CD8 Tumor-infiltrating Lymphocytes Research Articles

Abstract PO062: Isolation of tumor-infiltrating lymphocytes for higher sensitivity gene expression profiling

Abstract Gene expression profiling is a powerful approach to address the mechanism of action (MOA) of cancer immunotherapy-driven reprogramming of tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment. However, TILs, particularly T and NK cells, are a very rare population within the tumor. Gene expression profiling using Nanostring or RNAseq is often not sensitive enough to detect expression changes in rare populations, and it is difficult to separate gene expression profiles between different cell subsets. Although single-cell RNAseq can distinguish gene expression profiles between different cell subsets, it is very costly and would require many cells to be sequenced before obtaining the rare cell type of interest. Classically, fluorescence-activated cell sorting (FACS) has been used to isolate rare populations, but this method is a long process that can affect cell phenotype and results in dramatic cell loss. Thus, we developed a protocol for rapid and reproducible magnetic bead-based isolation of tumor-infiltrating lymphocytes (TILs) for higher sensitivity MOA studies. CD8 and CD4 TILs were isolated from both MC38 and CT26.KSA tumor models with high purity and sufficient yield for further Nanostring gene expression analysis. As expected, CD8 and CD4 T cell gene expression profiles were dramatically enriched in the isolated TILs compared to the whole tumor. Thus, our protocol for isolating TILs from mouse tumors can be applied broadly to multiple tumor types and provides sufficient yield to perform downstream high-sensitivity gene expression profiling. Citation Format: Lindsay M. Webb, Bartholomew Naughton, Sireesha Yalavarthi, Clotilde Bourin, Jacques Moisan, Chunxiao Xu. Isolation of tumor-infiltrating lymphocytes for higher sensitivity gene expression profiling [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO062.

Atezolizumab and nab-Paclitaxel in Advanced Triple-Negative Breast Cancer: Biomarker Evaluation of the IMpassion130 Study.

BackgroundUnderstanding the impact of the tumor immune microenvironment and BRCA1/2-related DNA repair deficiencies on the clinical activity of immune checkpoint inhibitors may help optimize both patient and treatment selection in metastatic triple-negative breast cancer. In this substudy from the phase 3 IMpassion130 trial, immune biomarkers and BRCA1/2 alterations were evaluated for association with clinical benefit with atezolizumab and nab-paclitaxel (A+nP) vs placebo and nP in unresectable (P+nP) locally advanced or metastatic triple-negative breast cancer.MethodsPatients were randomly assigned 1:1 to nab-paclitaxel 100 mg/m2 (days 1, 8, and 15 of a 28-day cycle) and atezolizumab 840 mg every 2 weeks or placebo until progression or toxicity. Progression-free survival and overall survival were evaluated based on programmed death-ligand 1 (PD-L1) expression on immune cells (IC) and tumor cells, intratumoral CD8, stromal tumor-infiltrating lymphocytes, and BRCA1/2 mutations.ResultsPD-L1 IC+ in either primary or metastatic tumor tissue was linked to progression-free survival and overall survival benefit with A+nP. PD-L1 IC+ low (26.9%; 243 of 902 patients) and high (13.9%; 125 of 902 patients) populations had improved outcomes that were comparable. Intratumoral CD8 and stromal tumor-infiltrating lymphocytes positivity (sTIL+) were associated with PD-L1 IC+ status; improved outcomes were observed with A+nP vs P+nP only in CD8+ and sTIL+ patients who were also PD-L1 IC+. BRCA1/2 mutations (occurring in 14.5% [89 of 612 patients]) were not associated with PD-L1 IC status, and PD-L1 IC+ patients benefited from A+nP regardless of BRCA1/2 mutation status.ConclusionsAlthough A+nP was more efficacious in patients with richer tumor immune microenvironment, clinical benefit was only observed in patients whose tumors were PD-L1 IC+.

Case Report: Therapeutic Response to Chemo-Immunotherapy in an Advanced Large Cell Lung Carcinoma Patient With Low Values of Multiple Predictive Biomarkers

