control was strictly CD8+ T-cell dependent, as shown by experiments utilizing CD8 antibody depletion in combination with DXR treatment. Finally, using a MC38 colon adenocarcinoma model, where MC38 cells were engrafted into the flanks of WT and S1PR4-/- mice, we demonstrate a synergistic effect of S1PR4 depletion and anti-PD-1 immunotherapy reflected by reduced tumor growth and an increased survival rate of tumor-bearing S1PR4-/- compared to WT mice. A similar effect of additional tumor growth reduction by combinatorial anti-PD-1 treatment was seen in S1PR4-/- PyMT mice compared to their WT counterpart, although less pronounced compared to the MC38 allograft model. Taken together, our data indicate that S1PR4 depletion restores antitumor immunity by, among other mechanisms, increasing CD8+ T-cell abundance, translating into enhanced response to chemotherapy and anti-PD-1 immunotherapy. Thus, we reveal the potential of S1PR4 as a target to improve cancer therapy. Citation Format: Catherine Olesch, Evelyn Sirait-Fischer, Bernhard Brüne, Andreas Weigert. Targeting immune cell-specific sphingosine-1-phosphate receptor 4 to restore antitumor immunity resulting in improved therapy response [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A209.

Abstract A083: Inflammasome-independent IL-1β release by myeloid cells promotes vessel destabilization and immune suppression in the tumor microenvironment

Abstract Background: Chronic inflammation in the tumor microenvironment (TME) sustained by immune cells has a crucial role both in tumor initiation and progression. One of the central cytokines of inflammation, IL-1β, is produced as a biologically inactive precursor that requires proteolytic processing by caspase-1. Activation of caspase-1 is triggered by the formation of inflammasomes, multiprotein complexes that detect microbial and endogenous danger signals primarily via NOD-like receptors, such as NLRP3 and NLRC4. Biologically active IL-1β is believed to be released through membrane pores formed by gasdermin D during a lytic form of cell death called pyroptosis. Although IL-1β-mediated inflammation has been shown to have a detrimental role in tumor progression, the signaling pathway controlling IL-1β release in the TME and the exact effect of the cytokine on antitumor T-cell responses have not been fully elucidated. A better understanding of how IL-1β release is controlled in tumors will also pave the way towards the therapeutic utilization of small-molecule inhibitors available to target NOD-like receptors and caspase-1. Methods: First, we characterized the impact of IL-1β in the TME by assessing the immune cell composition and vasculature of Lewis lung carcinomas (LLC) and E0771 breast carcinomas in IL-1β-deficient mice using flow cytometry and histologic analysis. Next, we used mice deficient in different inflammasome components, including NLRP3, NLRC4 and caspase-1, to investigate the involvement of these proteins in controlling IL-1β release in LLC and E0771 tumors. Using immunoblots and small-molecule inhibitors, we further characterized the activation of alternative enzymatic pathways and their involvement in IL-1β release by tumor-associated myeloid cells. Finally, we examined the role of pyroptosis and necroptosis in IL-1β release using gasdermin D- and MLKL-deficient mice, respectively. Release of IL-1β was assessed using ELISA and immunoblots. Results: We found that IL-1β secretion was restricted to myeloid cells and promoted tumor progression in mouse models of lung and breast carcinoma. IL-1β deletion abrogated the tumor-induced mobilization of immunosuppressive neutrophils and normalized the tumor vasculature, thereby alleviating hypoxia. Consequently, proliferation of effector T-cells in the TME was enhanced, leading to higher CD4+ and CD8+ T-cell abundance in the absence of IL-1β. We observed that, although the NLRP3 inflammasome was active in tumor-infiltrating myeloid cells, NLRP3 and caspase-1 were not essential for the proteolytic maturation of pro-IL-1β and secretion of biologically active IL-1β in the TME. Inhibition or genetic deletion of caspase-8 reduced inflammasome-independent IL-1β release, indicating that caspase-8 provides an alternative pathway for proteolytic activation and secretion of IL-1β in tumor-infiltrating myeloid cells. Moreover, IL-1β release by tumor-infiltrating myeloid cells was independent of lytic cell death modalities including gasdermin D-mediated pyroptosis and MLKL-mediated necroptosis, suggesting an alternative release mechanism for the cytokine in the TME. Conclusions: Overall, our results demonstrate that tumor-infiltrating myeloid cells are able to release IL-1β independently of inflammasomes. We show that proteolytic maturation of IL-1β via caspase-8 in myeloid cells acts as an important driver of immune suppression in the TME through vascular destabilization, recruitment of immunosuppressive neutrophils and consequential inhibition of antitumor T-cell responses. We also show, that, unlike in autoinflammation, gasdermin D-mediated pyroptosis is not essential for the release of IL-1β in tumors. These results suggest that therapeutic inhibition of inflammasomes or pyroptosis will likely not be beneficial in certain tumor types due to the presence of an alternative caspase-8-mediated IL-1β release pathway in tumor-associated myeloid cells. Citation Format: Máté Kiss, Lieselotte Vande Walle, Helena Van Damme, Aleksandar Murgaski, Evangelia Bolli, Jiri Keirsse, Maria Solange Martins, Yvon Elkrim, Amelie Fossoul, Jens Serneels, Massimiliano Mazzone, Mohamed Lamkanfi, Jo A. Van Ginderachter, Damya Laoui. Inflammasome-independent IL-1β release by myeloid cells promotes vessel destabilization and immune suppression in the tumor microenvironment [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A083.

