Tumor-infiltrating Lymphocytes Subsets Research Articles

Abstract 6732: Immunomodulatory and anti-tumor effect of a CCR2-targeted STING agonist iADC in human lung cancer

Abstract Introduction: Antibody drug conjugates (ADCs) targeting cancer-cell antigens can improve therapeutic index and show prominent clinical impact. Limited studies have used ADCs to target antigens present in non-malignant cells in the tumor microenvironment or to deliver immunomodulatory agents. We developed an immune-cell directed antibody drug conjugate (iADC) TAK-500 that selectively activates STING in C-C chemokine receptor type 2 (CCR2) expressing cells to achieve enhanced efficacy over non-targeted STING agonists. Here, we spatially mapped CCR2 protein in human NSCLC and studied the immunomodulatory role and anti-tumor effect of the novel iADC TAK-500 on human lung cancer tumor explants ex vivo. Methods: Using multiplexed quantitative immunofluorescence (mQIF) panels we measured the levels of CCR2 protein, myeloid cell populations and tumor infiltrating lymphocyte (TIL) subsets in 411 primary NSCLC carcinomas from treatment-naïve patients in 2 independent cohorts and determined their clinical significance. In addition, we longitudinally studied CCR2 levels, myeloid cells (DAPI/CCR2/CD68/CD11b), TILs (DAPI/CK/CD4/CD8/CD20), T-cell functional markers (DAPI/CK/CD3/Ki-67/GZMB [Granzyme B]) and cancer-cell death/proliferation markers (DAPI/CK/TFR1/Ki-67/CC3 [Cleaved Caspase 3]) in 314 primary cultured tissue fragments from paired tumor and non-tumor specimens resected from 11 lung cancer patients at Yale New Haven Hospital between 2021-2023 (8 adenocarcinomas, 2 small cell carcinomas, and 1 squamous cell carcinoma). Each biopsy was divided into 4-mm2 fragments, cultured in extracellular matrix for 5 days with or without TAK-500 treatment. Samples were processed into FFPE tissue and stained with mQIF panels using a tissue microarray format. Results: CCR2 protein was detected in 386 of 411 (94%) NSCLCs from 2 independent cohorts predominantly localized in CD11b+ intratumor myeloid cells and CD68+ tumor-associated macrophages (TAMs). Elevated CCR2 was associated with adenocarcinoma histology, increased PD-L1, activating KRAS mutations and longer survival. Treatment with TAK-500 for 5 days reduced CCR2 levels in myeloid cells and CD68+ TAMs, without significant changes in TIL or T-cell GZMB/Ki-67 levels. TAK-500 also significantly reduced tumor proliferation (Ki-67 expression) and increased tumor cell apoptosis (CC3 levels) after 5 days of treatment. Notably, these TAK-500-induced changes were observed only in tumor specimens with high baseline CCR2 levels above the median in retrospective NSCLC cohorts. No significant effect was observed in the paired non-tumor fragments. Conclusion: Our results reveal prominent myeloid CCR2 expression in a subset of human NSCLCs with distinct clinicopathologic/molecular characteristics. Using a novel lung cancer explant ex vivo system, we demonstrate a CCR2-dependent immunomodulatory and anti-tumor effect of TAK-500 iADC. Citation Format: Angelo Porciuncula, Vicky A. Appleman, Alex Parent, Jeff Raizer, Richard C. Gregory, Neil Lineberry, Adnan O. Abu-Yousif, Kurt A. Schalper. Immunomodulatory and anti-tumor effect of a CCR2-targeted STING agonist iADC in human lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6732.

Abstract 1053: The Influence of PD1 and TIM3 Expression on CD4+CD103+ and CD8+CD103+ Cells in Glioblastoma Progression

