Correlation of IFNγ, CD107 and CD137 at the Single Cell Level Can Be Used To Monitor T Cell Responses in Patients after Immunotherapy.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2306-2306
Author(s):  
Debra K. Czerwinski ◽  
Joshua D. Brody ◽  
Ronald Levy
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cd8 T Cells ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cell Responses ◽  
Ifn Γ

Abstract As immunotherapies become increasingly important in the treatment of various cancers, monitoring the immune response to reflect the efficacy of the therapy also becomes increasingly important. Previously, tumor antigen-specific humoral responses in patients receiving vaccines for low-grade follicular lymphoma (FL) correlated with clinical outcomes, including tumor regression, molecular remission, progression free survival (PFS) and overall survival (OS). By contrast, T cell immune responses have been difficult to validate. T cell proliferation assays, mostly, measure CD4 T cell responses; whereas, CD8 T cells may be the important effectors generated by immunotherapies. However, assays designed to measure CD8 T cells, i.e. chromium release CTL assays, and IFN-γ ELISPOT and intracellular flow cytometry assays, are difficult to make reproducible. To address this issue, PBL were obtained from FL patients, cryopreserved, and thawed, then used to design a standardized method for detection of intracellular IFN-γ by flow cytometry. The combined stimulus of soluble anti-CD3 and anti-CD28 antibodies provides a robust stimulation, typically about 5% of normal PBL CD8+ T cells respond. By using a panel of irradiated B cell lymphoma cell lines as stimulators, we demonstrated that, on average, 1 – 2% of these T cells were capable of mounting a response in this assay. Surprisingly, CD8+ PBL T cells from several patients with FL were more responsive to combined anti-CD3 and anti-CD28 stimulation as well as to allo-stimulation, 15 – 22% and 2 – 6%, respectively. This response was accompanied by surface expression of CD107, a surrogate marker for CTL degranulation, in the same population of cells as demonstrated by multi-color flow cytometry. Both the IFN-γ and the CD107 responses were inhibited by an anti-class I antibody, W6/32, suggesting a class I restricted T cell receptor-mediated response. Furthermore, at later time points, these T cells also up-regulated CD137 on their surface. This activation molecule is upregulated on CD8 T cells in response to specific antigen recognition and provides an anti-apoptotic signal to the cells. In conclusion, immune competency of CD8 T cells isolated from FL patients can be assessed through allo-stimulation by a panel of B cell lymphoma cell lines. More importantly, correlation by flow cytometry of 3 independent indicators of response (IFN-γ, CD107 and CD137) within single populations of cells to both allo-stimulation and to the specific target, may lead to better understanding of the role of T cells in the immune response. Ultimately, these responses will need to be validated with patient outcomes in clinical trials of vaccines in lymphoma.

scholarly journals Beyond PD-1: Investigating the Therapeutic Potential of TIGIT Blockade in DLBCL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 391-391 ◽  
Author(s):  
Nicole Sunseri ◽  
Xiufen Chen ◽  
Noemie Wald ◽  
Julie Preillon ◽  
Sonali M. Smith ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Ex Vivo ◽  
B Cell Lymphoma ◽  
Isotype Control

