scholarly journals PD-1 Blockade Modulates Functional Activities of Exhausted-Like T Cell in Patients With Cutaneous Leishmaniasis

2021 ◽  
Vol 12 ◽  
Author(s):  
Renan Garcia de Moura ◽  
Luciana Polaco Covre ◽  
Carlos Henrique Fantecelle ◽  
Vitor Alejandro Torres Gajardo ◽  
Carla Baroni Cunha ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cutaneous Leishmaniasis ◽  
Cell Function ◽  
Parasite Clearance ◽  
Skin Lesions ◽  
T Cell Subsets ◽  
Leishmania Braziliensis ◽  
T Cell Function

Patients infected by Leishmania braziliensis develop debilitating skin lesions. The role of inhibitory checkpoint receptors (ICRs) that induce T cell exhaustion during this disease is not known. Transcriptional profiling identified increased expression of ICRs including PD-1, PDL-1, PDL-2, TIM-3, and CTLA-4 in skin lesions of patients that was confirmed by immunohistology where there was increased expression of PD-1, TIM-3, and CTLA-4 in both CD4+ and CD8+ T cell subsets. Moreover, PDL-1/PDL-2 ligands were increased on skin macrophages compared to healthy controls. The proportions PD1+, but not TIM-3 or CTLA-4 expressing T cells in the circulation were positively correlated with those in the lesions of the same patients, suggesting that PD-1 may regulate T cell function equally in both compartments. Blocking PD-1 signaling in circulating T cells enhanced their proliferative capacity and IFN-γ production, but not TNF-α secretion in response to L. braziliensis recall antigen challenge in vitro. While we previously showed a significant correlation between the accumulation of senescent CD8+CD45RA+CD27- T cells in the circulation and skin lesion size in the patients, there was no such correlation between the extent of PD-1 expression by circulating on T cells and the magnitude of skin lesions suggesting that exhausted-like T cells may not contribute to the cutaneous immunopathology. Nevertheless, we identified exhausted-like T cells in both skin lesions and in the blood. Targeting this population by PD-1 blockade may improve T cell function and thus accelerate parasite clearance that would reduce the cutaneous pathology in cutaneous leishmaniasis.

scholarly journals Reversal of the CD8+ T-Cell Exhaustion Induced by Chronic HIV-1 Infection Through Combined Blockade of the Adenosine and PD-1 Pathways

2021 ◽  
Vol 12 ◽  
Author(s):  
Jing Li ◽  
Hui-Huang Huang ◽  
Bo Tu ◽  
Ming-Ju Zhou ◽  
Wei Hu ◽  
...  
Keyword(s):  
T Cells ◽  
Viral Load ◽  
T Cell ◽  
Cell Function ◽  
T Cell Subsets ◽  
Cell Counts ◽  
T Cell Function ◽  
Treatment Naïve ◽  
Hiv 1

BackgroundTargeting immune checkpoints for HIV treatment potentially provides a double benefit resulting from the ability to restore viral-specific CD8+ T-cell functions and enhance HIV production from reservoir cells. Despite promising pre-clinical data, PD-1 blockade alone in HIV-1-infected patients with advanced cancer has shown limited benefits in controlling HIV, suggesting the need for additional targets beyond PD-1. CD39 and PD-1 are highly co-expressed on CD8+ T cells in HIV-1 infection. However, the characteristics of CD39 and PD-1 dual-positive CD8+ T-cell subsets in chronic HIV-1 infection remain poorly understood.MethodsThis study enrolled 72 HIV-1-infected patients, including 40 treatment naïve and 32 ART patients. A total of 11 healthy individuals were included as controls. Different subsets of CD8+ T cells defined by CD39 and/or PD-1 expression were studied by flow cytometry. The relationships between the frequencies of the different subsets and parameters indicating HIV-1 disease progression were analyzed. Functional (i.e., cytokine secretion, viral inhibition) assays were performed to evaluate the impact of the blockade of adenosine and/or PD-1 signaling on CD8+ T cells.ResultsThe proportions of PD-1+, CD39+, and PD-1+CD39+ CD8+ T cells were significantly increased in treatment naïve patients but were partially lowered in patients on antiretroviral therapy. In treatment naïve patients, the proportions of PD-1+CD39+ CD8+ T cells were negatively correlated with CD4+ T-cell counts and the CD4/CD8 ratio, and were positively correlated with viral load. CD39+CD8+ T cells expressed high levels of the A2A adenosine receptor and were more sensitive to 2-chloroadenosine-mediated functional inhibition than their CD39- counterparts. In vitro, a combination of blocking CD39/adenosine and PD-1 signaling showed a synergic effect in restoring CD8+ T-cell function, as evidenced by enhanced abilities to secrete functional cytokines and to kill autologous reservoir cells.ConclusionIn patients with chronic HIV-1 infection there are increased frequencies of PD-1+, CD39+, and PD-1+CD39+ CD8+ T cells. In treatment naïve patients, the frequencies of PD-1+CD39+ CD8+ T cells are negatively correlated with CD4+ T-cell counts and the CD4/CD8 ratio and positively correlated with viral load. Combined blockade of CD39/adenosine and PD-1 signaling in vitro may exert a synergistic effect in restoring CD8+ T-cell function in HIV-1-infected patients.

