scholarly journals Chronic Lymphocytic Leukemia Actively Induces T-Cell Dysfunction By Contact-Dependent Signaling Via CD24 and CD52

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3714-3714
Author(s):  
Jaco A. C. Van Bruggen ◽  
Fleur Peters ◽  
Gaspard Cretenet ◽  
J. Joseph Melenhorst ◽  
Eric Eldering ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Research Funding ◽  
Cell Activation ◽  
Cell Function ◽  
Cell Dysfunction ◽  
T Cell Function ◽  
Car T ◽  
T Cell Dysfunction

Abstract Introduction Success rates of autologous T cell-based therapies, such as CAR-T cell therapy, in chronic lymphocytic leukemia (CLL) have been suboptimal and correlate with failure of activation and proliferation of T cells in vitro and in vivo. Previous data showing that impaired CD8 T-cell activation, proliferation and metabolic reprogramming could be restored by purifying CLL T cells via cell-sorting (van Bruggen et al., Blood, 2019) indicating that an as yet unknown, CLL-derived factor is responsible for acquired T-cell dysfunction. In this study we aim to elucidate the mechanistic basis of CLL-mediated T-cell dysfunction. Results Dynamic analysis of αCD3/CD28 stimulated autologous T cells in presence of CLL cells over a period of 9 days revealed that T-cell activation (CD25, CD71, CD95 and PD-1) in CLL is in fact not impaired but occurs in a delayed fashion. CLL T cells reached peak activation after 5-6 days in contrast to 2-3 days for age-matched healthy donors. (Fig. 1A). This delayed T cell receptor-induced T cell activation was largely normalized with tumor cell depletion by flow-sorting prior to activation. Accordingly, in absence versus presence of autologous CLL cells, CAR-T cells derived from CLL patients showed enhanced proliferation, cytokine production and cytotoxicity, indicating potential clinical relevance. These findings show that T cells in CLL are not (terminally) exhausted but that a CLL-derived factor interferes with proper T-cell activation, leading to a delay in activation and impaired proliferation and cytotoxicity. We attempted to identify the mechanism of action in which CLL cells induce T cell dysfunction and whether these suppressive effects are mediated through a soluble factor secreted by CLL cells or by contact-dependent mechanisms. Previous studies have shown that CD40 activation of CLL cells results in increased expression of key surface-expressed adhesion and costimulatory molecules, but also in alterations of immune-modulatory cytokines secretion. This model was therefore used to decipher mechanisms of CLL-mediated T cell dysfunction. CD40-activation of CLL cells resulted in improved T-cell activation and proliferation upon αCD3/CD28 stimulation in a contact-dependent manner (based on trans-well experiments; Fig. 1B ). Several clinically approved kinase inhibitors were tested to identify signaling cascades involved in CD40-mediated alleviation of T-cell dysfunction. Only pre-treatment of CLL cells with the SRC-inhibitor dasatinib (100nM) abrogated the enhanced T-cell activation induced by CD40-activated CLL cells. Additional control experiments excluded direct effects of dasatinib on T cell function. Dasatinib did not reduce expression of co-stimulatory markers on CD40-activated CLL cells, indicating that lack of co-stimulation was not the sole explanation for CLL-mediated T cell dysfunction. RNA sequencing of CD40-stimulated CLL cells treated with or without dasatinib and filtered for membrane-bound factors revealed the Sialic acid-binding Ig-like lectin 10 (Siglec-10) ligands CD24 and CD52 as potential candidates responsible for inhibiting T-cell function in CLL, which we confirmed at the protein level. We also found increased expression of Siglec-10 on CLL T cells, suggesting a role for Siglec-10 ligation in inhibition of the TCR signaling cascade. Indeed, inhibition of Siglec-10 ligation by blocking CD24, and CD52 antibodies subsequently improved T-cell activation despite presence of CLL cells (Fig. 1C). Conclusion These results demonstrate that T cells derived from CLL patients are not terminally dysfunctional and can be revived. Our observations indicate that CLL cells actively suppress (CAR) T-cell function in a contact-dependent fashion through CD24- and CD52-mediated Siglec-10 ligation. These proteins might represent targets for therapeutic intervention aimed at enhancing T-cell function in CLL. Figure 1 Figure 1. Disclosures Kater: Genmab, LAVA: Other: Ad Board, Steering Committee; Abbvie: Honoraria, Other: Ad Board, Research Funding; Janssen, AstraZeneca: Other: Ad Board, steering committee, Research Funding; BMS, Roche/Genentech: Other: Ad Board, , Research Funding.

