scholarly journals Characterisation of Immune Cell Subsets of Tumour Infiltrating Lymphocytes in Brain Metastases

2021 ◽  
Author(s):  
Priyakshi Kalita-de Croft ◽  
Haarika Chittoory ◽  
Tam Nguyen ◽  
Jodi Saunus ◽  
Woo Gyeong Kim ◽  
...  
Keyword(s):  
T Cells ◽  
Brain Metastasis ◽  
Cd8 T Cells ◽  
Immune Cell ◽  
Immune Surveillance ◽  
Tumor Infiltrating Lymphocytes ◽  
Targeted Analysis ◽  
Immune Cell Subsets ◽  
Tumour Infiltrating Lymphocytes ◽  
Infiltrating Lymphocytes

The heterogeneity of tumor infiltrating lymphocytes is not well characterized in brain metastasis. To address this, we performed a targeted analysis of immune cell subsets in brain metastasis tissues to test which immunosuppressive routes are involved in brain metastasis. We performed multiplex immunofluorescence (mIF), using commercially available validated antibodies on twenty formalin-fixed paraffin embedded whole sections. We quantitated the subsets of immune cells utilizing a targeted panel of proteins including PanCK, CD8, CD4, VISTA and Iba1, and analyzed an average of 15000 cells per sample. We classified tumours as either high (>30%) or low (<30%) tumour infiltrating lymphocytes (TILs) and found that increased TILs density correlated with survival. We next sought out to phenotype these TILs using mIF. The tumours with low TILs (n=9) had significantly higher expression of the immune-checkpoint molecule VISTA in tumor cells (p<0.01) as well as in their microenvironment (p<0.001). Contrastingly, the brain metastatic tumours with high TILs (n=8) displayed higher levels of activated microglia, as measured by Iba1 expression. Low TILs-tumours displayed CD8+ T-cells that co-express VISTA (p<0.01) significantly more compared to high TILs group, where CD8+ T-cells significantly co-express Iba1 (p<0.05). Interestingly, no definite phenotypes of CD4+ subsets were observed. These results were supported by RNA analysis of a publicly available, independent cohort. In conclusion, our work contributes to a growing understanding of the immune surveillance escape routes active in brain metastasis.

scholarly journals Characterisation of immune cell subsets of tumour infiltrating lymphocytes in brain metastases

2021 ◽  
Author(s):  
Priyakshi Kalita-de Croft ◽  
Haarika Chittoory ◽  
Tam Nguyen ◽  
Jodi M Saunus ◽  
Woo Gyeong Kim ◽  
...  
Keyword(s):  
T Cells ◽  
Brain Metastasis ◽  
Cd8 T Cells ◽  
Immune Cell ◽  
Immune Surveillance ◽  
Tumor Infiltrating Lymphocytes ◽  
Activated Microglia ◽  
Immune Cell Subsets ◽  
Tumour Infiltrating Lymphocytes ◽  
Infiltrating Lymphocytes

AbstractThe heterogeneity of tumor infiltrating lymphocytes is not well characterized in brain metastasis. To address this, we performed a targeted analysis of immune cell subsets in brain metastasis tissues to test which immunosuppressive routes are involved in brain metastasis. We performed multiplex immunofluorescence (mIF), using commercially available validated antibodies on twenty formalin-fixed paraffin embedded whole sections. We quantitated the subsets of immune cells utilizing a targeted panel of proteins including PanCK, CD8, CD4, VISTA and Iba1, and analyzed an average of 15000 cells per sample. We classified tumours as either high (>30%) or low (<30%) tumour infiltrating lymphocytes (TILs) and found that increased TILs density correlated with survival. We next sought out to phenotype these TILs using mIF. The tumours with low TILs (n=9) had significantly higher expression of the immune-checkpoint molecule VISTA in tumor cells (p<0.01) as well as in their microenvironment (p<0.001). Contrastingly, the brain metastatic tumours with high TILs (n=8) displayed higher levels of activated microglia, as measured by Iba1 expression. Low TILs-tumours displayed CD8+ T-cells that co-express VISTA (p<0.01) significantly more compared to high TILs group, where CD8+ T-cells significantly co-express Iba1 (p<0.05). Interestingly, no definite phenotypes of CD4+ subsets were observed. These results were supported by RNA analysis of a publicly available, independent cohort. In conclusion, our work contributes to a growing understanding of the immune surveillance escape routes active in brain metastasis.Graphical AbstractGraphical abstract:Brain metastasis patients with low TILs have high VISTA expression and patients with high TILs have significantly more activated microglia

scholarly journals Characterization of Immune Cell Subsets of Tumor Infiltrating Lymphocytes in Brain Metastases