scholarly journals The Cysteine-Containing Cell-Penetrating Peptide AP Enables Efficient Macromolecule Delivery to T Cells and Controls Autoimmune Encephalomyelitis

Pharmaceutics ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1134
Author(s):  
Won-Ju Kim ◽  
Gil-Ran Kim ◽  
Hyun-Jung Cho ◽  
Je-Min Choi
Keyword(s):  
Drug Delivery ◽  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Fluorescent Protein ◽  
Autoimmune Encephalomyelitis ◽  
Optimal Sequence ◽  
T Cell Function

T cells are key immune cells involved in the pathogenesis of several diseases, rendering them important therapeutic targets. Although drug delivery to T cells is the subject of continuous research, it remains challenging to deliver drugs to primary T cells. Here, we used a peptide-based drug delivery system, AP, which was previously developed as a transdermal delivery peptide, to modulate T cell function. We first identified that AP-conjugated enhanced green fluorescent protein (EGFP) was efficiently delivered to non-phagocytic human T cells. We also confirmed that a nine-amino acid sequence with one cysteine residue was the optimal sequence for protein delivery to T cells. Next, we identified the biodistribution of AP-dTomato protein in vivo after systemic administration, and transduced it to various tissues, such as the spleen, liver, intestines, and even to the brain across the blood–brain barrier. Next, to confirm AP-based T cell regulation, we synthesized the AP-conjugated cytoplasmic domain of CTLA-4, AP-ctCTLA-4 peptide. AP-ctCTLA-4 reduced IL-17A expression under Th17 differentiation conditions in vitro and ameliorated experimental autoimmune encephalomyelitis, with decreased numbers of pathogenic IL-17A+GM-CSF+ CD4 T cells. These results collectively suggest the AP peptide can be used for the successful intracellular regulation of T cell function, especially in the CNS.

CD3 limits the efficacy of TCR gene therapy in vivo

Blood ◽  
2011 ◽  
Vol 118 (13) ◽  
pp. 3528-3537 ◽  
Author(s):  
Maryam Ahmadi ◽  
Judith W. King ◽  
Shao-An Xue ◽  
Cécile Voisine ◽  
Angelika Holler ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Cell Receptor ◽  
Surface Expression ◽  
T Cell Function ◽  
Engineered T Cells ◽  
Rate Limiting

Abstract The function of T-cell receptor (TCR) gene modified T cells is dependent on efficient surface expression of the introduced TCR α/β heterodimer. We tested whether endogenous CD3 chains are rate-limiting for TCR expression and antigen-specific T-cell function. We show that co-transfer of CD3 and TCR genes into primary murine T cells enhanced TCR expression and antigen-specific T-cell function in vitro. Peptide titration experiments showed that T cells expressing introduced CD3 and TCR genes recognized lower concentration of antigen than T cells expressing TCR only. In vivo imaging revealed that TCR+CD3 gene modified T cells infiltrated tumors faster and in larger numbers, which resulted in more rapid tumor elimination compared with T cells modified by TCR only. After tumor clearance, TCR+CD3 engineered T cells persisted in larger numbers than TCR-only T cells and mounted a more effective memory response when rechallenged with antigen. The data demonstrate that provision of additional CD3 molecules is an effective strategy to enhance the avidity, anti-tumor activity and functional memory formation of TCR gene modified T cells in vivo.

scholarly journals Primary CLL Xenograft: A Model System to Study the Role of T-Cells in CLL Biology and Therapeutic Response

