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Invariant natural killer T cells (iNKT cells) express a semi-invariant T cell receptor that recognizes certain glycolipids (including α-galactosylceramide, αGC) bound to CD1d, and can induce potent antitumor responses. Here, we assessed whether αGC could enhance the efficacy of a GM-CSF-producing tumor cell vaccine in the transgenic SV40 T antigen-driven TRAMP prostate cancer model. In healthy mice, we initially found that optimal T cell responses were obtained with αGC-pulsed TRAMP-C2 cells secreting GM-CSF and milk fat globule epidermal growth factor protein-8 (MFG-E8) with an RGD to RGE mutation (GM-CSF/RGE TRAMP-C2), combined with systemic low dose IL-12. In a therapeutic model, transgenic TRAMP mice were then castrated at ~ 20 weeks, followed by treatment with the combination vaccine. Untreated mice succumbed to tumor by ~ 40 weeks, but survival was markedly prolonged by vaccine treatment, with most mice surviving past 80 weeks. Prostates in the treated mice were heavily infiltrated with T cells and iNKT cells, which both secreted IFNγ in response to tumor cells. The vaccine was not effective if the αGC, IL-12, or GM-CSF secretion was eliminated. Finally, immunized mice were fully resistant to challenge with TRAMP-C2 cells. Together these findings support further development of therapeutic vaccines that exploit iNKT cell activation.

This study was aimed at assessing the associations of sarcopenia, muscle density, adiposity, and inflammation with overall survival (OS) after cytoreductive nephrectomy (CN) for metastatic renal cell carcinoma.In all, 158 patients undergoing CN from 2001 to 2014 had digitized preoperative imaging for tissue segmentation via Slice-O-Matic software (version 5.0) at the mid-L3 level. The skeletal muscle index was calculated with the skeletal muscle area (cmSeventy-six of the 158 patients (48%) were sarcopenic. Sarcopenia was associated with elevated neutrophil to lymphocyte ratios (NLRs; P = .02), increased age (P = .001), lower body mass indices (P = .009), greater modified Motzer scores (P = .019), and lower SMD (P = .006). The median OS was 15.0 and 29.4 months for sarcopenic and nonsarcopenic patients, respectively (P = .04). Elevated inflammation (NLR or C-reactive protein), in addition to sarcopenia, was independently associated with OS, with an elevated NLR ≥ 3.5 and sarcopenia associated with the poorest OS at 10.2 months. No associations were observed between measurements of muscle density or adiposity and OS.Sarcopenia and measures of high systemic inflammation are additively associated with inferior OS after CN and may be of use in preoperative risk stratification.Body composition and sarcopenia (a deficiency in skeletal musculature) have been shown to affect outcomes in cancer. We found that sarcopenic patients had poor survival in comparison with nonsarcopenic patients in the setting of metastatic renal cell carcinoma (mRCC). Patients with both elevated inflammation and sarcopenia had the poorest survival. Sarcopenia is an objective measure of nutrition that can assist in therapeutic counseling and decision-making for individualized treatment in mRCC.

Increased demand for novel, highly effective vaccination strategies necessitates a better understanding of long-lived memory CD8 T cell differentiation. To achieve this understanding, we used the mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection. We reexamined classical memory CD8 T cell subsets and performed in-depth, longitudinal analysis of their phenotype, transcriptional programming, and anatomic location within the spleen. All analyses were performed at multiple time points from 8 days to 1 year postinfection. Memory subsets are conventionally defined by their expression of KLRG1 and IL-7Rα, as follows: KLRG1(+)IL-7Rα(−) terminal effectors (TEs) and KLRG1(−)IL-7Rα(+) memory precursors (MPs). But we also characterized a third KLRG1(+)IL-7Rα(+) subset which we refer to as KLRG1(+) MPs. In these analyses, we defined a comprehensive memory phenotype that is associated with higher levels of CD28 expression. We also demonstrated that MPs, KLRG1(+) MPs, and TEs have distinct localization programs within the spleen. We found that MPs became preferentially enriched in the white pulp as early as 1 to 2 weeks postinfection, and their predominance in the white pulp was maintained throughout the course of a year. On the other hand, KLRG1(+) MPs and TEs localized to the red pulp just as early, and they consistently localized to the red pulp thereafter. These findings indicate that location may be crucial for memory formation and that white pulp-derived signals may contribute to long-term memory survival. Achieving robust memory responses following vaccination may require more deliberate consideration of which memory phenotypes are induced, as well as where they traffic, as these factors could impact their longevity. IMPORTANCE CD8 T cells play a critical role in viral immunity and it is important to understand how memory cells are formed and what processes lead to their long-term maintenance. Here, we use a mouse model of acute infection to perform an in-depth, longitudinal analysis of memory CD8 T cell differentiation, examining the phenotype and location of memory cells out to 1 year postinfection.

T cells are important in tumour immunity but a better understanding is needed of the differentiation of antigen-specific T cells in human cancer1,2. Here we studied CD8 T cells in patients with human papillomavirus (HPV)-positive head and neck cancer and identified several epitopes derived from HPV E2, E5 and E6 proteins that allowed us to analyse virus-specific CD8 T cells using major histocompatibility complex (MHC) class I tetramers. HPV-specific CD8 T cells expressed PD-1 and were detectable in the tumour at levels that ranged from 0.1% to 10% of tumour-infiltrating CD8 T lymphocytes (TILs) for a given epitope. Single-cell RNA-sequencing analyses of tetramer-sorted HPV-specific PD-1+ CD8 TILs revealed three transcriptionally distinct subsets. One subset expressed TCF7 and other genes associated with PD-1+ stem-like CD8 T cells that are critical for maintaining T cell responses in conditions of antigen persistence. The second subset expressed more effector molecules, representing a transitory cell population, and the third subset was characterized by a terminally differentiated gene signature. T cell receptor clonotypes were shared between the three subsets and pseudotime analysis suggested a hypothetical differentiation trajectory from stem-like to transitory to terminally differentiated cells. More notably, HPV-specific PD-1+TCF-1+ stem-like TILs proliferated and differentiated into more effector-like cells after in vitro stimulation with the cognate HPV peptide, whereas the more terminally differentiated cells did not proliferate. The presence of functional HPV-specific PD-1+TCF-1+CD45RO+ stem-like CD8 T cells with proliferative capacity shows that the cellular machinery to respond to PD-1 blockade exists in HPV-positive head and neck cancer, supporting the further investigation of PD-1 targeted therapies in this malignancy. Furthermore, HPV therapeutic vaccination efforts have focused on E6 and E7 proteins; our results suggest that E2 and E5 should also be considered for inclusion as vaccine antigens to elicit tumour-reactive CD8 T cell responses of maximal breadth. An analysis of human papillomavirus (HPV)-specific CD8 T cells in patients with head and neck cancer identifies functional PD-1+TCF-1+CD8 T cells in the tumour with implications for therapeutic vaccination and PD-1 directed immunotherapy.

Background Several immune checkpoint inhibitors (ICIs) are approved for the treatment of advanced urothelial carcinoma (UC). There are limited biomarkers for ICI-treated patients with UC. We investigated the association between body composition and clinical outcomes in ICI-treated UC patients. Materials and Methods We conducted a retrospective analysis of 70 ICI-treated patients with advanced UC at Winship Cancer Institute from 2015 to 2020. Baseline computed tomography images within 2 months of ICI initiation were collected at mid-L3 and muscle and fat compartments (subcutaneous, intermuscular, and visceral) were segmented using SliceOMatic v5.0 (TomoVision, Magog, Canada). A prognostic body composition risk score (high: 0–1, intermediate: 2–3, or low-risk: 4) was created based on the β coefficient from the multivariate Cox model (MVA) following best-subset variable selection. Our body composition risk score was skeletal muscle index (SMI) + 2 × attenuated skeletal muscle (SM) mean + visceral fat index (VFI). Concordance statistics (C-statistics) were used to quantify the discriminatory magnitude of the predictive model. Results Most patients (70%) were men and the majority received ICIs in the second- (46%) or third-line (21%) setting. High-risk patients had significantly shorter overall survival (OS; hazard ratio [HR], 6.72; p Conclusion Body composition variables such as SMI, SM mean, and VFI may be prognostic and predictive of clinical outcomes in ICI-treated patients with UC. Larger, prospective studies are warranted to validate this hypothesis-generating data. Implications for Practice This study developed a prognostic body composition risk scoring system using radiographic biomarkers for patients with bladder cancer treated with immunotherapy. The study found that the high-risk patients had significantly worse clinical outcomes. Notably, the study's model was better at predicting outcomes than body mass index. Importantly, these results suggest that radiographic measures of body composition should be considered for inclusion in updated prognostic models for patients with urothelial carcinoma treated with immunotherapy. These findings are useful for practicing oncologists in the academic or community setting, particularly given that baseline imaging is routine for patients starting on treatment with immunotherapy.

