Cytotoxic CD8+ T cells and CD138+ plasma cells prevail in cerebrospinal fluid in non-paraneoplastic cerebellar ataxia with contactin-associated protein-2 antibodies

Abstract Introduction Blocking inhibitory immune checkpoints holds promise to treat multiple myeloma (MM) patients. However, currently available checkpoint inhibitors have not shown significant clinical benefits for MM patients, warranting the need for alternative checkpoint blockers. The immune checkpoint TIGIT was recently shown to be the most upregulated immune inhibitory receptor on CD8+ T cells in MM patients' bone marrow (BM), compared to other checkpoints (Guillerey C., Blood. 2018). Preclinical models demonstrated the dominant effects of TIGIT blockade or depletion, by significantly improving mice survival, reducing myeloma cell numbers and exhausted T cell hallmarks (Minnie S., Blood. 2018). As a result, several clinical trials using anti-TIGIT monoclonal antibodies have been recently initiated in MM patients. The DNAM-1 family, in addition to TIGIT, also includes the inhibitory receptor PVRIG, that competes with the co-activating receptor DNAM-1 for the binding to the shared ligand PVRL2, similarly to the TIGIT/PVR/DNAM-1 interaction. Accordingly, TIGIT and PVRIG co-blockade were shown to synergize in enhancing T cell activity and anti-tumor activity in preclinical models (Whelan S., Cancer Immunol. Res. 2019). PVRL2 together with PVR (ligand of TIGIT) were shown to be highly expressed on plasma cells and on CD14+ cells in BM of MM patients (Lozano E., Clin. Cancer Res. 2020). This study aimed at evaluating DNAM-1 axis receptors expression in MM patients' BM. Methods Fresh BM aspirates were collected from 21 MM patients with progressive disease (PD) or in complete response (CR) after obtaining IRB approval. BM mononuclear cells were isolated and single cell suspensions were obtained followed by staining with anti-human antibodies to evaluate DNAM-1 axis members and PD-1 expression. BM biopsies from 6 MM patients (each patient had 4 core on the Tissue Micro-Array T291 USBiomax) were stained for PVRL2 expression by immuno-histochemistry (IHC). Results Flow cytometry analysis of PD-1 and DNAM-1 axis receptors revealed a significant lower fraction of PD1+ cells among cell populations examined compared with other receptors. TIGIT expression was the highest on NK, CD8+ and NKT cells compared to CD4+ T cells, which is in line with previous published data (Lozano E. Clin. Cancer Res. 2020). In contrast, DNAM-1 was expressed on CD8+ T, NK and NKT cells with prominent high expression on CD4+ T cells (Fig 1A). The highest expression among the receptors was of PVRIG on all lymphoid populations, except CD4+ where DNAM-1 was more highly expressed. Importantly, 50% of CD8+ T cells co-expressed TIGIT and PVRIG, supporting a combinatorial therapeutic approach (Fig. 1B). Additionally, the expression of the PVRL2 ligand on MM plasma and endothelial cells was demonstrated by IHC. FACS analysis further supported PVRL2 expression on plasma cells in MM BM (Fig 2). A higher expression of PVRIG, TIGIT and PD-1 was present on DNAM-1 negative CD8+ T cells (Fig 3A, B), suggesting accumulation of exhausted cells in MM tumor microenvironment (TME) as previously described (Minnie S., Blood. 2018). PVRIG had significantly higher expression on DNAM+ cells, compared to PD-1 and TIGIT (Fig 3C), suggesting the potential of its blockade to enhance DNAM-1 activation and subsequent proliferation of earlier differentiated memory cells in MM TME. Finally, CR patients had a trend for higher DNAM-1 expression on CD8+ T cells compared to those with PD (Fig 3D). This is consistent with other reports in mice showing that the expression of DNAM-1 negatively correlates with BM myeloma cell numbers (Minnie S., Blood. 2018). Conclusions DNAM-1 axis receptors are dominantly expressed on lymphocytes in BM of MM patients, with PVRIG exhibiting the most prominent expression. The reduced expression of DNAM-1 in PD patients' TME, compared to CR patients, suggests a link between DNAM-1 axis and clinical outcomes. Recent data suggest TIGIT is an attractive target for blockade in MM. Our new findings highlight for the first time the dominant expression of PVRIG, as well as TIGIT, and suggest that combined blockade of TIGIT with PVRIG may potentially benefit MM patients, placing the DNAM-1 axis as a dominant pathway in MM therapy. Figure 1 Figure 1. Disclosures Frenkel: Compugen Ltd.: Current Employment, Other: in the event of frontal participation, I will be reimbursed for my travel expenses by Compugen Ltd.. Alteber: Compugen Ltd.: Current Employment. Cojocaru: Compugen Ltd.: Current Employment. Ophir: Compugen Ltd.: Current Employment.