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3284-3284
Author(s):  
Ceri E Oldreive ◽  
Anna Skowronska ◽  
Angelo Agathanggelou ◽  
Helen M Parry ◽  
Sergey Krysov ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Xenograft Model ◽  
Cell Number ◽  
Biological Properties ◽  
T Cell Subsets ◽  
Cell Phenotype

Abstract The interaction between chronic lymphocytic leukaemia (CLL) cells and T-cells is an important aspect of CLL biology. CLL cells require T-cell support for their proliferation and in addition induce proliferation of regulatory and cytotoxic (CD8+) T-cells. T-cell number and repertoire are both markedly affected by CLL therapy and there is considerable interest in how current treatments modulate the interaction between T-cells and the tumour clone. In this study we investigated whether this relationship was maintained in a xenotransplantation model. CLL engraftment in NOG mice was facilitated by humanisation of the murine microenvironment by allogeneic CD34+ umbilical cord cells or CD14+ monocytes. Accelerated engraftment of both CLL and T-cell compartments was observed in xenografts derived from patients with progressive CLL, suggesting that the biological properties of both subsets are maintained in the murine model. Furthermore, the distribution of helper (CD4+), cytotoxic (CD8+) and regulatory (CD4+CD25+FoxP3+) T-cells was maintained within the xenografts, including retention of the CD4:CD8 ratio. Interestingly, the anergic PD-1+CD160+CD244+TIM3+ T-cell phenotype reported in CLL patients was also evident in T-cells expanded in xenograft models. Consistent with an anergic T-cell phenotype, T-cells from CLL xenografts lacked anti-tumour activity in vitro. Importantly, such anergic cells were observed when T-cells were reconstituted from allogeneic cord blood cells as well as autologous cells, suggesting that CLL cells have the ability to shape T-cell populations of different origin in diverse microenvironments. Finally, to investigate the interaction between specific T-cell subsets and engrafted CLL cells, CD4+, CD8+, and CD25+ T-cells were depleted prior to generation of xenografts. CD8+ T-cell depletion significantly prolonged CLL engraftment (p≤0.01) whereas neither depletion of CD4+ nor CD25+cells had a significant impact. In summary, our results demonstrate that the relationship between CLL tumour cells and reactive T-cells is accurately maintained in a murine xenograft model. Such models will be of great value for investigation of aspects of T-cell function in CLL biology. Disclosures No relevant conflicts of interest to declare.

scholarly journals Glycolysis Inhibition Induces Functional and Metabolic Exhaustion of CD4+ T Cells in Type 1 Diabetes

2021 ◽  
Vol 12 ◽  
Author(s):  
Christina P. Martins ◽  
Lee A. New ◽  
Erin C. O’Connor ◽  
Dana M. Previte ◽  
Kasey R. Cargill ◽  
...  
Keyword(s):  
T Cells ◽  
Type 1 Diabetes ◽  
T Cell ◽  
Cell Function ◽  
T Cell Responses ◽  
Metabolic Reprogramming ◽  
Cell Responses ◽  
T Cell Function

In Type 1 Diabetes (T1D), CD4+ T cells initiate autoimmune attack of pancreatic islet β cells. Importantly, bioenergetic programs dictate T cell function, with specific pathways required for progression through the T cell lifecycle. During activation, CD4+ T cells undergo metabolic reprogramming to the less efficient aerobic glycolysis, similarly to highly proliferative cancer cells. In an effort to limit tumor growth in cancer, use of glycolytic inhibitors have been successfully employed in preclinical and clinical studies. This strategy has also been utilized to suppress T cell responses in autoimmune diseases like Systemic Lupus Erythematosus (SLE), Multiple Sclerosis (MS), and Rheumatoid Arthritis (RA). However, modulating T cell metabolism in the context of T1D has remained an understudied therapeutic opportunity. In this study, we utilized the small molecule PFK15, a competitive inhibitor of the rate limiting glycolysis enzyme 6-phosphofructo-2-kinase/fructose-2,6- biphosphatase 3 (PFKFB3). Our results confirmed PFK15 inhibited glycolysis utilization by diabetogenic CD4+ T cells and reduced T cell responses to β cell antigen in vitro. In an adoptive transfer model of T1D, PFK15 treatment delayed diabetes onset, with 57% of animals remaining euglycemic at the end of the study period. Protection was due to induction of a hyporesponsive T cell phenotype, characterized by increased and sustained expression of the checkpoint molecules PD-1 and LAG-3 and downstream functional and metabolic exhaustion. Glycolysis inhibition terminally exhausted diabetogenic CD4+ T cells, which was irreversible through restimulation or checkpoint blockade in vitro and in vivo. In sum, our results demonstrate a novel therapeutic strategy to control aberrant T cell responses by exploiting the metabolic reprogramming of these cells during T1D. Moreover, the data presented here highlight a key role for nutrient availability in fueling T cell function and has implications in our understanding of T cell biology in chronic infection, cancer, and autoimmunity.

