Dasatinib-Induced Reduction of Tumor Growth Is Accompanied By the Changes in the Immune Profile in a Melanoma B16.OVA Mouse Model

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1408-1408
Author(s):  
Mette Matilda Ilander ◽  
Can Hekim ◽  
Markus Vähä-Koskela ◽  
Paula Savola ◽  
Siri Tähtinen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Cd8 T Cells ◽  
Research Funding ◽  
K562 Cells ◽  
Cell Cytotoxicity ◽  
Cd8 Cells

Abstract Background: Dasatinib is a 2nd generation tyrosine kinase inhibitor (TKI) used in the treatment of chronic myeloid leukemia (CML). Its kinase inhibition profile is broad and includes several kinases important in the immune cell function such as SRC kinases. Furthermore, it is known that dasatinib has immunomodulatory effects in vivo. Recently, we observed that dasatinib induces a rapid and marked mobilization of lymphocytes, which closely follows the drug plasma concentration. The phenomenon is accompanied by an increase of NK-cell cytotoxicity. In addition, we have shown that dasatinib alters T-cell responses long-term favoring Th1 type of responses. Interestingly, the dasatinib induced immune effects have been associated with better treatment responses. We now aimed to characterize the dasatinib-induced antitumor immune responses in a syngeneic murine melanoma model to address whether dasatinib-induced immunoactivation affects tumor growth. Methods: Direct cytotoxic effect of dasatinib on B16.OVA melanoma cells in vitro was assessed with an MTS cell viability assay. T-cell cytotoxicity was assessed by preincubating splenocytes isolated from naïve and OT-I mouse spleen with 100 nM dasatinib and measured their cytotoxic capacity against B16.OVA cells. To further evaluate the dasatinib induced antitumor immune effects in vivo, B16.OVA cells were implanted subcutaneously in C57BL/6J mice. The mice (n=6/group) were treated daily i.g. either with 30 mg/kg dasatinib or vehicle only. Blood was collected before tumor transplantation, before treatment, and on treatment days 4, 7 and 11. Tumor volumes were measured manually and specific growth rate was calculated based on the first and the last day of the treatment. In addition to white blood cell differential counts, immunophenotyping of blood and tumor homogenate was performed by flow cytometry using antibodies against CD45.1, CD3, CD4, CD8b, NK1.1, CTLA4, PD-1 and CD107. Immunohistochemical staining of CD8+ T-cells was performed from the paraffin embedded tumor samples. Results: In vitro incubation of B16.OVA cells with dasatinib showed only a moderate unspecific cytotoxicity with the two highest concentrations of dasatinib (1- and 10 µM), whereas in K562 cells (a CML blast crisis cell line) almost complete killing was observed already with the 100nM concentration. The cell viability of B16.OVA cells was 90% with at 100 nM of dasatinib concentration (as compared to 21% of K562 cells) suggesting that there was no direct dasatinib sensitive target oncokinase in this cell line. In contrast, a significant enhancement in the cytotoxic capacity of splenocytes was observed when they were pretreated with 100nM dasatinib (60% of target cells were alive when incubated with dasatinib pretreated naïve splenocytes compared to 100% with control treated splenocytes, p=0.004). The in vivo tumor experiments demonstrated that the tumor volumes were smaller in dasatinib group, and there was a significant decrease in the specific tumor growth rate (0.06 vs. 0.18, p=0.01) on the 11th day of treatment. Interestingly, dasatinib treated mice had increased proportion of CD8+cells in the circulation (17.9% vs. 14.4%, p=0.005) and the CD4/CD8 ratio was significantly decreased (1.39 vs. 1.52, p= 0.04). During the tumor growth the mean CTLA-4 expression on CD8+ cells in PB increased from 1.2% to 9% in the control group, whereas, in dasatinib group the increase was more modest (1.2% to 5.7%). When the tumor content was analyzed, dasatinib treated mice had significantly more tumor infiltrated CD8+ T-cells (median 17 vs. 4/counted fields, p=0.03). In dasatinib group 80% of the tumor infiltrating CD8+ cells expressed PD-1 antigen compared to <5% of PD1 positive CD8+ cells in the peripheral blood suggesting either tumor induced CD8 T-cell exhaustion or the presence of tumor-reactive effector cells. Lastly, when CD4 and CD8 cells were depleted before tumor inoculation, dasatinib was no longer able to slow down the tumor growth. Conclusions: Dasatinib treatment slowed the tumor growth in a B16.OVA mouse model. The growth retardation was due to immunomodulatory properties of dasatinib as the drug was not directly cytotoxic and depletion of T-cells abolished the effect. Dasatinib may be a therapeutically useful immunomodulatory agent for targeting tumor-associated anergy, particularly in combination with novel checkpoint inhibitors and tumor-targeting drugs. Disclosures Hemminki: Oncos Therapeutics Ltd: Shareholder Other; TILT BioTherapeutics Ltd: Employment, Shareholder, Shareholder Other. Porkka:BMS and Novartis: Honoraria, Research Funding; Pfizer: Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

scholarly journals CD28 Costimulation Is Required for In Vivo Induction of Peripheral Tolerance in CD8 T Cells

2002 ◽  
Vol 197 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Melanie S. Vacchio ◽  
Richard J. Hodes
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Tolerance Induction ◽  
Peripheral Tolerance ◽  
Cd8 Cells ◽  
Costimulatory Signal ◽  
Cd28 Costimulation