Background The modified Glasgow prognostic score (mGPS), a clinical tool that incorporates albumin and C-reactive protein, has proven useful in the prognostication of multiple cancers. Several immune checkpoint inhibitors (ICIs) have been approved for the treatment of metastatic urothelial cell carcinoma (mUC), but a prognostic biomarker is needed. We investigated the impact of mGPS on survival outcomes in patients with mUC receiving ICIs. Materials and Methods We retrospectively reviewed patients with mUC treated with ICIs (programmed cell death protein 1 or programmed cell death ligand 1 inhibitors) at Winship Cancer Institute from 2015 to 2018. Overall survival (OS) and progression-free survival (PFS) were measured from the start date of ICI until death or clinical or radiographic progression, respectively. mGPS was defined as a summary score with one point given for C-reactive protein >10 mg/L and/or albumin Results A total of 53 patients were included with a median follow-up 27.1 months. The median age was 70 years, with 84.9% male and 20.8% Black. Baseline mGPS was 0 in 43.4%, 1 in 28.3% and 2 in 28.3%. Increased mGPS at the time of ICI initiation was associated with poorer OS and PFS in UVA, MVA, and Kaplan-Meier analyses. Conclusion The mGPS may be a useful prognostic tool in patients with mUC when treatment with ICI is under consideration. These results warrant a larger study for validation. Implications for Practice The ideal prognostic tool for use in a busy clinical practice is easy-to-use, cost-effective, and capable of accurately predicting clinical outcomes. There is currently no universally accepted risk score in metastatic urothelial cell carcinoma (mUC), particularly in the immunotherapy era. The modified Glasgow prognostic score (mGPS) incorporates albumin and C-reactive protein and may reflect underlying chronic inflammation, a known risk factor for resistance to immune checkpoint inhibitors (ICIs). This study found that baseline mGPS is associated with survival outcomes in patients with mUC treated with ICIs and may help clinicians to prognosticate for their patients beginning immunotherapy.

Tumours often contain B cells and plasma cells but the antigen specificity of these intratumoral B cells is not well understood1-8. Here we show that human papillomavirus (HPV)-specific B cell responses are detectable in samples from patients with HPV-positive head and neck cancers, with active production of HPV-specific IgG antibodies in situ. HPV-specific antibody secreting cells (ASCs) were present in the tumour microenvironment, with minimal bystander recruitment of influenza-specific cells, suggesting a localized and antigen-specific ASC response. HPV-specific ASC responses correlated with titres of plasma IgG and were directed against the HPV proteins E2, E6 and E7, with the most dominant response against E2. Using intratumoral B cells and plasma cells, we generated several HPV-specific human monoclonal antibodies, which exhibited a high degree of somatic hypermutation, consistent with chronic antigen exposure. Single-cell RNA sequencing analyses detected activated B cells, germinal centre B cells and ASCs within the tumour microenvironment. Compared with the tumour parenchyma, B cells and ASCs were preferentially localized in the tumour stroma, with well-formed clusters of activated B cells indicating ongoing germinal centre reactions. Overall, we show that antigen-specific activated and germinal centre B cells as well as plasma cells can be found in the tumour microenvironment. Our findings provide a better understanding of humoral immune responses in human cancer and suggest that tumour-infiltrating B cells could be harnessed for the development of therapeutic agents.

Recent studies have defined a novel population of PD-1(+) TCF-1(+) stem-like CD8 T cells in chronic infections and cancer. These quiescent cells reside in lymphoid tissues, are critical for maintaining the CD8 T cell response under conditions of persistent antigen, and provide the proliferative burst after PD-1 blockade. Here we examined the role of TGF-beta in regulating the differentiation of virus-specific CD8 T cells during chronic LCMV infection of mice. We found that TGF-beta signaling was not essential for the generation of the stem-like CD8 T cells but was critical for maintaining the stem-like state and quiescence of these cells. TGF-beta regulated the unique transcriptional program of the stem-like subset, including upregulation of inhibitory receptors specifically expressed on these cells. TGF-beta also promoted the terminal differentiation of exhausted CD8 T cells by suppressing the effector-associated program. Together, the absence of TGF-beta signaling resulted in significantly increased accumulation of effector-like CD8 T cells. These findings have implications for immunotherapies in general and especially for T cell therapy against chronic infections and cancer.

Introduction: T cells lacking costimulatory receptors CD27/28, with high levels of differentiation markers CD57 and KLRG1, may accumulate with aging. These T cells have been described as “senescent” (unable to proliferate) with high cytolytic enzyme expression. However, robust functional studies on these cells are lacking. Further, emerging studies on the implications of these cells in the context of prognosis and responses to immunotherapy have shown mixed results. To better understand the role of these cells in the pathogenesis of cancer, a better understanding of their functional potential is needed. Methods and Results: We collected blood from 46 patients with previously untreated, locally advanced head and neck squamous cell carcinoma (HNSCC), aged 47-89, undergoing surgery. Matched tumor tissue was collected from 29 patients. Samples were analyzed by flow cytometry. There was correlation between the percentage of CD27/28-CD57+ (CD28neg) T cells in the blood vs. tumor (R2 = 0.51 for CD8+, 0.41 for CD4+; p Citation Format: Brendan L.C. Kinney, Connor Parrish, Vikash Kansal, Zachary S. Buchwald, Haydn T. Kissick, Amy Y. Chen, Nicole C. Schmitt. CD28-negative T cells from head and neck cancer patients are functionally frail but not senescent [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B025.

Invariant natural killer T cells (iNKT cells) express a semi-invariant T cell receptor that recognizes certain glycolipids (including α-galactosylceramide, αGC) bound to CD1d, and can induce potent antitumor responses. Here, we assessed whether αGC could enhance the efficacy of a GM-CSF-producing tumor cell vaccine in the transgenic SV40 T antigen-driven TRAMP prostate cancer model. In healthy mice, we initially found that optimal T cell responses were obtained with αGC-pulsed TRAMP-C2 cells secreting GM-CSF and milk fat globule epidermal growth factor protein-8 (MFG-E8) with an RGD to RGE mutation (GM-CSF/RGE TRAMP-C2), combined with systemic low dose IL-12. In a therapeutic model, transgenic TRAMP mice were then castrated at ~ 20 weeks, followed by treatment with the combination vaccine. Untreated mice succumbed to tumor by ~ 40 weeks, but survival was markedly prolonged by vaccine treatment, with most mice surviving past 80 weeks. Prostates in the treated mice were heavily infiltrated with T cells and iNKT cells, which both secreted IFNγ in response to tumor cells. The vaccine was not effective if the αGC, IL-12, or GM-CSF secretion was eliminated. Finally, immunized mice were fully resistant to challenge with TRAMP-C2 cells. Together these findings support further development of therapeutic vaccines that exploit iNKT cell activation.

Previous studies have shown that plasmablasts expand massively in dengue patients as compared to many other situations such as influenza infection or vaccination. However, a detailed understanding of the phenotypes and transcriptional features of these cells is lacking. Moreover, despite India having nearly a third of global dengue disease burden, there is virtually no information on plasmablasts responses in dengue patients from India. Here, we provide a detailed characterization of plasmablast responses from dengue confirmed febrile children in India. Immunophenotyping and RNA seq analysis showed that in addition to secreting dengue specific antibodies, these massively expanding cells expressed several adhesion molecules, chemokines and chemokine receptors that are involved in endothelial interactions, homing to skin or mucosal tissues including intestine. Surprisingly, we found that these cells also upregulated expression of several cytokine genes that are involved in angiogenesis, leukocyte extravasation and vascular permeability. These transcriptional features were qualitatively similar to plasmablasts from influenza vaccinees. Interestingly, the expansion of the plasmablasts in dengue patients was significantly lower in patients with primary dengue infection compared to those with secondary dengue. Moreover, within the primary dengue patients, their expansion was significantly lower in patients with mild dengue infection (DI) compared to patients with dengue with warning signs (DW) or severe dengue (SD). These results significantly improve our understanding of human plasmablast responses in dengue.ImportanceDengue is a globally spreading with over 100 million clinical cases annually with symptoms ranging from mild self-limiting febrile illness to more severe and sometimes life-threatening dengue hemorrhagic fever or shock, especially among children. India contributes nearly a third of global dengue disease burden. The pathophysiology of dengue is complex and remains poorly understood despite many advances indicating a key role for antibody dependent enhancement of infection. While serum antibodies have been extensively studied, the characteristics of the cellular factories responsible for antibody production, i.e., plasmablasts, are only beginning to emerge. This study provides a comprehensive understanding of the magnitude, phenotype, functional and transcriptional profiles of human plasmablasts from dengue patients in India.