scholarly journals Combination rhIL-15 and Anti-PD-L1 (Avelumab) Enhances HIVGag-Specific CD8 T-Cell Function

2020 ◽  
Vol 222 (9) ◽  
pp. 1540-1549
Author(s):  
Bruktawit A Goshu ◽  
Hui Chen ◽  
Maha Moussa ◽  
Jie Cheng ◽  
Marta Catalfamo
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Function ◽  
Cd8 T Cell ◽  
Peripheral Tissues ◽  
T Cell Function ◽  
Interleukin 15 ◽  
Checkpoint Receptors

Abstract In chronic HIV infection, virus-specific cytotoxic CD8 T cells showed expression of checkpoint receptors and impaired function. Therefore, restoration of CD8 T-cell function is critical in cure strategies. Here, we show that in vitro blockade of programmed cell death ligand 1 (PD-L1) by an anti-PD-L1 antibody (avelumab) in combination with recombinant human interleukin-15 (rhIL-15) synergistically enhanced cytokine secretion by proliferating HIVGag-specific CD8 T cells. In addition, these CD8 T cells have a CXCR3+PD1−/low phenotype, suggesting a potential to traffic into peripheral tissues. In vitro, proliferating CD8 T cells express PD-L1 suggesting that anti-PD-L1 treatment also targets virus-specific CD8 T cells. Together, these data indicate that rhIL-15/avelumab combination therapy could be a useful strategy to enhance CD8 T-cell function in cure strategies.

scholarly journals IMMU-15. A UNIQUE SELF-STIMULATIVE PROPERTY OF CD70 CAR ADVANCES T CELL FUNCTION AND ANTI-TUMOR RESPONSE IN GLIOBLASTOMA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi122-vi122
Author(s):  
Linchun Jin ◽  
Alicia Hou ◽  
Haipeng Tao ◽  
Aida Karachi ◽  
Meng Na ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mouse Models ◽  
Tumor Response ◽  
Cell Function ◽  
Antitumor Efficacy ◽  
Car T Cells ◽  
T Cell Function ◽  
Car T

Abstract BACKGROUND Glioblastoma (GBM) is a refractory brain tumor that desperately needs new therapeutic interventions. Our group identified CD70 as a novel target of CAR-T therapy for this malignancy. We demonstrate that CD70 is overexpressed by low-/high-grade gliomas and associated with poor survival for patients; CD70 promotes CD8 specific cell death and tumor-associated macrophage infiltration in gliomas. The CD70 CAR (using CD27, a natural costimulatory receptor of T cells as an antigen-binding region) T cells can efficiently eradicate CD70 positive tumors in syngeneic and xenograft mouse models. OBJECTIVE To evaluate the properties of CD70 CAR-transduced T cells in GBM treatment. METHODS CD70 CAR or IL13Rα2 CAR was linked with fluorescent reporter gene EGFP, and cloned into a retroviral vector (pMSGV8). In vitro T cell culture and flow cytometry were used to evaluate the self-enrichment property and susceptibility to TCR stimulation of the CAR T cells. KI67, Bcl-2, CD70 gene expression was tested by qPCR to measure the proliferation/apoptosis properties of the CAR T cells. Cytokine profile was analyzed by ELISA. The anti-tumor response was evaluated using Xenograft mouse models. RESULTS Compared with IL13Rα2 CAR T cells, the frequency of CD70 CAR T cells was significantly increased 3 weeks post transduction, and approximately 100 to 150-fold CD70 CAR T cell expansion without additional stimuli was achieved in vitro. The expanded CD70 CAR T cells were mostly (up to 85%) CD8+ T cells three weeks post CAR transduction. Enhanced proliferative capacity and production of IL-2, IFN-γ, and TNF-α of the CD70 CAR-transduced T cells upon anti-CD3/CD28 stimulation were also revealed. Results from animal models show that the CD70 CAR T cells present superior in vivo persistence and antitumor efficacy. CONCLUSION We show the auto-stimulative property, as well as superior T cell function and antitumor efficacy of CD70 CAR T cells in GBM models.