Abstract Glioblastoma (GB), IDH-wildtype, is the most common and aggressive high-grade primary malignant brain tumor with extremely poor outcome. Although the use of immune checkpoint inhibitors (ICIs) in GB has shown disappointing results, the identification of a small subgroup of responders underpins the role of the intratumoral immune contexture, in particular tumour-infiltrating lymphocytes (TILs), in determining disease outcome. This study aims to define the features of TIL subpopulations correlating with GB prognosis, and to establish in parallel the predictive roles of peripheral T cell subsets. A single cohort observational study, including patients undergoing GB resection and standard therapeutic and follow-up approaches in Neuro-Oncology, was conducted. Of the 45 patients, histologically confirmed WHO grade 4, 26 had MGMT promoter methylation (60.0%), 24 were male (53.3%) and the median age of young patients (≤63 years) vs elderly patients (>63 years) was 57 vs 71, respectively. Predictors of OS were KPS (≥70 vs <70: 14.7 vs 7.5 months, p=0.0007) and GTR (GTR vs non-GRT: 15 vs 9 months, p=0.004) whereas patients with methylated MGMT promoter had non-significant longer median OS compared to those with unmethylated promoter (14.5 vs 10.5 months). Patients were characterized for the expression of lineage, differentiation, memory, activation, and inhibition markers on intratumoral and peripheral CD4+ and CD8+ T cell subpopulations, and the correlation of an array of CD4+ and CD8+ T cell subsets with OS or PFS was evaluated by multiparametric flow cytometry and uni/multivariate analyses. Intratumoral immune infiltrate was positively associated with CD8+CD103+ tissue-resident memory T cells (Trm), and in particular with CD8+CD103+ Trm expressing PD1 and TIM3 rather than CD4+ Trm. Low CD8+CD103+PD1+ Trm resulted significantly associated with better OS and PFS (p=0.02 and 0.0031, respectively) and even higher significant was the correlation with better OS and PFS of low CD8+CD103+TIM3+ Trm (p=0.0002 and 0.0033, respectively) and CD8+CD103+PD1+TIM3+ Trm (p<0.0001, both). On multivariate analysis, low CD8+CD103+PD1+TIM3+ Trm and KPS ≥70 were confirmed to be the most predictive independent factors associated with longer OS (hazard ratios - HR [95%CI]: 0.14 [0.04 - 0.52] p˂0.001, 0.39 [0.16 - 0.96] p=0.04, respectively). Moreover, CD8+CD103+ Trm subpopulations resulted age-related predictors for GB survival. Consistent with this finding, in vitro PD1 and TIM3 blockade released the capability of intratumoral T cells to produce the effector cytokines IFN-γ and TNF-α, and the cytotoxic factor GrzB. Conversely, T cells expressing PD1 and TIM3 of peripheral blood did not show any prognostic significance. In conclusion, low frequency of Trm expressing PD1 or TIM3 or both markers defined TIL subsets as independent positive prognostic factors for patient survival. Citation Format: Lucia Gabriele, Giulia Romagnoli, Quintino Giorgio D'Alessandris, Imerio Capone, Andrea Tavilla, Irene Canini, Caterina Lapenta, Mariachiara Buccarelli, Martina Giordano, Valentina Tirelli, Massimo Sanchez, Alessandra Fragale, Stefano Giannetti, Rina Di Bonaventura, Liverana Lauretti, Mauro Biffoni, Lucia Ricci-Vitiani, Roberto Pallini. The Influence of PD1 and TIM3 Expression on CD4+CD103+ and CD8+CD103+ Cells in Glioblastoma Progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1053.

Tumor immunity: A brief overview of tumor‑infiltrating immune cells and research advances into tumor‑infiltrating lymphocytes in gynecological malignancies (Review).

Tumor immunity is a promising topic in the area of cancer therapy. The 'soil' function of the tumor microenvironment (TME) for tumor growth has attracted wide attention from scientists. Tumor-infiltrating immune cells in the TME, especially the tumor-infiltrating lymphocytes (TILs), serve a key role in cancer. Firstly, relevant literature was searched in the PubMed and Web of Science databases with the following key words: 'Tumor microenvironment'; 'TME'; 'tumor-infiltrating immunity cells'; 'gynecologic malignancies'; 'the adoptive cell therapy (ACT) of TILs'; and 'TIL-ACT' (https://pubmed.ncbi.nlm.nih.gov/). According to the title and abstract of the articles, relevant items were screened out in the preliminary screening. The most relevant selected items were of two types: All kinds of tumor-infiltrating immune cells; and advanced research on TILs in gynecological malignancies. The results showed that the subsets of TILs were various and complex, while each subpopulation influenced each other and their effects on tumor prognosis were diverse. Moreover, the related research and clinical trials on TILs were mostly concentrated in melanoma and breast cancer, but relatively few focused on gynecological tumors. In conclusion, the present review summarized the biological classification of TILs and the mechanisms of their involvement in the regulation of the immune microenvironment, and subsequently analyzed the development of tumor immunotherapy for TILs. Collectively, the present review provides ideas for the current treatment dilemma of gynecological tumor immune checkpoints, such as adverse reactions, safety, personal specificity and efficacy.

Prognostic Value of Immune Cells Subsets Within the Tumor Microenvironment in Patients With Rectal Adenocarcinoma.

One-third of newly diagnosed colorectal cancer cases are rectal cancers. Multimodal treatment regimens including surgery, radiotherapy, and chemotherapy improve local control and survival outcome and decrease tumor relapse for patients with rectal adenocarcinoma (READ). However, stratification of patients to predict their responses is urgently needed to improve therapeutic responses. Immunostainings of CD3+, CD8+, and CD45RO+ immune cell subsets within the tumor microenvironment were evaluated using immunohistochemistry in two hundred seventy-nine READ patients. In this study, we found that examination of the adaptive immune response by quantifying CD3+, CD8+, and CD45RO+ immune cell subsets, provides improved and independent prognostic value for patients with READ. Regardless of conventional clinical and pathologic parameters, the densities of T cell subsets were strongly related to a better prognosis in patients with READ. High density of intratumoral immune cells is associated with absence of nodal metastasis, lymphovascular invasion, and perineural invasion. Moreover, high tumor-infiltrating lymphocyte (TIL) subsets were associated with favorable survival outcome in patients with READ, especially high-risk patients with advanced READ. Immune cell subsets including CD3, CD8, and CD45RO within the tumor microenvironment were independent prognostic factors for patients with READ.