EMT- and stroma-related gene expression and resistance to PD-1 blockade in urothelial cancer

Cancers infiltrated with T-cells are associated with a higher likelihood of response to PD-1/PD-L1 blockade. Counterintuitively, a correlation between epithelial–mesenchymal transition (EMT)-related gene expression and T-cell infiltration has been observed across tumor types. Here we demonstrate, using The Cancer Genome Atlas (TCGA) urothelial cancer dataset, that although a gene expression-based measure of infiltrating T-cell abundance and EMT-related gene expression are positively correlated, these signatures convey disparate prognostic information. We further demonstrate that non-hematopoietic stromal cells are a major source of EMT-related gene expression in bulk urothelial cancer transcriptomes. Finally, using a cohort of patients with metastatic urothelial cancer treated with a PD-1 inhibitor, nivolumab, we demonstrate that in patients with T-cell infiltrated tumors, higher EMT/stroma-related gene expression is associated with lower response rates and shorter progression-free and overall survival. Together, our findings suggest a stroma-mediated source of immune resistance in urothelial cancer and provide rationale for co-targeting PD-1 and stromal elements.

Leukocyte Dynamics during the Evolution of Human Coronary Atherosclerosis: Conclusions from a Sevenfold, Chromogen-Based, Immunohistochemical Evaluation

Atherosclerosis is a complex process with strong inflammatory component. We developed a straightforward sevenfold staining protocol for simultaneous assessment of dominant leukocyte classes, vascularization, and expression of the putative foam cell maker CD36. The method was applied on human coronaries covering the full spectrum of atherosclerotic disease. Results confirm the progressive association of macrophages and T cells with the process and a global presence of mast cells. B cells are exclusively present in adventitial follicles that accompany the process plaque destabilization (thin cap and ruptured lesions) and are otherwise absent. Neutrophils are only present as part of the hemorrhage that accompanies plaque rupture. This study does not classify CD36 as a key factor in foam cell formation. Observed macrophage accumulation in the neointima of stabilized fibrous calcified plaques is consistent with a process of neoatherosclerosis. This study on human coronaries shows a progressive association of macrophage and T-cell abundance with plaque progression. Follicle-like structures are transiently present during the process of plaque destabilization. Plaque healing is accompanied by cessation of the inflammatory response but followed by a new cycle of atherosclerosis.

Abstract 2672: Response to anti-PD-1 based therapy in metastatic melanoma patients is associated with the diversity and composition of the gut microbiome