Background: The PD-1/PD-L1 axis is a dominant cancer immune escape pathway, and PD-1 blockade therapy has greatly benefited patients with select solid tumors and lymphomas. Unfortunately, anti-PD-1 monotherapy has limited efficacy against relapsed/refractory (r/r) diffuse large B cell lymphoma (DLBCL) - a disease where new therapies are needed. Because numerous inhibitory checkpoint receptors have been implicated in driving tumor-specific T cell dysfunction, we hypothesized that combinatorial checkpoint blockade therapy (CBT) would enhance the activity of PD-1-based therapy in r/r DLBCL. T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) is a recently identified co-inhibitory receptor expressed on dysfunctional tumor-infiltrating T cells. PD-1 and TIGIT co-blockade therapy has demonstrated impressive activity in pre-clinical solid tumor and myeloma models. However, the degree to which TIGIT is involved in mediating T cell suppression in DLBCL is not fully known. Methods: TIGIT expression on lymphoma-infiltrating T cells (LITs) from 18 fresh lymphoma samples was analyzed by flow cytometry. Multiplex IHC on tissue microarrays (TMAs) was also performed to investigate TIGIT expression in DLBCL samples. The syngeneic murine A20 B cell lymphoma model was employed to study: 1) the kinetics of TIGIT and other co-receptor expression on LITs, 2) the association of TIGIT expression with effector function among LITs, and 3) the effectiveness of anti-TIGIT mono- and combination CBT in mice with established A20 lymphomas. A20 lymphoma tumors were established in groups of Balb/c mice by subcutaneous (SQ) injections of 5 x 106 cells. Expression of TIGIT and other co-receptors in A20 LITs was examined by flow cytometry at various time points. Function of TIGIT+ LITs was assessed by examining cytokine production following ex vivo stimulation with PMA and ionomycin. To test the efficacy of TIGIT blockade, mice received intraperitoneal injections of anti-TIGIT, anti-PD-1, anti-4-1BB, or combinations of these antibodies. Treatments began once tumors reached a diameter of 10 mm and were continued every 3 days for 5 doses. Tumor growth was monitored and compared to that in A20-bearing mice treated with isotype control antibodies. In some experiments, mice that achieved complete tumor rejection following single or dual CBT were re-challenged with A20 cells to investigate immunological memory responses. Results: Across a variety of human lymphomas, flow cytometric analysis revealed that TIGIT was broadly upregulated on LITs, including regulatory T cells and conventional CD4+and CD8+ T cells (Figure A and B). TIGIT expression on LITs in DLBCL was particularly high. Nearly all TIGIT+ LITs were also PD-1+, suggesting that these receptors co-orchestrate a T cell dysfunctional state in the lymphoma environment. Multiplex immunofluorescence staining of DLBCL samples demonstrated that TIGIT was most highly expressed on CD8+ T cells and that TIGIT+ T cells tended to be localized near, and in some cases, surrounding CD20+ lymphoma cells. Consistent with observations in human lymphomas, LITs isolated from murine A20 lymphoma commonly co-expressed TIGIT and PD-1, and the degree of expression correlated directly with tumor volume. This correlation was also present for other co-receptors, including 4-1BB, TIM3, and CTLA-4. Ex vivo restimulation of A20 LITs revealed that TIGIT+ T cells produced lower levels of effector cytokines, such as TNF-α, compared with TIGIT- T cells. In mice with established A20 lymphomas, both TIGIT and PD-1 mono-blockade led to modest delays in tumor outgrowth compared with mice treated with isotype control antibodies. Strikingly, however, combined PD-1 and TIGIT blockade resulted in complete rejection of A20 lymphomas in most mice and led to significantly prolonged survival compared to mice treated with single agent CBT (Figure C). Combination TIGIT and 4-1BB CBT was also remarkably effective in driving rejection of A20 lymphomas, and led to remarkable memory responses. Conclusions: TIGIT promotes immune tolerance in the DLBCL environment. While TIGIT monotherapy has anti-lymphoma activity, combinatorial CBT incorporating anti-TIGIT antibodies drives extremely potent rejection of established lymphomas in mice. These results provide rationale for further study of TIGIT blockade as a therapeutic strategy in r/r lymphomas, including DLBCL. Disclosures Sunseri: iTeos Therapeutics: Research Funding. Wald:iTeos Therapeutics: Employment. Preillon:iTeos Therapeutics: Employment. Smith:Portola Pharmaceuticals: Research Funding. Driessens:iTeos Therapeutics: Employment. Kline:Merck: Honoraria; Merck: Research Funding.

scholarly journals Accelerated, but not conventional, radiotherapy of murine B-cell lymphoma induces potent T cell–mediated remissions

2018 ◽  
Vol 2 (19) ◽  
pp. 2568-2580 ◽  
Author(s):  
Suparna Dutt ◽  
Michelle B. Atallah ◽  
Yoshitaka Minamida ◽  
Alexander Filatenkov ◽  
Kent P. Jensen ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
B Cell ◽  
Cd8 T Cells ◽  
Hematopoietic Cell Transplantation ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Tumor Irradiation ◽  
Total Radiation Dose