Objective Analysis and Clinical Significance of the Spatial Tumor-Infiltrating Lymphocyte Patterns in Non-Small Cell Lung Cancer.

The spatial arrangement of lymphocytes in the tumor bed (e.g., immune infiltrated, immune excluded, immune desert) is expected to reflect distinct immune evasion mechanisms and to associate with immunotherapy outcomes. However, data supporting these associations are scant and limited by the lack of a clear definition for lymphocyte infiltration patterns and the subjective nature of pathology-based approaches. We used multiplexed immunofluorescence to study major tumor-infiltrating lymphocyte (TIL) subsets with single-cell resolution in baseline whole-tissue section samples from NSCLC patients treated with immune checkpoint inhibitors (ICI). The spatial TIL patterns were analyzed using a qualitative pathologist-based approach, and an objective analysis of TIL density ratios in tumor/stromal tissues. The association of spatial patterns with outcomes was studied for different TIL markers. The analysis of CD8+ TIL patterns using qualitative assessment identified prominent limitations including the presence of a broad spectrum of phenotypes within most tumors and limited association with outcomes. The utilization of an objective method to classify NSCLCs showed the existence of at least three subgroups with partial overlap with those defined using visual patterns. Using this strategy, a subset of cases with "immune excluded-like" tumors showed prominently worse outcomes, suggesting reduced sensitivity to ICI; however, these results need to be validated. The analysis for other TIL subsets showed different results, underscoring the relevance of the marker selected for spatial TIL pattern evaluation and opportunities for market integration. Our results identified major challenges associated with the qualitative spatial TIL pattern evaluation. We devised a novel objective strategy to overcome some of these limitations that has strong biomarker potential.

A2AR eGFP reporter mouse enables elucidation of A2AR expression dynamics during anti-tumor immune responses

There is significant clinical interest in targeting adenosine-mediated immunosuppression, with several small molecule inhibitors having been developed for targeting the A2AR receptor. Understanding of the mechanism by which A2AR is regulated has been hindered by difficulty in identifying the cell types that express A2AR due to a lack of robust antibodies for these receptors. To overcome this limitation, here an A2AR eGFP reporter mouse is developed, enabling the expression of A2AR during ongoing anti-tumor immune responses to be assessed. This reveals that A2AR is highly expressed on all tumor-infiltrating lymphocyte subsets including Natural Killer (NK) cells, NKT cells, γδ T cells, conventional CD4+ and CD8+ T lymphocytes and on a MHCIIhiCD86hi subset of type 2 conventional dendritic cells. In response to PD-L1 blockade, the emergence of PD-1+A2AR- cells correlates with successful therapeutic responses, whilst IL-18 is identified as a cytokine that potently upregulates A2AR and synergizes with A2AR deficiency to improve anti-tumor immunity. These studies provide insight into the biology of A2AR in the context of anti-tumor immunity and reveals potential combination immunotherapy approaches.

Prognostic and Predictive Significance of Tumor Immune Microenvironment in Breast Ductal Carcinoma In Situ

This study aims to identify the role of different subtypes of tumor infiltrating lymphocytes (TILs) in ductal carcinoma in situ (DCIS) in predicting risk of recurrence and benefit of whole breast irradiation (WBI). Immunohistochemical stain for CD3, CD4, CD8, FOXP3 and CD20 were carried in a well characterized DCIS cohort who received breast-conserving surgery (BCS) from Jan 2009 to Dec 2018. All the TILs subtypes were evaluated by the average numbers of touching-TILs which defined as TILs touching or within one lymphocyte cell thickness from the malignant ducts' basement membrane. The optimal cut-off values of TILs subtypes were selected by the X-tile. In total, 167 patients were enrolled in this analysis with 114 patients received WBI. After a median follow-up of 67 months, 15 IBTR events occurred with 6 invasive-IBTRs. Nine out of 15 IBTRs occurred outside of the original quadrant (elsewhere failure event, EFE). CD3+ lymphocytes were the predominant cell subtype while Treg showed the lowest levels. High abundance of TILs subtypes was associated with high tumor grade, presence of microinvasion, high Ki67 index, ER negativity and HER2 positivity. For various TILs subtypes, the multivariate analyses showed that dense CD4+ TILs (HR = 9.84, 95% CI 2.43-39.91, p<0.01) and dense Treg (HR = 4.22, 95% CI 1.24-14.36, p = 0.02) were independent prognostic factors for higher IBTR. As the infiltration of TIL subsets was correlated with one another, we also analyzed the relationship between IBTR and the ratios of different TILs subtypes. By adjusted by clinicopathological parameters, high ratios of CD4+/CD8+, Treg/CD4+ and Treg/CD8+ were found to be independent prognostic factors for higher IBTR (HR = 11.31, 95% CI 3.14-40.76, p<0.01; HR = 3.09, 95% CI 1.05-9.11, p = 0.04; HR = 7.14, 95% CI 1.98-25.73, p<0.01). Consistent with the results of IBTR, the 5-y rate of invasive-IBTR and EFE was both significantly associated with the high CD4+/CD8+, Treg/CD4+ and Treg/CD8+ TILs ratios (all p<0.01). WBI reduced the rate of 5y-IBTR risk from 8.4% to 1.3% (p = 0.02) in the low Treg/CD8+ group, but there was no benefit of WBI in the high group. With respect to EFE, WBI significantly reduced the rate from 2.8% to 0.0% (p = 0.03) in the low Treg/CD8+ group while not in the high group. The benefits of WBI in reducing IBTR and EFE were not significant difference between different CD4+/CD8+ and Treg/CD4+ groups. Assessment of overall TILs provides a tool for comprehensive evaluation of the DCIS immune microenvironment. Patients with pro-tumoral immune infiltrate (high Treg, high ratios of CD4+/CD8+, Treg/CD4+ and Treg/CD8+) in tumor microenvironment show an increased risk of IBTR and less benefit from breast radiotherapy.

