scholarly journals BET Inhibition As a Targeted Epigenetic Approach to Reverse T Cell Dysfunction in Chronic Lymphocytic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3715-3715
Author(s):  
Audrey L Smith ◽  
Alexandria P Eiken ◽  
Sydney A Skupa ◽  
Dalia Y Moore ◽  
Avyakta Kallam ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Surface Expression ◽  
Lymphocytic Leukemia ◽  
T Cell Proliferation ◽  
Cell Dysfunction ◽  
T Cell Dysfunction

Abstract Introduction : Chronic Lymphocytic Leukemia (CLL) is characterized by the clonal expansion of mature CD19+/CD5+ lymphocytes in the peripheral blood and secondary lymphoid organs. The accumulation of B-CLL cells yields profound immune defects in the CLL tumor microenvironment (TME), promoting evasion of immune surveillance that contributes to tumor persistence and thus relapsed/refractory disease. The bromodomain and extra-terminal domain (BET) family of proteins are epigenetic readers that bind acetylated histone residues to regulate transcription of numerous genes involved in critical CLL protumor pathways. Of the BET family proteins, BRD4 is overexpressed in CLL and highly enriched at super-enhancers of genes that regulate CLL-TME interactions such as B cell receptor pathway components, chemokine/cytokine receptors, and immune checkpoint molecules. Pan BET inhibitors (BET-i), such as PLX51107 (Plexxikon Inc.) significantly improve survival in aggressive CLL murine models. Here we demonstrate that blocking BRD4 function with PLX51107 (PLX5) can alleviate the inherent immune defects observed in CLL, hence reducing B-CLL induced T cell dysfunction and allowing for robust B-CLL cell elimination. This therapeutic strategy may be vital in overcoming frequent drug resistance and/or bolstering the anti-tumor effect of current CLL therapies. Methods : Primary leukemic B cells were isolated from the peripheral blood of CLL patients and co-cultured with healthy donor T cells to evaluate the effect of PLX5 (0.1-0.5μM) on CLL-induced T cell immunosuppression ex vivo via an array of flow cytometry assays. T cell proliferation was assessed using CFSE after 96 h co-culture with α-CD3/α-CD28 stimulation. Effector cytokine production was evaluated after 48 h co-culture in the presence of PMA/ionomycin (final 6 h) and brefeldin A (final 5 h). Immune inhibitory molecule surface expression was measured following 48 h co-culture with α-CD3/α-CD28 stimulation. To further validate our ex vivo findings, the E μ-TCL1 adoptive transfer model was used. Once disease onset was confirmed in recipient WT B6 mice (>10% CD45+/CD19+/CD5+ peripheral blood lymphocytes), mice were randomized to receive either PLX5 (20 mg/kg) or vehicle (VEH) equivalent daily by oral gavage for 4 weeks. Following treatment, mouse spleens were processed to evaluate exhaustion marker expression, T cell proliferation (CellTrace™ Violet, 72 h a-CD3/α-CD28 stimulation ex-vivo), and T-cell effector function (ex-vivo mitogenic stimulation, 6 h). Results : T cell proliferation indices were reduced following ex vivo co-culture with primary B-CLL cells (mean ± SEM for T cells vs. co-culture, 2.0 ± 0.13 vs. 1.57 ± 0.05; P<0.01). This suppression was significantly alleviated in 0.5μM PLX5-treated co-cultures (1.84 ± 0.08; P<0.01). In a similar fashion, the percentage of polyfunctional TNF-α+/IFN-γ+ CD4+ T cells markedly increased in PLX5-treated co-cultures (VEH vs. 0.5μM PLX5, 10.0% ± 0.76% vs. 15.2% ± 0.92%; P<0.01). Notably, BET inhibition with PLX5 also bolstered T cell inflammatory function (%TNF-α+/IFN-γ+) in the absence of B-CLL cells (VEH vs. PLX5, 12.9% ± 1.0% vs. 15.3% ± 0.69%; P<0.05). Remarkably, the expression of numerous immune inhibitory molecules (e.g., PDL1, PD1, CTLA4, LAG3) was consistently reduced between 1.8- and 3-fold in PLX5-treated co-cultures (0.1μM). In the adoptive transfer E μ-TCL1 model, mice receiving PLX5 displayed reduced expansion of B-CLL cells and increased T cell infiltration in the spleen (Fig. 1A). Splenic CD4+ T cells from PLX5-treated mice had significantly greater proliferative capacity (Fig. 1B) and pro-inflammatory functionality (Fig. 1C). Finally, PLX5 treatment markedly reduced the surface expression of immune inhibitory molecules (e.g., PDL1, LAG3, VISTA) on CD4+ and CD8+ T cells in the spleen (Fig. 1D). Studies to evaluate the effects of PLX5 on malignant B-CLL and T cells within the bone marrow niche and soluble factors in the plasma are ongoing. Collectively, our data indicate that the novel BET-i, PLX5, exerts beneficial immunomodulatory effects on T cells within the CLL TME. Conclusion : Epigenetic-targeted therapies such as BET-i have the potential to alleviate CLL-induced T cell dysfunction while eliminating B-CLL cells and preventing tumor expansion. Future profiling studies are pending to further illuminate how BET proteins regulate immune function in CLL. Figure 1 Figure 1. Disclosures Lunning: AstraZeneca: Consultancy; Legend: Consultancy; Acrotech: Consultancy; ADC Therapeutics: Consultancy; Kyowa Kirin: Consultancy; Myeloid Therapeutics: Consultancy; Beigene: Consultancy; Celgene, a Bristol Myers Squibb Co.: Consultancy; Verastem: Consultancy; Janssen: Consultancy; Daiichi-Sankyo: Consultancy; Morphosys: Consultancy; TG Therapeutics: Consultancy; Novartis: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Spectrum: Consultancy; Kite, a Gilead Company: Consultancy. Vose: Kite, a Gilead Company: Honoraria, Research Funding. Powell: Plexxikon Inc.: Current Employment.