Abstract Conventional local tumor irradiation (LTI), delivered in small daily doses over several weeks, is used clinically as a palliative, rather than curative, treatment for chemotherapy-resistant diffuse large B-cell lymphoma (DLBCL) for patients who are ineligible for hematopoietic cell transplantation. Our goal was to test the hypothesis that accelerated, but not conventional, LTI would be more curative by inducing T cell–mediated durable remissions. We irradiated subcutaneous A20 and BL3750 lymphoma tumors in mice with a clinically relevant total radiation dose of 30 Gy LTI, delivered in 10 doses of 3 Gy over 4 days (accelerated irradiation) or as 10 doses of 3 Gy over 12 days (conventional irradiation). Compared with conventional LTI, accelerated LTI resulted in more complete and durable tumor remissions. The majority of these mice were resistant to rechallenge with lymphoma cells, demonstrating the induction of memory antitumor immunity. The increased efficacy of accelerated LTI correlated with higher levels of tumor cell necrosis vs apoptosis and expression of “immunogenic cell death” markers, including calreticulin, heat shock protein 70 (Hsp70), and Hsp90. Accelerated LTI–induced remissions were not seen in immunodeficient Rag-2−/− mice, CD8+ T-cell–depleted mice, or Batf-3−/− mice lacking CD8α+ and CD103+ dendritic cells. Accelerated, but not conventional, LTI in immunocompetent hosts induced marked increases in tumor-infiltrating CD4+ and CD8+ T cells and MHCII+CD103+CD11c+ dendritic cells and corresponding reductions in exhausted PD-1+Eomes+CD8+ T cells and CD4+CD25+FOXP3+ regulatory T cells. These findings raise the possibility that accelerated LTI can provide effective immune control of human DLBCL.

Generation of CD8 T Cell-Mediated Protective Immunity against Tumor Escapees

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2623-2623 ◽  
Author(s):  
Bindu Varghese ◽  
Behnaz Taidi ◽  
Adam Widman ◽  
James Do ◽  
R. Levy
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Cell Lymphoma ◽  
Ex Vivo ◽  
B Cell Lymphoma ◽  
Cd8 T Cell

Abstract Introduction: Anti-idiotype antibodies against B cell lymphoma have shown remarkable success in causing tumor regression in the clinic. In addition to their known ability to mediate ADCC, anti-idiotype antibodies have also been shown to directly inhibit the proliferation of tumor cells by sending negative growth signals via the target idiotype. However, further studies to investigate this mechanism have been hindered by the failure of patient tumor cells to grow ex vivo. Methods and Results: In order to study this phenomenon further, we developed an antibody against the idiotype on an A20 mouse B lymphoma cell line. A radioactive thymidine incorporation assay showed decreased A20 cell proliferation in the presence of the anti-id antibody ex vivo. In vivo, when mice were treated intraperitoneally (i.p.) with 100 μg of antibody 3 hours post-tumor inoculation (1×106 A20 subcutaneously (s.c.)), tumor growth was delayed for greater than 40 days after which the tumor began to grow once again. Further analysis of these escaping tumor cells by flow cytometry showed that that the tumor cells escaped the antibody-mediated immune response by down-regulating expression of idiotype and IgG on their surfaces although the cells retained idiotype expression intracellularly. This down-regulation of surface idiotype rendered the tumor cells resistant to both ADCC and signaling-induced cell death. The addition of an immunostimulatory bacterial mimic (CpG-DNA; 100 μg × 5 intratumoral (i.t.) injections; Days 2, 3 4, 6 & 8) to antibody therapy (Day 0; 100 μg i.p.) cured large established tumors (Day 0 = 1 cm2) and prevented the occurrence of tumor escapees (p<0.0001). Antibody plus CpG combination therapy in tumor-bearing mice deficient for CD8+ T cells demonstrated the critical role of CD8+ T cells in A20 tumor eradication (p<0.005). Depletion of CD4+ T cells was found to have no significant impact on the therapy. We also found that when mice were inoculated with two tumors and treated with anti-idiotype antibody (i.p.) followed by intratumoral CpG in just one tumor (Day 0=1 cm2; anti-idiotype antibody 100 μg Day 0; 100 μg CpG Days 2, 3, 4, 6 & 8), untreated tumors regressed just as well as CpG-treated tumors indicating a systemic anti-tumor immune response was generated. Conclusion: Anti-idiotype therapy, although effective in delaying tumor growth, frequently generates antigen-loss variants. However, we found that when anti-idiotype antibodies were combined with CpG, even large established tumors were cured due to systemic CD8+ T cell-dependent tumor immunity. Rather than simply mediating ADCC against a single tumor antigen, which requires the constant infusion of antibody to hamper tumor growth, we hypothesize a cytotoxic T-cell response against many tumor antigens was also generated. Such a diverse T-cell repertoire can prevent the emergence of tumor escapees and collectively provide long-lasting tumor protection. These pre-clinical results suggest that anti-tumor antibodies combined with CpG warrant further study in patients with B cell lymphoma.

scholarly journals The role of dendritic cells for therapy of B-cell lymphoma with immune checkpoint inhibitors