Intratumoral Microbiota Changes with Tumor Stage and Influences the Immune Signature of Oral Squamous Cell Carcinoma.

Characterization of the oral microbiota profile through various studies has shown an association between the microbiome and oral cancer; however, stage-specific determinants of dynamic changes in microbial communities of oral cancer remain elusive. Additionally, the influence of the intratumoral microbiota on the intratumoral immune system remains largely unexplored. Therefore, this study aims to stratify microbial abundance in the early-onset and subsequent stages of oral cancer and analyze their influence on clinical-pathological and immunological features. The microbiome composition of tissue biopsy samples was identified using 16S rRNA amplicon sequencing, while intratumoral and systemic immune profiling was done with flow cytometry and immunohistochemistry-based analysis. The bacterial composition differed significantly among precancer, early cancer, and late cancer stages with the enrichment of genera Capnocytophaga, Fusobacterium, and Treponema in the cancer group, while Streptococcus and Rothia were enriched in the precancer group. Late cancer stages were significantly associated with Capnocytophaga with high predicting accuracy, while Fusobacterium was associated with early stages of cancer. A dense intermicrobial and microbiome-immune network was observed in the precancer group. At the cellular level, intratumoral immune cell infiltration of B cells and T cells (CD4+ and CD8+) was observed with enrichment of the effector memory phenotype. Naive and effector subsets of tumor-infiltrating lymphocytes (TILs) and related gene expression were found to be distinctly associated with bacterial communities; most importantly, highly abundant bacterial genera of the tumor microenvironment were either negatively correlated or not associated with the effector lymphocytes, which led to the conclusion that the tumor microenvironment favors an immunosuppressive and nonimmunogenic microbiota. IMPORTANCE The gut microbiome has been explored extensively for its importance in the modulation of systemic inflammation and immune response; in contrast, the intratumoral microbiome is less studied for its influence on immunity in cancer. Given the established correlation between intratumoral lymphocyte infiltration and patient survival in cases of solid tumors, it was pertinent to explore the extrinsic factor influencing immune cell infiltration in the tumor. Modulation of intratumoral microbiota could have a beneficial effect on the antitumor immune response. This study stratifies the microbial profile of oral squamous cell carcinoma starting from precancer to late-stage cancer and provides evidence for their immunomodulatory role in the tumor microenvironment. Our results suggest combining microbiome study with immunological signatures of tumors for their prognostic and diagnostic application.

The expression profiles of signature genes from CD103+LAG3+ tumour-infiltrating lymphocyte subsets predict breast cancer survival.

Tumour-infiltrating lymphocytes (TILs), including T and B cells, have been demonstrated to be associated with tumour progression. However, the different subpopulations of TILs and their roles in breast cancer remain poorly understood. Large-scale analysis using multiomics data could uncover potential mechanisms and provide promising biomarkers for predicting immunotherapy response. Single-cell transcriptome data for breast cancer samples were analysed to identify unique TIL subsets. Based on the expression profiles of marker genes in these subsets, a TIL-related prognostic model was developed by univariate and multivariate Cox analyses and LASSO regression for the TCGA training cohort containing 1089 breast cancer patients. Multiplex immunohistochemistry was used to confirm the presence of TIL subsets in breast cancer samples. The model was validated with a large-scale transcriptomic dataset for 3619 breast cancer patients, including the METABRIC cohort, six chemotherapy transcriptomic cohorts, and two immunotherapy transcriptomic cohorts. We identified two TIL subsets with high expression of CD103 and LAG3 (CD103+LAG3+), including a CD8+ T-cell subset and a B-cell subset. Based on the expression profiles of marker genes in these two subpopulations, we further developed a CD103+LAG3+ TIL-related prognostic model (CLTRP) based on CXCL13 and BIRC3 genes for predicting the prognosis of breast cancer patients. CLTRP-low patients had a better prognosis than CLTRP-high patients. The comprehensive results showed that a low CLTRP score was associated with a high TP53 mutation rate, high infiltration of CD8 T cells, helper T cells, and CD4 T cells, high sensitivity to chemotherapeutic drugs, and a good response to immunotherapy. In contrast, a high CLTRP score was correlated with a low TP53 mutation rate, high infiltration of M0 and M2 macrophages, low sensitivity to chemotherapeutic drugs, and a poor response to immunotherapy. Our present study showed that the CLTRP score is a promising biomarker for distinguishing prognosis, drug sensitivity, molecular and immune characteristics, and immunotherapy outcomes in breast cancer patients. The CLTRP could serve as a valuable tool for clinical decision making regarding immunotherapy.