BackgroundCD4 T cells can differentiate into multiple effector subsets that can mediate variable functions. In this work we aim to understand how CD4 T cells differentiate in response to tumor antigens and their respective function in the anti-tumor response.MethodsTumor tissue was collected from patients undergoing surgery at Emory University Hospital. Activated PD1+ CD45RA- tumor infiltrating CD4 T cells were sent for 10X single cell RNA-seq. Tumor samples were also processed for flow cytometry and ex vivo functional analyses. For in vivo studies, prostate cancer mouse model expressing the LCMV glycoprotein (TRAMPC1-GP) was used, as well as LCMV Armstrong infection.ResultsTo characterize the heterogeneity of CD4 T cells infiltrating kidney tumors, we performed single cell RNAseq. We found three distinct activated (PD1+ CD45RA-) CD4 T cell populations. Two effector clusters consisting of Th1-like (EOMES+) and Treg (FOXP3+) cells, and a third cluster expressing TCF1, and genes associated with stemness and survival that did not fit defined CD4 effector lineages. We further confirmed these data by flow cytometry and found the same tumor infiltrating CD4 subsets in 100 kidney cancer patients. When placed in culture under different polarization conditions, tumor TCF1+ CD4 T cells proliferated and differentiated into the Th1-like and Treg effector populations found in the tumor, in addition to other effector lineages (Th1, Tfh) given the appropriate conditions, while the Th1-like and Treg cells underwent no proliferation or phenotype changes. These data suggests that the TCF1+ CD4s act as activated unpolarized precursors to the effector subsets in the tumor. To further test this hypothesis in vivo, we adoptively transferred tumor specific (SMARTA) CD4 T cells into mice followed by TRAMPC1-GP tumor inoculation. Transferred SMARTAs activated and first acquired a TCF1+ phenotype in the TDLN prior to predominantly differentiating into Tregs in the tumor. Given their plasticity in vitro, we asked whether TCF1+ SMARTAs primed in tumors were destined to differentiate into Tregs. To test this, we transferred 4-week activated TCF1+ SMARTAs from TDLNs of TRAMPC1-GP mice into naïve mice that were immediately infected with LCMV Armstrong. We found that the transferred SMARTAs differentiated into Th1 and Tfh cells in response to the virus, similar to the endogenous virus specific CD4 T cells.ConclusionsOverall, this work shows that CD4 T cells remain in an activated phenotype in the tumor with the capacity to differentiate into non-suppressive effector lineages given the appropriate conditions that may benefit the anti-tumor response.

BackgroundTumor infiltrating T-cells have a prognostic benefit in many tumor types,1–8 and we recently sought to determine whether the level of T-cell infiltration into renal tumors predicts clinical outcomes. In our recent publication,9 we showed that patients with high of CD8 T-cell infiltration have improved progression free survival (PFS). Further, we found that this T-cell response is supported by TCF1+ stem-like CD8 T-cells, which reside within dense regions of closely clustered antigen presenting cells within the tumor. Interestingly, aggregations of immune cells have also been described in other tumor types and termed ‘tertiary lymphoid structures’ (TLS), which are typically defined as B-cell-dominant aggregates, containing high endothelial venules and reactive germinal centers.10–12 Together, these findings raise several important questions, which we explore here—(1) what additional cell types comprise these niches?9 and (2) how are these niches similar to or different from TLS?MethodsTumor tissue was collected from patients with renal tumors undergoing surgery at Emory University Hospital. Intraoperative tumor samples were analyzed by flow cytometry, RNA sequencing, immunofluorescence, and immunohistochemistry. Immunofluorescence data was analyzed using our custom quantitative analysis pipelines, which allows for delineation of cell type and location, cell-cell distance, and density of cellular aggregation.ResultsThe proportion of CD8 T-cells infiltration human renal tumors varied widely, consistent with our previous reports.9 TCF1+ stem-like CD8 T-cells were identifiable by both flow cytometry and immunofluorescence and resided in dense antigen presenting niches. Quantitative immunofluorescence revealed the location of aSMA+ fibroblasts within tumor tissue, in relation to antigen presenting niches, and in tumors with many infiltrating T-cells. Pathologist scored hematoxylin and eosin-stained slides were delineated TLS+ or TLS-. Quantitative immunofluorescence imaging analysis revealed the detailed composition of tumor infiltrating immune cell populations and the contrasting cellular organization in TLS as compared to in antigen presenting niches.ConclusionsAs we have shown CD8 T-cell infiltration to predict PFS in renal tumors and that antigen presenting niches containing stem-like cells maintain the anti-tumor T-cell response,9 it is critical to understand the additional cell types present in these niches and to understand how these niches relate to previously described phenomena of immune organization, such as TLS.10–12 This mechanistic understanding of the anti-tumor immune response represents an opportunity to inform development of enhanced prognostic tools and innovative therapeutic possibilities.ReferencesAzimi F, et al. Tumor-infiltrating lymphocyte grade is an independent predictor of sentinel lymph node status and survival in patients with cutaneous melanoma. J Clin Oncol 2012;30(21):2678–83. Epub 2012/06/20. doi: 10.1200/jco.2011.37.8539. PubMed PMID: 22711850.Galon J, et al. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science 2006;313(5795):1960–4. Epub 2006/09/30. doi: 10.1126/science.1129139. PubMed PMID: 17008531.Mlecnik B, et al. Integrative analyses of colorectal cancer show immunoscore is a stronger predictor of patient survival than microsatellite instability. Immunity 2016;44(3):698–711. Epub 2016/03/18. doi: 10.1016/j.immuni.2016.02.025. PubMed PMID: 26982367.Mlecnik B, et al. Histopathologic-based prognostic factors of colorectal cancers are associated with the state of the local immune reaction. J Clin Oncol 2011;29(6):610–8. Epub 2011/01/20. doi: 10.1200/JCO.2010.30.5425. PubMed PMID: 21245428.Pagès F, et al. Immune infiltration in human tumors: a prognostic factor that should not be ignored. Oncogene 2009;29:1093. doi: 10.1038/onc.2009.416.Peranzoni E, et al. Macrophages impede CD8 T cells from reaching tumor cells and limit the efficacy of anti-PD-1 treatment. Proceedings of the National Academy of Sciences of the United States of America 2018;115(17):E4041–E50. Epub 2018/04/11. doi: 10.1073/pnas.1720948115. PubMed PMID: 29632196.Savas P, et al. Single-cell profiling of breast cancer T cells reveals a tissue-resident memory subset associated with improved prognosis. Nat Med 2018;24(7):986–93. Epub 2018/06/27. doi: 10.1038/s41591-018-0078-7. PubMed PMID: 29942092.Tosolini M, et al. Clinical impact of different classes of infiltrating T cytotoxic and helper cells (Th1, th2, treg, th17) in patients with colorectal cancer. Cancer Res 2011;71(4):1263–71. Epub 2011/02/10. doi: 10.1158/0008-5472.Can-10-2907. PubMed PMID: 21303976.Jansen CS, et al. An intra-tumoral niche maintains and differentiates stem-like CD8 T cells. Nature 2019;576(7787):465–70. doi: 10.1038/s41586-019-1836-5.Dieu-Nosjean MC, et al. Tertiary lymphoid structures in cancer and beyond. Trends Immunol 2014;35(11):571–80. Epub 2014/12/03. doi: 10.1016/j.it.2014.09.006. PubMed PMID: 25443495.Goc J, et al. Characteristics of tertiary lymphoid structures in primary cancers. Oncoimmunology 2013;2(12):e26836. Epub 2014/02/06. doi: 10.4161/onci.26836. PubMed PMID: 24498556; PMCID: PMC3912008.Sautes-Fridman C, et al. Tertiary lymphoid structures in the era of cancer immunotherapy. Nature reviews Cancer 2019;19(6):307–25. Epub 2019/05/17. doi: 10.1038/s41568-019-0144-6. PubMed PMID: 31092904.Ethics ApprovalSamples are collected under an approved IRB protocol (The Urological Satellite Specimen Bank at Emory University, IRB00055316). All patients provided informed consent.ConsentSamples are collected under an approved IRB protocol (The Urological Satellite Specimen Bank at Emory University, IRB00055316). All patients provided informed consent.

