Negative and Positive Separation Techniques for the Isolation of Antigen-Specific CD8+ T Cells from Blood and Tumor Tissue

5581 Background: Checkpoint inhibitors have shown to be effective in different tumors and are under investigation in HGSOC. Methods: INSPIRE (NCT02644369) is a prospective multi-cohort study investigating tumor genomic and immune landscapes in pts treated with Pembro at 200 mg IV Q3W. Patients underwent tumor biopsy pre, on-treatment and at progression for DNA/RNA sequence, immune-profile, and PD-L1 expression by immunohistochemistry (IHC). Serial blood samples for immunophenotyping were collected. Correlative data are available for 6 pts: 3 with shrinkage in target lesion and 3 with progressive disease (PD). Results: At interim analysis as of January 2017, 18 pts with HGSOC have been enrolled and 16 have platinum-resistant disease, with median 3 prior lines of treatment (range 1-7). Of 14 evaluable pts, best response by RECIST 1.1 was stable disease (SD) in 5 (36%) and PD in 9 (64%). Mean Tumor Proportion Score of PD-L1 by IHC (Qualtek) was 6.4% (range 0-30%). Grade 3/4 adverse events possibly related to Pembro were observed in 4/18 (22%) pts; none was fatal and the most common were fatigue and hyponatremia. Preliminary correlative data showed no significant change in CD4, CD8 and myeloid-derived suppressor cells in peripheral blood after Pembro treatment. Mean PD-1 expression on CD4 and CD8 T cells on baseline tumor tissue (measured as product of PD-1+ cells and the per cell expression of PD-1 [% of mean fluorescence intensity]) was significantly higher in pts with tumor shrinkage compared to pts with PD (CD4: 2658 vs 678, p = .02; CD8: 1999 vs 451, p = .048). Genomic analysis of baseline tumor tissue was available for 3 pts with tumor shrinkage and 2 with PD. Mean mutation burden was higher for pts with tumor shrinkage (2.38 vs 1.0 mutations/Mb covered). The pt with the longest SD in our cohort (6 months) had the highest mutation burden (2.72), including somatic POLE (c.6331-6C > G) and germline BRCA2mutations. Conclusions: In HGSOC, pts with higher PD-1 level on tumor CD4 and CD8 T cells and higher mutation burden at baseline may have a better outcome following treatment with Pembro. POLE mutation is rare in HGSOC but may correlate with checkpoint inhibitor activity. Clinical trial information: NCT02644369.

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Abstract 2260: Radiotherapy enhances newly infiltrating CD8+T cells into tumor tissue to increase intratumor CD8+T cells and exert an anti-tumor effects

10.1158/1538-7445.am2020-2260 ◽

2020 ◽

Author(s):

Kimihiro Yamashita ◽

EIJI Fukuoka ◽

Yutaka Sugita ◽

Akira Arimoto ◽

Akihiro Watanabe ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Tumor Tissue

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CD4 + and CD8 + T cells in sentinel nodes exhibit distinct pattern of PD‐1, CD69, and HLA‐DR expression compared to tumor tissue in oral squamous cell carcinoma

Cancer Science ◽

10.1111/cas.14816 ◽

2021 ◽

Author(s):

Krzysztof Piersiala ◽

Pedro Farrajota Neves da Silva ◽

Eric Hjalmarsson ◽

Aeneas Kolev ◽

Åsa Kågedal ◽

...

Keyword(s):

Squamous Cell Carcinoma ◽

T Cells ◽

Oral Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Squamous Cell ◽

Cd8 T Cells ◽

Tumor Tissue ◽

Distinct Pattern ◽

Sentinel Nodes ◽

Hla Dr

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Treatment with pembrolizumab in combination with the oncolytic virus pelareorep promotes anti-tumor immunity in patients with advanced pancreatic adenocarcinoma.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.4144 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. 4144-4144

Author(s):

Devalingam Mahalingam ◽

Siqi Chen ◽

Ping Xie ◽

Aparna Kalyan ◽

Sheetal Mehta Kircher ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Tumor Tissue ◽