Whereas ligation of CD28 is known to provide a critical costimulatory signal for activation of CD4 T cells, the requirement for CD28 as a costimulatory signal during activation of CD8 cells is less well defined. Even less is known about the involvement of CD28 signals during peripheral tolerance induction in CD8 T cells. In this study, comparison of T cell responses from CD28-deficient and CD28 wild-type H-Y–specific T cell receptor transgenic mice reveals that CD8 cells can proliferate, secrete cytokines, and generate cytotoxic T lymphocytes efficiently in the absence of CD28 costimulation in vitro. Surprisingly, using pregnancy as a model to study the H-Y–specific response of maternal T cells in the presence or absence of CD28 costimulation in vivo, it was found that peripheral tolerance does not occur in CD28KO pregnants in contrast to the partial clonal deletion and hyporesponsiveness of remaining T cells observed in CD28WT pregnants. These data demonstrate for the first time that CD28 is critical for tolerance induction of CD8 T cells, contrasting markedly with CD28 independence of in vitro activation, and suggest that the role of CD28/B7 interactions in peripheral tolerance of CD8 T cells may differ significantly from that of CD4 T cells.

Tumor-Specific Donor Lymphocyte Infusion for Tumor Relapse after MHC-Haploidentical Hematopoietic Stem Cell Transplantation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2157-2157
Author(s):  
Isao Tawara ◽  
Kana Okamori ◽  
Naoyuki Katayama ◽  
Hiroshi Shiku ◽  
Hiroaki Ikeda
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Cd8 T Cell ◽  
Donor Lymphocyte Infusion ◽  
Hematopoietic Stem ◽  
Tumor Relapse

Abstract Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for refractory hematologic malignancies such as leukemia and myelodysplastic syndrome. Tumor relapse, graft-vs host disease (GVHD) and infections are, however, major obstacles to successful allo-HSCT. Donor lymphocyte infusion (DLI) is carried out in some cases of tumor relapse and infections after allo-HSCT. DLI potentially induces or aggravates GVHD due to allo-reactivity of the lymphocytes used for infusion and the efficacy of DLI for tumor relapse is actually limited. Therefore, development of the method of DLI with enhanced anti-tumor effects without induction or aggravation of GVHD is expected to improve the results of DLI in the context of tumor relapse. Tumor antigen-specific T cell receptor (TCR)-expressing cell infusion is one of the potentially effective immunotherapies for refractory tumors. We previously reported that tumor antigen-specific TCR-gene-transduced human lymphocytes engineered with a novel retrovirus vector silencing endogenous TCRs showed reduced allo-reactivity (ASH annual meeting 2014). Utilizing this technology, we may be able to enhance anti-tumor effects without induction or aggravation of GVHD. In this study, we conducted a mouse pre-clinical model that mimics tumor-specific TCR-engineered DLI for tumor relapse after MHC-haploidentical HSCT and explored the effect of DLI on tumor growth and GVHD. We performed experimental study utilizing the following model: MHC-haploidentical BALB/c(H-2d)→CB6F1(H-2b/d) HSCT, BALB/c-derived sarcoma CMS5a and T cells from TCR-transgenic mouse DUC18. CMS5a has a unique mutated form of a mitogen-activated kinase ERK2, and a nonamer peptide, called 9m, incorporating the resulting amino acid substitution is presented on H-2Kd and recognized by the CD8+ CTL clone C18. DUC18 is a BALB/c-background TCR transgenic line that expresses the rearranged Vα10.1 and Vβ8.3 genes of the C18. CD8+ T cells of DUC18 mice can suppress CMS5a tumor growth in the syngeneic BALB/c hosts. Allo-reactivity of DUC18 T cells was reduced as compared to that of wild type BALB/c T cells. Based on the results of preliminary experiments, we set standard experimental conditions as follows: X-ray irradiation (10 Gy) for pre-conditioning, 5x106 whole bone marrow and 0.25x106 T cell transplant (BMT) for mild GVHD induction, 1x106 CMS5a inoculation for progressive tumor growth in BMT recipients, and 4x106 CD8+ T cells for observation of the effect of DLI on tumor growth and GVHD. In some experiment, CD4+ T cells were co-infused with CD8+ T cells. DLI was carried out 3 days after CMS5a inoculation at 2, 4 or 8 weeks after BMT. We first used CD8+ T cells from naïve BALB/c or DUC18 mice for DLI at 8 weeks after BMT. No GVHD aggravation was observed in either BALB/c or DUC18 CD8+ T cell recipients and, as expected, suppression of tumor growth was observed only in the DUC18 CD8+ T cell recipients. Next, DLIs using CD8+ T cells only, or both CD8+ and CD4+ (CD8+/CD4+) T cells from naïve mice were carried out at 4 or 2 weeks after BMT. No apparent effect of CD4+ T cell co-infusion on tumor growth and GVHD was observed in both types of donor T cell recipients when DLI was carried out at 4 weeks after BMT. However, GVHD was aggravated in CD8+/CD4+ T cell recipients from both types of donors when DLI was carried out at 2 weeks after BMT, and no CD4-driven additional anti-tumor effect was observed in DUC18 T cell recipients. We then performed DLI using in vitro activated/expanded T cells including both CD8+ and CD4+ as a more clinically relevant model. BALB/c and DUC18 splenocytes were activated and expanded in vitro and infused (adjusted CD8+ T cell number to 4x106/mouse) into recipients at 2 or 8 weeks after BMT. Aggravation of GVHD was observed in either BALB/c or DUC18 activated/expanded T cell recipients when DLI was carried out at 2 weeks after BMT. On the other hand, it was not observed when DLI was carried out at 8 weeks after BMT. Anti-tumor effect of DUC18 T cells was observed regardless of timings of DLI. Taken together, infusion of tumor-specific donor lymphocytes with reduced allo-reactivity for tumor relapse after MHC-haploidentical HSCT will be promising strategy. Timing of DLI and condition of recipients such as inflammatory status may affect GVHD. Multiple model studies are required for development of more effective DLI strategies without aggravation of GVHD. Disclosures Tawara: Astellas: Honoraria. Katayama:Nippon Shinyaku: Honoraria; Chugai: Honoraria, Research Funding; Alexion Pharmaceuticals: Honoraria; Dainippon Sumitomo Pharma: Honoraria; Pfizer: Honoraria; Celgene: Honoraria; Shionogi: Honoraria; Kyowa Hakko Kirin: Honoraria, Research Funding; Taisho Toyama Pharma: Honoraria; Shire: Honoraria; Astellas: Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Eisai: Honoraria; Takeda: Honoraria; Bristol-Myers Squibb Japan: Honoraria. Ikeda:Ono Pharmaceutical Co., Ltd: Honoraria; Daiichi Sankyo Co., Ltd: Honoraria.