Immune checkpoint inhibitors have revolutionized the treatments of lung cancers, and multiple predictive biomarkers alone or in combination help clinicians with the appropriate therapeutic selections. Recently, chemo-immunotherapy has been recommended for treating advanced non-small cell lung cancers in patients without driver mutations. However, the clinical relevance of predictive biomarkers and the treatment efficacy of chemo-immunotherapy in large cell lung carcinoma (LCLC) remain unclear. Here, we reported a rare case of LCLC with none driver gene mutations and low values of multiple predictive biomarkers. These biomarkers included a low PD-L1 expression of 5–10%, a low tumor mutational burden (TMB) of 2.5 muts/mb, a low CD8(+) tumor-infiltrating lymphocyte density of 147.91 psc/mm². After one-cycle chemotherapy, the patient progressed rapidly and then was switched to pembrolizumab combining paclitaxel plus cisplatin. Interestingly, he achieved a partial response after two cycles of chemo-immunotherapy, showing multiple lymph nodes obviously shrunk on CT scan, and other clinical symptoms were relieved when compared with the baseline findings. After five cycles of chemo-immunotherapy, this advanced patient still benefited and was changed to maintenance immunotherapy monotherapy. This case suggests that chemo-immunotherapy may provide an effective therapeutic option for those LCLC patients with low values of multiple predictive biomarkers, particularly for those who progressed from first-line classical treatments.

Meta-analysis of tumor- and T cell-intrinsic mechanisms of sensitization to checkpoint inhibition

SummaryCheckpoint inhibitors (CPIs) augment adaptive immunity. Systematic pan-tumor analyses may reveal the relative importance of tumor-cell-intrinsic and microenvironmental features underpinning CPI sensitization. Here, we collated whole-exome and transcriptomic data for >1,000 CPI-treated patients across seven tumor types, utilizing standardized bioinformatics workflows and clinical outcome criteria to validate multivariable predictors of CPI sensitization. Clonal tumor mutation burden (TMB) was the strongest predictor of CPI response, followed by total TMB and CXCL9 expression. Subclonal TMB, somatic copy alteration burden, and histocompatibility leukocyte antigen (HLA) evolutionary divergence failed to attain pan-cancer significance. Dinucleotide variants were identified as a source of immunogenic epitopes associated with radical amino acid substitutions and enhanced peptide hydrophobicity/immunogenicity. Copy-number analysis revealed two additional determinants of CPI outcome supported by prior functional evidence: 9q34 (TRAF2) loss associated with response and CCND1 amplification associated with resistance. Finally, single-cell RNA sequencing (RNA-seq) of clonal neoantigen-reactive CD8 tumor-infiltrating lymphocytes (TILs), combined with bulk RNA-seq analysis of CPI-responding tumors, identified CCR5 and CXCL13 as T-cell-intrinsic markers of CPI sensitivity.

PD-1 blockade restores helper activity of tumor-infiltrating, exhausted PD-1hiCD39+ CD4 T cells.

Tumor antigen–specific CD4 T cells accumulate at tumor sites, evoking their involvement in antitumor effector functions in situ. Contrary to CD8 cytotoxic T lymphocyte exhaustion, that of CD4 T cells remains poorly appreciated. Here, using phenotypic, transcriptomic, and functional approaches, we characterized CD4 T cell exhaustion in patients with head and neck, cervical, and ovarian cancer. We identified a CD4 tumor-infiltrating lymphocyte (TIL) population, defined by high PD-1 and CD39 expression, which contained high proportions of cytokine-producing cells, although the quantity of cytokines produced by these cells was low, evoking an exhausted state. Terminal exhaustion of CD4 TILs was instated regardless of TIM-3 expression, suggesting divergence with CD8 T cell exhaustion. scRNA-Seq and further phenotypic analyses uncovered similarities with the CD8 T cell exhaustion program. In particular, PD-1hiCD39+ CD4 TILs expressed the exhaustion transcription factor TOX and the chemokine CXCL13 and were tumor antigen specific. In vitro, PD-1 blockade enhanced CD4 TIL activation, as evidenced by increased CD154 expression and cytokine secretion, leading to improved dendritic cell maturation and consequently higher tumor-specific CD8 T cell proliferation. Our data identify exhausted CD4 TILs as players in responsiveness to immune checkpoint blockade.

The mutational load and a T-cell inflamed tumour phenotype identify ovarian cancer patients rendering tumour-reactive T cells from PD-1+ tumour-infiltrating lymphocytes

BackgroundAdoptive immunotherapy with tumour-infiltrating lymphocytes (TIL) may benefit from the use of selective markers, such as PD-1, for tumour-specific T-cell enrichment, and the identification of predictive factors that help identify those patients capable of rendering tumour-reactive TILs. We have investigated this in ovarian cancer (OC) patients as candidates for TIL therapy implementation.MethodsPD-1− and PD-1+ CD8 TILs were isolated from ovarian tumours and expanded cells were tested against autologous tumour cells. Baseline tumour samples were examined using flow cytometry, multiplexed immunofluorescence and Nanostring technology, for gene expression analyses, as well as a next-generation sequencing gene panel, for tumour mutational burden (TMB) calculation.ResultsTumour-reactive TILs were detected in half of patients and were exclusively present in cells derived from the PD-1+ fraction. Importantly, a high TIL density in the fresh tumour, the presence of CD137+ cells within the PD-1+CD8+ TIL subset and their location in the tumour epithelium, together with a baseline T-cell-inflamed genetic signature and/or a high TMB, are features that identify patients rendering tumour-reactive TIL products.ConclusionWe have demonstrated that PD-1 identifies ovarian tumour-specific CD8 TILs and has uncovered predictive factors that identify OC patients who are likely to render tumour-specific cells from PD-1+ TILs.