A Failure to Start: Aborted Activation of CAR T Cells in Chronic Lymphocytic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 681-681
Author(s):  
McKensie Collins ◽  
Weimin Kong ◽  
Inyoung Jung ◽  
Meng Wang ◽  
Stefan M Lundh ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Research Funding ◽  
Cell Dysfunction ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
T Cell Dysfunction ◽  
Low Levels

Introduction: Chronic Lymphocytic Leukemia (CLL) is a CD19+ B-cell malignancy that accounts for approximately 25% of adult leukemia diagnoses in the developed world. While conventional therapies have some efficacy, there are few curative therapeutic options and many patients ultimately progress to relapsed or refractory disease. CD19-targeting chimeric antigen receptor (CAR) T cell therapy has provided some hope, but induces complete remission in only 26% of patients. This suboptimal response rate is believed to be due to T cell dysfunction and immune-suppression by CLL cells, the mechanisms of which are poorly understood. Results: To understand the causes of CAR T cell dysfunction in CLL we investigated the defects that CLL cells induced in normal donor CD19-targeting CAR T cells. CAR T cells were repeatedly stimulated at 5-day intervals with either primary CLL cells from patients or a CD19-expressing control cell line (aAPC). Repeat stimulation of CAR T cells with aAPCs resulted in 5.36 ± .94 population doublings after three stimulations, whereas CLL cells only evoked 2.39 ± .92 population doublings. We performed phenotyping, proliferation analysis, and cytokine analysis of stimulated CAR T cells. CLL-stimulated T cells appeared un-activated, with low levels of PD-1, LAG3, and TIM3, low levels of cytokine production, and a high proportion of non-cycling cells as measured by Ki67 staining. We first hypothesized that CLL cells induce an altered epigenetic program that prevents effector function and is stabilized by successive stimulations. To test this, we stimulated CAR T cells with CLL cells or aAPCs as indicated in Fig. 1A. CLL-stimulated CAR T cells failed to proliferate or produce cytokines, but subsequent stimulation with aAPCs rescued these functions (Fig. 1B). Further, CLL-stimulated CAR T cells did not differentiate, suggesting that CLL cells do not induce stable defects but rather insufficiently activate CAR T cells (Fig. 1C). These cells also appeared un-activated as indicated by low levels of PD-1 and Ki67. We then used flow cytometry to assess expression of costimulatory and inhibitory molecules on the primary CLL samples. We found that the levels of co-stimulatory and adhesion molecules, namely CD80/CD86 and CD54/CD58 respectively were found at low frequencies, and where present were expressed at low levels. This suggested that one mechanism behind the lack of CAR T cell effector responses may be that a lack of co-stimulation prevents proper CAR T cell targeting of these cells. Towards this, we incubated CLL cells with a murine fibroblast line expressing CD40 ligand for 24 hours with IL-4 to activate the CLL cells. We found that this activation significantly increased expression of CD80, CD86, CD54, and CD58 on the CLL cells. We then used these cells to stimulate CAR T cells in our re-stimulation assay and found that CAR T cells were able to proliferate in response to these activated CLLs (Fig. 1D). In addition, CAR T cells stimulated with activated CLL cells formed more cell-to-cell conjugates than those stimulated with un-activated CLL cells. These data suggest not only that insufficient activation of CAR T cells may be responsible for the poor response rates to CAR T cell therapy in CLL, but also implicate a need for additional co-stimulation in this CAR T cell setting. Another contributing factor may be immune suppression by CLL cells. To determine if CLL cells are immune-suppressive, we used a co-culture assay to stimulate CAR T cells with aAPCs and CLL cells mixed at known ratios. Interestingly, all mixed cultures proliferated similarly, suggesting that CLL cells did not prevent T cell activation in the presence of a strong activation signal. We also found that CLL cells are responsive to IL-2, as addition of this cytokine to culture media prolongs survival of CLL cells out to 10 days depending on the dose. This suggests that CLL cells express a functional IL-2 receptor and may be taking up IL-2 from the culture media, further impairing T cell activation. In support of this, supplementing IL-2 into our CLL/CAR T cell co-cultures rescued T cell proliferative capacity. Taken together, these data suggest that T cell dysfunction in CLL is the result of insufficient activation rather than true functional defects. Disclosures June: Novartis: Research Funding; Tmunity: Other: scientific founder, for which he has founders stock but no income, Patents & Royalties. Melenhorst:National Institutes of Health: Research Funding; Parker Institute for Cancer Immunotherapy: Research Funding; Novartis: Research Funding, Speakers Bureau; IASO Biotherapeutics, Co: Consultancy; Simcere of America, Inc: Consultancy; Shanghai Unicar Therapy, Co: Consultancy; Colorado Clinical and Translational Sciences Institute: Membership on an entity's Board of Directors or advisory committees; Genentech: Speakers Bureau; Stand Up to Cancer: Research Funding; Incyte: Research Funding.