Biology ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 425
Author(s):  
Priyakshi Kalita-de Croft ◽  
Haarika Chittoory ◽  
Tam H. Nguyen ◽  
Jodi M. Saunus ◽  
Woo Gyeong Kim ◽  
...  
Keyword(s):  
Brain Metastasis ◽  
Immune Cell ◽  
Immune Surveillance ◽  
Tumor Infiltrating Lymphocytes ◽  
Cd8 Cells ◽  
Targeted Analysis ◽  
Immune Cell Subsets ◽  
Checkpoint Molecule ◽  
Formalin Fixed Paraffin Embedded ◽  
Infiltrating Lymphocytes

The heterogeneity of tumor infiltrating lymphocytes (TILs) is not well characterized in brain metastasis. To address this, we performed a targeted analysis of immune-cell subsets in brain metastasis tissues to test immunosuppressive routes involved in brain metastasis. We performed multiplex immunofluorescence (mIF), using commercially available validated antibodies on formalin-fixed paraffin embedded whole sections. We quantitated the subsets of immune-cells utilizing a targeted panel of proteins including PanCK, CD8, CD4, VISTA and IBA-1, and analyzed an average of 15,000 cells per sample. Classifying tumors as either high (>30%) or low (<30%) TILs, we found that increased TILs density correlated with survival. Phenotyping these TILs we found tumors with low TILs had significantly higher expression of the immune-checkpoint molecule VISTA in tumor cells (p < 0.01) as well as in their microenvironment (p < 0.001). Contrastingly, the tumors with high TILs displayed higher levels of microglia, as measured by IBA-1 expression. Low TILs-tumors displayed CD8+ T-cells that co-express VISTA (p < 0.01) significantly more compared to high TILs group, where CD8+cells significantly co-express IBA-11 (p < 0.05). These results were supported by RNA analysis of a publicly available, independent cohort. Our work contributes to a growing understanding of the immune surveillance escape routes active in brain metastasis.

scholarly journals Characterizing immune cell subsets of tumour infiltrating lymphocytes (TILs) in brain metastases

2021 ◽  
Vol 23 (Supplement_4) ◽  
pp. iv16-iv16
Author(s):  
Priyakshi Kalita-de Croft ◽  
Haarika Chittoory ◽  
Peter Simpson ◽  
Sunil Lakhani
Keyword(s):  
T Cells ◽  
T Cell ◽  
Brain Metastasis ◽  
Brain Metastases ◽  
Cd8 T Cells ◽  
Immune Cell ◽  
Immune Escape ◽  
Breast Cancers ◽  
Targeted Analysis ◽  
Immune Cell Subsets

Abstract Aims The heterogeneity of TILs are not well characterized in brain metastasis. To address this, we performed a targeted analysis of immune cell subsets in brain metastasis tissues. Method We performed multiplex immunofluorescence (mIF) on a limited cohort of brain metastases arising from breast cancers (n=20). Using RNA-interference validated antibodies, we quantitated the subsets of immune cells in formalin-fixed paraffin embedded whole sections. The panel of proteins analyzed included PanCK, CD8, CD4, Vista and Iba1 and an average of 15000 cells per sample were analysed. We also analysed an independent publicly available cohort at the RNA level to corroborate our findings. Results We found that increased density of tils (high&gt;30%; low &lt;30%) correlated with survival and they were two distinct phenotypes. The tumours with low tils had significantly higher expression of the immune-checkpoint molecule Vista in tumour cells (p&lt;0.01) as well as in their microenvironment (p&lt;0.001). Contrastingly, the brain metastatic tumours with high tils displayed higher expression of microglia. Low tils-tumours display CD8+ T-cells that exclusively co-express Vista (p&lt;0.01) compared to high tils group where CD8+ T-cells significantly co-express Iba1 (p&lt;0.05). Interestingly no definite phenotypes were observed in CD4+ T-cells. These results were also found in an independent cohort where Vista was a highly ranked gene within the CD8+ T-cell population. Conclusion Variety of immune escape routes may be involved in brain metastasis. This may be executed by increasing the expression of T-cell inhibitory molecule Vista or by increased activated microglia which may release immunosuppressive cytokines. Further studies are required to provide mechanistic insights into these phenomena.

773 Adoptive Cell Therapy Response in Melanoma is Mediated by Stem-like CD8 T cells

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A822-A822
Author(s):  
Sri Krishna ◽  
Frank Lowery ◽  
Amy Copeland ◽  
Stephanie Goff ◽  
Grégoire Altan-Bonnet ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Single Cell ◽  
Cd8 T Cells ◽  
Tumor Infiltrating Lymphocytes ◽  
High Dimensional ◽  
Tumor Control ◽  
Intrinsic Factors ◽  
Infiltrating Lymphocytes