Differences in TCR repertoire and T cell activation underlie the divergent outcomes of antitumor immune responses in tumor-eradicating versus tumor-progressing hosts

BackgroundAntitumor immunity is highly heterogeneous between individuals; however, underlying mechanisms remain elusive, despite their potential to improve personalized cancer immunotherapy. Head and neck squamous cell carcinomas (HNSCCs) vary significantly in...

Phased differentiation of γδ T and T CD8 tumor-infiltrating lymphocytes revealed by single-cell transcriptomics of human cancers

ABSTRACT γδ T lymphocytes diverge from conventional T CD8 lymphocytes for ontogeny, homing, and antigen specificity, but whether their differentiation in tumors also deviates was unknown. Using innovative analyses of our original and ~150 published single-cell RNA sequencing datasets validated by phenotyping of human tumors and murine models, here we present the first high-resolution view of human γδ T cell differentiation in cancer. While γδ T lymphocytes prominently encompass TCRVγ9 cells more differentiated than T CD8 in healthy donor’s blood, a different scenario is unveiled in tumors. Solid tumors and lymphomas are infiltrated by a majority of TCRVγnon9 γδ T cells which are quantitatively correlated and remarkably aligned with T CD8 for differentiation, exhaustion, gene expression profile, and response to immune checkpoint therapy. This cancer-wide association is critical for developing cancer immunotherapies.

4-1BB co-stimulation further enhances anti-PD-1-mediated reinvigoration of exhausted CD39+ CD8 T cells from primary and metastatic sites of epithelial ovarian cancers

BackgroundResponses to immunotherapy vary between different cancer types and sites. Here, we aimed to investigate features of exhaustion and activation in tumor-infiltrating CD8 T cells at both the primary and...

Abstract PO-54: Mechanistic consequences of histone-deacetylase inhibition towards sensitizing PD1 blockade-resistant B-cell lymphomas

Abstract PD1 blockade is effective in a subset of B-cell lymphoma patients (e.g., classical Hodgkin lymphomas); however, most patients do not respond to anti-PD1 therapy. To overcome PD1 resistance, we employ a newly developed isoform-selective histone-deacetylase-inhibitor (HDACi) (OKI-179) and a novel mouse mature B-cell lymphoma, G1XP lymphoma, that resembles immunosuppressive features of human B-cell lymphomas including downregulation of major histocompatibility complex (MHC) class I and II, exhaustion of CD8 and CD4 tumor-infiltrating lymphocytes (TILs), and PD1-blockade resistance. Using multiple lymphoma models, we show that combined treatment of OKI-179/anti-PD1 significantly inhibited growth of B-cell lymphomas refractory to PD1 blockade; furthermore, sensitivity to single or combined treatment required tumor-derived MHC class I, and positively correlated to MHC class II level. We conclude that OKI-179 sensitizes lymphomas to PD1 blockade by enhancing tumor immunogenicity. Additionally, we found that different HDACi exhibited distinct effects on tumors and T cells, yet the same HDACi could differentially affect HLA expression on different human B-cell lymphomas. Thus, our study highlights the importance of immunologic effects of HDACi on antitumor responses and suggests that optimal treatment efficacy requires personalized design and rational combination based on prognostic biomarkers (e.g., MHCs) and unique profiles of HDACi. Citation Format: Xiaoguang Wang, Brittany C. Waschke, Rachel A. Woolaver, Zhangguo Chen, Gan Zhang, Anthony D. Piscopio, Xuedong Liu, Jing H. Wang. Mechanistic consequences of histone-deacetylase inhibition towards sensitizing PD1 blockade-resistant B-cell lymphomas [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-54.

Obesity-Associated Myeloid-Derived Suppressor Cells Promote Apoptosis of Tumor-Infiltrating CD8 T Cells and Immunotherapy Resistance in Breast Cancer.