175 A Fas-4–1BB immunomodulatory fusion protein converts a pro-death to a pro-survival signal, enhancing T cell function and efficacy of adoptive cell therapy in murine models of AML and pancreatic cancer

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A189-A189
Author(s):  
Shannon Oda ◽  
Kristin Anderson ◽  
Philip Greenberg ◽  
Nicolas Garcia ◽  
Pranali Ravikumar ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
Fas Ligand ◽  
Cell Function ◽  
Adoptive Cell Therapy ◽  
Activated T Cells ◽  
T Cell Function

BackgroundAdoptive cell therapy (ACT) with genetically-modified T cells has shown impressive results against some hematologic cancers, but limited efficacy against tumors with restrictive tumor microenvironments (TMEs). FasL is a particular obstacle for ACT;1 it is expressed in many tumors and TMEs,1 including AML,2 ovarian3 and pancreatic cancers,4 and upregulated on activated T cells, where it can mediate activation-induced cell death (AICD).5MethodsWe engineered T cells to boost function with novel immunomodulatory fusion proteins (IFPs) that combine an inhibitory ectodomain with a costimulatory endodomain. Like current checkpoint-blocking therapies, IFPs can abrogate an inhibitory signal, but also provide an often absent costimulatory signal. Additionally, IFP-driven signals are delivered only to the T cells concurrently engineered to be tumor-specific, thereby avoiding systemic T cell activation. For FasL-expressing TMEs, we developed an IFP that replaces the Fas intracellular tail with costimulatory 4-1BB. We tested the the Fas-4-1BB IFP in primary human T cells and in immunocompetent murine models of leukemia and pancreatic cancer.ResultsFas-4-1BB IFP expression enhanced primary human T cell function and enhanced lysis of Panc1 pancreatic tumor cells in vitro. Fas-4-1BB IFP-engineered murine T cells exhibited increased pro-survival signaling, proliferation, antitumor function and altered metabolism in vitro. Notably, the Fas ectodomain is trimeric5 and the 4-1BB intracellular domain requires trimerization to signal.6 In contrast, the CD28 domain is dimeric and did not enhance function when paired with 4-1BB.In vivo, Fas-4-1BB increased T cell persistence and function, and Fas-4-1BB T cell ACT significantly improved survival in a murine AML model. When delivered with a mesothelin-specific TCR, Fas-4-1BB T cells prolonged survival in the autochthonous KPC pancreatic cancer model, increasing median survival to 65 from 37 days (with TCR-only, **P=0.0042). Single-cell RNA sequencing revealed differences in the endogenous tumor-infiltrating immune cells, included changes in cell frequency and programming.ConclusionsWe developed an engineering approach to enhance the in vivo persistence and antitumor efficacy of transferred T cells. Our targeted, two-hit strategy uses a single fusion protein to overcome a death signal prevalent in the TME of many cancers and on activated T cells, and to provide a pro-survival costimulatory signal to T cells. Our results suggest that this fusion protein can increase T cell function when combined with murine or human TCRs, and can significantly improve therapeutic efficacy in liquid and solid tumors, supporting clinical translation.ReferencesYamamoto, T.N., et al., T cells genetically engineered to overcome death signaling enhance adoptive cancer immunotherapy. J Clin Invest 2019.Contini P, et al., In vivo apoptosis of CD8(+) lymphocytes in acute myeloid leukemia patients: involvement of soluble HLA-I and Fas ligand. Leukemia 2007;21(2):p. 253–60.Motz GT, et al., Tumor endothelium FasL establishes a selective immune barrier promoting tolerance in tumors. Nat Med 2014;20(6):p. 607–15.Kornmann M, et al., Fas and Fas-ligand expression in human pancreatic cancer. Ann Surg 2000. 231(3): p. 368–79.Villa-Morales M and J Fernandez-Piqueras, Targeting the Fas/FasL signaling pathway in cancer therapy. Expert Opin Ther Targets 2012;16(1):p. 85–101.Wyzgol, A., et al., Trimer stabilization, oligomerization, and antibody-mediated cell surface immobilization improve the activity of soluble trimers of CD27L, CD40L, 41BBL, and glucocorticoid-induced TNF receptor ligand. J Immunol 2009;183(3):p. 1851–61.