TIGIT Induces (CD3+) T Cell Dysfunction by Inhibiting Glucose Metabolism and its Reversal by TIGIT and/or PD-1 Blockade in Colorectal Cancer

2021 ◽  
Author(s):  
qi shao ◽  
Lei Wang ◽  
maoling yuan ◽  
Xiaohong Jin ◽  
changping wu
Keyword(s):  
Colorectal Cancer ◽  
T Cells ◽  
T Cell ◽  
Glucose Metabolism ◽  
Tumor Growth ◽  
Tumor Cells ◽  
Peripheral Blood ◽  
Cancer Tissue ◽  
Cell Dysfunction ◽  
T Cell Dysfunction

Abstract Background: T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) is an immunosuppressive receptor expressed on the surface of immune cells, suppressing immune responses by activating the intracellular negative regulatory signals. TIGIT plays an important role in the pathogenesis of various tumors, but its immune escape in colorectal cancer remains unclear.Methods: In this study, TIGIT expression in the peripheral blood and tissue microarrays was detected flow cytometry and immunofluorescence and its relationship with prognosis was evaluated. The proliferation and cytokines of TIGIT+ T cells were measured. Glucose metabolism and key enzymes were detected by qPCR or western blot. After establishing the co-cultured system and xenotransplant models, TIGIT antibody alone or combined with PD-1 antibody was blocked to observe the tumor growth.Results: We found that the proportion of CD3+TIGIT+ T cells was increased in peripheral blood and cancer tissue in colorectal cancer patients when compared with the healthy donors. These cells exhibited functional defects, low proliferative activity, impaired cytokine production and reduced glucose metabolism. A strong association was also observed between the elevated TIGIT expression and poor prognosis. In the in vitro co-culture assays of T cells and tumor cells, the suppressed glucose metabolic activity of T cells was reversed by TIGIT blockade. In addition, this blockade induced the apoptosis and reduced G2/M transit in tumor cells. The antitumor efficacy of TIGIT Ab therapy was further demonstrated in a human colorectal xenograft mice model while co-blockers of TIGIT and PD-1 exhibited synergistic suppressing effects on tumor growth.Conclusions: It is suggest that while TIGIT induces CD3+ T cell dysfunction in colorectal cancer, co-targeting TIGIT and PD-1 can lead to an effective antitumor response and may serve as a novel therapeutic strategy for colorectal patients.