BackgroundPaucity of T cells in the immune privileged tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) is a major reason that PDAC is refractory to immune checkpoint blockade.1 In this study, we show that human PDAC tumors over-express vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, that inhibits effector T cell responses and regulates chemokine receptor expression on activated T cells.2 3 We thus hypothesized that pharmacological inhibition of VIP receptor signaling could enhance anti-tumor responses in PDAC.MethodsVIP levels in plasma were determined via VIP-specific enzyme immunoassay and confirmed with immunohistochemistry (IHC) of tissue sections. VIP receptor (VIP-R) signaling in C57BL/6 immunocompetent murine models of KPC, MT5 or Panc02 pancreatic cancer was inhibited by daily sub-cutaneous treatment with ANT008 or ANT308, two novel VIP-R antagonists with predicted high binding affinities to VIP receptors.4–7 Multiplex IHC or flow cytometry detected frequencies and phenotypes of intra-tumoral T cells across treatment groups.ResultsHuman PDAC tumors expressed VIP by immunohistochemistry, and PDAC patients had significantly elevated plasma VIP levels when compared to healthy volunteers (pConclusionsVIP-R antagonists represent a novel approach to treat PDAC. VIP and VIP-R sequences are highly conserved between humans and mice,8 and human T cells are activated in vitro following treatment with VIP-R antagonists. Thus, we predict comparable anti-tumor activity of the combination of VIP-R antagonist and anti-PD-1 MoAb in human PDAC patients. Further clinical development of this novel concept will require appropriate pre-clinical pharmacokinetic and toxicology studies.AcknowledgementsThe authors thank healthy volunteers and patients for blood and/or tissue samples. The authors also thank the shared resources at Emory University, namely the Emory Integrated Genomics Core (EIGC), Emory Flow Cytometry Core (EFCC), Cancer Animal Models Shared Resource (CAMS), Cancer Tissue Pathology Core (CTP), Biostatistics Shared Resource (BSR) and Integrated Cellular Imaging Core (ICI), that provided services or instruments at subsidized cost to conduct some of the reported experiments. BioRender was used to make figure 4A and 5C. This work was supported in part by Katz Foundation funding and Emory School of Medicine Dean's Imagine, Innovate and Impact (I3) venture award to Edmund K. Waller and NIH R01 CA207619 awarded to Susan N. Thomas. Part of the cost for the immunohistochemistry staining of tissues was covered by Winship Cancer Institute Development Discovery and Therapeutic Program Pilot funding to Sruthi Ravindranathan.Abstract 748 Figure 1VIP is over-expressed by PDAC. (A) VIP mRNA expression levels in various solid malignancies, as obtained from TCGA. (B) Representative images of human PDAC tumor stained with antibodies to VIP or CK19, showing VIP co-expression in islets (black arrow) and cancer epithelial cells (red arrow). Levels of VIP in (C) culture supernatants collected from murine and human PDAC cell lines cultured for 24 hours (n=3 per cell line) were compared to culture supernatants from B16F10 and D4M melanoma cells; (D) plasma of mice bearing melanoma or PDAC tumors (n=5) compared to plasma of non-tumor-bearing mice; (E) plasma of PDAC patients (n=19) compared to that from healthy volunteers (n=26). Statistical differences in C and D were performed by ANOVA followed by Dunnett's post-test and in E were performed by student's t-test. Error bars show mean ± SEM. *pAbstract 748 Figure 2VIP-R antagonists improve responses to anti-PD-1. KPC.Luc, MT5 or Panc02 cells were subcutaneously implanted in immunocompetent C57BL/6 mice. About one week after tumor implantation, when the tumors were palpable, mice were randomized into treatment groups and treated with VIP-R antagonist and/or anti-PD-1 as described in methods. (A) KPC.Luc, MT5 and Panc02 tumor volumes as measured by Vernier calipers on day 22 after subcutaneous tumor implantation. (B) Kaplan-Meier survival plots of C57BL/6 mice with subcutaneously implanted KPC.Luc, MT5 or Panc02 tumors stratified by treatment. Kaplan-Meier survival plots of (C) C57BL/6 mice receiving monoclonal CD4 and/or CD8 monoclonal antibodies (D) CD4KO or (E) CD8KO mice compared to wild-type CD57BL/6 mice with subcutaneously implanted KPC.Luc tumors, stratified by treatment. Statistical differences in A were calculated by ANOVA followed by Dunnett's post-test. Solid line shows mean with in each treatment group. Statistical differences in B-E are calculated via Log-rank test. *pAbstract 748 Figure 3Enhanced T cell response with combination therapy. mRNA expression in T cells isolated from subcutaneous KPC.Luc tumors in C57BL/6 mice treated with ANT008 and/or anti-PD-1 (n=3 per treatment group), were analyzed via Nanostring metabolism panel. Volcano plot showing differential expression of genes in T cells from (A) ANT008+ isotype IgG (IgG) vs scrambled peptide (Scram) + isotype IgG, (B) scrambled peptide +anti-PD-1 vs scrambled peptide + isotype IgG and (C) ANT008+anti-PD-1 vs scrambled peptide + isotype IgG (n=3 mice per treatment group). Genes that are associated with TCR activation and co-stimulation and are at levels significantly higher when compared to Scram+ isotype IgG (FDRAbstract 748 Figure 4Increased T cell density with combination therapy. KPC.Luc cells were orthotopically implanted in the tail of the pancreas of C57BL/6 mice and treated with ANT008 and/or anti-PD-1 with n=9, 10, 8 and 11 in scrambled+IgG, ANT008+IgG, scrambled+anti-PD-1 and ANT008+anti-PD-1, respectively. (A) Schematic showing orthotopic implantation of KPC.Luc cells and treatment strategy with ANT008 and/or anti-PD-1. (B) Waterfall plot showing % change in tumor flux on day 22 relative to day 7 prior to start of treatment. (C) Total flux as measured by IVIS bioluminescent imaging in the different treatment groups. Cross symbol represents mice that were euthanized before day 25 due to ulceration of the tumor and circle symbol represent mouse that were imaged on day 26 via MRI imaging shown in supplementary figure S5. (D) Bar graph showing weight of pancreas on day 25 when the mice were euthanized. 'Star' shaped data points indicate tumor free mice and dotted horizontal line represents the average weight of healthy pancreas from naïve mice. (E) Representative multiplex IHC images (right) showing pancreatic tumors stained for DAPI (blue), CD4 (yellow), CD8 (red) and Ki67 (cyan) and trichrome staining (left) with black arrows showing blue collagen stain in the tissue. XY plot showing the correlation between number of (F) CD4+ or (G) CD8+ T cells/mm2; and (H) Ki67+ CD4+ or (I) Ki67+ CD8+ T cells/mm2 with weight of the pancreas with n=4 to 6 mice per group. P values in panel D were calculated using student ANOVA followed by Dunnett's post hoc test (comparing each treatment group with Scram+IgG). Error bars show mean ± SEM. *pAbstract 748 Figure 5Increased T cell homing with combination therapy. KPC.Luc tumors were subcutaneously implanted in C57BL/6 mice and treated with VIP-R antagonist and/or anti-PD-1 checkpoint therapy for 10 days after the tumors were palpable. Tumor draining lymph nodes were then analyzed for percentage of (A) CXCR4+CD69+ and (B) CXCR4+Ki67+ cells in CD4+ (left) and CD8+ (right) subsets of T cells. In a separate experiment, on day 15 after subcutaneous implantation of KPC.Luc tumors, GFP+ T cells from enhanced GFP transgenic mice (C57BL/6 background) were adoptively transferred (via tail vein injections) and treated with ANT308± aPD-1 for 3 days. (C) Schematic showing GFP+ T cell transfer and treatment strategy in mice with subcutaneous KPC.Luc tumors. (D) Representative Hoescht (blue for nucleus) stained tumor tissues from tumors of each treatment group. Two regions of interest (ROI) in ANT308+aPD-1 treated tumors are shown at higher magnification. Statistical differences in A and B were determined via repeated measures ANOVA and Dunnett's post-test with n=4–5 mice per group. *pReferencesSahin IH, et al. Immunotherapy in pancreatic ductal adenocarcinoma: an emerging entity? Ann Oncol 2017;28(12):2950–2961.Gonzalez-Rey E, Anderson P, Delgado M. Emerging roles of vasoactive intestinal peptide: a new approach for autoimmune therapy. Ann Rheum Dis 2007;66(Suppl 3):p. iii70–6.Anderson P, Gonzalez-Rey E. Vasoactive intestinal peptide induces cell cycle arrest and regulatory functions in human T cells at multiple levels. Mol Cell Biol 2010;30(10):2537–51.Li JM, et al. VIPhyb, an antagonist of vasoactive intestinal peptide receptor, enhances cellular antiviral immunity in murine cytomegalovirus infected mice. PLoS One 2013;8(5):e63381.Moody TW, et al., VIP receptor antagonists and chemotherapeutic drugs inhibit the growth of breast cancer cells. Breast Cancer Res Treat 2001;68(1):55–64.Moody TW, et al. A vasoactive-Intestinal-Peptide antagonist inhibits nonsmall cell lung-cancer growth. Proceedings of the National Academy of Sciences of the United States of America 1993;90(10):4345–4349.Zia H, et al. Breast cancer growth is inhibited by vasoactive intestinal peptide (VIP) hybrid, a synthetic VIP receptor antagonist. Cancer Res 1996;56(15):3486–9.Sena M, et al. High conservation of upstream regulatory sequences on the human and mouse vasoactive intestinal peptide (VIP) genes. DNA Seq 1994;5(1):25–9.Ethics ApprovalAll experimental procedures involving mice were approved by the Institutional Animal Care and Use Committee (IACUC) at Emory University. De-identified blood samples from consented patients with PDAC (IRB 00087397) or healthy volunteers (IRB 00046063) were obtained with approval from Institutional Review Boards.