2020 ◽  
Author(s):  
Anne Scheuerpflug ◽  
Fatima Ahmetlić ◽  
Vera Bauer ◽  
Tanja Riedel ◽  
Martin Röcken ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
B Cell ◽  
Immune Checkpoint ◽  
Cell Lymphoma ◽  
T Cell Responses ◽  
B Cell Lymphoma ◽  
Cell Responses

Abstract Immune checkpoint blocking (ICB) is a promising new tool of cancer treatment. Yet, the underlying therapeutic mechanisms are not fully understood. Here we investigated the role of dendritic cells (DCs) for the therapeutic effect of ICB in a λ-MYC-transgenic mouse model of endogenously arising B-cell lymphoma. The growth of these tumors can be effectively delayed by antibodies against CTLA-4 and PD-1. Tumor-infiltrating DCs from mice having received therapy showed an upregulation of costimulatory molecules as well as an augmented IL-12/IL-10 ratio as compared to untreated controls. Both alterations seemed to be induced by interferon-γ (IFN-γ), which is upregulated in T cells and natural killer cells upon ICB. Furthermore, the enhanced IL-12/IL-10 ratio, which favors Th1-prone antitumor T-cell responses, was a consequence of direct interaction of ICB antibodies with DCs. Importantly, the capability of tumor-infiltrating DCs of stimulating peptide-specific or allogeneic T-cell responses in vitro was improved when DCs were derived from ICB-treated mice. The data indicate that ICB therapy is not only effective by directly activating T cells, but also by triggering a complex network, in which DCs play a pivotal role at the interface between innate and adaptive antitumor responses.

scholarly journals Analysis of CAR-T and Immune Cells within the Tumor Micro-Environment of Diffuse Large B-Cell Lymphoma Post CAR-T Treatment By Multiplex Immunofluorescence

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 678-678 ◽  
Author(s):  
Pei-Hsuan Chen ◽  
Mikel Lipschitz ◽  
Kyle Wright ◽  
Philippe Armand ◽  
Caron A. Jacobson ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract BACKGROUND: Axicabtagene ciloleucel is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy that shows efficacy in patients with refractory diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma and transformed follicular lymphoma after failure of conventional therapy. However, the exact mechanism of anti-tumor immunity is poorly understood, in part due to the dearth of data on the events in the tumor micro-environment (TME) that occur upon exposure to CAR-T cells. We sought to quantify and characterize both CAR-T cells and non-CAR T cells within the TME of DLBCL using tissue biopsy samples collected in the ZUMA-1 multicenter trial of CAR-T cell therapy for patients with refractory DLBCL. METHODS: Tumor samples obtained from patients 5-30 days (median 10 days) after CAR-T infusion ("CAR-treated", n=14) and randomly-selected untreated ("untreated ", n=15) archival DLBCL tissue samples were analyzed by multiplex immunofluorescence using formalin-fixed, paraffin embedded tissue sections, with successive labeling by the primary antibodies KIP-1 and/or KIP-3 (recognizing separate CD19 CAR epitopes), PAX5, PD-1, CD4, and CD8, followed by secondary amplification and tyramide-conjugated fluorophores. For each case, at least 3 representative 20x fields of view were selected and imaged using a multispectral imaging platform. Two specific image analysis algorithms were designed to accurately identify CD4 and CD8 T cells and PAX5+ DLBCL cells simultaneously, then to threshold PD-1 and KIP-1/-3 by relative fluorescent units (RFU) in each phenotype. RESULTS: We identified CAR T-cells within the fixed biopsy samples of CAR-treated DLBCLs by immunostaining with CAR T-cell specific antibody KIP-1; at the timepoints analyzed, CAR T-cells comprised only a small minority of total T- cells (<2%) and included CD4+ and CD8+ T-cells. Immunostaining with a second antibody, KIP-3, validated the presence of CAR T-cells in these cases and confirmed the KIP-1 results. Expression of the T cell activation marker PD-1 was detected among majority of KIP-1+ cells. Further analysis that included KIP1-negative cells revealed that the percentage of CD8+ cells co-expressing PD-1 across all CD8+ cells was higher in the CAR-treated DLBCLs compared to the untreated DLBCLs (mean 50.1% vs 17.5%, p<0.0001 with unpaired t test ), indicating CD8 T cell activation within the tumor environment. In contrast, PD-1 positivity across CD4+ T cells were equivalent between the two groups (mean 21.8% vs 21.6%, ns with unpaired t test). The percentages of total, CD4+, and CD8+ T-cell populations in the TME were similar between the CAR-treated DLBCL and untreated biopsies. CONCLUSIONS: CD4+ and CD8+ CAR-T cells can be detected in CAR-treated DLBCL patient tissue biopsies by multiplex immunofluorescence. At the time points analyzed to date, CAR-T cells comprise only a small percentage of all T-cells (<2%) within the TME. However, the presence of gene marked T cells with downregulated CAR protein expression is also possible. The activation marker PD-1 is preferentially expressed by KIP-1-negative CD8+ T cells compared to CD4+ T cells in CAR-T treated DLBCLs relative to untreated DLBCLs. These data implicate preferential activation of CD8+ non-CAR "by-stander" T-cells in the post CAR-T TME, and the possible benefit of combining PD-1 blockade with CAR-T therapy in DLBCL. *PH.C and M.L share equal contribution. Disclosures Armand: Otsuka: Research Funding; Affimed: Consultancy, Research Funding; Pfizer: Consultancy; Infinity: Consultancy; Adaptive: Research Funding; Merck: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Roche: Research Funding; Tensha: Research Funding. Roberts:KITE: Employment. Rossi:KITE: Employment. Bot:KITE: Employment. Go:KITE: Employment. Rodig:Merck: Research Funding; Bristol Myers Squibb: Research Funding; Affimed: Research Funding; KITE: Research Funding.