Pattern of CD8+ tissue resident memory T cells between local recurrence and distant metastasis in nasopharyngeal carcinoma.

e15161 Background: CD8+ T lymphocytes are believed to be the major anti-tumor immune subsets. Consequently, most cancer immunotherapeutic approaches seek to amplify cytotoxic T lymphocytes specific to malignant cells. A recently identified subpopulation of memory CD8+ T cells, named tissue-resident memory T (Trm) cells, persists in non-lymphoid tissues, including solid tumors. This T-cell subset is considered an independent memory T-cell lineage with cytotoxic features. However, their function, spatial location and influence on disease-related outcomes (local recurrence [LR], distant metastasis [DM] in nasopharyngeal carcinoma (NPC), a disease entity characterized by substantial immune infiltrate, remain poorly understood. We aim to close this gap through investigation on the abundance and spatial localization of CD8+ Trm in relation to tumor site (primary or recurrence [LR and DM]). Methods: This retrospective cohort analysis based on 20 NPC patient samples: primary (initial diagnosis; [I; n = 19], recurrence ([LR; n = 14] and [DM; n = 10]). We accessed the spatial localization in tumor tissues within tissue-segmented classifications of either cancer islands (pan-cytokeratin–positive [CK+] areas) or stromal areas (CK– areas). Tumor infiltrating lymphocytes (TILs) among patients from I, LR and DM group was quantified through multiplexed 5-colour opal staining using biomarkers against CD8, CD103, IL17, CK plus DAPI (nuclear cell counterstain). Results: While total CD8+ TILs (total tumor) as well as their spatially localized cell densities (tumor islet [TI] vs. tumor stroma [TS]) did not show any significant changes among I, LR and DM patient groups, a higher proportion of CD8+ TILs in the TI co-express the CD103 molecule, an indication of CD8+ Trm (CD103+CD8+) infiltration (mean CD103+CD8+/CD8+ in TI: 32.1% in I group, 44.8% in LR group, 46.0% in DM group; mean CD103+CD8+/CD8+ in TS: 19.0% in I group, 26.2% in LR group 26.2% in DM group). Notably, we observed that such augmentation only reached statistically significant within TI from patient in LR group (p = 0.0451). Furthermore, we found that CD8+ Trm in the LR group expressed higher level of cytokine IL-17, which was ~2.1-fold (p = 0.0788) and 3.6-fold upregulated (p = 0.0285) as compared to I and DM, respectively. Conclusions: Cancer islet–localized CD8+ TILs are composed of CD103+ Trms, which make up nearly 45% the total CD8+ T cell population within LR tumors of NPC patients, highlighting spatial localization of CD103+CD8+ Trms as a key phenotype of CD8+ TIL subset. The CD8+ Trm expressed with increased IL17 suggests that enrichment of this T-cell subset might exerts cytotoxic functions. Our results demonstrate that CD8+ Trm had a greater likelihood as an indicator of LR risk for NPC patients and help identify patient cohorts likely to benefit from immunotherapy.

CD4-/CD8- double-negative tumor-infiltrating lymphocytes expanded from solid tumor tissue suppress the proliferation of tumor cells in an MHC-independent way.

Tumor-infiltrating lymphocytes (TILs) have shown remarkable clinical responses in some patients with advanced solid tumors. As a rare subset of TILs, CD4-/CD8- double-negative T cells (DNTs) were poorly known. This study aims to investigate the characteristics and function of CD3+CD4-CD8- TILs (double-negative TIL, DN-TILs) derived from solid tumor. DN-TILs were derived and expanded ex vivo from resected gastric carcinoma tissue and phenotyped by flow cytometry. The cytotoxicity of DN-TILs was determined against established tumor cell lines in vitro or through in vivo adoptive transfer into xenograft models. K562 cells were transferred with the HLA gene to verify whether the cytotoxicity of DN-TILs was MHC-independent. Flow cytometric analysis revealed a high-purity population of DN-TILs (> 97%) within CD3+ TILs, which expanded more than 800-folds in 2weeks, consisting of a mixture of alpha-beta (αβ) and gamma-delta (γδ) T-cell receptor (TCR)-expressing cells (with the majority being αβ-TCR, > 95%). Using single-cell RNA sequencing, the expanded DN-TILs were categorized into four main subsets, Natural Killer T cells (approximately 80%, 5563 in 7028), Progenitor cells, Germ cells and T helper2 cells. DN-TILs exhibited a broad anticancer cytotoxicity in a donor-unrestricted manner against various cancer cell lines derived from pancreatic cancer (Panc-1), gastric cancer (HGC-27), ovarian cancer (SKOV-3), malignant melanoma (A375). The cytotoxicity was MHC-independent, which was not altered in K562 transferring with HLA gene or not. DN-TILs significantly reduced tumor volume in xenograft models with superior tumor-homing ability and low off-target toxicity. Gastric carcinoma derived DN-TIL can target tumor cells in vitro and in vivo. DN-TILs have the potential to be used as a adoptive cell therapy for solid cancers with both the advantages of DNT and TIL.