BackgroundCD8 T cell are a crucial part of the immune response to tumors, with CD8 infiltration predicting disease progression in numerous cancer types. Recently two subsets of CD8 T cells that respond to tumors have been described, a stem-like (TCF1+) CD8 T cell that can give rise to a more cytotoxic terminally differentiated (TD) (TCF1-Tim3+) CD8 T cell. In this study we aimed to understand the origin of stem-like TCF1+ CD8 T cells within tumors.MethodsHuman patient TDLN and tumor samples from kidney and prostate cancer were processed after resection and used for flow cytometry, RNA-seq, TCR-seq and whole genome DNA methylation analysis. We also used a prostate cancer mouse model that expresses the LCMV GP protein (TRAMPC1-LCMV-GP) to track tumor-specific CD8 T cells in both TDLNs and tumors.ResultsWe studied human prostate and kidney cancer tumor-draining lymph nodes (TDLN) and found that CD8 T cells are activated but fail to acquire an effector phenotype within the TDLN. Instead, they share functional, transcriptional, and epigenetic traits with stem-like cells in the tumor. We also found that activated CD8 T cells from TDLNs shared TCR overlap with both CD8 subsets within tumors. This suggests that these activated cells are a precursor to the stem-like CD8 T cells in tumors. To further test this hypothesis, we used our TRAMPC1-LCMV-GP tumor model to study tumor-specific CD8 T cell activation. We found that CD8 T cells are activated in TDLNs but fail to acquire an effector program. These cells then establish the stem-like CD8s within tumor where they require additional co-stimulation from antigen presenting cells to differentiate into TCF1- TD CD8 T cells. This is strikingly different from canonical CD8 T cell activation to acute viruses, where the effector program is acquired immediately. We also showed that human stem-like CD8 T cells require co-stimulation and TCR stimulation to divide and differentiate into terminally differentiated CD8s in-vitro, and DCs from autologous tumors can also induce this differentiation.ConclusionsOverall this work shows a model of CD8 T cell activation in response to tumors that has two distinct phases. The first occurs in the TDLN where CD8 T cells are initially activated, the second occurs in the tumor where CD8 T cells acquire an effector function after additional co-stimulation. This model of T cell differentiation adds to our understanding of basic CD8 T cell biology and has important implications to improve our current immunotherapies.

BackgroundThe modified Glasgow Prognostic Score (mGPS) is a composite biomarker that uses albumin and C reactive protein (CRP). There are multiple immune checkpoint inhibitor (ICI)-based combinations approved for metastatic renal cell carcinoma (mRCC). We investigated the ability of mGPS to predict outcomes in patients with mRCC receiving ICI.MethodsWe retrospectively reviewed patients with mRCC treated with ICI as monotherapy or in combination at Winship Cancer Institute between 2015 and 2020. Overall survival (OS) and progression-free survival (PFS) were measured from the start date of ICI until death or clinical/radiographical progression, respectively. The baseline mGPS was defined as a summary score based on pre-ICI values with one point given for CRP>10 mg/L and/or albuminResults156 patients were included with a median follow-up 24.2 months. The median age was 64 years and 78% had clear cell histology. Baseline mGPS was 0 in 36%, 1 in 40% and 2 in 24% of patients. In UVA, a baseline mGPS of 2 was associated with shorter OS (HR 4.29, 95% CI 2.24 to 8.24, pConclusionThe mGPS is prognostic in this cohort of patients with mRCC treated with ICI as monotherapy or in combination. These results warrant external and prospective validation.

Background The International Metastatic Renal Cell Carcinoma Database Consortium (IMDC) criteria are the gold standard for risk‐stratifying patients with metastatic renal cell cancer (mRCC). We developed a novel risk scoring system for patients with mRCC treated with immune checkpoint inhibitors (ICIs). Methods We performed a retrospective analysis of 100 ICI‐treated patients with mRCC at Winship Cancer Institute from 2015 to 2018. Several baseline variables were collected, including markers of inflammation, body mass index (BMI), and sites of metastatic disease, and all were considered for inclusion in our risk scoring system. Upon variable selection in multivariable model, monocyte‐to‐lymphocyte ratio (MLR), BMI, and number and sites of metastases at baseline were used for risk score calculation. Patients were categorized using four‐level risk groups as good (risk score = 0), intermediate (risk score = 1), poor (risk score = 2), or very poor (risk score = 3–4). Cox's proportional hazard model and the Kaplan‐Meier method were implemented for survival outcomes. Results Most patients were male (66%) with clear cell renal cell carcinoma (72%). The majority (71%) received anti–programmed cell death protein‐1 monotherapy. Our risk scoring criteria had higher Uno's concordance statistics than IMDC in predicting overall survival (OS; 0.71 vs. 0.57) and progression‐free survival (0.61 vs. 0.58). Setting good risk (MLR, Considering the increasing number of treatment options for patients with metastatic renal cell carcinoma, it is becoming more important to identify factors that increase the likelihood of responses to systemic therapy. This article describes a novel risk scoring system for patients with metastatic renal cell carcinoma treated with ICI, focusing on risk from systemic inflammation, number and sites of metastatic disease, and body composition.

Background Sarcopenia and inflammation have been associated with poor survival in patients with cancer. We explored the combined effects of these variables on survival in patients with cancer treated with immunotherapy. Methods We performed a retrospective review of 90 patients enrolled on immunotherapy‐based phase I clinical trials at Emory University from 2009 to 2017. Baseline neutrophil‐to‐lymphocyte ratio, monocyte‐to‐lymphocyte ratio, and platelet‐to‐lymphocyte ratio (PLR) were used as surrogates of inflammation. The skeletal muscle index (SMI) was derived from the skeletal muscle density calculated from baseline abdominal computed tomography images. Optimal cutoffs for continuous inflammation biomarkers and SMI were determined by bias‐adjusted log‐rank test. A four‐level risk stratification was used to create low‐risk (PLR, The interaction between chronic inflammation and body composition is particularly important in the era of immunotherapy, considering that immune checkpoint inhibitors rely on the host immune system for their efficacy. This article reports on the combined effects of inflammation and sarcopenia on clinical outcomes in patients with solid tumors treated with immunotherapy‐based regimens.

Molecular events activating the PI3K pathway are frequently detected in human tumors and the activation of PI3K signaling alters numerous cellular processes including tumor cell proliferation, survival, and motility. More recent studies have highlighted the impact of PI3K signaling on the cellular response to interferons and other immunologic processes relevant to antitumor immunity. Given the ability of IFNγ to regulate antigen processing and presentation and the pivotal role of MHC class I (MHCI) and II (MHCII) expression in T-cell–mediated antitumor immunity, we sought to determine the impact of PI3K signaling on MHCI and MHCII induction by IFNγ. We found that the induction of cell surface MHCI and MHCII molecules by IFNγ is enhanced by the clinical grade PI3K inhibitors dactolisib and pictilisib. We also found that PI3K inhibition increases STAT1 protein levels following IFNγ treatment and increases accessibility at genomic STAT1-binding motifs. Conversely, we found that pharmacologic activation of PI3K signaling can repress the induction of MHCI and MHCII molecules by IFNγ, and likewise, the loss of PTEN attenuates the induction of MHCI, MHCII, and STAT1 by IFNγ. Consistent with these in vitro studies, we found that within human head and neck squamous cell carcinomas, intratumoral regions with high phospho-AKT IHC staining had reduced MHCI IHC staining. Implications: Collectively, these findings demonstrate that MHC expression can be modulated by PI3K signaling and suggest that activation of PI3K signaling may promote immune escape via effects on antigen presentation.