Nearly 70% of adults in the US are currently overweight or obese. Despite such high prevalence, the impact of obesity on antitumor immunity and immunotherapy outcomes remains incompletely understood, particularly in patients with breast cancer. Here, we addressed these gaps in knowledge using two murine models of breast cancer combined with diet-induced obesity. We report that obesity increases CXCL1 concentrations in the mammary tumor microenvironment, driving CXCR2-mediated chemotaxis and accumulation of granulocytic myeloid-derived suppressor cells (G-MDSCs) expressing Fas ligand (FasL). Obesity simultaneously promotes hyperactivation of CD8 tumor-infiltrating lymphocytes (TILs), as evidenced by increased expression of CD44, PD-1, Ki-67, IFNγ, and the death receptor Fas. Accordingly, G-MDSCs induce Fas/FasL-mediated apoptosis of CD8 T cells ex vivo and in vivo. These changes promote immunotherapy resistance in obese mice. Disruption of CXCR2-mediated G-MDSC chemotaxis in obese mice is sufficient to limit intratumoral G-MDSC accumulation and improve immunotherapy outcomes. The translational relevance of our findings is demonstrated by transcriptomic analyses of human breast tumor tissues, which reveal positive associations between CXCL1 expression and body mass index, poor survival, and a MDSC gene signature. Further, this MDSC gene signature is positively associated with FASLG expression. Thus, we have identified a pathway wherein obesity leads to increased intratumoral CXCL1 concentrations, which promotes CXCR2-mediated accumulation of FasL+ G-MDSCs, resulting in heightened CD8 TIL apoptosis and immunotherapy resistance. Disruption of this pathway may improve immunotherapy outcomes in patients with breast cancer and obesity.

Metformin changes the immune microenvironment of colorectal cancer in patients with type 2 diabetes mellitus.

Accumulating evidence suggests that metformin reduces the incidence and mortality of colorectal cancer (CRC). However, underlying mechanisms have not been fully clarified. The aim of this study was to examine the pathological characteristics of resected CRC from patients treated with metformin for type 2 diabetes mellitus (DM). In total, 267 patients with DM underwent curative colectomy for Stage I‐III CRC and 53 (19.9%) patients had been treated medically including metformin. Pathological N‐stage was significantly lower in metformin‐treated patients (P < .05) with prolonged disease‐free survival (DFS) (P < .05). Immunohistochemistry showed that the densities of CD3(+) and CD8(+) tumor‐infiltrating lymphocytes (TILs) in the invasive front area were significantly higher in 40 patients treated with metformin compared with propensity score matched cases without metformin (P < .05). The density of tertiary lymphoid structures (TLS) in tumor stroma was markedly increased in metformin‐treated patients (P < .001). In those tumors, there were more CD68(+) tumor‐associated macrophages (TAM) infiltrated (P < .05), while the ratio of CD163(+) M2‐phenotype was markedly reduced (P < .001). Stromal fibrosis tended to be suppressed by metformin intake (P = .051). These findings suggested that metformin drastically changes the characteristics of infiltrating immune cells in CRC and reprograms the tumor microenvironment from immunosuppressive to immunocompetent status, which may lead to suppression of microscopic tumor spread and improve the outcomes of patients with CRC and type 2 DM.

Deletion of p53 and Hyper-Activation of PIK3CA in Keratin-15+ Stem Cells Lead to the Development of Spontaneous Squamous Cell Carcinoma.

Squamous cell carcinoma (SCC) is the second commonest type of skin cancer, and SCCs make up about 90% of head and neck cancers (HNSCCs). HNSCCs harbor two frequent molecular alterations, namely, gain-of-function alterations of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and loss-of-function mutations of tumor protein p53 (TP53). However, it remains poorly understood whether HNSCCs harboring different genetic alterations exhibit differential immune tumor microenvironments (TME). It also remains unknown whether PIK3CA hyperactivation and TP53 deletion can lead to SCC development spontaneously. Here, we analyzed the Cancer Genome Atlas (TCGA) datasets of HNSCCs and found that patients with both PIK3CA and TP53 alterations exhibited worse survival, significantly lower CD8 tumor infiltrating lymphocytes (TILs) and higher M0 macrophages than other controls. To better model human tumorigenesis, we deleted TP53 and constitutively activated PIK3CA in mouse keratin-15-expressing stem cells, which leads to the spontaneous development of multilineage tumors including SCCs, termed Keratin-15-p53-PIK3CA (KPPA) tumors. KPPA tumors were heavily infiltrated with myeloid-derived suppressor cells (MDSCs), with a drastically increased ratio of polymorphonuclear-MDSC (PMN-MDSC) versus monocytic-MDSC (M-MDSC). CD8 TILs expressed more PD-1 and reduced their polyfunctionality. Overall, we established a genetic model to mimic human HNSCC pathogenesis, manifested with an immunosuppressive TME, which may help further elucidate immune evasion mechanisms and develop more effective immunotherapies for HNSCCs.