scholarly journals Predictors of T Cell Expansion and Clinical Responses Following B-Cell Maturation Antigen-Specific Chimeric Antigen Receptor T Cell Therapy (CART-BCMA) for Relapsed/Refractory Multiple Myeloma (MM)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1974-1974 ◽  
Author(s):  
Adam D. Cohen ◽  
J. Joseph Melenhorst ◽  
Alfred L. Garfall ◽  
Simon F Lacey ◽  
Megan Davis ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Advisory Committees ◽  
Car T ◽  
Equity Ownership

Abstract Background: Relapsed/refractory (rel/ref) MM is associated with progressive immune dysfunction, including reversal of CD4:CD8 T cell ratio and acquisition of terminally-differentiated T cell phenotypes. BCMA-directed CAR T cells have promising activity in MM, but the factors that predict for robust in vivo expansion and responses are not known. In a phase 1 study of CART-BCMA (autologous T cells expressing a human BCMA-specific CAR with CD3ζ/4-1BB signaling domains) in refractory MM patients (median 7 priors, 96% high-risk cytogenetics), we observed partial response (PR) or better in 12/25 (47%) (Cohen et al, ASH 2017, #505). Recently, we demonstrated in CLL pts receiving CD19-directed CAR T cells that certain T cell phenotypes prior to generation of the CAR T product were associated with improved in vivo expansion and clinical outcomes (Fraietta et al, Nat Med 2018). We thus sought to identify pre-treatment clinical or immunological features associated with CART-BCMA expansion and/or response. Methods: Three cohorts were enrolled: 1) 1-5 x 108 CART cells alone; 2) cyclophosphamide (Cy) 1.5 g/m2 + 1-5 x 107 CART cells; and 3) Cy 1.5 g/m2 + 1-5 x 108 CART cells. Phenotypic analysis of peripheral blood (PB) and bone marrow (BM) mononuclear cells, frozen leukapheresis aliquots, and phenotype and in vitro kinetics of CART-BCMA growth during manufacturing were performed by flow cytometry. CART-BCMA in vivo expansion was assessed by flow cytometry and qPCR. Responses were assessed by IMWG criteria. Results: Responses (≥PR) were seen in 4/9 pts (44%, 1 sCR, 2 VPGR, 1 PR) in cohort 1; 1/5 (20%, 1 PR) in cohort 2; and 7/11 (64%, 1 CR, 3 VGPR, 3 PR) in cohort 3. As of 7/9/18, 3/25 (12%) remain progression-free at 11, 14, and 32 months post-infusions. As previously described, responses were associated with both peak in vivo CART-BCMA expansion (p=0.002) as well as expansion over first month post-infusion (AUC-28, p=0.002). No baseline clinical or MM-related characteristic was significantly associated with expansion or response, including age, isotype, time from diagnosis, # prior therapies, being quad- or penta-refractory, presence of del 17p or TP53 mutation, serum hemoglobin, BM MM cell percentage, MM cell BCMA intensity, or soluble BCMA concentration. Treatment regimen given before leukapheresis or CART-BCMA infusions also had no predictive value. We did find, however, that higher CD4:CD8 T cell ratios within the leukapheresis product were associated with greater in vivo CART-BCMA expansion (Spearman's r=0.56, p=0.005) and clinical response (PR or better; p=0.014, Mann-Whitney). In addition, and similar to our CLL data, we found that a higher frequency of CD8 T cells within the leukapheresis product with an "early-memory" phenotype of CD45RO-CD27+ was also associated with improved expansion (Spearman's r=0.48, p=0.018) and response (p=0.047); Analysis of manufacturing data confirmed that higher CD4:CD8 ratio at culture start was associated with greater expansion (r=0.41, p=0.044) and, to a lesser degree, responses (p=0.074), whereas absolute T cell numbers or CD4:CD8 ratio in final CART-BCMA product was not (p=NS). In vitro expansion during manufacturing did associate with in vivo expansion (r=0.48, p=0.017), but was not directly predictive of response. At the time of CART-BCMA infusion, the frequency of total T cells, CD8+ T cells, NK cells, B cells, and CD3+CD56+ cells within the PB or BM was not associated with subsequent CART-BCMA expansion or clinical response; higher PB and BM CD4:CD8 ratio pre-infusion correlated with expansion (r=0.58, p=0.004 and r=0.64, p=0.003, respectively), but not with response. Conclusions: In this study, we found that CART-BCMA expansion and responses in heavily-pretreated MM patients were not associated with tumor burden or other clinical characteristics, but did correlate with certain immunological features prior to T cell collection and manufacturing, namely preservation of normal CD4:CD8 ratio and increased frequency of CD8 T cells with a CD45RO-CD27+ phenotype. This suggests that patients with less dysregulated immune systems may generate more effective CAR T cell products in MM, and has implications for optimizing patient selection, timing of T cell collection, and manufacturing techniques to try to overcome these limitations in MM patients. Disclosures Cohen: Celgene: Consultancy; Novartis: Research Funding; Oncopeptides: Consultancy; Janssen: Consultancy; Poseida Therapeutics, Inc.: Research Funding; Bristol Meyers Squibb: Consultancy, Research Funding; Kite Pharma: Consultancy; GlaxoSmithKline: Consultancy, Research Funding; Seattle Genetics: Consultancy. Melenhorst:Parker Institute for Cancer Immunotherapy: Research Funding; novartis: Patents & Royalties, Research Funding; Casi Pharmaceuticals: Consultancy; Incyte: Research Funding; Shanghai UNICAR Therapy, Inc: Consultancy. Garfall:Amgen: Research Funding; Kite Pharma: Consultancy; Bioinvent: Research Funding; Novartis: Research Funding. Lacey:Novartis Pharmaceuticals Corporation: Patents & Royalties; Parker Foundation: Research Funding; Tmunity: Research Funding; Novartis Pharmaceuticals Corporation: Research Funding. Davis:Novartis Institutes for Biomedical Research, Inc.: Patents & Royalties. Vogl:Karyopharm Therapeutics: Consultancy. Pruteanu:Novartis: Employment. Plesa:Novartis: Research Funding. Young:Novartis: Patents & Royalties, Research Funding. Levine:Novartis: Consultancy, Patents & Royalties, Research Funding; CRC Oncology: Consultancy; Incysus: Consultancy; Tmunity Therapeutics: Equity Ownership, Research Funding; Brammer Bio: Consultancy; Cure Genetics: Consultancy. June:Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Stadtmauer:Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AbbVie, Inc: Research Funding; Janssen: Consultancy. Milone:Novartis: Patents & Royalties.

