IMMU-01. CHARACTERIZATION OF TUMOR-INFILTRATING CD8+ T CELLS IN BRAIN METASTASES

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi91-vi92
Author(s):  
Lisa Sudmeier ◽  
William Hudson ◽  
Kimberly Hoang ◽  
Edjah Nduom ◽  
Stewart Neill ◽  
...  
Keyword(s):  
T Cells ◽  
Brain Metastases ◽  
Cd8 T Cells ◽  
Metastatic Cancer ◽  
Cell Receptor ◽  
Immune Checkpoint Blockade ◽  
Effector Cells ◽  
Cluster A ◽  
Inhibitory Molecules ◽  
Terminal Effector

Abstract BACKGROUND Use of immune checkpoint blockade (ICB) therapy has prolonged overall survival in patients with metastatic cancer. One potential strategy to improve the effectiveness of ICB is to target additional inhibitory receptors on exhausted CD8+ T cells, which may promote rescue of CD8+ T cells that do not respond to PD-1 pathway blockade alone. This therapeutic strategy requires knowledge of the inhibitory molecules expressed on tumor-specific CD8+ T cells. Toward achieving this goal, we characterized the phenotype of CD8+ T cells infiltrating brain metastases. METHODS We performed flow cytometry on 45 brain metastases samples, single cell RNA sequencing with T cell receptor (TCR) sequencing on 5 samples, and spatially-resolved transcriptomics on 8 samples. RESULTS Analysis of our scRNA-seq data revealed 4 populations of PD-1+ CD8+ T cells infiltrating brain metastases. One of these populations (cluster A) has a terminal effector exhausted phenotype suggesting that this population contains tumor-specific CD8+ T cells. Two of the other populations (clusters B and C) have a transcriptional profile that suggests they may contain stem-like CD8+ T cells. TCR sequencing shows that cells in cluster A do not express the same TCRs as cells in clusters B and C, suggesting that stem-like cells in clusters B and C are not the progenitors of the terminal effector cells in cluster A. Bystander cells expressing TCRs specific for viral antigens are found predominantly in clusters B and C, further supporting the hypothesis that cluster A contains tumor-specific cells. Spatial transcriptomics reveals that cluster A cells are infiltrating the tumor parenchyma while cluster B and C cells are predominantly in peri-tumoral inflammatory tissue. CONCLUSIONS Brain metastases are infiltrated by a population of terminally-differentiated effector CD8+ T cells which express co-inhibitory molecules that may be potential therapeutic targets to improve control of metastatic disease in the brain.

Role of NKG2D signaling in the cytotoxicity of activated and expanded CD8+ T cells

Blood ◽  
2004 ◽  
Vol 103 (8) ◽  
pp. 3065-3072 ◽  
Author(s):  
Michael R. Verneris ◽  
Mobin Karami ◽  
Jeanette Baker ◽  
Anishka Jayaswal ◽  
Robert S. Negrin
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Interleukin 2 ◽  
Cell Receptor ◽  
Malignant Cell ◽  
Leukemic Cells ◽  
Target Cells ◽  
High Dose ◽  
Effector Cells ◽  
Nkg2d Expression

Abstract Activating and expanding T cells using T-cell receptor (TCR) cross-linking antibodies and interleukin 2 (IL-2) results in potent cytotoxic effector cells capable of recognizing a broad range of malignant cell targets, including autologous leukemic cells. The mechanism of target cell recognition has previously been unknown. Recent studies show that ligation of NKG2D on natural killer (NK) cells directly induces cytotoxicity, whereas on T cells it costimulates TCR signaling. Here we demonstrate that NKG2D expression is up-regulated upon activation and expansion of human CD8+ T cells. Antibody blocking, redirected cytolysis, and small interfering RNA (siRNA) studies using purified CD8+ T cells demonstrate that cytotoxicity against malignant target cells occurs through NKG2D-mediated recognition and signaling and not through the TCR. Activated and expanded CD8+ T cells develop cytotoxicity after 10 to 14 days of culture, coincident with the expression of the adapter protein DAP10. T cells activated and expanded in low (30 U/mL) and high (300 U/mL) concentrations of IL-2 both up-regulated NKG2D expression equally, but only cells cultured in high-dose IL-2 expressed DAP10 and were cytotoxic. Collectively these results establish that NKG2D triggering accounts for the majority of major histocompatibility complex (MHC)–unrestricted cytotoxicity of activated and expanded CD8+ T cells, likely through DAP10-mediated signaling. (Blood. 2004;103: 3065-3072)

scholarly journals Monoclonal T-cell expansions in asymptomatic individuals and in patients with large granular leukemia consist of cytotoxic effector T cells expressing the activating CD94:NKG2C/E and NKD2D killer cell receptors