scholarly journals Mapping of the autophagosomal degradome identifies IL-7Rα as key cargo in proliferating CD4+ T-cells

2021 ◽  
Author(s):  
Dingxi Zhou ◽  
Mariana Borsa ◽  
Daniel J. Puleston ◽  
Susanne Zellner ◽  
Jesusa Capera ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Surface Expression ◽  
T Cell Proliferation ◽  
Gamma Chain ◽  
Direct Identification ◽  
The Common ◽  
First Time

CD4+ T cells orchestrate both humoral and cytotoxic immune responses. While it is known that CD4+ T cell proliferation relies on autophagy, direct identification of the autophagosomal cargo involved is still missing. Here, we created a transgenic mouse model, which, for the first time, enables us to directly map the proteinaceous content of autophagosomes in any primary cell by LC3 proximity labelling. IL-7Rα, a cytokine receptor mostly found in naive and memory T cells, was reproducibly detected in autophagosomes of activated CD4+ T cells. Consistently, CD4+ T cells lacking autophagy showed increased IL-7Rα surface expression, while no defect in internalisation was observed. Mechanistically, excessive surface IL-7Rα sequestrates the common gamma chain, impairing the IL-2R assembly and downstream signalling crucial for T cell proliferation. This study provides proof-of-principle that key autophagy substrates can be reliably identified with this model to help mechanistically unravel autophagy's contribution to healthy physiology and disease.

A Nanobody Against Cytotoxic T-Lymphocyte Associated Antigen-4 Increases the Anti-Tumor Effects of Specific CD8+ T Cells

2019 ◽  
Vol 15 (11) ◽  
pp. 2229-2239 ◽  
Author(s):  
Zhuoran Tang ◽  
Fengzhen Mo ◽  
Aiqun Liu ◽  
Siliang Duan ◽  
Xiaomei Yang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Cytotoxic T Lymphocyte ◽  
Ex Vivo ◽  
T Cell Proliferation ◽  
Tumor Cell Apoptosis ◽  
Xenograft Models

Adoptive cell-based immunotherapy typically utilizes cytotoxic T lymphocytes (CTLs), expanding these cells ex vivo. Such expansion is traditionally accomplished through the use of autologous APCs that are capable of interactions with T cells. However, incidental inhibitory program such as CTLA-4 pathway can impair T cell proliferation. We therefore designed a nanobody which is specific for CTLA-4 (CTLA-4 Nb 16), and we then used this molecule to assess its ability to disrupt CTLA-4 signaling and thereby overcome negative costimulation of T cells. With CTLA-4 Nb16 stimulation, dendritic cell/hepatocellular carcinoma fusion cells (DC/HepG2-FCs) enhanced autologous CD8+ T cell proliferation and production of IFN-γ in vitro, thereby leading to enhanced killing of tumor cells. Using this approach in the context of adoptive CD8+ immunotherapy led to a marked suppression of tumor growth in murine NOD/SCID hepatocarcinoma or breast cancer xenograft models. We also observed significantly increased tumor cell apoptosis, and corresponding increases in murine survival. These findings thus demonstrate that in response to nanobody stimulation, DC/tumor cells-FC-induced specific CTLs exhibit superior anti-tumor efficacy, making this a potentially valuable means of achieving better adoptive immunotherapy outcomes in cancer patients.

scholarly journals Defective interleukin-2 production and responsiveness by T cells in patients with chronic lymphocytic leukemia of B cell variety

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 279-284 ◽  
Author(s):  
O Ayanlar-Batuman ◽  
E Ebert ◽  
SP Hauptman
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Proliferative Response ◽  
Interleukin 2 ◽  
Lymphocytic Leukemia ◽  
T Cell Proliferation ◽  
Activated T Cells