C5a Complement Depletion Suppresses In Vivo B Lymphoma Progression and Enhances Development Of Cellular Anti-Tumor Immune Response

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1837-1837
Author(s):  
Suresh Veeramani ◽  
George J. Weiner
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Tumor Growth ◽  
Tumor Progression ◽  
Cd8 T Cells ◽  
B Lymphoma

Abstract Background Proteins within the complement system have complex effects on cellular immune responses. In previous studies, we found that active complement components, especially C5a, can dampen the development of antigen-specific immune responses following vaccination with a model antigen, in part by promoting generation of APC-induced T regulatory (Treg) cells. These studies also demonstrated that B lymphoma cell lines exposed to complement can induce Treg generation in vitro. The current study was designed to address whether depletion of C5a could enhance development of a cellular anti-lymphoma immune response in vivo. Methods Immunocompetent Balb/C mice were inoculated subcutaneously with syngeneic A20 B lymphoma cells mixed with either 10 μg of rat anti-mouse C5a monoclonal antibody (mAb) or 10 μg of isotype-matched Rat IgG2a control mAb. Tumor growth was followed. In select experiments, mice were sacrificed and analyzed for the percentage and activity of tumor-infiltrating T cells and A20-specific splenic T cell responses. Results 1. Tumor progression. Lymphoma grew more slowly in mice treated with anti-C5a mAb compared to mice treated with control mAb (p<0.05) {Fig. 1). 2. Intratumoral T cells. Tumors from mice treated with anti-C5a mAb had higher CD8+ T cell infiltration compared to mice treated with control mAb (p=0.002) (Fig. 2). Tumor-infiltrating CD8+ T cells showed a trend towards higher intracellular IFNg production in mice treated with anti-C5a mAb compared to control mAb (p=0.051). 3. Splenic T cells. Splenic T cells from mice treated with anti-C5a mAb produced IFNg to a greater degree than did splenic T cells from control mice when splenocytes were cultured with irradiated A20 cells in vitro (p=0.041) (Fig. 3). There was a trend towards decreased numbers of splenic CD4+CD25highFoxp3+ Tregs in C5a-depleted mice compared to control mice. Conclusions Depletion of C5a at the site of tumor inoculation slows tumor growth and increases the number of tumor infiltrating CD8 T cells in a syngenic immunocompetent model of lymphoma. A trend towards enhanced production of IFNg in the tumor infiltrating T cells, increased numbers of tumor-specific splenic T cells, and reduced numbers of splenic Tregs, suggests intratumoral C5a depletion can enhance tumor-specific immune responses both within the tumor and systemically. Ongoing studies are exploring the molecular mechanisms involved in C5a-promoted tumor progression and the use of C5a depletion as a novel strategy to improve anti-tumor immunity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals In CLL, Myeloid-Derived Suppressor Cells and Their Monocytic and Granulocytic Varieties Differ in T-Cell Subset Association and Polarization Induction