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Landscape of glioblastoma niches reveals the prognostic effects of tumor-infiltrating cells

10.1101/2021.01.20.427411 ◽

2021 ◽

Author(s):

Zixuan Xiao ◽

Wei Zhang ◽

Guanzhang Li ◽

Wendong Li ◽

Lin Li ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

Tumor Cell ◽

Cd8 T Cells ◽

Plasma Cells ◽

Cell Types ◽

Enrichment Score ◽

Large Gene ◽

Mesenchymal Differentiation ◽

Positive Immune Response

AbstractA comprehensive characterization of non-tumor cells in the niches of primary glioblastoma is not fully established yet. This study aims to present an overview of tumor-infiltrating non-malignant cells in the complex microenvironment of glioblastoma with detailed characterizations of their prognostic effects. We curate 540 gene signatures covering a total of 64 non-tumor cell types. Cell type-specific expression patterns are interrogated by normalized enrichment score (NES) across four large gene expression profiling cohorts of glioblastoma with a total number of 967 cases. The GBMs in each cohort are hierarchically clustered into negative or positive immune response classes with significantly different overall survival. Our results show that astrocytes, macrophages, monocytes, NKTs, preadipocytes, smooth muscle cells, and MSC are risk factors, while CD8 T cells, CD8+ T cells, and plasma cells are protective factors. Moreover, we find that the immune system and organogenesis are uniformly enriched in negative immune response clusters, in contrast to the enrichment of nervous system in positive immune response clusters. Mesenchymal differentiation is also observed in the negative immune response clusters. High enrichment status of macrophages in negative immune response clusters are independently validated by analyzing scRNA-seq data from eight high-grade gliomas, revealing that negative immune response samples comprised 46.63% to 55.12% of macrophages, whereas positive immune response samples comprised only 1.70% to 8.12%, with IHC staining of samples from six short-term and six long-term survivors of GBMs confirming the results.Simple SummaryThe landscape of infiltrating non-tumor cells in glioblastoma niches remains unclear. In this study, we explore the enrichment status of a total of 64 non-tumor cell types predicted by applying 540 gene signatures curated from literature and normalized enrichment score (NES) across four large gene expression profiling cohorts of glioblastoma with 967 cases. Based on non-tumor cell type-based enrichment status, GBMs in each cohort are classified into positive or negative immune response clusters, showing a statistically significant different overall survival. Astrocytes, macrophages, monocytes, NKTs, preadipocytes, smooth muscle cells, and MSC are identified as risk factors, as well as protector factors of CD8 T cells, CD8+ T cells, and plasma cells. Our results also find that immune system- and organogenesis-related GO terms are uniformly enriched in negative immune response clusters, whereas positive immune response clusters are enriched with GO terms concerning the nervous system. The mesenchymal differentiation is observed in the negative immune response clusters. Particularly, the high presence of macrophages in the negative immune response clusters is further validated using scRNA-seq analysis and IHC staining of GBMs from independent cohorts.