scholarly journals Treatment-Free Intervals Mitigate T-Cell Exhaustion Induced By Continuous CD19xCD3-BiTE® Construct Stimulation in Vitro

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 44-45
Author(s):  
Nora Zieger ◽  
Alyssa Nicholls ◽  
Jan Wulf ◽  
Gerulf Hänel ◽  
Maryam Kazerani Pasikhani ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Cell Function ◽  
Granzyme B ◽  
T Cell Exhaustion ◽  
Specific Lysis ◽  
Cell Exhaustion ◽  
T Cell Function

The bispecific T-cell engager (BiTE®) blinatumomab is approved for treatment of relapsed/refractory B-cell precursor acute lymphoblastic leukemia and applied as continuous infusion over 28 days. The overall response rate to blinatumomab reported in clinical trials was 43 % and correlated to T-cell expansion (Zugmaier et al. 2015). In chronic viral infections, continuous antigen stimulation induces T-cell exhaustion, defined by phenotypic changes and functional impairment (Wherry 2011). Thus, we hypothesized that continuous BiTE® construct stimulation leads to T-cell exhaustion and that a treatment-free interval (TFI) reverses progressive T-cell dysfunction. To simulate continuous application of a BiTE® construct in vitro, T-cell long-term co-cultures were set up. Healthy donor T cells were stimulated in the presence of CD19+ OCI-Ly1 cells for 28 days with AMG 562, a half-life extended CD19 and CD3 specific BiTE® construct. T cells were harvested from the co-culture every 3-4 days between day 7 and 28 and assessed for markers of T-cell exhaustion: (1) AMG 562-mediated cytotoxicity of T cells was evaluated as specific lysis of CD19+ Ba/F3 target cells after 3 days, (2) T-cell expansion during the cytotoxicity assay was calculated as fold change (FC) of CD2+ counts, (3) Cytokine secretion of AMG 562-stimulated T cells was evaluated in co-culture supernatants by cytometric bead array (CBA) or after PMA/Ionomycine stimulation via intracellular cytokine staining (ICCS), (4) T-cell metabolic fitness was determined by Mito- and Glycolytic Stress Test using a Seahorse Analyzer, and (5) expression of the exhaustion-related transcription factor TOX was assessed by multiparameter flow cytometry. In order to assess the effect of a TFI on T-cell function, we cultured T cells and CD19+ OCI-Ly1 cells in the absence of AMG 562 from day 7-14 and 21-28 and compared their activity to T cells stimulated continuously with AMG 562. On day 7 of continuous (CONT) AMG 562 stimulation, we observed high cytotoxic and proliferative potential (% specific lysis=93±0.2, FC=2.9±0.2) as well as high IFN-g and TNF-a secretion analyzed by ICCS (% CD8+IFN-g+TNF-a+=23±6.7). However, cytotoxicity and proliferation decreased gradually until day 28 (% specific lysis=28±8.9; FC=0.6±0.1). CBA analysis confirmed decreasing secretion of IFN-g (day 3: 61113±12482, day 24: 3085±1351 pg/ml) and TNF-a (day 3: 1160±567, day 24: 43±7.6 pg/ml) as well as decreased IL-2 and granzyme B levels in culture supernatants. We furthermore observed highest mitochondrial fitness and basal glycolysis in T cells on day 7 of stimulation (basal OCR=2.2±0.6, maximal OCR=3.7±1.0, SRC=1.5±1.1 pmol/min/1000 cells, basal ECAR=2.0±0.4 mpH/min/1000 cells) which decreased until day 28 (basal OCR=0.4±0.2, maximal OCR=1.5±0.5, SRC=1.0±0.2 pmol/min/1000 cells, basal ECAR=0.5±0.2 mpH/min/1000 cells). In concordance, TOX increased during continuous stimulation (MFI ratio CD8+ day 7=6±0.8 to 12±0.8 on day 28). Strikingly, implementation of a TFI of 7 days led to superior cytotoxicity in T cells compared to continuously stimulated T cells (% specific lysis on day 14 CONT=34±4.2, TFI=99±2.2) and granzyme B production (CD8+; MFI ratio on day 14 CONT=124±11, TFI=303±34). Furthermore, increased proliferation during the cytotoxicity assay was observed in previously rested T cells (FC CONT=0.2±0.0, TFI=1.6±0.6). Although T cell function also decreased over time in TFI T cells, they maintained a strikingly higher cytotoxic potential (CONT=6±4.4, TFI=52±9.9) as well as higher granzyme B production (CONT=25±2, TFI=170±11) on day 28 compared to continuously stimulated T cells. In addition, TFI T cells showed increased IFN-g and TNF-a secretion after PMA/Ionomycine stimulation on day 28 (% CD8+IFN-g+TNF-a+ CONT=21±3.8, TFI=38±11.6). Our in vitro results demonstrate that continuous AMG 562 exposure negatively impacts T-cell function. Comprehensive analysis of T-cell activity in an array of functional assays suggests that continuous BiTE® construct exposure leads to T-cell exhaustion which can be mitigated through TFI. Currently, T cells from patients receiving blinatumomab are being analyzed to confirm the clinical relevance of our findings. Furthermore, RNA-Seq of continuously vs. intermittently AMG 562-exposed T cells will help us to understand underlying transcriptional mechanisms of BiTE® construct induced T-cell exhaustion. Disclosures Zieger: AMGEN Research Munich: Research Funding. Buecklein:Pfizer: Consultancy; Novartis: Research Funding; Celgene: Research Funding; Amgen: Consultancy; Gilead: Consultancy, Research Funding. Brauchle:AMGEN Inc.: Research Funding. Marcinek:AMGEN Research Munich: Research Funding. Kischel:AMGEN: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Subklewe:Gilead Sciences: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Morphosys: Research Funding; Seattle Genetics: Research Funding; AMGEN: Consultancy, Honoraria, Research Funding; Janssen: Consultancy; Roche AG: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Honoraria.