Targeting DKK1 for the Immunotherapy of B-Cell Lymphomas.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 465-465
Author(s):  
Jianfei Qian ◽  
Sungyoul Hong ◽  
Liang Zhang ◽  
Yuhuan Zheng ◽  
Haiyan Li ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cells ◽  
Specific Ctls

Abstract Abstract 465 Immunotherapy may complement the current treatments for lymphomas. The lack of suitable shared lymphoma-associated antigens limits its applicability. Therefore, identification and utilization of novel and more potent tumor-associated antigens, particularly those shared among patients, are urgently needed to improve the efficacy of immunotherapy in the diseases. Recent studies have shown that Dickkopf-1 (DKK1), a secreted protein and Wnt signaling pathway inhibitor, is highly expressed by myeloma and other tumor cells, and is absent from normal tissues and organs except placenta and prostate. In the present study we demonstrated that DKK1 is also overexpressed in mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Using DKK1 peptide-pulsed dendritic cells (DCs), we successfully generated HLA-A*0201+ DKK1-specific CTL lines and clones in vitro. These CTLs effectively lysed DKK1+/HLA-A*0201+ lymphoma cell lines Jeko-1 and Granta 519 cells, but not DKK1-/HLA-A*0201+ BJAB, RL and Mino cells nor DKK1+/HLA-A*020- CA46 and Daudi cells. Furthermore, the T-cell clones efficiently killed DKK1+/HLA-A*0201+ primary B-cell lymphoma cells from patients but not lymphoma cells from DKK1–/HLA-A*0201+ patients. HLA-ABC or HLA-A*0201 blocking mAbs significantly inhibited T cell-mediated cytotoxicity against peptide-pulsed T2 cells (P < .01, compared with medium control). No inhibitory effect was observed with mAb against HLA-DR and isotype control IgG. The results indicate that the cytotoxicity was attributed to MHC class I and more specifically, HLA-A*0201-restricted CD8+ CTLs. The CTLs did not kill DKK1–/HLA-A*0201+ DCs, B cells, or PBMCs, These results suggest that the CTLs recognized DKK1 peptides that are naturally processed and presented in the context of HLA-A*0201 molecules on lymphoma cells. To determine the in vivo antitumor activity, NOD-SCID and SCID-hu mice were used for lymphoma cell lines and primary lymphoma cells, respectively. Mice were treated with DKK1-specific CTLs after tumor established in NOD-SCID and SCID-hu mice. Control mice were treated with naïve CD8+ T cells or PBS alone. Tumor burden was measured according to levels of circulating human B2M, and survival rates were determined. Low levels (< 50 ng/ml) of circulating human B2M were detected in group treated DKK1-specific CTLs, while high levels (≥ 150 ng/ml) of circulating human B2M were detected in control mice. In SCID-hu model, X-ray examination showed that established tumors were eradicated in 60% mice treated with DKK1-specific CTLs, while large tumor burdens were found in all control mice. In NOD-SCID model, 40% of mice survived with the treatment of DKK1-specific CTLs. TUNEL assay further confirmed that tumor cells were lysed by DKK1-specific CTLs not naïve CD8+ T cells. These results indicate that DKK1-specific CTLs are able to eradicate established, patient-derived primary B- cell lymphoma in the hosts and adoptive transfer of DKK1-specific CTLs may be used for B-cell lymphoma therapy. Disclosures: No relevant conflicts of interest to declare.