BackgroundAdoptive T cell therapy (ACT) utilizing ex vivo-expanded autologous tumor infiltrating lymphocytes (TILs) can result in complete regression of human cancers.1 Successful immunotherapy is influenced by several tumor-intrinsic factors.2 3 Recently, T cell-intrinsic factors have been associated with immunotherapy response in murine and human studies.4 5 Analyses of tumor-reactive TILs have concluded that anti-tumor neoantigen-specific TILs are enriched in subsets defined by the expression of PD-1 or CD39.6 7 Thus, there is a lack of consensus regarding the tumor-reactive TIL subset that is directly responsible for successful immunotherapies such as ICB and ACT. In this study, we attempted to define the fitness landscape of TIL-enriched infusion products to specifically understand its phenotypic impact on human immunotherapy responses.MethodsWe compared the phenotypic differences that could distinguish bulk ACT infusion products (I.P.) administered to patients who had complete response to therapy (complete responders, CRs, N = 24) from those whose disease progressed following ACT (non-responders, NRs, N = 30) by high dimensional single cell protein and RNA analysis of the I.P. We further analyzed the phenotypic states of anti-tumor neoantigen specific TILs from patient I.P (N = 26) by flow cytometry and single cell transcriptomics.ResultsWe identified two CD8+ TIL populations associated with clinical outcomes: a memory-progenitor CD39-negative stem-like TIL (CD39-CD69-) in the I.P. associated with complete cancer regression (overall survival, P < 0.0001, HR = 0.217, 95% CI 0.101 to 0.463) and TIL persistence, and a terminally differentiated CD39-positive TIL (CD39+CD69+) population associated with poor TIL persistence post-treatment. Although the majority (>65%) of neoantigen-reactive TILs in both responders and non-responders to ACT were found in the differentiated CD39+ state, CR infusion products also contained a pool of CD39- stem-like neoantigen-specific TILs (median = 8.8%) that was lacking in NR infusion products (median = 23.6%, P = 1.86 x 10-5). Tumor-reactive stem-like T cells were capable of self-renewal, expansion, and persistence, and mediated superior anti-tumor response in vivo.ConclusionsOur results support the hypothesis that responders to ACT received infusion products containing a pool of stem-like neoantigen-specific TILs that are able to undergo prolific expansion, give rise to differentiated subsets, and mediate long-term tumor control and T cell persistence, in line with recent murine ICB studies mediated by TCF+ progenitor T cells.4 5 Our data also suggest that TIL subsets mediating ACT-response (stem-like CD39-) might be distinct from TIL subsets enriched for anti-tumor-reactivity (terminally differentiated CD39+) in human TIL.6 7AcknowledgementsWe thank Don White for curating the melanoma patient cohort, and J. Panopoulos (Flowjo) for helpful discussions on high-dimensional analysis, and NCI Surgery Branch members for helpful insights and suggestions. S. Krishna acknowledges funding support from NCI Director’s Innovation Award from the National Cancer Institute.Trial RegistrationNAEthics ApprovalThe study was approved by NCI’s IRB ethics board.ReferencesGoff SL, et al. Randomized, prospective evaluation comparing intensity of lymphodepletion before adoptive transfer of tumor-infiltrating lymphocytes for patients with metastatic melanoma. J Clin Oncol 2016;34:2389–2397.Snyder A, et al. Genetic basis for clinical response to CTLA-4 blockade in melanoma. N Engl J Med 2014;371:2189–2199.McGranahan N, et al. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science 2016;351:1463–1469.Sade-Feldman M, et al. Defining T cell states associated with response to checkpoint immunotherapy in melanoma. Cell 2019;176:404.Miller BC, et al. Subsets of exhausted CD8 T cells differentially mediate tumor control and respond to checkpoint blockade. Nat. Immunol 2019;20:326–336.Simoni Y, et al. Bystander CD8 T cells are abundant and phenotypically distinct in human tumour infiltrates. Nature 2018;557:575–579.Gros A, et al. PD-1 identifies the patient-specific CD8+ tumor-reactive repertoire infiltrating human tumors. J Clin Invest 2014;124:2246–2259.

scholarly journals Impaired enolase 1 glycolytic activity restrains effector functions of tumor-infiltrating CD8+T cells

2019 ◽  
Vol 4 (31) ◽  
pp. eaap9520 ◽  
Author(s):  
Lelisa F. Gemta ◽  
Peter J. Siska ◽  
Marin E. Nelson ◽  
Xia Gao ◽  
Xiaojing Liu ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Tumor Infiltrating Lymphocytes ◽  
Biochemical Basis ◽  
Effector Functions ◽  
Glycolytic Activity ◽  
Functional Exhaustion ◽  
Critical Insight ◽  
Infiltrating Lymphocytes ◽  
Insight Into