Abstract The present studies were designed to investigate the mechanism(s) of the defective T cell proliferative response to various stimuli in patients with B cell chronic lymphocytic leukemia B-CLL. In 14 patients with advanced B-CLL (stage III or IV) we found the T cell response in the autologous (auto) and allogeneic (allo) mixed lymphocyte reaction (MLR) to be 35.7% and 30% of the controls, respectively. Proliferation in the MLR depends upon the production of and response to interleukin 2 (IL 2), a T cell growth factor. IL 2 production in eight B-CLL patients was 22% of the control. The response to IL 2 was measured by the increase in the T cell proliferation in the MLR with the addition of IL 2. T cell proliferation in both the auto and allo MLR of CLL patients was significantly lower than in the controls after the addition of IL 2. The proliferative response of normal T cells to stimulation by CLL B cells was 50% of the control. This latter response was increased to control levels when cultures were supplemented with exogenous IL 2, suggesting that CLL B cells could stimulate IL 2 receptor generation in normal T cells in an allo MLR, but not IL 2 production. The presence of IL 2 receptors on activated T cells was directly determined using anti- Tac, a monoclonal antibody with specificity for the IL 2 receptor. Of the mitogen- or MLR-activated T cells in CLL patients, 6% and 10%, respectively, expressed Tac antigen, whereas identically stimulated control T cells were 60% and 47% Tac+, respectively. Our findings suggest that T cells in B-CLL are defective in their recognition of self or foreign major histocompatibility antigens as demonstrated by their impaired responsiveness in the MLR. Thus, these cells are unable to produce IL 2 or generate IL 2 receptors.

scholarly journals Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection

2008 ◽  
Vol 205 (12) ◽  
pp. 2763-2779 ◽  
Author(s):  
R. Brad Jones ◽  
Lishomwa C. Ndhlovu ◽  
Jason D. Barbour ◽  
Prameet M. Sheth ◽  
Aashish R. Jha ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Surface Expression ◽  
Cell Dysfunction ◽  
Cd38 Expression ◽  
Immunodeficiency Virus ◽  
T Cell Dysfunction ◽  
Novel Target ◽  
Hiv 1

Progressive loss of T cell functionality is a hallmark of chronic infection with human immunodeficiency virus 1 (HIV-1). We have identified a novel population of dysfunctional T cells marked by surface expression of the glycoprotein Tim-3. The frequency of this population was increased in HIV-1–infected individuals to a mean of 49.4 ± SD 12.9% of CD8+ T cells expressing Tim-3 in HIV-1–infected chronic progressors versus 28.5 ± 6.8% in HIV-1–uninfected individuals. Levels of Tim-3 expression on T cells from HIV-1–infected inviduals correlated positively with HIV-1 viral load and CD38 expression and inversely with CD4+ T cell count. In progressive HIV-1 infection, Tim-3 expression was up-regulated on HIV-1–specific CD8+ T cells. Tim-3–expressing T cells failed to produce cytokine or proliferate in response to antigen and exhibited impaired Stat5, Erk1/2, and p38 signaling. Blocking the Tim-3 signaling pathway restored proliferation and enhanced cytokine production in HIV-1–specific T cells. Thus, Tim-3 represents a novel target for the therapeutic reversal of HIV-1–associated T cell dysfunction.

scholarly journals Zoledronic Acid Induces the Proliferation of Human Cord Blood Gamma Delta T Cells Ex Vivo

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1427-1427
Author(s):  
Suzanne L Tomchuck ◽  
Jin He ◽  
Ross W. Perko ◽  
Scarlett Evans ◽  
Amy McKenna ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Fold Increase ◽  
T Cell Proliferation ◽  
Gamma Delta T Cells ◽  
Memory Cells ◽  
Gamma Delta ◽  
Gamma Delta T Cell