Abstract 602: Single cell evaluation of intratumoral B cells across solid tumor indications

Abstract The immune system is a critical component of the tumor microenvironment that has both tumor-promoting and tumor-eradicating activity based on the specific immune cell subtype and their activation status. T lymphocytes represent the largest lymphocyte population within the tumor, and numerous immunotherapies have been developed to specifically target this cellular subset. B cells are the next most abundant lymphocyte subset within the tumor microenvironment, and recent studies have identified crucial roles for intratumoral B cells in the production of tumor-reactive antibodies. Using human dissociated tumor cells (DTCs) samples, we have profiled intratumoral B cells to determine their frequency and subset breakdown across thirteen different solid tumor indications. Intratumoral B cells were prevalent in lung cancer and cancers of the gastrointestinal tract (esophageal, gastric, and colorectal), while they were rarer in cancers of the female reproductive tract (endometrial and ovarian). The transcriptional profile and B cell repertoire of the intratumoral B cell subsets were evaluated to provide a deeper understanding of the biology of these tumor-infiltrating lymphocyte subsets. Additionally, spatial transcriptomics were utilized to understand the spatiotemporal location of these subsets within the tumor microenvironment. These studies will provide critical insight for the development of the next generation of immunotherapies. Citation Format: Shawn P. Fahl, Kerri Colwell, Casey Smith, Cavin Ott, Alex Moore, Kayla Williams. Single cell evaluation of intratumoral B cells across solid tumor indications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 602.

CD47 and CD68 expression in breast cancer is associated with tumor-infiltrating lymphocytes, blood vessel invasion, detection mode, and prognosis.

CD47 expressed on tumor cells binds to signal regulatory protein alpha on macrophages, initiating inhibition of phagocytosis. We investigated the relationships between tumor expression of CD47 and CD68 macrophage content, subsets of tumor-infiltrating lymphocytes (TILs), and vascular invasion in breast cancer. A population-based series of 282 cases (200 screen detected and 82 interval patients) from the Norwegian Breast Cancer Screening Program was examined. Immunohistochemical staining for CD47 and CD68 was evaluated on tissue microarray (TMA) slides. For CD47 evaluation, a staining index was used. CD68 tumor-associated macrophages were counted and dichotomized. TIL subsets (CD45, CD3, CD4, CD8, and FOXP3) were counted and dichotomized using immunohistochemistry on TMA slides. Vascular invasion (both lymphatic and blood vessel) was determined on whole tissue slides. High CD47 tumor cell expression or high counts of CD68 macrophages were significantly associated with elevated levels of all TIL subsets (p < 0.02), CD163 macrophages (p < 0.001), blood vessel invasion (CD31 positive) (p < 0.01), and high tumor cell Ki67 (p < 0.004). High CD47 expression was associated with ER negativity (p < 0.001), HER2 positive status (p=0.03), and interval-detected tumors (p=0.03). Combined high expression of CD47-CD68 was associated with a shorter recurrence-free survival (RFS) by multivariate analysis (hazard ratio [HR]: 2.37, p=0.018), adjusting for tumor diameter, histologic grade, lymph node status, and molecular subtype. Patients with luminal A tumors showed a shorter RFS for CD47-CD68 high cases by multivariate assessment (HR: 5.73, p=0.004). This study demonstrates an association of concurrent high CD47 tumor cell expression and high CD68 macrophage counts with various TIL subsets, blood vessel invasion (CD31 positive), other aggressive tumor features, and interval-presenting breast cancer. Our findings suggest a link between CD47, tumor immune response, and blood vessel invasion (CD31 positive). Combined high expression of CD47-CD68 was an independent prognostic factor associated with poor prognosis in all cases, as well as in the luminal A category.

Characterization of the tumor-infiltrating lymphocyte landscape in sinonasal mucosal melanoma

Tumor-infiltrating lymphocytes (TILs) are important prognostic biomarkers in several types of cancers. The interplay between TIL subgroups and immune checkpoint molecules like programmed cell death ligand 1 (PD-L1) is a promising target for immunotherapy. However, the TIL landscape in sinonasal mucosal melanoma (SNMM) has not been sufficiently characterized yet and the prognostic value of TIL subgroups and PD-L1 expression remains uncertain. Here, we investigated subsets of TILs (CD3+, CD4+, CD8+, CD20+) and PD-L1 expression patterns in SNMM and assessed their prognostic value for recurrence-free and overall survival. Immunohistochemical staining for CD3, CD4, CD8, CD20 and PD-L1 was performed on tumor tissue from 27 patients with primary SNMM. Patient history was obtained and associations between TIL subgroups or PD-L1 expression and AJCC tumor stage, overall survival, and recurrence-free survival were retrospectively analyzed. Patients with high CD3+ and CD8+ TILs in the primary tumor survived significantly longer than patients with SNMMs with a low number of CD3+ and CD8+ TILs. High CD3+ and high CD8+ TILs were associated with the lower T3 stage and increased 5-year survival. PD-L1 positivity in tumor cells was associated with advanced tumor stage. Our results indicate that high densities of CD3+ and CD8+ TILs are strong positive prognostic biomarkers for survival in SNMM. Prospective studies with larger case numbers are warranted to confirm our findings.