BACKGROUND Body mass index (BMI) is used to define obesity, but it is an imperfect measure of body composition. In the current study, the authors explored the association between types of fat and survival in patients treated with immunotherapy. METHODS A retrospective analysis of 90 patients who were treated with immunotherapy on phase 1 clinical trials at the Winship Cancer Institute in Atlanta, Georgia, from 2009 through 2017 was performed. Overall survival (OS) and progression-free survival (PFS) were used to measure clinical outcomes. Baseline BMI and radiographic images at the middle of the third lumbar vertebrae were obtained. Fat densities were calculated and converted to indices (subcutaneous fat index [SFI], intermuscular fat index [IFI], and visceral fat index [VFI]) after dividing by height in meters squared. Risk groups were created using recursive partitioning and the regression trees method for SFI and IFI, which were selected by stepwise variable selection among all fat-related variables. The Cox proportional hazards model and Kaplan-Meier method were used for the association with OS and PFS. RESULTS The majority of patients (59%) were male and diagnosed with melanoma (33%) or gastrointestinal cancers (22%). The median BMI was 27.4 kg/m2 , the median SFI was 62.78, the median IFI was 4.06, and the median VFI was 40.53. Low-risk patients (those with an SFI ≥73) had a significantly longer OS (hazard ratio, 0.20; 95% CI, 0.09-0.46 [P

Background Immune checkpoint inhibitors (ICIs) are an important treatment for metastatic renal cell carcinoma (mRCC). These agents may cause immune-related adverse events (irAEs), and the relationship between irAEs and outcomes is poorly understood. We investigated the association between irAEs and clinical outcomes in patients with mRCC treated with ICIs. Methods We performed a retrospective study of 200 patients with mRCC treated with ICIs at Winship Cancer Institute from 2015 to 2020. Data on irAEs were collected from clinic notes and laboratory values and grades were determined using Common Terminology Criteria in Adverse Events version 5.0. The association with overall survival (OS) and progression-free survival (PFS) was modeled by Cox proportional hazards model. Logistic regression models were used to define odds ratios (ORs) for clinical benefit (CB). Landmark analysis and extended Cox models were used to mitigate lead-time bias by treating irAEs as a time-varying covariate. Results Most patients (71.0%) were male, and one-third of patients (33.0%) experienced at least one irAE, most commonly involving the endocrine glands (13.0%), gastrointestinal tract (10.5%), or skin (10.0%). Patients who experienced irAEs had significantly longer OS (hazard ratio [HR], 0.52; p = .013), higher chance of CB (OR, 2.10; p = .023) and showed a trend toward longer PFS (HR, 0.71; p = .065) in multivariate analysis. Patients who had endocrine irAEs, particularly thyroid irAEs, had significantly longer OS and PFS and higher chance of CB. In a 14-week landmark analysis, irAEs were significantly associated with prolonged OS (p = .045). Patients who experienced irAEs had significantly longer median OS (44.5 vs. 18.2 months, p = .005) and PFS (7.5 vs. 3.6 months, p = .003) without landmark compared with patients who did not. Conclusion We found that patients with mRCC treated with ICIs who experienced irAEs, particularly thyroid irAEs, had significantly improved clinical outcomes compared with patients who did not have irAEs. This suggests that irAEs may be effective clinical biomarkers in patients with mRCC treated with ICIs. Future prospective studies are warranted to validate these findings. Implications for Practice This study found that early onset immune-related adverse events (irAEs) are associated with significantly improved clinical outcomes in patients with metastatic renal cell carcinoma (mRCC) treated with immune checkpoint inhibitors (ICIs). In this site-specific irAE analysis, endocrine irAEs, particularly thyroid irAEs, were significantly associated with improved clinical outcomes. These results have implications for practicing medical oncologists given the increasing use of ICIs for the treatment of mRCC. Importantly, these results suggest that early irAEs and thyroid irAEs at any time on treatment with ICIs may be clinical biomarkers of clinical outcomes in patients with mRCC treated with ICIs.

Combination therapy with PD-1 blockade and IL-2 is highly effective during chronic lymphocytic choriomeningitis virus infection(1). Here we examine the underlying basis for this synergy. We show that PD-1 + IL-2 combination therapy, unlike PD-1 monotherapy, dramatically changes the differentiation program of the PD-1(+)TCF-1(+) stem-like CD8(+) T cells and results in the generation of transcriptionally and epigenetically distinct effector CD8(+) T cells that resemble highly functional effector CD8(+) T cells seen after an acute viral infection. The generation of these qualitatively superior CD8(+) T cells that mediate viral control underlies the synergy between PD-1 and IL-2. Our results show that the PD-1(+)TCF-1(+) stem-like CD8(+) T cells, also referred to as precursors of exhausted CD8(+) T cells, are not fate-locked into the exhaustion program and their differentiation trajectory can be changed by IL-2 signals. These virus-specific effector CD8(+) T cells emerging from the stem-like CD8(+) T cells after combination therapy expressed increased levels of the high affinity IL-2 trimeric (CD25, CD122, CD132) receptor. This was not seen after PD-1 blockade alone. Finally, we show that CD25 engagement with IL-2 plays an important role in the observed synergy between IL-2 cytokine and PD-1 blockade. Either blocking CD25 with an antibody or using a mutated version of IL-2 that does not bind CD25 but still binds CD122/CD132 almost completely abrogated the synergistic effects seen after PD-1 + IL-2 combination therapy. There is currently considerable interest in PD-1 + IL-2 combination therapy for patients with cancer(2,3) and our fundamental studies defining the underlying mechanisms of how IL-2 synergizes with PD-1 blockade should inform these human translational studies.

Expansion and differentiation of antigen-experienced PD-1

PurposeA subset of primary prostate cancer (PCa) expresses programmed death-ligand 1 (PD-L1), but whether they have unique tumor immune microenvironment (TIME) or genomic features is unclear.Experimental DesignWe selected PD-L1-positive high-grade and/or high-risk primary PCa, characterized tumor-infiltrating lymphocytes (TILS) with multiplex immunofluorescence, and identified genomic alterations in immunogenic and non-immunogenic tumor foci.ResultsOne-quarter of aggressive localized PCa cases (29/115) had tumor PD-L1 expression >5%. This correlated with increased density of CD8+ T cells, a large fraction co-expressing PD-1, versus absent PD-1 expression on sparse CD8 T cells in unselected cases. Most CD8+PD-1+ cells did not express terminal exhaustion markers (TIM-3 or LAG-3), while a subset expressed TCF1. Consistent with these CD8+PD-1+TCF1+ cells being progenitors, they were found in antigen-presenting-cell niches in close proximity to MHC II+ cells. CD8 T cell density in immunogenic PCa and renal cell carcinoma (RCC) was nearly identical. Shallow RB1 and BRCA2 losses, and deep deletions of CHD1, were prevalent; the latter being strongly associated with a dendritic cell gene set in TCGA. Tumor mutation burden was variable; neither high microsatellite instability nor CDK12 alterations were present.ConclusionsA subset of localized PCa is immunogenic, manifested by PD-L1 expression and CD8+ T cell content comparable to RCC. The CD8+ T cells include effector cells and exhausted progenitor cells, which may be expanded by ICIs. Genomic losses of RB1, BRCA2, and CHD1 may be drivers of this phenotype. These findings indicate that immunotherapies may be effective in biomarker-selected subpopulations of localized PCa patients.Statement of Translational RelevanceProstate cancer (PCa) is generally considered poorly immunogenic, with low expression of programmed death-ligand 1 (PD-L1) and low density of tumor-infiltrating immune cells. Accordingly, response rates to PD(L)-1 inhibition in unselected patients with advanced prostate cancer have been low. Here, we find that a substantial subset of aggressive primary PCa exhibits tumor PD-L1 expression and contains a high density of tumor-infiltrating lymphocytes. These lymphocytes contain sub-populations of exhausted progenitor CD8+ T cells and differentiated effector T cells, the hallmarks of ongoing anti-tumor immune response and a prerequisite for response to checkpoint inhibition. Furthermore, we identify genomic alterations that may be contributing to immunogenicity in these cases. These findings point to immune responses elicited in a subset of primary PCa, supporting the development of immune checkpoint blockade clinical trials in early-stage disease, such as biochemically recurrent PCa, that are driven by genomic features of the tumor or the immune microenvironment.

Purpose: A subset of primary prostate cancer expresses programmed death-ligand 1 (PD-L1), but whether they have a unique tumor immune microenvironment or genomic features is unclear. Experimental Design: We selected PD-L1–positive high-grade and/or high-risk primary prostate cancer, characterized tumor-infiltrating lymphocytes with multiplex immunofluorescence, and identified genomic alterations in immunogenic and nonimmunogenic tumor foci. Results: One quarter of aggressive localized prostate cancer cases (29/115) had tumor PD-L1 expression more than 5%. This correlated with increased density of CD8+ T cells, a large fraction coexpressing PD-1, versus absent PD-1 expression on sparse CD8 T cells in unselected cases. Most CD8+PD-1+ cells did not express terminal exhaustion markers (TIM3 or LAG3), while a subset expressed TCF1. Consistent with these CD8+PD-1+TCF1+ cells being progenitors, they were found in antigen-presenting cell niches in close proximity to MHC-II+ cells. CD8 T-cell density in immunogenic prostate cancer and renal cell carcinoma (RCC) was nearly identical. Shallow RB1 and BRCA2 losses, and deep deletions of CHD1, were prevalent, the latter being strongly associated with a dendritic cell gene set in The Cancer Genome Atlas. Tumor mutation burden was variable; neither high microsatellite instability nor CDK12 alterations were present. Conclusions: A subset of localized prostate cancer is immunogenic, manifested by PD-L1 expression and CD8+ T-cell content comparable with RCC. The CD8+ T cells include effector cells and exhausted progenitor cells, which may be expanded by immune checkpoint inhibitors (ICI). Genomic losses of RB1, BRCA2, and CHD1 may be drivers of this phenotype. These findings indicate that immunotherapies may be effective in biomarker-selected subpopulations of patients with localized prostate cancer.