Blood ◽  
2003 ◽  
Vol 101 (8) ◽  
pp. 3198-3204 ◽  
Author(s):  
Valérie Bigouret ◽  
Till Hoffmann ◽  
Lionel Arlettaz ◽  
Jean Villard ◽  
Marco Colonna ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Clinical Symptoms ◽  
Killer Cell ◽  
Cell Receptor ◽  
Effector Cells ◽  
Size Characteristic ◽  
Asymptomatic Individuals ◽  
Cytotoxic Effector

Abstract We have analyzed the phenotype, cytokine profile, and mitotic history (telomere length) of monoclonal T-cell expansions in 5 CD3+ T-cell large granular lymphocyte (TLGL) leukemia patients by fluorescence activated cell sorting (FACS) and single-cell polymerase chain reaction (PCR). We confirm that the common phenotype of TLGL leukemia is CD3+CD8+CD45RA+CD27−CD94+(CD57+). Interestingly, the C-type lectin-like type killer cell receptor CD94 was invariably associated with the activating form of its signal-transducing molecule NKG2. Furthermore, when judged by criteria such as interferon gamma (IFN-γ)/tumor necrosis factor (TNF) production, expression of granzyme, FasL, and NKG2D, the TLGL cells had all the features of a cytotoxic effector T cell. Telomere shortening in TLGL cells was in the normal range for CD8+ T cells, indicating that they had not divided significantly more than chronically stimulated CD8+ T cells in healthy individuals. In 25 of 27 controls, cells with a TLGL phenotype occurred at low (1%-3%) frequencies. However, in the other 2 individuals (ages 28-36 years), large stable (> 3 years) monoclonal expansions of CD3+CD8+CD45RA+CD27−CD57+CD94+ NKG2C+ were found which rendered these controls phenotypically indistinguishable from TLGL leukemia patients. We believe that the TLGL clonopathy, rather than being of a neoplastic nature, is more likely an extreme manifestation of the large and stable clonal size characteristic of CD8+ effector cells. Such a TLGL clone consisting of cells without any particular pathologic trait might exist in a considerable number of individuals. Clinical symptoms may occur in individuals in whom the TLGL clone encounters antigen and is triggered to produce large amounts of effector molecules that dysregulate the immune system, which could manifest itself as autoimmunity or as a FasL-mediated neutropenia.

scholarly journals The CD8+ T cell landscape of human brain metastases

2021 ◽  
Author(s):  
Lisa J. Sudmeier ◽  
Kimberly B. Hoang ◽  
Edjah K. Nduom ◽  
Andreas Wieland ◽  
Stewart G. Neill ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Brain Metastasis ◽  
Brain Metastases ◽  
Immune Checkpoint ◽  
Expression Patterns ◽  
Immune Checkpoint Blockade ◽  
Checkpoint Blockade ◽  
Antigen Specificity ◽  
Inhibitory Molecules