Increased Peripheral T Cell Responses to EBV-Infected Cells with Frequent Detection of EBV-DNA In Plasma and Viral mRNA In Peripheral B-Cells In Immunocompetent EBV-Positive Diffuse Large B-Cell Lymphoma Patients

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4163-4163 ◽  
Author(s):  
Satoko Morishima ◽  
Kazuhito Yamamoto ◽  
Hiroshi Kimura ◽  
Seiko Iwata ◽  
Tomohiro Kinoshita ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
B Cell Lymphoma ◽  
Viral Mrna ◽  
Ebv Dna ◽  
Ifn Γ

Abstract Abstract 4163 Introduction: It is well known that immunocompromised patients(pts) such as post-transplantation or HIV infection are at increased risk of EBV-associated B-cell lymphoproliferaitive disorders. However, the immunological status of immunocompetent EBV-positive diffuse large B-cell Lymphoma (DLBCL) has not been well elucidated. For example, EBV-positive DLBCL in the elderly is defined as an EBV-positive clonal B-cell lymphoid proliferation occurring in pts more than 50 years of age and without any known immunodeficiency in WHO classification 2008, accounting for 5 – 10% of DLBCL in Japan. This disease is speculated to be related to the immunological deterioration accompanying the aging process. We conducted a multi-center prospective study to assess EBV status and the T cell response to EBV of peripheral blood (PB) in EBV-positive DLBCL in comparison with EBV-negative DLBCL pts and normal old-healthy persons (old-HPs). Patients and Methods: Fifteen newly-diagnosed pts with EBV-positive DLBCL, 8 pts with EBV-negative DLBCL, and 16 old-HPs were enrolled. Median ages of pts with EBV-positive DLBCL, pts with EBV-negative DLBCL, and old-HPs were 74.5 years (range 29–82 years), 71 years (48-79), and 65 years (60-74), respectively. Among the 15 pts with EBV-positive DLBCL, 2 were pyothorax-associated lymphoma, 2 were treated with methotrexate for rheumatoid arthritis, and the remaining 11 pts were without past histories predisposing to immunodeficiency. The diagnosis of EBV-positive DLBCL was made by positive signals for in situ hybridization using EBV-encoded small RNA (EBER) on paraffin section. To analyze T cell reactivity to autologous EBV-infected B-cell lymphoblastoid cell lines (LCL), CFSE-labeled peripheral blood mononuclear cells (PBMCs) were co-cultured with irradiated autologous LCL, and the division indices (DI) of CD4+ and CD8+ T cells were determined on day 5 (Cytometry 34:143, 1998). Percentage of proliferating CFSElow IFN-γ+CD4+ T cells and CFSElow IFN-γ+CD8+ T cells was assessed on day 7. EBV DNA load in PBMCs and plasma was determined by quantitative real-time PCR. CD19+ B cells were separated from of PBMCs by immunomagnetic sorting, and viral mRNA expression of B cells was quantified by one-step multiplex real-time RT-PCR. Results: (1) Plasma EBV-DNA was detectable in 13 of the 15 EBV-positive DLBCL pts but in none of the 8 EBV-negative DLBCL pts or the 16 old-HPs. Copy number of EBV-positive DLBCL was significantly higher than EBV-negative DLBCL (p<0.00001) or old-HPs (p=0.0001). EBV-DNA in PBMC was positive in 10 of the 15 EBV-positive DLBCL pts, 2 of 8 EBV-negative DLBCL pts, and 2 of 15 old-HPs. (2) EBER1 of PB B cells was detected in all 10 EBV-positive DLBCL pts, 4 of 7 EBV-negative DLBCL pts, and 7 of 14 old-HPs. BamHI A rightward transcripts (BARTs) of B-cells was detected in 8 of 10 EBV-positive DLBCL pts, 3 of 10 EBV-negative DLBCL pts, and 2 of 14 old-HPs. Latent membrane protein 2 (LMP2) of B cells was detected in 2 of 10 EBV-positive DLBCL pts, 2 of 14 old-HPs, and none of 5 EBV-negative DLBCL pts. LMP1 of B cells was detected in 1 of 10 EBV-positive DLBCL pts, none of 7 EBV-negative DLBCL pts, and none of 14 old-HPs. EBV-encoded nuclear antigen 1 (EBNA1) or EBNA2 were not detected in any of the pts nor old-HPs. (3) DI of CD4+ T cells for 5 days in EBV-positive DLBCL (median 1.41) was significantly higher than in old-HPs (median 0.53) (p=0.0009), and DI of CD8+ T cells of EBV-positive DLBCL (median 1.94) showed a higher tendency than old-HP (median 1.35). (4) Percentage of CFSE-low IFN-γ+CD4+ T cells in EBV-positive DLBCL (median 9.8%) was significantly higher than in old-HPs (median 2.2%) (p=0.002), and percentage of CFSE-low IFN-γ+CD8+ T cells in EBV-positive DLBCL (median 12.0%) was significantly higher than in old-HPs (median 6.6%)(p=0.025). Conclusions: Measurement of the plasma EBV-DNA copy number is a good indicator for the diagnosis of EBV-positive DLBCL, and the frequent detection of EBV viral mRNA of peripheral B-cells in EBV-positive DLBCL pts might be associated with greater viral load. Proliferative and INF-γ secreting responses of both peripheral CD4+ T cells and CD8+ T cells to EBV-infected cells were increased in EBV-positive DLBCL compared to old-HPs, which might be driven by an elevated viral load. These findings suggest that systemic immunological reaction to EBV might be intact in EBV-positive DLBCL. Disclosures: No relevant conflicts of interest to declare.