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Selective Targeting of Multiple Myeloma By Bcma-Specific Central Memory CD8+ cytotoxic T Lymphocytes: A Potential Immunotherapeutic Application in Multiple Myeloma and Other Plasma Cell Disorders

Blood ◽

10.1182/blood-2018-99-117282 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3196-3196

Author(s):

Jooeun Bae ◽

Teru Hideshima ◽

Nikhil Munshi ◽

Kenneth C. Anderson

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

Immune Responses ◽

Cd8 T Cells ◽

Plasma Cells ◽

Checkpoint Inhibitor ◽

Central Memory ◽

Cytotoxic Activities ◽

Specific Memory ◽

Equity Ownership

Abstract Background: Multiple myeloma (MM) is a B-cell malignancy characterized by the clonal proliferation and accumulation of malignant plasma cells in the bone marrow and development of osteolytic bone lesions. Despite recent advances in treatment using novel therapeutics, MM remains incurable with high mortality rates. We have demonstrated in preclinical studies that CD8+ cytotoxic T lymphocytes (CTL) generated with immunogenic HLA-A2 or HLA-A24 peptides targeting XBP1(X-box binding protein 1), CD138 (Syndecan-1) and CS1 (SLAMF7) antigens induces robust cytotoxic activities against MM. The Phase 1/2a trials were completed or are in progress in the patients with smoldering multiple myeloma or triple negative breast cancer, respectively, using the HLA-A2 XBP1/CD138/CS1 multipeptide vaccine. The clinical data demonstrated that the multipeptide vaccine is safe and induces the XBP1/CD138/CS1-specific immune responses, evidenced by expansion of peptides-specific Tetramer+/CD45RO+memory CTL and Th-1 specific immune responses. Moreover, clinical trials combining with Lenalidomide or checkpoint inhibitor enhanced the CD8+ CTL activities induced by multipeptide vaccine, indicating the benefit of combination therapy. To expand the breadth and extent of the antigens-specific immunotherapy beyond XBP1/CD138/CS1, we have recently identified additional tumor-associated antigens (TAA) on tumor cells obtained from newly diagnosed MM patients (N=616). Here, we introduce a novel heteroclitic peptides specific to BCMA, the receptor forbinding of B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL). Due to its restricted expression pattern on MM cells and plasma cells along with its critical role in promoting MM cell growth, survival and drug resistance, we are currently in development of novel immunotherapeutic to target BCMA on MM cells with different therapeutic approaches. Objective: The aims of current study were to target BCMA as a TAA by generating the antigen-specific memory CD8+ CTL to induce effective and long-lasting immune response against MM. Findings: We report on immunogenic HLA-A2-restricted peptides derived from BCMA, which are capable of evoking antigen-specific immune responses against MM. The heteroclitic BCMA peptides displayed improved binding affinity/stability to HLA-A2 molecules from their native BCMA peptides. To define immunogenicity of the selected peptides, we generated BCMA-specific CTL (BCMA-CTL) by repeated stimulationof CD3+ T cells with respective heteroclitic peptide. The BCMA-specific engineered peptides evoked the expansion of antigen-specific CD8+ CTL and generated BCMA-CTL displaying high T cell activation (CD38, CD69) and co-stimulatory (CD40L, OX40, GITR) molecules expression. Additionally, a gradual expansion was observed in BCMA-specific memory CD8+ T cells, with a corresponding decrease in naïve CD8+ T cells. The BCMA-CTL demonstrated robust poly-functional immune responses with Th1-specific anti-MM activities [high IFN-g/IL-2/TNF-aproduction, CD8+ T cells proliferation, cytotoxicity] in antigen-specific and HLA-A2-restricted manner. The functional activities were directly correlated with the expansion of central memory CD8+ CTL in the BCMA-CTL generated from different HLA-A2+ individuals (Donor 1 BCMA-CTL: 81.0%, Donor 2 BCMA-CTL: 82.6%, Donor 3 BCMA-CTL: 67.0%). Finally, the combination with checkpoint inhibitor (anti-LAG3) or immune agonist (anti-OX40) enhanced the anti-tumor activities of BCMA-CTL, along with the induction of cytotoxic activities by central memory CD8+ T cell subset. Therefore, these studies suggest that heteroclitic BCMA peptides offer a therapeutic potential to effectively generate BCMA-specific CD8+ CTL targeting MM. Significance: Here, we introduce novel immunogenic engineered heteroclitic BCMA peptides capable of inducing antigen-specific memory CD8+ CTL with robust poly-functional immune responses against MM. These results provide the framework for therapeutic application of heteroclitic BCMA peptides in MM patients. They further support combination treatment options incorporating BCMA peptides-specific vaccine or BCMA peptides-specific adoptive T cells immunotherapy with anti-LAG3 and/or anti-OX40for patients with myeloma or other diseases expressing BCMA. Disclosures Munshi: OncoPep: Other: Board of director. Anderson:Celgene: Consultancy; Takeda Millennium: Consultancy; Bristol Myers Squibb: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncopep: Equity Ownership; C4 Therapeutics: Equity Ownership.