Despite improved outcomes with checkpoint blockade immunotherapy, patients with brain metastases have the worst prognosis among patients with metastatic cancer. Immune checkpoint blockade agents target inhibitory receptors, such as PD-1, on exhausted CD8+ T cells to restore their anti-cancer function. Many patients, however, either do not respond or progress after an initial response to immune checkpoint blockade, and distant intracranial failure is common despite excellent options for local treatment of brain metastasis. To develop more effective therapeutic strategies for the treatment of brain metastases, an understanding of the phenotype of brain metastasis-infiltrating CD8+ T cells is essential. Here we performed a detailed characterization of the CD8+ T cells contained in brain metastases. Brain metastases were densely infiltrated by CD8+ T cells; blood contamination of tumor samples was rare. Compared to patient-matched circulating cells, brain metastasis-infiltrating CD8+ T cells had a distinct phenotype characterized by more frequent expression of PD-1, with subpopulations defined by expression of additional co-inhibitory molecules and the residence marker CD69. Single cell RNA-sequencing identified four phenotypic subpopulations within brain metastasis-infiltrating PD-1+ CD8+ T cells. Two of these populations - a terminally-differentiated and a dividing population - were characterized by high expression of co-inhibitory molecules and lacked expression of progenitor markers such as TCF-1. There was significant T cell receptor (TCR) overlap between the terminally-differentiated and dividing populations, suggesting that the dividing cells give rise to the terminally-differentiated cells. There was minimal TCR overlap between these two populations and other brain metastasis-infiltrating PD-1+ CD8+ T cells. T cell clones from brain metastasis-infiltrating CD8+ T cells were rare in circulation, particularly clones from the terminally-differentiated and dividing populations. We systematically identified bystander CD8+ T cells specific for microbial antigens; these cells infiltrated brain metastases and expressed genes shared with exhausted progenitor CD8+ T cells, such as TCF7 and IL7R. We performed spatial transcriptomics on brain metastases and used a novel method to obtain TCR sequences from spatial transcriptomics data. These data revealed distinct niches within the TME defined by their gene expression patterns and cytokine profiles. Terminally-differentiated CD8+ T cells preferentially occupied niches within the tumor parenchyma. Together, our results show that antigen-specificity restricts the spatial localization, phenotypic states, and differentiation pathways available to CD8+ T cells within the brain metastasis TME.

KS01.4.A NK cells as key orchestrators in mediating the anti-tumour response to immune checkpoint blockade in melanoma brain metastases

2021 ◽  
Vol 23 (Supplement_2) ◽  
pp. ii3-ii3
Author(s):  
C M Fife ◽  
J Williams ◽  
R Brownlie ◽  
T Andreou ◽  
A Sunderland ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Brain Metastases ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Nk Cell ◽  
Immune Checkpoint Blockade ◽  
Cell Depletion

Abstract BACKGROUND Brain metastases (BrM) are an unmet clinical need with poor prognosis. 60% of melanoma patients develop BrM. BrM are strongly understudied due to frequent exclusion from clinical trials, and hence treatment options commonly lag behind. Antibodies targeting the immune-inhibitory receptors cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) have demonstrated efficacy against melanoma BrM. Despite this, therapeutic responses are highly variable, and it is unknown why therapy fails in a high proportion of patients. Improved therapeutic strategies require a thorough understanding of potentially exploitable mechanisms of therapeutic efficacy. Our data previously implicated different immune cells, foremost CD8+ T cells, but also NK cells, in the intracranial efficacy and enhanced survival benefit of immune checkpoint blockade (ICB). Our aim here is to investigate the role of NK cells in mediating the response to ICB in melanoma BrM. MATERIAL AND METHODS To study the role of NK cells in the response to ICB in melanoma BrM, a tumour transplantation model of B16 melanoma with simultaneous extracranial (i.e., flank) and brain tumours in C57BL/6 mice was utilised. NK cells were depleted through administration of anti-asialo-GM1 NK cell-depleting antibodies. Confirmation of NK cell depletion and quantification of intratumoral immune cell populations was performed using flow cytometry. Intratumoral gene expression of key chemokines and immune mediator genes was assessed using RT-qPCR and mRNA-seq. RESULTS Highly variable response to ICB with respect to intratumoral accumulation of CD8+ T cells allowed separation of mice into responders and non-responders and revealed genes and pathways associated with response to ICB. NK cell depletion reversed the ICB-mediated increase in the accumulation of CD8+ T cells and significantly reduced the expression of genes associated with response in intracranial and extracranial tumours. The ICB-mediated significant increase in gene expression of various chemokines (i.e., Cxcl9/10) and immune mediators (i.e., Ifng, Prf1 and Gzmb) was significantly abrogated upon NK cell depletion. CONCLUSION NK cells play a critical role in the underlying mechanisms of ICB efficacy through their modulation of the tumour microenvironment and enhancement of CD8+ T cell accumulation in intracranial tumours. Targeting of NK cells may allow potentiation of ICB therapy in the brain, as well as at extracranial sites.