CD8+ /ICOS+ /CXCR5+ Tumor Infiltrating T-CELLS (CD8fc) Sharing Functional Similarities With TFH CELLS ARE Present In Classical Hodgkin’S Lymphoma Tissues Displaying A Specific “MIXED NODULARITY” Pattern

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4233-4233 ◽  
Author(s):  
Suong Le Thi ◽  
Florence Broussais ◽  
Reda Bouabdallah ◽  
Françoise gondois-Rey ◽  
Luc Xerri ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cd8 T Cells ◽  
Cell Lymphoma ◽  
Cell Subset ◽  
B Cell Lymphoma ◽  
Cd8 T Cell ◽  
Tissue Samples ◽  
Tfh Cells

Abstract We have previously reported that some classical Hodgkin’s Lymphoma (cHL) tissues display a gene signature evocative of a Th1 immune reaction. In order to better characterize this process, immune cell subsets were isolated from cHL tissue samples (n=21) using a powerful multicolor flow cytometry method, in parallel with cell sorting. Fresh tissue samples from follicular B cell lymphoma (FL, n=8), diffuse large cell B cell lymphoma (n=8) and reactive lymphadenitis (n= 5) were used as controls. In 4 cLH cases, we observed a significant proportion of activated CD8+ T-cells expressing ICOS and CXCR5 at high levels. The presence of either CD8+/ICOS+/CXCR5- T cells or CD8+/ICOS +/ CXCR5+ T-cells was a specific feature of HL tissues since it was absent from B-cell lymphomas, T-cell lymphomas and reactive tissues. In contrast, CD8+/CXCR5+ T-cells were found not only in cHL, but also in most other samples analyzed. Further phenotypic characterization showed that the CD8+/ICOS +/ CXCR5+ T cells expressed markers associated with CD4 TFH cells, like PD1, BTLA, bcl-6 and IL-21. Under stimulation, they expressed only low levels of IFNG, granzyme B and perforin, and thus do not fulfill the criteria of activated cytotoxic effectors. Co-culture experiments showed a dramatic enhancement of CD86 expression on stimulated B-cells in contact with CD8+/ICOS +/ CXCR5+ T cells. This effect was similarly observed after co-culture with CD4+TFH cells. The 4 cHL cases associated with CD8+/ICOS +/ CXCR5+ T-cells contained CD30+ CD15+ EBV+ Reed Sternberg cells (RSC). They were characterized a nodular non-sclerotic pattern reminiscent of the nodular lymphocyte-rich classical HL (NLRCHL) subtype, but also displayed a specific “mixed nodularity” feature. Various nodules were indeed observed, including reactive germinal centers (GC) partly colonized by RSC co-localizing with CD8+/ICOS+ T-cells, suggesting an early GC invasion triggering an intra-follicular CD8 T-cell reaction. Other nodules were composed of a high number of RS cells admixed with numerous CD8+/ICOS+ T-cells. This “mixed nodularity” pattern was absent in the other HL cases. Altogether, our results point out a previously unrecognized intra- follicular CD8 T-cell subset sharing phenotypic and functional features with CD4 TFH, that we have thus considered as putative “follicular cytotoxic” CD8 T-cells (TFC). This cell subset appears to be specifically associated with EBV+ cHL tissues with unusual histo-phenotypic features, which may probably reflect a strong CD8 activation process. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Intratumoral Injection of CpG-ODN Plus Systemic Ibrutinib Induces an Anti-Tumor Immune Response Affecting T Cell Subsets in the Microenvironment of Both Injected and Non-Injected Tumor Sites in Patients with Low-Grade Lymphoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1612-1612
Author(s):  
Debra K. Czerwinski ◽  
Matthew J. Frank ◽  
Tanaya Shree ◽  
Michael S Khodadoust ◽  
Steven R. Long ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Lymphoma ◽  
Granzyme B ◽  
B Cell Lymphoma ◽  
Low Grade ◽  
Effector Cells ◽  
Treated Site