Prognostic Significance of Tumor-infiltrating Lymphocytes on Survival Outcomes of Patients With Resected Pancreatic Ductal Adenocarcinoma: A Systematic Review and Meta-Analysis.

Tumor-infiltrating lymphocytes (TILs) play an important role in mediating treatment response in pancreatic cancer. This meta-analysis investigated the prognostic significance of TIL subsets on overall survival (OS) and disease-free survival (DFS) of patients with pancreatic cancer. Studies were gathered via search of PubMed, Google Scholar, and Cochrane Library databases up to August 1, 2019. Using Review Manager version 5.3.5, pooled hazard ratios and 95% confidence intervals (CIs) were calculated using random or fixed-effects models, depending on the heterogeneity of studies. A total of 11 studies comprising 1760 patients were included in the meta-analysis. Pooled analysis revealed that high CD8 TILs were associated with improved OS [hazard ratio (HR)=0.59, 95% CI=0.51-0.69, P<0.00001] and DFS (HR=0.60, 95% CI=0.50-0.73, P<0.00001). Similarly, high CD3 TILs correlated with better OS (HR=0.64, 95% CI=0.54-0.75, P<0.00001) and DFS (HR=0.53, 95% CI=0.31-0.92, P<0.0001). In contrast, high FoxP3 TILs were associated with worse OS (HR=1.39, 95% CI=1.03-1.88, P=0.03). Finally, high CD4 TILs showed significant improvement in OS (HR=0.74, 95% CI=0.63-0.86, P=0.0001). TILs are a promising prognostic biomarker in pancreatic cancer. Prospective studies evaluating TILs are recommended as well as the establishment of standards in the assessment of TILs.

Abstract 5110: Cancer-associated fibroblast phenotypes vary across colorectal cancers and correlate with CD8+ T-cell infiltration

Abstract BACKGROUND: Cancer-associated fibroblasts (CAFs) play an important role in the tumor microenvironment (TME). This has been correlated with changes in cancer biology, including tumor progression and treatment response. CAFs exhibit divergent phenotypes differentiated by their expression profiles and function. Across cancer types, two CAF subsets have been consistently identified: myofibroblastic and non-myofibroblastic. Here we examine podoplanin (PDPN), α-smooth muscle actin (αSMA), and collagen as potential CAF subtype markers in the stromal compartment of colorectal cancers (CRCs). These markers were compared with versican (VCAN), an immunoregulatory proteoglycan associated with an immune excluded TME. METHODS: A tissue microarray containing representative tumor and normal tissue from 122 CRC patients was stained using Masson's Trichrome (collagen) or immunohistochemistry for αSMA, PDPN, VCAN and versikine (Vkine, a proteolytic fragment of VCAN). Quantification of the stains (0 to 3+) was performed by at least two observers in consultation with a surgical pathologist. Smooth muscle from the muscularis propria was excluded from quantification in the αSMA stain. CD8 staining was quantified as the number of tumor infiltrating CD8+ lymphocytes (TILs) per high power field (HPF). Separate tissue cores were sequenced for 107 of these patients using the Qiagen Comprehensive Cancer Panel. Mutations were identified using Strelka; SNPs and indels were cross-referenced to ClinVar. RESULTS: Stromal staining for αSMA, PDPN, and collagen varied across CRC sections. The abundance and intensity of staining was independent of disease stage. Those cancers with 3+ collagen or αSMA had reduced TILs (collagen 0-1+: 8.0 TILs/HPF vs 2-3+: 3.0, p=0.02; αSMA 0-1+: 7.4 vs 2-3+: 3.6, p=0.04). There was a similar trend observed for PDPN (0-1+: 6.5 vs 2-3+: 3.5, p=0.1). VCAN proteolysis predominant (VPP; VCAN 0-1+ and Vkine 2-3+) samples, previously reported to associate with higher CD8+ TILs, had lower collagen vs VCAN proteolytic weak (VPW; mean collagen score 1.6 vs 2.2, p=0.002) and lower αSMA (VPP: 1.7 vs VPW: 2.1, p=0.02). PDPN was not associated with proteolysis (p=0.4). In VPP tumors, stromal marker abundance did not correlate with CD8+ TILs; however, in VPW tumors, CD8+ TILs were higher in low PDPN (4.1 vs 1.5 TILs/HPF, p = 0.02) and low αSMA tumors (4.8 vs 1.6; p = 0.009). The trend was similar for low collagen tumors (3.7 vs 1.9, p = 0.1). Collagen was more abundant in TP53-mutant cancers (p=0.03, Chi-Square Test). CONCLUSIONS: The stromal microenvironment of CRC is highly variable across patients with diverse CAF densities and phenotypes. The makeup of this microenvironment has potential clinical impacts, as it correlates with immune cell infiltration. Further investigation into CAF biology in the TME could reveal therapeutic targets to alter immune surveillance of cancers. Citation Format: Katherine A. Johnson, Philip B. Emmerich, Cheri A. Pasch, Linda Clipson, Kristina A. Matkowskyj, Dustin A. Deming. Cancer-associated fibroblast phenotypes vary across colorectal cancers and correlate with CD8+ T-cell infiltration [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5110.