High CD4+/CD8+ Intratumour Ratio is Associated with Favourable Outcome in Triple-negative Breast Cancer

Triple-negative breast cancer (TNBC) is a heterogenous breast cancer subtype which accounts for 10-15% among all diagnosed breast cancers. There is increased resistance of TNBC to conventional chemotherapy and hormonal therapy due to lack of oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) expression. Mutual relationship and interactions between both CD4+ and CD8+ T lymphocytes are very crucial for eliciting adaptive immune system during anti-cancer immune response. This study aimed to determine the ratio of CD4+ and CD8+ T lymphocytes in TNBC by immunohistochemistry assay and to investigate the association of CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs) with survival outcome. Quantification of both immunostaining TILs subset was done using conventional light microscope. A wide range of CD4+ and CD8+ TILs subset was found within intratumor stroma of TNBC with population mean score of 0.93 and 0.53, respectively. However, the difference of mean population between both TILs subsets was insignificant overall (P-value of CD4+= 0.484; CD8+= 0.835) when compared statistically using independent t-test. The ratio of CD4+/CD8+ in intratumor stroma ranged from 0.50-5.88 among all TNBC subtypes. The high CD4+/CD8+ ratio within intratumor stroma showed favorable association with survival outcome during the 2 years’ follow-up.

CD3 high and FoxP3 - tumor-infiltrating lymphocytes in the invasive margin as a favorable prognostic marker in patients with invasive urothelial carcinoma of the bladder.

Tumor-infiltrating lymphocytes (TILs) have been extensively explored as prognostic biomarkers and cellular immunotherapy methods in cancer patients. However, the prognostic significance of TILs in bladder cancer remains unresolved. We evaluated the prognostic effect of TILs in bladder cancer patients. Sixty-four bladder cancer patients who underwent surgical resection between 2018 and 2020 in Zhejiang Provincial People's Hospital were analyzed in this study. Immunohistochemistry was used to evaluate CD3, CD4, CD8, and FoxP3 expression on TILs in the invasive margin of tumor tissue, and the presence of TIL subsets was correlated with the disease-free survival (DFS) of bladder cancer patients. The relationship between clinical-pathological features and DFS were analyzed. A high level of CD3 + TILs (CD3 high TILs) ( P = 0.027) or negative expression of FoxP3 TILs (FoxP3 - TILs) ( P = 0.016) was significantly related to better DFS in bladder cancer patients. Those with CD3 high FoxP3 - TILs had the best prognosis compared to those with CD3 high FoxP3 + TILs or CD3 low FoxP3 - TILs ( P = 0.0035). Advanced age [HR 4.57, (1.86-11.25); P = 0.001], CD3 low TILs [HR 0.21, (0.06-0.71); P = 0.012], CD8 low TILs [HR 0.34, (0.12-0.94); P = 0.039], and FoxP3 + TILs [HR 10.11 (1.96-52.27); P = 0.006] in the invasive margin were associated with a worse prognosis (DFS) by multivariate analysis. In conclusion, we demonstrated that CD3 high , FoxP3 - , and CD3 high FoxP3 - TILs in the invasive margin were significantly associated with better DFS. CD8 high and CD4 high TILs in the invasive margin tended to predict better DFS in bladder cancer. Patients with CD4 high CD8 high TILs in the invasive margin were likely to have a better prognosis.

Biomarkers of tumor-reactive CD4+ and CD8+ TILs associate with improved prognosis in endometrial cancer.

BackgroundDespite the growing interest in immunotherapeutic interventions for endometrial cancer (EC), the prevalence, phenotype, specificity and prognostic value of tumor infiltrating lymphocytes (TILs) in this tumor type remains unclear.MethodsTo better...

Expression of HLA class I is associated with immune cell infiltration and patient outcome in breast cancer

Downregulation of human leukocyte antigen (HLA) class I is one mechanism of escaping anti-tumor immunity by tumor cells. This study was conducted to compare HLA class I expression in ductal carcinoma in situ (DCIS) and invasive breast carcinoma (IBC) and to evaluate its association with immune cell infiltration of the tumors and clinical outcome of the patients. A total of 830 cases comprising 288 DCIS and 542 IBC were included in this study. Immunohistochemistry for HLA class I expression was performed using HLA-ABC in tissue microarrays and was analyzed in relation to clinicopathologic characteristics of tumors and infiltration of CD4+, CD8+, and FOXP3+ tumor-infiltrating lymphocyte (TIL) subsets and PD-L1+ immune cells. As a whole, there was no difference in HLA class I expression between DCIS and IBC when dichotomized into high or low expression. However, in the HR-negative group, a high level of HLA class I expression was more frequent in IBC than DCIS. On the contrary, in the HR-positive group, a complete loss of HLA class I expression was more frequently observed in IBC than DCIS. High HLA class I expression level was generally associated with aggressive clinicopathologic features of IBC and was associated with high CD4+, CD8+, and FOXP3+ TIL and PD-L1+ immune cell infiltration in both DCIS and IBC. In survival analyses, HLA class I expression was not associated with clinical outcome in DCIS and IBC as a whole; however, low HLA class I expression was associated with poor clinical outcome in HR-negative IBC, especially in triple-negative subtype. In conclusion, this study showed that HLA class I expression increased in association with increased immune cell infiltration during in situ to invasive transition of HR-negative breast cancer, and HLA class I down-regulation had a prognostic value in HR-negative breast cancer.