Abstract BACKGROUND: Low-grade B cell lymphoma is often characterized by an infiltration of immune effector cells including T, NK and dendritic cells. But, despite the presence of effector cells within the tumor, these cells fail to control tumor growth. In a preclinical mouse model, we showed that Ibrutinib, an inhibitor of Bruton's tyrosine kinase (BTK), but also ITK (IL-2-inducible T cell kinase), synergized with intratumoral CpG to facilitate complete regression of tumors at the treated site as well as a distal, non-treated site, curing all the mice. This was accompanied by a potent anti-tumor memory T cell response by both CD4 and CD8 T cells that rejected the tumor on re-challenge. (Sagiv-Barfi I, et al. Blood. 2015 March:125(13):2079-2086) STUDY: In an ongoing clinical trial (NCT02927964), patients with previously treated low-grade lymphoma receive low dose (2Gyx2) radiotherapy to a single tumor site followed by 5 weekly intratumoral injections of 3 mg CpG-ODN (SD-101, Dynavax Technologies) into the same site. 1 day after the second injection, patients begin taking a daily 560 mg dose of Ibrutinib. A fine needle aspirate (FNA) of the injected site and a non-injected site outside the radiation field is performed prior to radiotherapy, one week after the first injection, and at week 6, 1 week after the final injection of CpG. FNA samples are stained for flow cytometry with panels of antibodies to delineate all major cell populations and their subsets. Cellular activation as well as T cell exhaustion, inhibition and function are also characterized. When feasible, a biopsy is performed prior to treatment providing tumor cells to be used in an immune response assay to evaluate induced anti-tumor responses by circulating peripheral blood T cells obtained throughout the study. RESULTS: To date, 12 patients have been entered onto the study. Of these patients, 4 had excisional biopsies and subsequent immune response assays performed. All 4 patients exhibited CD8 anti-tumor responses as determined by an increase in the activation marker CD137 as well as the functional marker granzyme B above pretreatment CD8 T cells. At week 12, 7 weeks after the last CpG injection, the average increase above pretreatment baseline for CD137 and granzyme B was 6.1% and 9.3%, respectively. In 3 of these patients, we observed an increase in CD8 T cells expressing CD137 and granzyme B from the FNA of the non-injected site. The 4th patient did not have adequate number of cells for staining. Two patients exhibited CD4 immune responses as characterized by an up-regulation of CD137 and CD278. FNA of 7 patients produced enough cells to analyze the tumor microenvironment from both the injected and the non-injected sites over all 3 time points; 1 patient was evaluable through week 2 for both sites. In 7 out of these 8 patients, CD3 T cells increased at the injected and non-injected sites by week 6. The proportion of CD4 and CD8 T cells did not stay constant, however, as reflected by the changes in CD4:CD8 ratios. This suggests that the increase in T cells was not purely the result of a loss of tumor B cells in the samples. Notably, we observed a significant increase in the effector CD4 T cells in all patients at the injected site by week 2 (14.1% ± 6.5%, p = 0.005) and 5 of 8 patients in the non-injected site by week 6 (12.4% ± 10.4%). Moreover, the proportion of T follicular helper cells significantly decreased in all patients at the treated site by week 2 (17.7% ± 9.7% of T cells, p = 0.0012) and in 5 of 8 patients at the non-treated site by week 6 (8.1% ± 5.5% of T cells). Tregs were more variable, decreasing in 5 of 8 patients in the treated and 3 of 8 patients in the non-treated site by week 6. CONCLUSION: CpG is known to activate antigen-presenting cells such as dendritic cells and macrophages. Ibrutinib is a small molecule that has been shown to have direct anti-tumor effects in B cell lymphoma and may skew an immune response towards that of a TH1 type. Here we show that together, they can effect changes in the tumor microenvironment in both treated and in untreated sites of disease. This clinical trial is ongoing and open to accrual. Disclosures Khodadoust: Innate Pharma: Research Funding.

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