Abstract Background: Melanoma therapy has benefitted greatly from immune checkpoint blockade, although responses are variable and not always durable. There is a growing appreciation of the role of the microbiome in cancer-related outcomes and recent evidence in murine models suggests that modulation of the gut microbiome may enhance responses to immune checkpoint blockade in melanoma. However this has not been investigated in patients. Here, we demonstrate that differential bacterial “signatures” exist in the gut microbiome of responders (R) and non-responders (NR) to anti-PD-1 therapy, and that insights gained could be used to derive actionable strategies to enhance responses. Methods: We collected buccal (n=105) and stool (n=53) samples from a cohort of anti-PD-1 treated metastatic melanoma patients (n=110). Patients were classified as either R or NR based on RECIST criteria, and 16S rDNA, and whole-genome shotgun sequencing was performed to characterize the diversity, composition and functional capabilities of the microbiomes. Immune profiling (via 7-marker IHC panel of CD3, CD8, PD-1, PD-L1, Granzyme B, RORγT and FoxP3) and cytokine analyses were also performed on available tumors and serum samples at baseline. Results: In these studies, we observed significant differences in the diversity and composition of the gut microbiome in R versus NR to PD-1 blockade at baseline, but no clear differences in buccal microbiomes. Specifically, R had a significantly higher alpha diversity compared to NR (p=0.017), and the Ruminococcaceae family of the Clostridiales order was enriched in R whereas Prevotellaceae family of the Bacteroidales order was enriched in NR. Immune profiling demonstrated significantly increased immune infiltrates in baseline tumor samples of R, with a positive correlation between CD8, CD3, PD-1 and FoxP3 T-cell density and abundance of specific bacteria enriched in R (e.g. Faecalibacterium). Low diversity was also associated with elevated levels of chronic inflammation markers in the serum at baseline. Lastly, we saw differentially abundant metabolic pathways in the gut microbiomes of R (pyrimidine nucleotide biosynthesis, fatty acid biosynthesis, shikimate pathway) vs NR (Tricarboxylic acid cycle, assimilatory sulphate and nitrate reduction, tryptophan biosynthesis). Conclusion: Differences exist in the diversity and composition of the gut microbiome in R vs NR to anti-PD-1 therapy and these microbiota could bridge the gap between host metabolism and anti-tumor immunity. These results have far-reaching implications and suggest that modifications to the gut microbiome could potentially enhance therapeutic responses to immune checkpoint blockade. Citation Format: Vancheswaran Gopalakrishnan, Christine Spencer, Alexandre Reuben, Peter Prieto, Diego Vicente, Tatiana V. Karpinets, Courtney W. Hudgens, Diane S. Hutchinson, Michael Tetzlaff, Alexander Lazar, Michael A. Davies, Jeffrey E. Gershenwald, Robert Jenq, Patrick Hwu, Padmanee Sharma, James Allison, Andrew Futreal, Nadim Ajami, Joseph Petrosino, Carrie Daniel-MacDougall, Jennifer A. Wargo. Response to anti-PD-1 based therapy in metastatic melanoma patients is associated with the diversity and composition of the gut microbiome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2672. doi:10.1158/1538-7445.AM2017-2672

Sirolimus Increases T-Cell Abundance in the Sun Exposed Skin of Kidney Transplant Recipients.

Kidney transplant recipients (KTRs) receiving the mammalian target of rapamycin inhibitor sirolimus may display a reduced risk of skin cancer development compared to KTRs receiving calcineurin inhibitors. Despite studies investigating the effects of these 2 drug classes on T cells in patient blood, the effect these drugs may have in patient skin is not yet known. Fifteen patients with chronic kidney disease (not recipients of immunosuppressive drugs), and 30 KTRs (15 receiving a calcineurin inhibitor, and 15 receiving sirolimus) provided matched samples of blood, sun exposed (SE) and non-SE skin. The abundance of total CD8+ and CD4+ T cells, memory CD8+ and CD4+ T cells, and regulatory T (Treg) cells in each sample was then assessed by flow cytometry. Sirolimus treatment significantly increased absolute numbers of CD4+ T cells, memory CD8+- and CD4+ T cells, and Treg cells in SE skin versus paired samples of non-SE skin. No differences were found in the absolute number of any T cell subset in the blood. Correlation analysis revealed that the percentage of T cell subsets in the blood does not always accurately reflect the percentage of T-cell subsets in the skin of KTRs. Furthermore, sirolimus significantly disrupts the balance of memory CD4+ T cells in the skin after chronic sun exposure. This study demonstrated that immunosuppressive drug class and sun exposure modify the abundance of multiple T-cell subsets in the skin of KTRs. Correlation analysis revealed that the prevalence of Treg cells in KTR blood does not accurately reflect the prevalence of Treg cells in KTR skin.