Revealing the mechanisms that underlie the expansion of antitumor CD8+ T cells that are associated with improved clinical outcomes is critical to improving immunotherapeutic management of melanoma. How the lymphatic system, which orchestrates the complex sensing of antigen by lymphocytes to mount an adaptive immune response, facilitates this response in the context of malignancy is incompletely understood. To delineate the effects of lymphatic transport and tumor-induced lymphatic and lymph node (LN) remodeling on the elicitation of CD8+ T cell immunity within LNs, we designed a suite of nanoscale biomaterial tools enabling the quantification of antigen access and presentation within the LN and resulting influence on T cell functions. The expansion of antigen-specific stem-like and cytotoxic CD8+ T cell pools was revealed to be sensitive to the mechanism of lymphatic transport to LNs, demonstrating the potential for nanoengineering strategies targeting LNs to optimize cancer immunotherapy in eliciting antitumor CD8+ T cell immunity.

Background Several immune checkpoint inhibitors (ICI) have been approved for the treatment of advanced urothelial carcinoma (UC). There are limited predictive biomarkers for UC patients treated with ICI. In this study, we investigated the association between body composition and clinical outcomes in ICI-treated advanced UC patients. Methods We conducted a retrospective analysis of 70 ICI-treated advanced UC patients at Winship Cancer Institute from 2015–2020. Inclusion criteria was available computed tomography (CT) scans within 2 months of ICI-initiation. Baseline CT images were collected at mid-L3 and muscle and fat compartments were segmented using SliceOMatic v5.0 (TomoVision). The density of subcutaneous fat, inter-muscular fat, visceral fat, and skeletal muscle (SM) were measured and converted to indices by dividing by height(m)2 (SFI, IFI, VFI and SMI, respectively). Attenuated SM mean (Hounsfield Units) was also collected. Myosteatosis was calculated by IFI/SMI*100%. Gender-specific optimal cuts were used to dichotomize patients as high or low for each variable using OS as the primary outcome. A prognostic body composition risk score was created based on the beta coefficient from the multivariable Cox model (MVA) following best-subset variable selection. Our body composite risk score was SMI + 2*SM mean + VFI and patients were categorized as high (0–1), intermediate (2–3), or low-risk (4). Comparison of OS and PFS between the risk groups was performed via Kaplan-Meier method and Log-rank test. Concordance statistics (C-statistics) were used to quantify the discriminatory magnitude of the predictive model. Results Most patients (70%) were male and more than one-quarter (26%) had an ECOG PS ≥ 2. The majority received ICI in the second (46%) or third-line (21%) setting. Body composite poor-risk patients had significantly shorter OS (HR: 6.18, p Conclusions Body composition variables such as SMI, SM mean, VFI and myosteatosis may be predictive of clinical outcomes in ICI-treated advanced UC patients. Larger, prospective studies are warranted to validate this hypothesis-generating data. Acknowledgements Research reported in this publication was supported in part by the Biostatistics and Bioinformatics Shared Resource of Winship Cancer Institute of Emory University and NIH/NCI under award number P30CA138292. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Trial Registration Not applicable Ethics Approval This retrospective study was approved by the Emory University Institutional Review Board. Consent Not applicable References Not applicable

Systemic administration of immune checkpoint blockade (ICB) monoclonal antibodies (mAbs) can unleash antitumor functions of T cells but is associated with variable response rates and off-target toxicities. We hypothesized that antitumor efficacy of ICB is limited by the minimal accumulation of mAb within tissues where antitumor immunity is elicited and regulated, which include the tumor microenvironment (TME) and secondary lymphoid tissues. In contrast to systemic administration, intratumoral and intradermal routes of administration resulted in higher mAb accumulation within both the TME and its draining lymph nodes (LNs) or LNs alone, respectively. The use of either locoregional administration route resulted in pronounced T cell responses from the ICB therapy, which developed in the secondary lymphoid tissues and TME of treated mice. Targeted delivery of mAb to tumor-draining lymph nodes (TdLNs) alone was associated with enhanced antitumor immunity and improved therapeutic effects compared to conventional systemic ICB therapy, and these effects were sustained at reduced mAb doses and comparable to those achieved by intratumoral administration. These data suggest that locoregional routes of administration of ICB mAb can augment ICB therapy by improving immunomodulation within TdLNs.

Tumor-infiltrating CD8 T cells are associated with improved patient survival and response to immunotherapy in various cancers. Persistent antigen leads to CD8 T-cell exhaustion, where proliferation/self-renewal and killing are divided within distinct subsets of CD8 T cells in the tumor. CD8 T-cell responses in chronic antigen settings must be maintained for long periods of time, suggesting that mechanisms that regulate chronic CD8 T-cell responses may differ from those in acute settings. Currently, factors that regulate the maintenance of stem-like CD8 T cells in the tumor or their differentiation into terminally differentiated cells are unknown. In this review, we discuss the role of dendritic cells in the activation and differentiation of CD8 T-cell subsets within secondary lymphoid tissue and tumors. In addition, we examine changes in CD4 T-cell differentiation in response to chronic antigens and consider how subset-specific mechanisms could assist the stem-like and terminally differentiated CD8 T-cell subsets. Finally, we highlight how tumor-infiltrating CD4 T cells and dendritic cells interact with CD8 T cells within organized lymphoid-like areas in the tumor and propose a CD8 T-cell differentiation model that requires the collaboration of CD4 T cells and dendritic cells. These organized interactions coordinate the anti-tumor response and control disease progression by mechanisms that regulate CD8 T-cell differentiation, which permit the maintenance of an effective balance of stem-like and terminally differentiated CD8 T cells.

Recent studies on chronic viral infections have defined a novel programmed cell death 1-positive (PD-1(+)) T cell factor 1-positive (TCF1(+)) stem-like CD8 T cell subset that gives rise to the terminally differentiated exhausted CD8 T cells. In this study, we performed T cell receptor beta (TCRβ) sequencing of virus-specific CD8 T cells during chronic lymphocytic choriomeningitis virus (LCMV) infection to examine the TCR diversity and lineage relationship of these two functionally distinct subsets. We found that >95% of the TCR repertoire of the exhausted CD8 T cell subset was shared with the stem-like CD8 T cells. The TCR repertoires of both CD8 T cell subsets were composed mostly of a few dominant clonotypes, but there was slightly more breadth and diversity in the stem-like CD8 T cells than their exhausted counterpart (∼40 versus ∼15 GP33(+) clonotypes; ∼20 versus ∼7 GP276(+) clonotypes). Interestingly, the breadth of the TCR repertoire was broader during the early stages (day 8) of the chronic infection than the later stages (days 45 to 60), showing that there was a narrowing of the TCR repertoire during chronic infection (∼2-fold GP33(+) and GP276(+) stem-like subset; ∼10-fold GP33(+) and ∼5-fold GP276(+) exhausted subset). In contrast, during acute LCMV infection, the TCR repertoire was much broader in both GP33-specific effector (∼160 clonotypes) and memory CD8 T cells (∼160 clonotypes). Overall, our data demonstrate that the virus-specific CD8 T cell TCR repertoire is broad and remains stable after acute LCMV infection, but it contracts and is narrower during chronic infection. Our study also shows that the repertoire of the exhausted CD8 T cell subset is almost completely derived from the stem-like CD8 T cell subset during established chronic LCMV infection. IMPORTANCE CD8 TCR repertoires responding to chronic viral infections (HIV, hepatitis C virus [HCV], Epstein-Barr virus [EBV], and cytomegalovirus [CMV]) have limited breadth and diversity. How these repertoires change and are maintained throughout the chronic infection are unknown. We thus characterized the LCMV-specific CD8 TCR repertoires of stem-like and terminally exhausted subsets generated during chronic LCMV infections. During chronic LCMV infections, the repertoires started as diverse but became more clonal at the late time point. Further, the exhausted subset was composed of dominant clonotypes that were shared with the stem-like subset. Together, we demonstrate that the TCR repertoire contracts over time and is almost exclusively derived from the stem-like subset late during the persistent viral infection. Our data suggest that dominant clonotypes in the exhausted subset are derived from a diverse pool of stem-like clonotypes, which may be contributing to the clonality observed during chronic viral infections.

Tumour-infiltrating lymphocytes are associated with a survival benefit in several tumour types and with the response to immunotherapy1–8. However, the reason some tumours have high CD8 T cell infiltration while others do not remains unclear. Here we investigate the requirements for maintaining a CD8 T cell response against human cancer. We find that CD8 T cells within tumours consist of distinct populations of terminally differentiated and stem-like cells. On proliferation, stem-like CD8 T cells give rise to more terminally differentiated, effector-molecule-expressing daughter cells. For many T cells to infiltrate the tumour, it is critical that this effector differentiation process occur. In addition, we show that these stem-like T cells reside in dense antigen-presenting-cell niches within the tumour, and that tumours that fail to form these structures are not extensively infiltrated by T cells. Patients with progressive disease lack these immune niches, suggesting that niche breakdown may be a key mechanism of immune escape. The authors examine the immune cell infiltrates of human tumours and provide evidence for a population of CD8 T cells with stem-cell characteristics and proliferative capacity that reside in an antigen-presenting niche within tumours.