Abstract Cord blood (CB) T cells are known to be naïve cells, but can be activated to respond similar to adult peripheral blood (PB) T cells. Reports indicate that culture with aminobisphosphonate (NBP) stimulates CB gamma delta T cell proliferation ex vivo, specifically the TCRγ9δ2 subset, which has been extensively studied in PB gamma delta T cells. As CB gamma delta T cells are not well described, we compared CB gamma delta T cell proliferation, phenotype and genotype to PB gamma delta T cells when culturing cells with the NBP, Zometa (zoledronic acid), and IL-2. Fourteen days in culture resulted in significant fold increase in the proliferation of gamma delta T cells and in the percent of lymphocytes in both sample types. PB gamma delta T cells proliferated more robustly than CB with a 288.60 versus 21.32 fold increase, respectively. Additionally, in freshly isolated samples, CB gamma delta T cells comprised an average of 1.404% of the lymphocyte population, which was similar to PB gamma delta T cells, with an average of 2.319%. However, by day 14, PB gamma delta T cells increased to 70.15% of lymphocytes whereas CB gamma delta T cells increased to 12.49%. Phenotypically, both CB and PB had similar percent of CD45RA+ and CD45RO+ gamma delta T cell memory subsets in freshly isolated samples. Following culture, PB gamma delta T cells were mostly CD45RO+ memory cells, with significantly fewer CD45RA+ naïve cells, whereas more CB gamma delta T cells were of the intermediate CD45RA+CD45RO+ subset. Further phenotypic analysis of the memory subsets indicated that cultured PB gamma delta T cells were either effector memory cells (CD27-CD45RA-) or central memory cells (CD27+CD45RA-), while CB gamma delta T cells were mostly naïve (CD27+CD45RA+). The cytokines secreted by these cells were also assessed and the culture of PB and CB gamma delta T cells resulted in differing cytokine secretion profiles. After 14 days of culture, PB gamma delta T cells secreted more IFNγ and TNFα, while CB gamma delta T cells secreted more IL-10 and RANTES. We also examined TCRγ9 and TCRδ2 phenotypic expression and found that the TCRγ9δ2 was a common clone in freshly isolated PB gamma delta T cells, which predominated after 14 days in culture. However, while the TCRγ9δ2 variant was expressed in CB gamma delta T cells, it was low before and after culture, suggesting that Zometa may not stimulate gamma delta T cells in CB the same as PB. As limited TCRγδ phenotypic reagents are available, we developed a single cell PCR assay for genotypic analysis of the TCRγδ repertoire. PCR analysis suggests that the TCRγδ repertoire is diverse in both samples, yet TCRγ9δ2 is most prevalent. Further analysis of the variant subsets is warranted and may give insight into how each of these receptor pairings affects function. Disclosures No relevant conflicts of interest to declare.

scholarly journals Circulating Extracellular Vesicles Induce Chimeric Antigen Receptor T Cell Dysfunction in Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 679-679
Author(s):  
Michelle J. Cox ◽  
Fabrice Lucien-Matteoni ◽  
Reona Sakemura ◽  
Justin C. Boysen ◽  
Yohan Kim ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Ex Vivo ◽  
Data Safety Monitoring Board ◽  
Lymphocytic Leukemia ◽  
Cell Dysfunction