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4350-4350
Author(s):  
Gerardo Ferrer ◽  
Brendan Franca ◽  
Pui Yan Chiu ◽  
Stefano Vergani ◽  
Andrea Nicola Mazzarello ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Th17 Cells ◽  
Research Funding ◽  
Suppressor Cells ◽  
Myeloid Derived Suppressor Cells ◽  
Cd8 Cells

Abstract In chronic lymphocytic leukemia (CLL) monoclonal B cells expand and progressively accumulate in the bone marrow, eventually migrating to secondary lymphoid organs for even greater proliferation. At both sites, CLL cells engage in complex, incompletely defined cellular and molecular interactions involving multiple cell types such as T cells, myeloid cells, mesenchymal stromal cells, and matrix, collectively referred to as the "tumor microenvironment". This microenvironment is critical for the survival and proliferation of CLL cells, and data indicate that T cells and myeloid cells have an important role in these processes. In this study, we focus on two cells types: CD4+ T lymphocytes and myeloid-derived suppressor cells (MDSCs). In CLL patients, these populations are altered and impact on clinical outcome. CD4+ T cells comprise several subtypes, and CLL patients often have expanded Th2 and Tregs populations, consistent with the immunosuppressive status of these patients. Moreover, patients with higher numbers of another CD4+ subset, Th17 cells that produce IL-17 and other pro-inflammatory cytokines, can have longer survival times. Although studied minimally in CLL, MDSCs are known suppressors of T cell proliferation in vitro, and expand along with malignant cells in several cancers. However, no information is available about their effects on CD4+ T cell differentiation or on B-cell biology in CLL. In a cohort of 56 untreated CLL patients, we first explored correlation of the numbers of MDSCs and autologous T cells, using flow cytometry. CD3+ cell numbers significantly paralleled total MDSCs and monocyte-like MDSCs (mMDSCs) (P = 0.002, Spearman r = 0.44; P = 0.004, Spearman r = 0.41, respectively). Interestingly, MDSCs correlated with CD4+ and CD8+ T-cells (P < 0.001, Spearman r = 0.646; P < 0.001, Spearman r = 0.61, respectively). However, the correlation of MDSC subpopulations with CD4+ and CD8+ cells differed: mMDSCs associated significantly with CD4+ cells (P < 0.001, Spearman r = 0.73) and granulocyte-like MDSCs (gMDSCs) with CD8+ cells (P= 0.008; Spearman r = 0.45). Furthermore, although gMDSCs did not correlate with the numbers of CD4+ T-cells, we observed that they positively paralleled Tregs defined as CD3+/CD4+/CD25+/CD127-/FoxP3+ cells (P = 0.020, Spearman r = 0.44). Other subpopulations are currently under study. To address the effect of MDSCs on CD4+ cell differentiation, we FACS sorted CD3+/CD45RO- naïve CD4+ lymphocytes and stimulated them in vitro with anti-CD3/CD28 beads and IL2 in the presence or absence of mMDSCs (HLA-DRlo/CD11b+/CD33+/CD14+), gMDSCs (HLA-DRlo/CD11b+/CD33+/CD15+) or monocytes (HLA-DRhi/CD11b+/CD14+); these studies involved samples from 3 CLLs and 3 healthy controls (HCs). On day 7, cells were harvested and cytokine production was quantified by intracellular flow cytometry as the percentages of the following populations: Th1 (INFγ), Th2 (IL-4), Tregs (FoxP3), Th17 (IL-17A and IL-17F), Th9 (IL-9) and Th22 (IL-22). Culturing CLL or HC T cells in the absence of MDSCs revealed a lower percentage of cytokine-producing cells (24% vs. 55%; P = 0.017) in CLL, which was mainly due to a reduction in IL-4+ cells (P = 0.066). However, when analyzing the effects of MDSC subsets on the polarization of CLL or HC T cell, gMDSCs promoted significantly more FoxP3+ and less IL-22+ cells in CLL than in HC (P = 0.025 and P = 0.048, respectively). When analyzing only CLL T cells, supplementation with mMDSCs induced a reduction in IL-22+ cells (P = 0.027) and an insignificant increase of IL-4+ and IL-17+ cells. Conversely monocytes supported an expansion of INFγ+ T-cells (P=0.066), and gMDSCS promoted an increase of IL-9+ cells (P = 0.046) and a reduction of FoxP3+ cells (P = 0.019). In summary, in CLL the absolute numbers of total MDSCs and T cells are tightly linked. There is a significant correlation between CD4+ T cells and mMDSCs, and between CD8+ T cells and gMDSCs. Additionally, in CLL naïve CD4+ differentiation appears reduced compared to HC, in concordance with lower T-cell responses previously reported. Moreover, the preliminary aspects of the study suggest that CLL mMDSCs promote an expansion of Th2, Th17 cells and a reduction of Th22 cells, and monocytes enhance Th1s. Unexpectedly, since we observed a significant positive correlation in the PBMCs, gMDSCs may reduce Tregs and augment Th9. These findings depict differential consequences of CLL T cell - MDSC / mMDSC / gMDSC interactions. Disclosures Stamatopoulos: Abbvie: Honoraria, Other: Travel expenses; Gilead: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding.