Abstract 2662: Mitochondrial genetics appear to alter immune cell development/trafficking

Abstract Background Metastatic burden is the leading cause of cancer deaths; however, it remains unclear why some patients are more susceptible to metastatic disease. While the nuclear genome's role in tumor progression and metastasis is known, the role of mitochondrial DNA (mtDNA) polymorphisms (SNP) has only recently been explored. Using mitochondrial nuclear exchange (MNX) mice, we previously showed that mtDNA strongly influences mammary carcinoma progression and metastasis both intrinsically and via non-cell autonomous mechanisms. We hypothesized that mtDNA SNP alter immune cell development/trafficking which, in turn, could influence metastasis efficiency. Methods Peritoneal exudate, and splenocytes were collected from male and female wild-type C57BL/6J (CC) and C3H/HeN (HH), and MNX mice - C57BL/6-mtMNX(C3H/HeN) (CH) and C3H/HeN-mtMNX(C57BL/6J) (HC) mice [first letter=nuclear; second letter=mitochondrial]. Lung metastases were established from tail-vein injection of E0771 (CC/CH) or K1735-M2 (HH/HC) cell lines. Results Lung metastases derived from histocompatible (i.e. nDNA matched) tumor cell injection into wild-type or MNX mice increased in C3H/HeN mtDNA backgrounds (HH and CH). No significant differences were observed in seeding, suggesting that mtDNA mediated differences in metastatic microenvironments likely impact metastatic outgrowth. No significant changes in broad immune cell populations were observed in naïve animals, but selective changes in differentiation markers were observed. The most significant change was lower CD11c+ peritoneal macrophages in HH (3%) versus CC (16%) mice (p &amp;lt; 0.001). C57BL/6 mtDNA (HC) increased the percentage of macrophages (9%) compared to wild-type (HH) (p &amp;lt; 0.001). We next confirmed a role for mitochondrial derived ROS mediating immune microenvironmental regulation of metastasis, as mtDNA mediated metastatic differences are abrogated upon treatment with the anti-oxidant MitoTEMPO. Tumor infiltrating CD8+ lymphocytes (TIL) increased 1.5-fold in CH (1.7%) compared to wild-type CC (1.2%) (p = 0.07) while no differences were observed between HH and HC mice. Consistent with changes in metastasis in the HH background, MitoTempo treatment reduced CD4+ TIL &amp;gt;1.5-fold (1.5%), compared to the vehicle control (2.8%) (p = 0.029). Discussion Our data support the hypothesis that mitochondrial SNP modulate immune cell development and/or trafficking, providing a plausible explanation for how metastatic potentials of syngeneic tumor cells injected into MNX mice are altered. That is, mtDNA contributions to immune function affect metastasis and may provide insight as to why some immune therapies succeed/fail. Support: DOD BCRP BC171381 and Kansas INBRE P20 GM103418 (TCB); Susan G. Komen for the Cure SAC110037; National Foundation for Cancer Research and NIH CA168524 (DRW) Citation Format: Thomas C. Beadnell, Amanda E. Brinker, Cori Fain, Carolyn J. Vivian, Danny R. Welch. Mitochondrial genetics appear to alter immune cell development/trafficking [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2662.

Abstract 2051: Precision immune phenotyping from primary tumor and metastatic lesions reveals novel insights into therapeutic intervention in cancer immunotherapy