Tumor Response ◽

Effector Memory ◽

Epigenetic Mark ◽

Immunological Changes ◽

Tumor Biopsies

4144 Background: Pelareorep is an intravenously delivered oncolytic reovirus that can induce a T-cell-inflamed phenotype in pancreatic ductal adenocarcinoma (PDAC). In prior studies, tumor tissue analysis from patients treated with pelareorep shows pelareorep replication, increased T cell infiltration, and upregulation of PD-L1. We hypothesized that pelareorep in combination with pembrolizumab in patients with PDAC would lead to improved responses and anti-tumor immunological changes within peripheral blood and tumor biopsies in responding patients. Methods: PDAC patients who progressed after first-line treatment received pelareorep at a dose of 4.5x1010 TCID50 IV on Days 1, 2, 3 & 8 of Cycle (C) 1, and Days 1 & 8 with C2 onwards. Pembrolizumab was administered on Day 1 of each 21-day cycle at 200 mg IV. The primary objective was overall response rate by RECIST v 1.1 criteria. Secondary objectives included evaluating immunological changes within tumor tissue and peripheral blood, performed by multi-plex immunohistochemistry and spectral flow cytometry (Cytek), respectively. Results: Thirteen patients were enrolled. Disease control was achieved in 33% of the 12 efficacy-evaluable patients. One patient achieved a partial response (PR). Three additional patients achieved stable disease (SD). On-treatment tumor biopsies, collected during C1, showed pelareorep replication, increased infiltration of CD8+ T cells and PD-L1+ cells, and decreased expression of VDAC1, a mitochondrial gatekeeper for tumor promotion, relative to archival tissue. Reduced infiltration of Foxp3+ regulatory T cells (Treg) was observed in patients showing tumor response. Peripheral blood was collected at day 1 of each cycle and on C1 day 8. Relative to pretreatment samples, the number of CD8+ effector memory T cells and B cells tend to increase while the number of Treg cells declined in C2 onwards in patients with tumor response. Furthermore, these patients had increased expression of the mitochondrial protein TOMM20 in CD8+ T cells and decreased expression of PD-1 and the H3K27me3 epigenetic mark in Treg. Treatment was well tolerated with most treatment-related adverse events, including flu-like symptoms, being grade 1 or 2. Conclusions: The combination of pelareorep and pembrolizumab showed a manageable safety profile and modest efficacy in an unselected PDAC population. Additional correlation analyses between treatment efficacy and immunological changes will be presented. The anti-tumor activity of pelareorep and checkpoint blockade therapy is being evaluated further in additional ongoing studies. Clinical trial information: NCT03723915.

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Tumor Localization and Quantitation of Adoptively Transfered T Lymphocytes in a Murine Model.

Blood ◽

10.1182/blood.v104.11.1343.1343 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1343-1343

Author(s):

Marianne Hokland ◽

Mikkel S. Petersen ◽

Charlotte C. Fleischer ◽

Hans Stødkilde-Jørgensen ◽

Søren B. Hansen ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Lymphocytes ◽

Cd8 T Cells ◽

Tumor Tissue ◽

Cellular Therapy ◽

Mean Value ◽

Tumor Localization ◽

Toxic Substances

Abstract Tracking adoptively transferred antigen-specific T lymphocytes is an important prerequisite for devising better protocols for cellular therapy. To this end we have developed a highly sensitive method for “in situ” visualization of labelled lymphocytes in vivo by combined PET and magnetic resonance imaging (MRI) to monitor the distribution of adoptively transferred tumour-specific T cells in a mouse model system. Moreover, quantitation of the adoptively transferred cells in tumor was performed by flow cytometry. C57BL/6J mice carrying subcutaneous tumours of the ovalbumin (OVA)-expressing malignant melanoma cell line B16-OVA were adoptively transferred with OVA-specific CD8+ T cells labelled with 124IdU. Five days after transfer of T cells, mice were killed and subjected to PET and MR imaging. Using a newly developed method for co-registration of the two image modalities, the anatomical localisation of the transferred cells was visualised and the amount of radioactivity in various anatomical locations very accurately determined. For quantitation of tumor infiltrating non-labelled OVA-specific CD8+ T cells by flow cytometry (using AbsoluteCount Beads), tumors were removed from mice day 1 until day 8 following adoptive transfer (6 mice/group) and prepared for single cell suspension before labeled with anti-CD8-FITC and SIINFEKL-Tetramer-PE. Results showed a clear tumor localization of the adoptively transferred OVA-specific T cells in the tumours. In two independent experiments comprising 12 and 13 evaluable mice, respectively, we found a mean value of 0.909 +/− 0.468 Bq and 0.926 +/− 0.553 Bq in the tumours, and only 0.182 +/− 0.479 Bq and 0.026 +/− 0.480 Bq in the corresponding contralateral control volumes. The difference in activity between the tumour regions and the control regions was statistically highly significant with 2p-values of 0.002 and 0.006 for the two experiments. Using flow cytometry it was shown that the number of OVA specific T lymphocytes accumulating in tumor gradually increased until day 5 after transfer when an average of 3.3 million SIINFEKL-specific cells per gram tumor tissue was found. From day 5 until day 8 the number of SIINFEKL-specific cells per gram tumor tissue fluctuated at a fairly constant level. This method presented for tracking adoptively transfered tumor specific T lymphocytes represent a significant advancement for studies of adoptively transferred specific T cells, and could potentially be developed for diagnostic purposes. Moreover, since these studies show that tumor-specific T cells home to subcutaneous tumours in substantial numbers, we suggest that these migrating cells could be employed in a new form of therapy as carriers of toxic substances to tumors.