scholarly journals TCF-1 regulates the stem-like memory potential of HIV-specific CD8+ T cells in elite controllers

2020 ◽  
Author(s):  
Rachel L. Rutishauser ◽  
Christian Deo T. Deguit ◽  
Joseph Hiatt ◽  
Franziska Blaeschke ◽  
Theodore L. Roth ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Cell Receptor ◽  
Elite Controllers ◽  
Direct Role ◽  
Human Virus ◽  
Effector Cells ◽  
Expansion Capacity

AbstractAlthough many HIV cure strategies seek to expand HIV-specific CD8+ T cells to control the virus, all are likely to fail if cellular exhaustion is not prevented. A loss in stem-like memory properties (i.e., the ability to proliferate and generate secondary effector cells) is a key feature of exhaustion; little is known, however, about how these properties are regulated in human virus-specific CD8+ T cells. We found that virus-specific CD8+ T cells from humans and non-human primates naturally controlling HIV/SIV infection express more of the transcription factor, TCF-1, than non-controllers. HIV-specific CD8+ T cell TCF-1 expression correlated with memory marker expression and proliferative capacity and declined with antigenic stimulation. CRISPR-Cas9 editing of TCF-1 in human primary T cells demonstrated a direct role in regulating expansion capacity. Collectively, these data suggest that TCF-1 controls the stem-like memory properties of HIV-specific CD8+ T cells and provides a rationale for enhancing this pathway in T cell-based therapeutic strategies for HIV.One Sentence SummaryTCF-1 is highly expressed in HIV-specific CD8+ T cells from elite controllers and directly regulates human CD8+ T cell expansion capacity in response to T cell receptor stimulation.

Human Telomerase Reverse Transcriptase-Specific T-Cell Receptor Gene Transfer Redirects T-Lymphocytes to Exert Effective Antitumor Reactivity Against Adult T-Cell Leukemia,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4169-4169
Author(s):  
Yukihiro Miyazaki ◽  
Hiroshi Fujiwara ◽  
Toshiki Ochi ◽  
Hiroaki Asai ◽  
Kozo Nagai ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Mononuclear Cells ◽  
Treatment Options ◽  
Cell Receptor ◽  
Human Telomerase Reverse Transcriptase ◽  
Effector Cells ◽  
Adult T Cell Leukemia