Treatment with CD19-directed chimeric antigen receptor T cell (CART19) therapy has resulted in unprecedented clinical outcomes and was FDA-approved in acute lymphoblastic leukemia and non-Hodgkin B-cell lymphoma. However, its success in chronic lymphocytic leukemia (CLL) has been modest to date. An increasing body of evidence indicates that impaired CART cell fitness is the predominant mechanism of the relative dysfunction in CLL. The immunosuppressive microenvironment in CLL is well known and in part may be related to the abundance of circulating extracellular vesicles (EVs) bearing immunomodulatory properties. We hypothesized that CLL-derived EVs contribute to CART cell dysfunction. In this study, we aimed to investigate the interaction between circulating EVs isolated from CLL patient plasma (designated as CLL-derived EVs) and CART19 cells. We enumerated and immunophenotyped circulating EVs from platelet free plasma in untreated patients with CLL. We determined their interaction with CART19 cells using second generation, 41BB co-stimulated, lentiviral transduced CART19 cells generated in the laboratory from normal donors (FMC63-41BBζ CART cells). Our findings indicate that CLL-derived EVs impair normal donor CART19 antigen-specific proliferation against the CD19+ mantle cell lymphoma cell line Jeko-1 (Figure 1A). Next, we characterized CLL-derived EVs using nanoscale flow cytometric analysis of surface proteins and compared to healthy controls. Although the total EV particle count was not different between CLL and healthy controls (Figure 1B), there were significantly higher PD-L1+ EVs in patients with CLL (Figure 1C). Based on these results, we sought to assess the physical interaction between CLL-derived EVs and CART cells from normal individuals. When CLL-derived EVs were co-cultured with CART19 and CLL B cells and imaged with super-resolution microscopy, EVs were localized at the T cell-tumor junction (Figure 1D). Furthermore, CLL-derived EVs are captured by T cells as indicated by a significant reduction in the absolute count of EVs when co-cultured with resting T cells (Figure 1E). Having demonstrated that 1) there is an excess of PD-L1+ EVs in patients with CLL (Figure 1C) and 2) CLL-derived EVs physically interact with CART cells (Figures 1D-E), we sought to establish their functional impact on CART19 cells. Here, CART19 cells were stimulated with irradiated CD19+ JeKo-1 cells at a 1:1 ratio in the presence of increasing concentrations of CLL-derived EVs. There was a significant upregulation of inhibitory receptors such as PD-1 and CTLA-4 on the T cells (Figure 1F). This is associated with a reduction in CART effector cytokines (i.e., TNFβ) at higher concentrations of EVs (Figure 1G), suggesting a state of exhaustion in activated CART19 cells in the presence of CLL-derived EVs. This was further supported by transcriptome interrogation of CART19 cells. Here, CART19 cells were stimulated via 24-hour co-culture with the irradiated CD19+ cell line JeKo-1, in the presence of CLL-derived EVs at ratios of 10:1 and 1:1 EV:CART19 and then isolated by magnetic sorting. RNA sequencing of these activated CART19 cells indicated a significant upregulation of AP-1 (FOS-JUN) and YY1 (Figures 1H), known critical pathways in inducing T cell exhaustion. Finally, to confirm the impact of CLL-derived EVs on CART19 functions in vivo, we used our xenograft model for relapsed mantle cell lymphoma. Here, immunocompromised NOD-SCID-ɣ-/- mice were engrafted with the CD19+ luciferase+ cell line JeKo-1 (1x106 cells I.V. via tail vein injection). Engraftment was confirmed through bioluminescent imaging and mice were randomized to treatment with 1) untreated, 2) CART19 cells, or 3) CART19 cells co-cultured ex vivo with CLL-derived EVs for six hours prior to injection. A single low dose of CAR19 (2.5x105) was injected, to induce relapse. Treatment with CART19 cells that were co-cultured ex vivo with CLL-derived EVs resulted in reduced anti-tumor activity compared to treatment with CART19 alone (Figure 1I). Our results indicate that CLL-derived EVs induce significant CART19 cell dysfunction in vitro and in vivo, through a direct interaction with CART cells resulting in a downstream alteration of their exhaustion pathways. These studies illuminate a novel way through which circulating and potentially systemic EVs can lead to CART cell dysfunction in CLL patients. Disclosures Cox: Humanigen: Patents & Royalties. Sakemura:Humanigen: Patents & Royalties. Parikh:Ascentage Pharma: Research Funding; Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Genentech: Honoraria; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding. Kay:Agios: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; MorphoSys: Other: Data Safety Monitoring Board. Kenderian:Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Lentigen: Research Funding; Novartis: Patents & Royalties, Research Funding; Tolero: Research Funding; Morphosys: Research Funding; Kite/Gilead: Research Funding.

scholarly journals p16INK4a Regulates Cellular Senescence in PD-1-Expressing Human T Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Valérie Janelle ◽  
Mathieu Neault ◽  
Marie-Ève Lebel ◽  
Dave Maurice De Sousa ◽  
Salix Boulet ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cellular Senescence ◽  
Ex Vivo ◽  
Cell Dysfunction ◽  
Human T Cells ◽  
Car T ◽  
P38mapk Signaling ◽  
T Cell Dysfunction