scholarly journals The Absence of NLRP6 in Donor T Cells Exacerbates Gvhd

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2766-2766
Author(s):  
Masahiro Suto ◽  
Eri Matsuki ◽  
Masahiro Miyata ◽  
Erika Sekiguchi ◽  
Hiroya Tamaki ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Cell Responses ◽  
Donor T Cells ◽  
Induced Apoptosis

Abstract The Nlrp6 (NOD-like receptor family pyrin domain containing 6) inflammasome is important for intestinal epithelial cell innate immune responses and for maintaining gut homeostasis by preventing microbial dysbiosis. Contrary to its role in epithelial cell inflammasome-mediated responses, we recently showed that Nlrp6 in gut epithelial cells exacerbates GVHD in a manner independent of the inflammasome or gut microbiota. However, donor allogeneic T cells are also critical for GVHD development, yet, the function of Nlrp6 in allogeneic T cells is unknown. We hypothesized that Nlrp6 deficient donor T cells would ameliorate experimental GVHD. To test our hypothesis, WT-BALB/crecipients were lethally irradiated and transplanted on day 0 with 5x10 6 bone marrow and 1.0x10 6 splenic CD90 +T cells from either syngeneic WT-BALB/c, allogeneic MHC-mismatched WT-B6 or Nlrp6 -/- donors. Contrary to our hypothesis, the survival of allogeneic recipients of Nlrp6 -/- donor T cells was significantly worse than those receiving WT-B6 T cells (p&lt;0.05). Nlrp6 -/- donor T cells also caused greater GVHD mortality and morbidity in an MHC mismatched haploidentical B6 into B6D2F1 model (p&lt;0.05) and an MHC mismatched B10.BR into B6 model. Similar results were obtained using B6 into BALB/c and B6 into B6D2F1 models performed at the University of Michigan, suggesting our results were not unique to local environmental factors. By contrast, GVHD severity and mortality were similar in an MHC matched multiple minor antigen mismatched B6 into C3H.sw model. Because the B6 into C3H.sw model is largely driven by CD8+ T cells whereas the previous models are mediated by both CD4+ and CD8+ T cells, we examined whether Nlrp6 separately regulates CD4+ and CD8+ T cell-mediated GVHD. In order to test this, we transplanted C3H.sw recipients as above except we infused either 1x10 6 CD4+ or CD8+ T cells from B6-WT or Nlrp6 -/- animals. GVHD severity and mortality (P&lt;0.05) were enhanced only when Nlrp6 -/- CD4+ T cells were transplanted. These data suggested that Nlrp6 regulates allogeneic T cell responses in a subset-specific manner. To explore how Nlrp6 regulates intrinsic responses in donor T cell subsets, we tested naïve T cell proliferation in vitro after allogeneic or non-specific TCR stimulation. Consistent with the lack of increased GVHD induced by CD8+ Nlrp6 -/- donor T cells in the B6 into C3H.sw model, Nlrp6 -/- CD4+ but not CD8+ T cells proliferated more than WT-B6 CD4+ or CD8+ T cells, respectively, when stimulated with either anti-CD3/CD28 antibodies or lethally irradiated allogeneic antigen presenting cells in a mixed lymphocyte reaction. In addition, activation-induced apoptosis was decreased in Nlrp6 -/- CD4+ T cells compared to WT T cells. Importantly, Treg suppressive function was not altered in Nlrp6 -/- T cells. Therefore, increased proliferative responses and resistance to activation-induced apoptosis may have contributed to the enhanced GVHD caused by Nlrp6 -/- donor T cells. Increased Th1 and Th17 polarization is associated with worse GVHD. Because only CD4+ Nlrp6 -/- T cells enhanced GVHD, we tested whether Nlrp6 influenced T helper cell differentiation into Th1, Th17, and Th2 subsets. Consistent with our in vivo data, Th1 in vitro differentiation was enhanced in Nlrp6 -/- CD4+ T cells. To determine the molecular signaling events altered by Nlrp6 deficiency, we tested various T cell activation signaling pathways and found that phosphorylation of ZAP-70 was increased in Nlrp6 -/- T cells. These data suggested that Nlrp6 in donor T cells may regulate allo-immune responses via ZAP-70 pathway. GVH and graft-versus-tumor (GVT) responses are intricately linked. Because CD8+ responses were not affected by Nlrp6 deficiency, we hypothesized that GVT responses would be unaltered in Nlrp6 -/- donor T cells. Indeed, Nlrp6 -/- T cells showed equivalent in vivo GVL responses to MLL-AF4 leukemia cells as WT-T cells. Hence Nlrp6in donor T cells is not required for GVT responses. Altogether our data suggested that Nlrp6 negatively-regulates allogeneic donor CD4+ T cell responses, possibly via negative regulation of ZAP-70 signaling, resulting in mitigation of GVHD and maintenance of robust GVT responses. Disclosures Ishizawa: AbbVie: Research Funding; Eisai: Honoraria; Chugai: Honoraria; Ono: Honoraria; Celgene: Honoraria; Takeda: Honoraria; Bayer: Research Funding; Bristol Myers Squibb: Speakers Bureau; Pfizer: Research Funding; Kyowa Kirin: Consultancy; SymBio: Honoraria, Research Funding; Otsuka: Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau; Sanofi: Research Funding; IQVIA: Research Funding.