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Tigit, CD226 and PD-L1/PD-1 Are Highly Expressed By Marrow-Infiltrating T Cells in Patients with Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.2102.2102 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2102-2102 ◽

Cited By ~ 5

Author(s):

Mahesh Yadav ◽

Cherie Green ◽

Connie Ma ◽

Alberto Robert ◽

Andrew Glibicky ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Nk Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Plasma Cells ◽

Color Flow

Abstract Introduction:TIGIT (T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif [ITIM] domain) is an inhibitory immunoreceptor expressed by T and natural killer (NK) cells that is an important regulator of anti-tumor and anti-viral immunity. TIGIT shares its high-affinity ligand PVR (CD155) with the activating receptor CD226 (DNAM-1). We have recently shown that TIGIT blockade, together with PD-L1/PD-1 blockade, provides robust efficacy in syngeneic tumor and chronic viral infection models. Importantly, CD226 blockade abrogates the benefit of TIGIT blockade, suggesting additional benefit of TIGIT blockade through elaboration of CD226-mediated anti-tumor immunity, analogous to CTLA-4/CD28 regulation of T-cell immunity. Whether TIGIT and CD226 are expressed in patients with multiple myeloma (MM) and how TIGIT expression relates to PD-L1/PD-1 expression is unknown. Here we evaluate expression of TIGIT, CD226, PD-1 and PD-L1 in patients with MM to inform novel immunotherapy combinations. Methods:We performed multi-color flow cytometry (n = 25 patients), and multiplex qRT-PCR (n = 7) on bone marrow specimens from patients with MM to assess expression of TIGIT, CD226, PD-1, and PD-L1 on tumor and immune cells. Cells were stained with fluorescently conjugated monoclonal antibodies to label T cells (CD3, CD4, CD8), NK cells (CD56, CD3), plasma cells (CD38, CD45, CD319, CD56), inhibitory/activating receptors (PD-1, TIGIT, PD-L1, CD226), and an amine-reactive viability dye (7-AAD). Stained and fixed cells were analyzed by flow cytometry using BD FACSCanto™ and BD LSRFortessa™. Results:TIGIT, CD226 and PD-L1/PD-1 were detectable by flow cytometry in all patients with MM who were tested, with some overlapping and distinct expression patterns. TIGIT was commonly expressed by marrow-infiltrating CD8+ T cells (median, 65% of cells), CD4+ T cells (median, 12%) and NK cells. In contrast, CD226 was more commonly expressed by marrow-infiltrating CD4+ T cells (median, 74%) compared with CD8+ T cells (median, 38%). PD-1 was expressed by marrow-infiltrating CD8+ T cells (median 38%) and CD4+ T cells (median, 16%). TIGIT was co-expressed with PD-1 on CD8+ T cells (67%-97% TIGIT+ among PD-1+), although many PD-1-negative CD8+ T cells also expressed TIGIT (39%-78% of PD-1-negative). PD-L1 was also expressed by CD8+ (median, 23%) and CD4+ (median, 8%) T cells in addition to MM plasma cells (median, 95%), albeit with significantly lower intensity on T cells compared with plasma cells. The expression of TIGIT and PD-L1 mRNA was highly correlated (R2 = 0.80). Analysis of PVR expression will also be presented. Conclusions: TIGIT, CD226, PD-1, and PD-L1 were commonly expressed in MM bone marrow, but with different patterns. Among CD8+ T cells, the frequency of TIGIT+ T cells was almost twice that of PD-1+ T cells, whereas the majority of CD4+ T cells expressed CD226. TIGIT blockade may complement anti-PD-L1/PD-1 immunotherapy by activating distinct T-cell/NK-cell subsets with synergistic clinical benefit. These results provide new insight into the immune microenvironment of MM and rationale for targeting both the PD-L1/PD-1 interaction and TIGIT in MM. Disclosures Yadav: Genentech, Inc.: Employment. Green:Genentech, Inc.: Employment. Ma:Genentech, Inc.: Employment. Robert:Genentech, Inc.: Employment. Glibicky:Makro Technologies Inc.: Employment; Genentech, Inc.: Consultancy. Nakamura:Genentech, Inc.: Employment. Sumiyoshi:Genentech, Inc.: Employment. Meng:Genentech, Inc.: Employment, Equity Ownership. Chu:Genentech Inc.: Employment. Wu:Genentech: Employment. Byon:Genentech, Inc.: Employment. Woodard:Genentech, Inc.: Employment. Adamkewicz:Genentech, Inc.: Employment. Grogan:Genentech, Inc.: Employment. Venstrom:Roche-Genentech: Employment.