IMMU-42. ADOPTIVE HEMATOPOIETIC STEM CELL AND APD1 THERAPY CONVERGE IN THE THYMUS TO PROMOTE ANTI-GLIOBLASTOMA IMMUNITY

Abstract Our lab previously published that adoptive hematopoietic stem cell (HSC) therapy administered in conjunction with aPD1 increases median survival in multiple models of CNS malignancies. Tumor-infiltrating lymphocytes (TIL) abundance is independently associated with better outcomes in many cancers. Impressively, approximately 25% of TILs are HSC-derived in early disease. Importantly, we show HSC therapy generates de novo TILs in a thymus-dependent fashion and offer evidence of altered central tolerance following aPD1 therapy, which may permit immune activation against a larger pool of tumor-associated antigens. Therapeutic HSCs were prepared from constitutive GFP-expressing (CD45.2+) and PD1KO (CD45.2+) mice and transferred into CD45.1+ hosts. Sublethal 5Gy total body irradiation was administered day 5 post-intracranial tumor implantation. The next day, 1E6 HSCs from a 1:1 – GFP:PD1KO cocktail were injected intravenously. Descriptive statistics are reported as mean ± SD. Transferred HSCs engraft, expand, differentiate, and migrate to form approximately 25% of the TIL compartment in only 15 days. We found that, 56.2% ± 20.3 of CD3+ TILs display a thymocyte-like CD4+CD8+ (DP) phenotype in early disease, and 43.8% ± 17.4 of these DP-TILs are HSC-derived. Interestingly, DP-TILs were virtually absent in advanced disease 29 days after treatment. At this stage, HSC-derived TILs most commonly fell within the single-positive (SP) TIL subsets, comprising 9.8% ± 5.6 of SP-CD8+ TILs. Notably, HSCPD1KO-derived cells were 4x more abundant amongst SP-CD8 TILs when compared to HSCGFP-derived cells (6.5% vs 1.6%, p &amp;lt; 0.05). SP-CD8 thymocytes were similarly enriched for HSCPD1KO progeny (41.1% vs 24.7%, p &amp;lt; 0.05) which may suggest PD-1 blockade directly promotes thymopoiesis. The privileged maturation of PD1-deficient HSCs is intriguingly reversed when GFP:PD1KO HSCs are co-transferred into non-tumor-bearing mice. Thus, we show the thymus is an active site where PD-1-blocking agents function, and that HSC + aPD1 therapy is uniquely positioned to leverage thymus-dependent anti-tumor immunity.

IMMU-31. PROTEASOME REARRANGED PEPTIDES ARE A NOVEL SOURCE OF ANTIGENS FOR THE PEPTIDE VACCINE IMMUNOTHERAPY OF GLIOBLASTOMAS

Abstract pepVIII, an anticancer vaccine targeting EGFRvIII, has been tested in several trials for glioblastoma including ACT IV. a randomized Phase III trial for newly diagnosed patients. While results from ACT IV were equivocal, available data suggests the elimination of EGFRvIII was not robust. Despite its essential role in antigen presentation, enhancing proteasomal processing is an unexploited strategy for improving vaccines. We examined 20 peptides in silico and experimentally, which showed that a tyrosine substitution (Y6-pepVIII) maximizes proteasome cleavage and survival in a subcutaneous tumor model. In an intracranial glioma model, Y6-pepVIII showed a 62 and 31% improvement in median survival compared to control animals and pepVIII-vaccinated mice. Y6-pepVIII vaccination altered tumor-infiltrating lymphocyte subsets and expression of PD-1 on intratumoral T cells. Combination with anti–PD-1 therapy cured 45% of the Y6-pepVIII–vaccinated mice but was ineffective for pepVIII-treated mice. LC-MS/MS analysis of proteasome-digested pepVIII and Y6-pepVIII revealed that most fragments were similar but more abundant inY6-pepVIII digests and 77% resulted from proteasome-catalyzed peptide splicing (PCPS). We identified 10 peptides that bound human and murine MHC class I. Nine were PCPS products and only one peptide was colinear with EGFRvIII, indicating that PCPS fragments may be a component of MHC class I recognition. Despite not being colinear with EGFRvIII, two of three PCPS products tested were capable of increasing survival when administered independently as vaccines. We hypothesize that the immune response to a vaccine represents the collective contribution from multiple PCPS and linear products. Our work suggests a strategy to increase proteasomal processing of a vaccine that results in an augmented immune response and enhanced survival in mice.