Abstract Abstract 4169 Background and Purpose Currently adult T-cell leukemia (ATL) is one of the most chemotherapy-resistant T cell malignancies and the prognosis of ATL patients still remains very poor. Although several novel treatment options, for example anti-chemokine receptor 4 (CCR4) monoclonal antibody, look promise, development of other novel treatment options still remains warranted. Currently activated status of human telomerase reverse transcriptase (hTERT) in ATL cells has been underscored. Thus, in this study, in order to develop a novel T cell-based immunotherapy for the treatment of ATL, we investigated the feasibility of redirected T-cell using hTERT-specific T-cell receptor (TCR) gene transfer. Methods Approval for this study was obtained from the Institutional Review Board of Ehime University Hospital. HLA-A*24:02-restricted and hTERT461–469epitope (residues: VYGFVRACL)-specific TCR -a/b genes (Tajima K et al. Int J Cancer 2004) cloned from our established hTERT-specific CTL clone were inserted into a novel GaLV-pseudotyped retroviral vector encoding built-in siRNAs for constant regions of endogenous TCR a /b genes (hTERT-siTCR vector). hTERT-siTCR vector was transfected into normal CD8+ T cells in RetroNectin (Takara Bio Inc.) coated-plates and these redirected CD8+ T cells (hTERT-siTCR CD8) were used as effector cells. HLA-A*24:02+ or A*24:02− ATL cell lines, HTLV-1 infected T-cell lines and freshly isolated ATL cells from patients were examined as target cells. All patients gave written informed consents in accordance with the declaration of Helsinki. Normal HLA-A*24:02+CD4+ T cells andHLA-A*24:02+cord blood CD34+ mononuclear cells (CB-CD34+) were similarly obtained from healthy volunteers. Expression of hTERT mRNA and hTERT protein in ATL cell lines, freshly isolated ATL cells, and HTLV-1 infected T cells were evaluated by quantitative real time PCR (QRT-PCR) using DCt method and Western blotting. Antileukemia reactivity mediated by hTERT-siTCR CD8 against ATL cells were examined by standard 51chromium release assay and flow-based CD107a assay. For the safety assessment, effector cells-mediated cytotoxicity against CB-CD34+ cells as normal hematopoietic progenitors was similarly examined. Peripheral blood mononuclear cells (PBMCs) from ATL patients were obtained on admission and in stable disease or remission after receiving treatments, and hTERT461–469 epitope responsive CD8+ T cells were detected using hTERT461–469peptide/ HLA-A*2402 tetramer or ELISPOT assay. Results QRT-PCR revealed that both freshly isolated ATL cells and ATL cell lines markedly overexpressed hTERT mRNA, while that in normal CD4+ cells and CB-CD34+ cells was less than undetectable. hTERT-siTCR CD8 cells were more than 40% positive for hTERT/HLA-A*2402 tetramer and successfully displayed HLA-A*2402-restricted and hTERT461–469-specific cytocidal effect against target-loaded C1R-A24 cells. Additionally, hTERT-siTCR CD8 cells successfully discriminated A*2402+ ATL cell lines (ATN-1, TL-Su) from A*2402− ones (TLO-m1, HUT-102); those all cell lines similarly overexpressed hTERT. Furthermore, those effector cells successfully killed freshly isolated HLA-A*2402+ ATL cells, but not A*2402− ones. Finally, hTERT461–469-specific cytotoxic T-lymphocyte precursors were detected at variable levels in PBMCs obtained from HLA-A*2402+ ATL patients, which shows hTERT overexpressed by ATL cells may be naturally processed in vivo. Conclusion Although much further invetigations are warranted, in our preliminary study, we have shown for the first time that hTERT could be a potential therapeutic target of redirected T cell-based immunotherapy for the treatment of ATL. We are now in detail examining efficacy and safety of those redirected T cells using xenograft mouse model. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CD27 Expression Promotes Long-Term Survival of Functional Effector–Memory CD8+Cytotoxic T Lymphocytes in HIV-infected Patients

2004 ◽  
Vol 200 (11) ◽  
pp. 1407-1417 ◽  
Author(s):  
Adrian F. Ochsenbein ◽  
Stanley R. Riddell ◽  
Michele Brown ◽  
Lawrence Corey ◽  
Gabriela M. Baerlocher ◽  
...  
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Cd8 T Cells ◽  
Interleukin 2 ◽  
Tumor Necrosis Factor Receptor ◽  
Cell Receptor ◽  
Effector Memory ◽  
Term Survival ◽  
Functional Consequences

Human immunodeficiency virus (HIV)-specific CD8+ T cells persist in high frequencies in HIV-infected patients despite impaired CD4+ T helper response to the virus, but, unlike other differentiated effector cytotoxic T lymphocytes, most continue to express the tumor necrosis factor receptor family member CD27. Because the ligand for CD27 (CD70) is also overexpressed in HIV-infected hosts, we examined the nature of expression and potential functional consequences of CD27 expression on HIV-specific CD8+ T cells. Analysis of CD27+ and CD27− T cells derived from the same HIV-specific clone revealed that retention of CD27 did not interfere with acquisition of effector functions, and that after T cell receptor stimulation, CD27+ cells that concurrently were triggered via CD27 exhibited more resistance to apoptosis, interleukin 2 production, and proliferation than CD27− T cells. After transfer back into an HIV-infected patient, autologous HIV-specific CD27− T cells rapidly disappeared, but CD27+ T cells derived from the same clone persisted at high frequency. Our findings suggest that the CD27–CD70 interaction in HIV infection may provide CD27+ CD8+ T cells with a survival advantage and compensate for limiting or absent CD4+ T help to maintain the CD8 response.

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