Abstract 004: Aberrant CD4 + T Cell Function in Stroke-prone Hypertensive Rats Due to a Mutation in Stim1

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Isha S Dhande ◽  
Mykola Mamenko ◽  
Yaming Zhu ◽  
Oleh Pochynyuk ◽  
Scott Wenderfer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cytokine Production ◽  
Cell Function ◽  
Spontaneously Hypertensive Rat ◽  
Flow Cytometric Analysis ◽  
T Cell Subsets ◽  
Organ Injury ◽  
Truncating Mutation ◽  
T Cell Function

The genetic mechanism of end organ injury in hypertension may involve gene variation in genes participating in inflammation and its regulation. We identified a novel truncating mutation in the hypertensive end organ injury-prone spontaneously hypertensive rat (SHR-A3/SHRSP) line affecting the C-terminus of STIM1, a protein involved in the store-operated Ca 2+ entry (SOCE) pathway. The genomes of injury-resistant SHR lines, including SHR-B2 used here, encode the ‘wild-type’ STIM1. SOCE is required by T cells to activate the transcription factor NFAT and regulate T cell proliferation and cytokine production. T cell receptor (TCR) stimulation depletes intracellular Ca 2+ stores, activates the ER Ca 2+ -sensor STIM1 and results in SOCE. We tested the effect of STIM1 mutation on lymphocyte SOCE and found it was dramatically reduced in SHR-A3, but not in SHR-B2 (maximal [Ca 2+ ] i : 150.5±34.9 vs 521.5±42.6 nM, p<0.0001). Flow cytometric analysis of circulating T cell subsets revealed comparable levels of CD4 + and CD8 + T cells in both lines, however circulating CD4 + CD25 + FoxP3 + T regs were reduced in SHR-A3 compared to SHR-B2 (4.34±0.59 vs 7.12±0.33%, p=0.01). TCR-induced lymphocyte proliferation was similar in both SHR-A3 and SHR-B2. T cell cytokine production in response to TCR stimulation was markedly impaired CD4 + T cells from SHR-A3 compared with SHR-B2 (IL-2: 168 ±83.4 vs 1385±377.0 pg/mL, p=0.01; IFNγ: 235±69.5 vs 2119±434.7 pg/mL, p=0.002). IL-2 and IFNγ production was completely inhibited by Pyr6, an inhibitor of Stim1-dependent SOCE. However, circulating levels of IL-2 and IFNγ were not different between the two lines. Based on our findings, we conclude that TCR-mediated effector signaling is impaired due to defective SOCE in SHR-A3 rats. Defects in SOCE in SHR-A3 attributable to STIM1 mutation alter T cell function, reduce T reg numbers and may disturb regulatory interactions between T cells and other immune cells involved in end organ injury.

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