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Responsibility of immune microenvironment for poor response to immune checkpoint inhibitors in CRC patients with KRAS mutation.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.e15588 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. e15588-e15588

Author(s):

Qiyu Liu ◽

Hushan Zhang ◽

Shenggang Cai ◽

Lin Xu ◽

Rong Zhao ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Cd8 T Cells ◽

Immune Checkpoint ◽

Kras Mutation ◽

Immune Checkpoint Inhibitors ◽

Tumor Tissue ◽

Checkpoint Inhibitors ◽

Immune Microenvironment ◽

Significant Difference

e15588 Background: The clinical trial Keynote 177 is a breakthrough research in colon rectal cancer (CRC), however, the results of subgroups revealed that immunotherapy did not benefit more than chemotherapy in patients with KRAS mutation. Here we assessed characteristics of tumor immune microenvironment in patients with or without KRAS mutation, to find out the underlying mechanisms that prevent KRAS-mutated CRC patients from benefiting from immunotherapy. Methods: Tumor tissue samples of 77 CRC patients were collected from Janary 2019, several kinds of immune cells including CD8+ T cells, M1/M2 tumor associated macrophages (TAM), CD56bright NK cells and CD56dim NK cells in the tumor tissue were evaluated by multiple fluorescence immunohistochemistry (mIHC), and expression of PD-L1 were detected by using Dako PD-L1 IHC 22C3 pharmDx, Tumor Proportion Score (TPS) was used to determine expression of PD-L1. In addition, gene mutation was detected by means of next generation squesing (NGS). All these detections were performed in a College of American Pathologists (CAP)-certified and Clinical Laboratory Improvement Amendments (CLIA)-accredited lab for gene mutation analysis (3D Medicines Inc.,Shanghai, China). Statistical analysis was performed using GraphPad Prism (version 7.01) and SPSS version 21.0 (SPSS,Inc.). Results: According to whether KRAS is mutated, these 77 patients were divided into two groups, KRAS wild type (WT) (n = 49) and KRAS mutation group (n = 28). There was no significant difference in the CD8+T cells infiltrated in tumor tissue of KRAS WT and KRAS mutation samples, however, less CD8+ T cells were found in stroma of tumor tissue of KRAS WT samples than in KRAS mutation. What’s more, both the ratio of M1:M2 and CD56dim NK: CD56bright NK cells in the tumor tissues of KRAS WT were significantly higher than in KRAS mutation samples(p < 0.05). No significant difference was found between two groups, while higher MSI-H proportion was found in KRAS mutation group than in KRAS WT (33.4% and 12.5%, respectively). The proportion of positive PD-L1expression (TPS≥1) in both groups is very low, especially, proportion of strong positive (TPS≥50). Conclusions: The different response to immune checkpoint inhibitors therapy for CRC patient with or without KRAS mutation mainly associated with factors of TIME, not only CD8+T cells, but tumor associated macrophages and different sub-population of NK cells should be put into consideration.

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Comparative evaluation of T cell receptors in experimental glioma-draining lymph nodes

Neuro-Oncology Advances ◽

10.1093/noajnl/vdab147 ◽

2021 ◽

Author(s):

Jens Blobner ◽

Michael Kilian ◽

Chin Leng Tan ◽

Katrin Aslan ◽

Khwab Sanghvi ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Lymph Nodes ◽

Cd8 T Cells ◽

Tumor Tissue ◽

Tumor Model ◽

Cell Receptor ◽

Cervical Lymph Nodes ◽

Inguinal Lymph Nodes ◽

Draining Lymph Nodes

Abstract Background Glioblastomas, the most common primary malignant brain tumors, are considered immunologically cold malignancies due to growth in an immune sanctuary site. While peptide vaccines have shown to generate intra-tumoral antigen-specific T cells, the identification of these tumor-specific T cells is challenging and requires detailed analyses of tumor tissue. Several studies have shown that CNS antigens may be transported via lymphatic drainage to cervical lymph nodes, where antigen-specific T cell responses can be generated. Therefore, we investigated whether glioma-draining lymph nodes (TDLN) may constitute a reservoir of tumor-reactive T cells. Methods We addressed our hypothesis by flow cytometric analyses of chicken ovalbumin (OVA)-specific CD8 + T cells as well as T cell receptor beta (TCRβ) next-generation-sequencing (TCRβ-NGS) of T cells from tumor tissue, TDLN, spleen, and inguinal lymph nodes harvested from experimental mouse GL261 glioma models. Results Longitudinal dextramer-based assessment of specific CD8 + T cells from TDLN did not show tumor model antigen reactivity. Unbiased immunogenomic analysis revealed a low overlap of TCRβ sequences from glioma-infiltrating CD8 + T cells between mice. Enrichment-scores, calculated by the ratio of productive frequencies of the different TCRβ-CDR3 amino-acid (aa) rearrangements of CD8 + T cells derived from tumor, TDLN, inguinal lymph nodes, and spleen demonstrated a higher proportion of tumor-associated TCR in the spleen compared to TDLN. Conclusions In experimental glioblastoma, our data did not provide evidence that glioma-draining cervical lymph nodes are a robust reservoir for spontaneous glioma-specific T cells highlighting the requirement of detailed analyses of glioma-infiltrating T cells for the discovery of tumor-specific TCR.

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