T-cell dysfunction arising upon repeated antigen exposure prevents effective immunity and immunotherapy. Using various clinically and physiologically relevant systems, we show that a prominent feature of PD-1-expressing exhausted T cells is the development of cellular senescence features both in vivo and ex vivo. This is associated with p16INK4a expression and an impaired cell cycle G1 to S-phase transition in repeatedly stimulated T cells. We show that these T cells accumulate DNA damage and activate the p38MAPK signaling pathway, which preferentially leads to p16INK4a upregulation. However, in highly dysfunctional T cells, p38MAPK inhibition does not restore functionality despite attenuating senescence features. In contrast, p16INK4a targeting can improve T-cell functionality in exhausted CAR T cells. Collectively, this work provides insights into the development of T-cell dysfunction and identifies T-cell senescence as a potential target in immunotherapy.

Sirolimus Inhibits Proliferation but Enhances Suppressive Activity of Rhesus Macaque Natural CD4 Regulatory T Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1008-1008
Author(s):  
Karnail Singh ◽  
Natalia Kozyr ◽  
Linda Stempora ◽  
Allan D Kirk ◽  
Christian P Larsen ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Cd8 T Cell ◽  
T Cell Proliferation ◽  
Suppressive Activity ◽  
Suppressive Capacity

Abstract Abstract 1008 Regulatory T cells (Tregs) have been shown to be potent inhibitors of autoimmunity, and to be capable of suppressing alloimmune responses that occur during both allograft rejection and graft-versus host disease. However, they have yet to gain widespread use clinically, due in part to the fact that it remains extremely costly and difficult to produce them in sufficient numbers and with sufficient suppressive capacity to significantly impact the alloimmune response. Here we have used our established non-human primate model to demonstrate that significant Treg expansion (up to 600-fold in 21 days) can be maintained, and suppressive capacity enhanced by exposing Treg cultures to a short burst of sirolimus at the end of the culture period. Using a highly sensitive and specific in vitro CFSE-MLR assay we show that Tregs significantly inhibit allo-proliferation of multiple T cell subpopulations including both CD4+ and CD8+ T cells (3.2 and 2.7-fold inhibition of proliferation, respectively), as well as their CD28+CD95+ and CD28-CD95+ subpopulations (2.2 and 2.1 and 1.9 and 2.7-fold inhibition of CD4+ and CD8+ subpopulation proliferation, respectively). Tregs were able to combine in vitro with the newly FDA-approved CTLA4-Ig analog belatacept to enhance the inhibition of alloproliferation that occurred with either agent alone (4.8-fold inhibition of CD8 T cell proliferation with Tregs + belatacept, compared to 3.0-fold or 1.9-fold inhibition of CD8 T cell proliferation with Tregs or belatacept alone, respectively). Importantly, we have found that the suppressive activity of ex-vivo expanded Tregs could be further enhanced by pulsing with sirolimus. Thus, while long-term culture of Tregs in the presence of sirolimus (1–1000 nM) profoundly inhibited Treg expansion (50–800 fold inhibition of expansion when cultured in the presence of 1–1000 nM sirolimus), a 48 hour pulse of sirolimus (100 nM) on days 20–21 of culture completely preserved Treg yields while doubling their suppressive function against CD8 proliferation when compared to unpulsed Tregs, p<0.01) A mechanistic evaluation of the increase potency observed with sirolimus pulsed Tregs (SPTs) has revealed several key differences that distinguish these cells from the less-potent unpulsed Tregs: SPTs were found to undergo fewer rounds of proliferation in an MLR when compared with unpulsed Tregs (14% proliferation in SPTs versus 37% proliferation in un-pulsed Tregs, p= 0.015), suggesting that the suppressive capability of Tregs may be inversely related to their proliferative capacity. SPTs were also shown to have significantly increased expression of CD25 (p=0.04) and total CTLA4 (p= 0.009) compared to unpulsed Tregs, implicating signaling through both of these molecules in their enhanced function. Our results suggest that the creation of SPTs may provide a novel avenue by which to achieve enhanced Treg-based suppression of alloimmunity, in a manner that is amenable to large-scale ex-vivo expansion and to combinatorial therapy with novel, costimulation-blockade-based immunosuppression strategies. Disclosures: No relevant conflicts of interest to declare.

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