Abstract A key question in cancer immunotherapy is the general immune status of the patient's tumor-microenvironment (TME) prior to therapy. A patient's underlying tumor immune-contexture may therefore a) guide the therapeutic intervention and b) help to identify potential resistance mechanisms to immunotherapies. The most commonly used classification of the patients' immune status is based on the CD8 tumor infiltrating lymphocytes (TIL) status referring to either “cold”- or “hot” tumors. Patients in early clinical development programs will mostly present with late metastatic disease and therefore the immune phenotypes from these lesions may more relevant to determine the actual tumor-immune contexture status than the currently used characterization based on the primary tumor type. In this study, we systematically analyzed the CD8 immune phenotype (CD8 IP) in 454 patients from various Phase 1 clinical trials using the commonly applied CD8 immnohistochemistry (IHC)-based classification into cold (CD8 deserts) and hot (CD8 excluded and inflamed) tumors and matched gene expression profiling. In total, we observed 116 inflamed, 95 excluded, and 243 desert phenotypes from 25 different indications. The main sources of tumor biopsy tissues were liver metastasis (N=151), lymph node (N=73) as well as lung (N=59). Overall, we identified a significant overrepresentation of the desert phenotype in liver metastasis (p=1e-05). In patients with metastatic disease, we compared the predominant CD8 IP in metastatic lesions to that of the primary tumor. The overall distribution for tumor types such as UBC, NSCLC or OvCa did not change, whereas PDAC, GC, and CRC revealed a significant change towards the desert phenotype for metastatic lesions. Beyond the predominant CD8 IP assessment via IHC, we characterized each CD8 subtype further using specific immune gene signatures. We could identify a reverse correlation of inflammatory and suppressive immune signatures along the CD8 phenotypes in metastatic lesions such as M2-like macrophages, MDSC, or Dendritic Cells. Further zooming into the desert phenotype, we were also able to show that liver metastasis have a more significant immunosuppressive tumor environment compared to other tumor biopsies. In summary, our data provide essential insights about the importance of the origin of the tissue biopsy to reduce impaired decision making based on the underlying tumor-immune contexture of cancer patients. Precision immune phenotyping beyond CD8 TIL infiltration is needed to identify specific resistance mechanisms to cancer immunotherapy and can be used for efficient patient enrichment and thus increase the probability of success for early clinical trials in immuno-oncology. Citation Format: Andreas Roller, Claudia Ferreira, Laura Jarassier, Astrid Heller, Gabriele Dietmann, Konstanty Korski, Bruno Gomes, Michael A. Cannarile. Precision immune phenotyping from primary tumor and metastatic lesions reveals novel insights into therapeutic intervention in cancer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2051.

Abstract 2198: The anti-tumor enhancement of a dendritic-cell targeting MIP3α-Gp100-Trp2 DNA vaccine by IFNα and 5-Aza-2'-deoxycytidine treatments correlates with intratumoral CCL19 but not CCL21 expression

Abstract Background: The chemokine MIP-3α (CCL20) binds to CCR6 on immature dendritic cells (DCs). DNA vaccines fusing MIP-3α to melanoma-associated antigens gp100 and/or tyrosinase-related protein 2 (Trp2) have delayed tumor growth and increased survival time in the B16F10 mouse melanoma model system compared to vaccines lacking the chemokine. To further enhance the therapeutic effects of the vaccine, our laboratory has added type-I interferon (IFNα) and 5-Aza-2'-deoxycitidine (5Aza) to the therapy. Here, we report that the enhancement previously seen by the combination of IFNα, 5Aza, and a MIP-3α-Gp100-Trp2 (MGpTrp2) DNA vaccine correlates with increases of tumor-infiltrating CD8+T-cells (CD8 TILs) and intratumoral expression of CCL19 but not CCL21. Methods: Beginning on day five post-transplantation of B16F10 melanoma, vaccine was administered intramuscularly (i.m.) by electroporation. CpG adjuvant was given two days later. 5Aza was given intraperitoneally at 1mg/kg and IFNα therapy either intratumorally or i.m. as noted. Tumor sizes, tumor growth, and mouse survival were assessed. Tumor lysate gene expression levels and tumor-infiltrating lymphocytes (TILs) were assessed by qRT-PCR and flow cytometry with intracellular cytokine staining, respectively. Results: Previous work has shown that the combination of IFNα, 5Aza, and MGpTrp2 led to significantly reduced tumor burden and overall increases in mouse survival dependent upon all three components. The addition of 5Aza and IFNα to the vaccine affected T-cell tumor infiltration, increasing the proportion of CD3+CD8+ cells in gated TILs by 92% over vaccine alone (p&amp;lt;0.0001), which correlated with tumor size (r2: 0.507; p&amp;lt;0.0001). Interferon stimulated genes such as Mx1, MHC1, CXCL10, and GranzymeB were modestly upregulated in the tumor lysate. However, CCL19, normally expressed constitutively in lymphoid tissues, was upregulated 10-fold compared to vaccine alone (p&amp;lt;0.0001) and was correlated with tumor size (r2: 0.195; p=0.002). The CCL19 receptor, CCR7, was upregulated to a lesser degree (5-fold over vaccine; p&amp;lt;0.001), and interestingly its partner chemokine CCL21 did not have significantly different expression patterns across groups (p=0.14 to vaccine). Conclusions: Efficient targeting of antigen to immature dendritic cells with a chemokine-fusion vaccine offers a potential alternative approach to classic and dendritic cell-based vaccines. Combining this approach with IFNα and 5Aza treatments significantly improved vaccine efficacy. This enhancement was correlated with CD8+ TIL recruitment and with the expression of the T-cell trafficking chemokine CCL19. Further potential therapy optimization currently undergoing investigation offers promise for this line of investigation to become a novel melanoma therapy. Citation Format: James T. Gordy, Avinaash K. Sandhu, Samuel K. Ayeh, Aakanksha Kapoor, Emily Kim, Petros C. Karakousis, Richard B. Markham. The anti-tumor enhancement of a dendritic-cell targeting MIP3α-Gp100-Trp2 DNA vaccine by IFNα and 5-Aza-2'-deoxycytidine treatments correlates with intratumoral CCL19 but not CCL21 expression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2198.