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Alterations in the antigen processing-presenting machinery of transformed plasma cells are associated with reduced recognition by CD8+ T cells and characterize the progression of MGUS to multiple myeloma

Blood ◽

10.1182/blood-2009-06-228676 ◽

2010 ◽

Vol 115 (6) ◽

pp. 1185-1193 ◽

Cited By ~ 33

Author(s):

Vito Racanelli ◽

Patrizia Leone ◽

Maria Antonia Frassanito ◽

Claudia Brunetti ◽

Federico Perosa ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

Cd8 T Cells ◽

Antigen Processing ◽

Plasma Cells ◽

Disease Status ◽

Transcriptional Level ◽

Proteasome Subunits ◽

Polymerase Chain ◽

Lower Expression

Abstract We hypothesized that progression of monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) reflects the escape of transformed plasma cells from T-cell recognition because of impaired antigen processing-presenting machinery (APM). We studied plasma cells and CD8+ T cells from bone marrow of 20 MGUS patients, 20 MM patients, and 10 control patients. Immunofluorescence and flow cytometry revealed significantly different patterns of APM component expression in plasma cells from the 3 groups. Compared with control patients, MM samples had lower expression of proteasome subunits and peptide transporters and greater expression of chaperones, considering both percentages of stained cells and molecular equivalents of soluble fluorochrome. MGUS samples had intermediate percentages of stained cells but molecular equivalents of soluble fluorochrome similar to control patients. Real-time polymerase chain reaction documented that APM changes occurred at the transcriptional level. Cytotoxicity assays demonstrated that MGUS CD8+ T cells lysed autologous transformed plasma cells more than MM CD8+ T cells did. MGUS progression correlated directly with calnexin, calreticulin, and tapasin and indirectly with δ, LMP2, and LMP10 expression levels; MM disease status did not correlate with APM levels. APM changes may allow transformed plasma cells to elude immunesurveillance in the MGUS-MM pathogenetic sequence.

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HLA-G expression defines a novel regulatory T-cell subset present in human peripheral blood and sites of inflammation

Blood ◽

10.1182/blood-2006-11-057125 ◽

2007 ◽

Vol 110 (2) ◽

pp. 568-577 ◽

Cited By ~ 122

Author(s):

Ute Feger ◽

Eva Tolosa ◽

Yu-Hwa Huang ◽

Anne Waschbisch ◽

Tilo Biedermann ◽

...

Keyword(s):

Skeletal Muscle ◽

T Cells ◽

Cerebrospinal Fluid ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Inflammatory Responses ◽

Human Peripheral Blood ◽

Cell Subset ◽

Single Positive

Abstract Regulatory T cells can inhibit harmful immunopathologic responses directed against self and foreign antigens and play a major role in controlling autoimmunity. Here we have identified and characterized a subpopulation of CD4 and CD8 T cells in human peripheral blood expressing the immune tolerizing molecule HLA-G. HLA-G–expressing T cells are hypoproliferative, are CD25- and FOXP3-negative, and exhibit potent suppressive properties that are partially mediated by HLA-G. HLA-G–positive (HLA-Gpos) T cells are found at low percentages among CD4 and CD8 single-positive thymocytes, suggesting a thymic origin. The presence of HLA-Gpos T cells at sites of inflammation such as inflamed skeletal muscle in myositis or the cerebrospinal fluid of patients with acute neuroinflammatory disorders suggests an important function in modulating parenchymal inflammatory responses in vivo.

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