627 The DLL3-targeted half-life extended bispecific T cell engager (HLE BiTE®) immune-oncology therapy AMG 757 has potent antitumor activity in neuroendocrine cancer

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A663-A663
Author(s):  
Keegan Cooke ◽  
Juan Estrada ◽  
Jinghui Zhan ◽  
Jonathan Werner ◽  
Fei Lee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Mouse Models ◽  
T Cell Activation ◽  
Phase 1 ◽  
Phase 1 Study ◽  
Human T Cells

BackgroundNeuroendocrine tumors (NET), including small cell lung cancer (SCLC), have poor prognosis and limited therapeutic options. AMG 757 is an HLE BiTE® immune therapy designed to redirect T cell cytotoxicity to NET cells by binding to Delta-like ligand 3 (DLL3) expressed on the tumor cell surface and CD3 on T cells.MethodsWe evaluated activity of AMG 757 in NET cells in vitro and in mouse models of neuroendocrine cancer in vivo. In vitro, co-cultures of NET cells and human T cells were treated with AMG 757 in a concentration range and T cell activation, cytokine production, and tumor cell killing were assessed. In vivo, AMG 757 antitumor efficacy was evaluated in xenograft NET and in orthotopic models designed to mimic primary and metastatic SCLC lesions. NSG mice bearing established NET were administered human T cells and then treated once weekly with AMG 757 or control HLE BiTE molecule; tumor growth inhibition was assessed. Pharmacodynamic effects of AMG 757 in tumors were also evaluated in SCLC models following a single administration of human T cells and AMG 757 or control HLE BiTE molecule.ResultsAMG 757 induced T cell activation, cytokine production, and potent T cell redirected killing of DLL3-expressing SCLC, neuroendocrine prostate cancer, and other DLL3-expressing NET cell lines in vitro. AMG 757-mediated redirected lysis was specific for DLL3-expressing cells. In patient-derived xenograft and orthotopic models of SCLC, single-dose AMG 757 effectively engaged human T cells administered systemically, leading to a significant increase in the number of human CD4+ and CD8+ T cells in primary and metastatic tumor lesions. Weekly administration of AMG 757 induced significant tumor growth inhibition of SCLC (figure 1) and other NET, including complete regression of established tumors and clearance of metastatic lesions. These findings warranted evaluation of AMG 757 (NCT03319940); the phase 1 study includes dose exploration (monotherapy and in combination with pembrolizumab) and dose expansion (monotherapy) in patients with SCLC (figure 2). A study of AMG 757 in patients with neuroendocrine prostate cancer is under development based on emerging data from the ongoing phase 1 study.Abstract 627 Figure 1AMG 757 Significantly reduced tumor growth in orthotopic SCLC mouse modelsAbstract 627 Figure 2AMG 757 Phase 1 study designConclusionsAMG 757 engages and activates T cells to kill DLL3-expressing SCLC and other NET cells in vitro and induces significant antitumor activity against established xenograft tumors in mouse models. These preclinical data support evaluation of AMG 757 in clinical studies of patients with NET.Ethics ApprovalAll in vivo work was conducted under IACUC-approved protocol #2009-00046.

scholarly journals IMMU-31. QUANTITATIVE EVALUATION OF ROUTES OF ADMINISTRATION OF IMMUNOTHERAPEUTIC T CELLS TO ORTHOTOPIC GLIOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii111-ii111
Author(s):  
Lan Hoang-Minh ◽  
Angelie Rivera-Rodriguez ◽  
Fernanda Pohl-Guimarães ◽  
Seth Currlin ◽  
Christina Von Roemeling ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Ex Vivo ◽  
Adoptive T Cell Therapy ◽  
Whole Body ◽  
T Cell Therapy ◽  
Clinical Responses

Abstract SIGNIFICANCE Adoptive T cell therapy (ACT) has emerged as the most effective treatment against advanced malignant melanoma, eliciting remarkable objective clinical responses in up to 75% of patients with refractory metastatic disease, including within the central nervous system. Immunologic surrogate endpoints correlating with treatment outcome have been identified in these patients, with clinical responses being dependent on the migration of transferred T cells to sites of tumor growth. OBJECTIVE We investigated the biodistribution of intravenously or intraventricularly administered T cells in a murine model of glioblastoma at whole body, organ, and cellular levels. METHODS gp100-specific T cells were isolated from the spleens of pmel DsRed transgenic C57BL/6 mice and injected intravenously or intraventricularly, after in vitro expansion and activation, in murine KR158B-Luc-gp100 glioma-bearing mice. To determine transferred T cell spatial distribution, the brain, lymph nodes, heart, lungs, spleen, liver, and kidneys of mice were processed for 3D imaging using light-sheet and multiphoton imaging. ACT T cell quantification in various organs was performed ex vivo using flow cytometry, 2D optical imaging (IVIS), and magnetic particle imaging (MPI) after ferucarbotran nanoparticle transfection of T cells. T cell biodistribution was also assessed in vivo using MPI. RESULTS Following T cell intravenous injection, the spleen, liver, and lungs accounted for more than 90% of transferred T cells; the proportion of DsRed T cells in the brains was found to be very low, hovering below 1%. In contrast, most ACT T cells persisted in the tumor-bearing brains following intraventricular injections. ACT T cells mostly concentrated at the periphery of tumor masses and in proximity to blood vessels. CONCLUSIONS The success of ACT immunotherapy for brain tumors requires optimization of delivery route, dosing regimen, and enhancement of tumor-specific lymphocyte trafficking and effector functions to achieve maximal penetration and persistence at sites of invasive tumor growth.