In the context of solid tumors, there is a positive correlation between the accumulation of cytotoxic CD8+tumor-infiltrating lymphocytes (TILs) and favorable clinical outcomes. However, CD8+TILs often exhibit a state of functional exhaustion, limiting their activity, and the underlying molecular basis of this dysfunction is not fully understood. Here, we show that TILs found in human and murine CD8+melanomas are metabolically compromised with deficits in both glycolytic and oxidative metabolism. Although several studies have shown that tumors can outcompete T cells for glucose, thus limiting T cell metabolic activity, we report that a down-regulation in the activity of ENOLASE 1, a critical enzyme in the glycolytic pathway, represses glycolytic activity in CD8+TILs. Provision of pyruvate, a downstream product of ENOLASE 1, bypasses this inactivity and promotes both glycolysis and oxidative phosphorylation, resulting in improved effector function of CD8+TILs. We found high expression of both enolase 1 mRNA and protein in CD8+TILs, indicating that the enzymatic activity of ENOLASE 1 is regulated posttranslationally. These studies provide a critical insight into the biochemical basis of CD8+TIL dysfunction.

Immunocytokines are a promising immunotherapeutic approach against glioblastoma

2020 ◽  
Vol 12 (564) ◽  
pp. eabb2311 ◽  
Author(s):  
Tobias Weiss ◽  
Emanuele Puca ◽  
Manuela Silginer ◽  
Teresa Hemmerle ◽  
Shila Pazahr ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Microenvironment ◽  
Cd8 T Cells ◽  
Tumor Necrosis ◽  
Interleukin 2 ◽  
Blood Perfusion ◽  
Tumor Infiltrating Lymphocytes ◽  
Infiltrating Lymphocytes ◽  
Necrosis Factor ◽  
New Strategies

Glioblastoma is a poorly immunogenic cancer, and the successes with recent immunotherapies in extracranial malignancies have, so far, not been translated to this devastating disease. Therefore, there is an urgent need for new strategies to convert the immunologically cold glioma microenvironment into a hot one to enable effective antitumor immunity. Using the L19 antibody, which is specific to a tumor-associated epitope of extracellular fibronectin, we developed antibody-cytokine fusions—immunocytokines—with interleukin-2 (IL2), IL12, or tumor necrosis factor (TNF). We showed that L19 accumulated in the tumor microenvironment of two orthotopic immunocompetent mouse glioma models. Furthermore, intravenous administration of L19-mIL12 or L19-mTNF cured a proportion of tumor-bearing mice, whereas L19-IL2 did not. This therapeutic activity was abolished in RAG−/− mice or upon depletion of CD4 or CD8 T cells, suggesting adaptive immunity. Mechanistically, both immunocytokines promoted tumor-infiltrating lymphocytes and increased the amounts of proinflammatory cytokines within the tumor microenvironment. In addition, L19-mTNF induced tumor necrosis. Systemic administration of the fully human L19-TNF fusion protein to patients with glioblastoma (NCT03779230) was safe, decreased regional blood perfusion within the tumor, and was associated with increasing tumor necrosis and an increase in tumor-infiltrating CD4 and CD8 T cells. The extensive preclinical characterization and subsequent clinical translation provide a robust basis for future studies with immunocytokines to treat malignant brain tumors.

Anti-tumor immunity in mismatch repair-deficient colorectal cancers requires type I IFN–driven CCL5 and CXCL10

2021 ◽  
Vol 218 (9) ◽  
Author(s):  
Courtney Mowat ◽  
Shayla R. Mosley ◽  
Afshin Namdar ◽  
Daniel Schiller ◽  
Kristi Baker
Keyword(s):  
T Cells ◽  
Mismatch Repair ◽  
Tumor Immunity ◽  
Cd8 T Cells ◽  
Tumor Infiltrating Lymphocytes ◽  
Colorectal Cancers ◽  
Type I ◽  
Type I Ifn ◽  
Dna Mismatch ◽  
Infiltrating Lymphocytes

Colorectal cancers (CRCs) deficient in DNA mismatch repair (dMMR) contain abundant CD8+ tumor-infiltrating lymphocytes (TILs) responding to the abundant neoantigens from their unstable genomes. Priming of such tumor-targeted TILs first requires recruitment of CD8+ T cells into the tumors, implying that this is an essential prerequisite of successful dMMR anti-tumor immunity. We have discovered that selective recruitment and activation of systemic CD8+ T cells into dMMR CRCs strictly depend on overexpression of CCL5 and CXCL10 due to endogenous activation of cGAS/STING and type I IFN signaling by damaged DNA. TIL infiltration into orthotopic dMMR CRCs is neoantigen-independent and followed by induction of a resident memory-like phenotype key to the anti-tumor response. CCL5 and CXCL10 could be up-regulated by common chemotherapies in all CRCs, indicating that facilitating CD8+ T cell recruitment underlies their efficacy. Induction of CCL5 and CXCL10 thus represents a tractable therapeutic strategy to induce TIL recruitment into CRCs, where local priming can be maximized even in neoantigen-poor CRCs.

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