Infiltrating tumor neutrophils and myeloid-derived suppressor cells represent major populations in the tumor microenvironment that contribute to tumor progression. However, the phenotype of circulating and tumor-associated neutrophils, and the impact of cancer patients’ metabolic state on neutrophil function need further characterization. Here we show that in kidney cancer patients, circulating neutrophils display an altered immature-like phenotype, and an activated/primed metabolic state. Circulating immature-like neutrophils acquire an activated phenotype upon migration into the tumor tissue, characterized by high expression of the immunosuppressive enzyme arginase-1, and active granule release. Interestingly, obesity and adipose tissue distribution were significantly associated with this activated phenotype of neutrophils, including the release of arginase-1 in the tumor tissue. These results provide a possible functional relationship between the metabolic status of the patients and disease progression, through an active immunosuppressive role of neutrophils within the kidney tumor microenvironment.

BACKGROUND Optimal prognostic and predictive biomarkers for patients with advanced-stage cancer patients who received immunotherapy (IO) are lacking. Inflammatory markers, such as the neutrophil-to-lymphocyte ratio (NLR), the monocyte-to-lymphocyte ratio (MLR), and the platelet-to-lymphocyte ratio (PLR), are readily available. The authors investigated the association between these markers and clinical outcomes of patients with advanced-stage cancer who received IO. METHODS A retrospective review was conducted of 90 patients with advanced cancer who received treatment on phase 1 clinical trials of IO-based treatment regimens. NLR, MLR, and PLR values were log-transformed and treated as continuous variables for each patient. Overall survival (OS), progression-free survival (PFS), and clinical benefit were used to measure clinical outcomes. For univariate associations and multivariable analyses, Cox proportional-hazards models or logistic regression models were used. RESULTS The median patient age was 63 years, and most were men (59%). The most common histologies were melanoma (33%) and gastrointestinal cancers (22%). High baseline NLR, MLR, and PLR values were associated significantly with worse OS and PFS (P < .05) and a lower chance of benefit (NLR and PLR; P < .05). Increased NLR, MLR, and PLR values 6 weeks after baseline were associated with shorter OS and PFS (P ≤ .052). CONCLUSIONS Baseline and early changes in NLR, MLR, and PLR values were strongly associated with clinical outcomes in patients who received IO-based treatment regimens on phase 1 trials. Confirmation in a homogenous patient population treated on late-stage trials or outside of trial settings is warranted. These values may warrant consideration for inclusion when risk stratifying patients enrolled onto phase 1 clinical trials of IO agents.

BACKGROUND: Biomarkers to guide treatment in metastatic renal cell carcinoma (mRCC) are lacking. We aimed to investigate the association between pretreatment neutrophil-to-lymphocyte ratio (NLR) and outcome of patients with mRCC receiving nivolumab. METHODS: Through retrospective chart review, we identified 38 patients with mRCC treated with standard of care nivolumab between 2015 – 2016 at Winship Cancer Institute of Emory University. NLR was determined from complete blood count collected prior to starting treatment and imaging was performed to assess progression. The NLR cutoff value of 5.5 was determined by log rank test, and the univariate association with overall survival (OS) or progress free survival (PFS) was assessed by Cox proportional hazard model and Kaplan-Meier method. RESULTS: The 38 patients had a median age of 68.5 years. The PFS and OS for all patients at 12 months was 54% and 69%, respectively. The median PFS was 2.6 months in the high NLR group but not reached in the low NLR group. Low NLR was strongly associated with increased PFS with hazard ratio of 0.20 (95%CI 0.07–0.64; p=0.006). The median OS was 2.7 months in the high NLR group but not reached in the low NLR group. Low NLR was significantly associated with a prolonged OS with hazard ratio of 0.06 (95% CI 0.01–0.55; p=0.012). CONCLUSION: Pre-treatment NLR less than 5.5 is associated with superior PFS and OS. NLR is a biomarker that can inform prognosis for patients with mRCC and should be further validated in larger cohorts and in prospective studies.

Pancreatic ductal adenocarcinoma (PDAC) is unresponsive to immune checkpoint therapy largely due to a paucity of T cells within the tumor microenvironment (TME) and abundant immunosuppressive signaling pathways. In this study we show that human PDAC tumors over-express vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide that suppresses T cell effector properties and promotes the generation of regulatory T cells (Tregs). Therefore, we treated tumor-bearing mice with VIP receptor peptide antagonists and measured T cell homing, activation, and anti-tumor responses in preclinical murine models of PDAC. Pharmacological inhibition of VIP receptor (VIP-R) signaling using daily subcutaneous injections of peptide antagonists had no discernable toxicity in healthy and tumor-bearing mice. Treatment with VIP-R antagonists in combination with anti-PD-1 checkpoint blockade significantly decreased tumor burden, and improved survival in subcutaneous and orthotopic murine PDAC models. Combination therapy significantly enhanced T cell activation and proliferation and decreased frequencies of Tregs within the TME. Anti-tumor responses were T cell dependent, as the combination therapy failed to improve survival in CD4 or CD8 deficient mice using knock-out strains and antibody depletion. Furthermore, combination therapy significantly increased frequencies of tumor specific T cells (measured with a tetramer reagent) and provided protective immunity against tumor rechallenge. Combination therapy led to significant increases in the infiltration of adoptively transferred GFP+ T cells into PDAC tumors and decreased CXCR4 expression levels on T cells. Encouragingly, peptide-based VIP-R antagonists enhanced the in vitro activation of human T cells isolated from peripheral blood of PDAC patients. Human T cells cultured with VIP-R antagonists had increased proliferation, activation, and decreased proportions of T regs and exhausted T cells co-expressing PD-1, Tim-3 and Lag-3. Taken together, our findings show that VIP is a targetable mechanism of immune escape in PDAC. Inhibiting VIP receptor signaling improves T cell effector properties and synergistically improves anti-tumor responses to checkpoint inhibitors in mouse PDAC models. Additionally, as the VIP sequence is identical between human and mice, and since VIP-R antagonists have similar effects on human and murine T cells in culture, clinical translation is highly feasible. Citation Format: Sruthi Ravindranathan, Passang Tenzin, Jian Ming Li, Rohan Dhamsania, Michael Ware, Mohammad Zaidi, Shuhua Wang, Jingru Zhu, Maria Cardenas, Yuan Liu, Gaurav Joshi, Sanjeev Gumber, Brian Robinson, Anish Sen-Majumdar, Shanmuganathan Chandrakasan, Haydn Kissick, Alan Frey, Susan Thomas, Bassel El-Rayes, Gregory Lesinski, Edmund K. Waller. Inhibiting vasoactive intestinal peptide receptor signaling elicits T cell dependent anti-tumor response of pancreatic ductal adenocarcinoma to immune checkpoint therapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-072.

Memory CD8 T cells that circulate in the blood and are present in lymphoid organs are an essential component of long-lived T cell immunity. These memory CD8 T cells remain poised to rapidly elaborate effector functions upon re-exposure to pathogens, but also have many properties in common with naive cells, including pluripotency and the ability to migrate to the lymph nodes and spleen. Thus, memory cells embody features of both naive and effector cells, fuelling a long-standing debate centred on whether memory T cells develop from effector cells or directly from naive cells. Here we show that long-lived memory CD8 T cells are derived from a subset of effector T cells through a process of dedifferentiation. To assess the developmental origin of memory CD8 T cells, we investigated changes in DNA methylation programming at naive and effector cell-associated genes in virus-specific CD8 T cells during acute lymphocytic choriomeningitis virus infection in mice. Methylation profiling of terminal effector versus memory-precursor CD8 T cell subsets showed that, rather than retaining a naive epigenetic state, the subset of cells that gives rise to memory cells acquired de novo DNA methylation programs at naive-associated genes and became demethylated at the loci of classically defined effector molecules. Conditional deletion of the de novo methyltransferase Dnmt3a at an early stage of effector differentiation resulted in reduced methylation and faster re-expression of naive-associated genes, thereby accelerating the development of memory cells. Longitudinal phenotypic and epigenetic characterization of the memory-precursor effector subset of virus-specific CD8 T cells transferred into antigen-free mice revealed that differentiation to memory cells was coupled to erasure of de novo methylation programs and re-expression of naive-associated genes. Thus, epigenetic repression of naive-associated genes in effector CD8 T cells can be reversed in cells that develop into long-lived memory CD8 T cells while key effector genes remain demethylated, demonstrating that memory T cells arise from a subset of fate-permissive effector T cells.