scholarly journals DNAM-1 promotes activation of cytotoxic lymphocytes by nonprofessional antigen-presenting cells and tumors

2008 ◽  
Vol 205 (13) ◽  
pp. 2965-2973 ◽  
Author(s):  
Susan Gilfillan ◽  
Christopher J. Chan ◽  
Marina Cella ◽  
Nicole M. Haynes ◽  
Aaron S. Rapaport ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Adhesion Molecules ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Cd8 T Cell ◽  
Antigen Presenting

Natural killer (NK) cells and CD8 T cells require adhesion molecules for migration, activation, expansion, differentiation, and effector functions. DNAX accessory molecule 1 (DNAM-1), an adhesion molecule belonging to the immunoglobulin superfamily, promotes many of these functions in vitro. However, because NK cells and CD8 T cells express multiple adhesion molecules, it is unclear whether DNAM-1 has a unique function or is effectively redundant in vivo. To address this question, we generated mice lacking DNAM-1 and evaluated DNAM-1–deficient CD8 T cell and NK cell function in vitro and in vivo. Our results demonstrate that CD8 T cells require DNAM-1 for co-stimulation when recognizing antigen presented by nonprofessional antigen-presenting cells; in contrast, DNAM-1 is dispensable when dendritic cells present the antigen. Similarly, NK cells require DNAM-1 for the elimination of tumor cells that are comparatively resistant to NK cell–mediated cytotoxicity caused by the paucity of other NK cell–activating ligands. We conclude that DNAM-1 serves to extend the range of target cells that can activate CD8 T cell and NK cells and, hence, may be essential for immunosurveillance against tumors and/or viruses that evade recognition by other activating or accessory molecules.

scholarly journals 708 Novel ways to exploit IL-21 to augment adoptive T cell transfer therapy against tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A737-A737
Author(s):  
Anna Cole ◽  
Guillermo Rangel RIvera ◽  
Aubrey Smith ◽  
Megan Wyatt ◽  
Brandon Ware ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Striking Difference ◽  
T Cell Therapy ◽  
Cell Immunity ◽  
Emory University

BackgroundIL-21 enhances the anti-tumor capacity of adoptively transferred CD8+ T cells, while IL-2 and IL-15 impair T cell immunity by driving their expansion to a more differentiated status. Yet, these cytokines can act on many different immune cells. Given the potency of IL-21, we tested if this cytokine directly augments T cells or rather if it enhances other immune cells in the culture that indirectly improves T cell therapy.MethodsTo test this question, splenocytes from pmel-1 transgenic mice were used, as all CD8+ T cells express a transgenic TCR specific for tumor-antigen gp10025–33 overexpressed on melanoma. We then peptide activated naïve CD8+ T cells enriched or not from the spleen of pmel-1 mice and expanded them in the presence of IL-21 or IL-2 (10 ng/mL) for four days. Expanded pmel-1 from these various cultures were then restimulated with irradiated splenocytes pulsed with gp10025–33 and grown an additional seven days with IL-2 (10 ng/mL), irrespective of their initial cytokine condition. The in vitro memory phenotype, exhaustion profile, and cytokine secretion of these cultures were then assayed. Furthermore, mice bearing B16KVP melanoma tumors were infused with pmel-1 T cells expanded via these various approaches and compared for their relative capacity to engraft, persist, and regress tumor in vivo.ResultsInterestingly, we discovered that IL-21-treated T cells generated from bulk splenocytes are phenotypically and functionally distinct from IL-21-treated isolated T cells. Upon restimulation, IL-21-treated T cells from bulk splenocytes exhibited an exhausted phenotype that was like anergic IL-2-treated T cells. Moreover, few cells expressed CD62L but expressed heightened markers of suppression, including TIM3, PD-1, and EOMES. Moreover, they produced more effector molecules, including granzyme B and IFN-gamma. In vivo IL-21-treated T cells expanded from bulk splenocytes engrafted and persisted poorly, in turn mediating suboptimal regression of melanoma. Conversely, IL-21 dramatically bolstered the engraftment and antitumor activity of T cells only if they were first isolated from the spleen prior to their expansion and infusion into the animal.ConclusionsCollectively, our data shows that IL-21 may improve ACT therapy best when used directly on antitumor CD8+ T cells. Further studies will illuminate the mechanism behind this striking difference and determine whether other cell subsets reactive to IL-21 cause T cell dysfunction and/or reduced bioavailability. These findings are important for defining the best culture conditions in which to use IL-21 for ACT.AcknowledgementsWe would like to acknowledge Emory University, The Winship Cancer Institute, and the Pediatrics/Winship Flow Cytometry Core.Ethics ApprovalAll animal procedures were approved by the Institutional Animal Care and Use Committee of Emory University, protocol number 201900225.

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