Why the Outcome of Anti-Tumor Immune Responses is Heterogeneous: A Novel Idea in the Context of Immunological Heterogeneity in Cancers.

The question as to why some hosts can eradicate their tumors while others succumb to tumor-progression remains unanswered. Here, a provocative concept is proposed that intrinsic differences in the T cell receptor (TCR) repertoire of individuals may influence the outcome of anti-tumor immunity by affecting the frequency and/or variety of tumor-reactive CD8 and/or CD4 tumor-infiltrating lymphocytes. This idea implicates that the TCR repertoire in a given patient might not provide sufficiently different TCR clones that can recognize tumor antigens, namely, "a hole in the TCR repertoire" might exist. This idea may provide a novel perspective to further dissect the mechanisms underlying heterogeneous anti-tumor immune responses in different hosts. Besides tumor-intrinsic heterogeneity and host microbiome, the various factors that may constantly shape the dynamic TCR repertoire are also discussed. Elucidating mechanistic differences in different individuals' immune systems will allow to better harness immune system to design new personalized cancer immunotherapy.

Abstract A18: Characterization of checkpoint inhibitors in the tumor microenvironment (TME) and peripheral blood in endemic Burkitt Lymphoma

Abstract Introduction: The tumor microenvironment and the interaction between immune checkpoint inhibitors (ICIs) and tumor cells are essential to understand for designing future immunotherapy strategies. In this study, we aim to characterize the tumor microenvironment (TME) in children diagnosed with endemic Burkitt lymphoma (eBL), the most common pediatric cancer in Africa. A history of chronic malaria (Plasmodium falciparum) and Epstein-Barr virus (EBV) coinfections are both risk factors in the etiology of eBL. Methods: Using multicolor flow cytometry and single-cell RNA sequencing, we analyzed the expression of immune checkpoint inhibitors in T cells from the tumor microenvironment and from peripheral blood mononuclear cells (PBMC) of eBL patients, along with age-matched controls with different malaria exposure. Additionally, CD8 T-cell infiltration was confirmed by immunohistochemistry. Results: PD-1+ CD4 T cells from eBL PBMCs were increased in number compared to healthy controls from the malaria-endemic area (p=0.03). Both CD4 and CD8 T cells from eBL PBMCs reveal higher frequency of TIGIT expressers compared to healthy controls (non-malaria endemic) (p=0.002, p=0.007 respectively). PD1+ TIGIT+ IFNG- memory CD4 T cells phenotype from eBL PBMCs was increased upon short stimulation with PMA/ionomycin, as well as “naive-like” CD95+ PD1high TIGIT+ CTLA4+ IFNG+ CD8T cells. Tumor samples from 4 eBL patient were used on single-cell analysis; we observed activated T-cells profile that highly upregulated TIGIT within the tumor microenvironment. We also noticed relatively low expression of other ICIs such as PD1, TIM3, LAG3, and CTLA4. Base on all these data, we calculated an exhaustion score that was higher than the cytotoxicity score in our eBL-derived tumor-infiltrating lymphocytes (TILs). In addition, we use immunohistochemistry staining to confirm CD8 T-cell infiltration and ICI expression in eBL tumor fine needle aspirates. Our future directions will employ TIGIT blockade inhibitor studies of CD8 T cells, in order to increase expansion and function of CD8 TILs. Our results will help support future development of ICI therapies in endemic Burkitt lymphoma. Citation Format: Priya Saikumar-Lakshmi, Cliff Oduor, Catherine Forconi, Patrick Marsh, John Michael Ong’echa, Jeffrey Bailey, Ann Moormann. Characterization of checkpoint inhibitors in the tumor microenvironment (TME) and peripheral blood in endemic Burkitt Lymphoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A18.