The CXCR4/STAT3/IL-10 Pathway Controls the Immunoregulatory Function of Chronic Lymphocytic Leukemia (CLL) and Can be Modulated By Lenalidomide

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1709-1709 ◽  
Author(s):  
Hila Shaim ◽  
Zeev Estrov ◽  
Takuya Sekine ◽  
Kayo Kondo ◽  
Peter P. Ruvolo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Cell Function ◽  
Blocking Antibody ◽  
Cell Dysfunction ◽  
T Cell Suppression ◽  
T Cell Function ◽  
Stat3 Phosphorylation ◽  
T Cell Dysfunction

Abstract Patients with CLL experience generalized immune suppression, susceptibility to infections and secondary malignancies that likely involve complex bi-directional interactions between leukemic cells, components of the tumor microenvironment and immune effectors. CLL cells are capable of secreting IL-10 and exhibit regulatory functions comparable to that of normal B10 cells, a regulatory B cell subset that suppresses effector T-cell function through STAT3-mediated production of IL-10. However, the underlying mechanisms governing IL-10 production by CLL cells are not fully understood. The chemokine CXCL12 is constitutively secreted by bone marrow stroma in CLL, and binds CXCR4 to direct chemotaxis, support tumor survival and activate various signaling pathways, including STAT3. Thus, we investigated if CXCL12 can enhance IL-10 production by activating the STAT3 pathway in CLL. Using peripheral blood mononuclear cells (PBMC) from 24 CLL patients who had not received therapy for ≥2 years, we showed that CXCL12 can enhance IL-10 production by CLL cells by activating S727-STAT3. This effect was CXCR4-mediated since blocking the CXCR4-CXCL12 interaction with a blocking antibody abolished CXCL12-induced IL-10 production. Addition of the STAT3 inhibitor curcubitacin to the culture also abrogated CXCL12-induced IL-10 production, confirming an important role for S727-STAT3 as a mediator of CXCL12-CXCR4-induced IL-10 production by CLL cells. We next determined if activation of the CXCR4-CXCL12-STAT3-IL10 pathway in CLL is important in mediating their immunoregulatory function. Culture of primary CLL withCXCL12 induced significantly more suppression of CD3+ T cell function, including TNF-α, IFN-γ and IL-2 production, and CD107a degranulation, compared to CD3+ T cells cultured with untreated CLL cells or with CXCL12 alone. The addition of IL-10 blocking antibody to the co-culture completely reversed T cell dysfunction, supporting an important for IL-10 in CLL-mediated T-cell suppression. IL-10 has been reported to induce T cell suppression through phosphorylation of Y705-STAT3.. Blocking IL-10 also prevented CLL-induced phosphorylation of Y705-STAT3 in T cells, confirming an important role for CLL-derived IL-10 in activation of Y705-STAT3 and induction of T cell dysfunction. Lenalidomide is an immune-modulatory drug with clinical efficacy in CLL and was recently reported to inhibit STAT3 phosphorylation. To investigate if lenalidomide can inhibit CXCL12-induced STAT3 phosphorylation, we treated CLL cells with lenalidomide and measured p-S727-STAT3 levels. Exposure of CLL cells to 10µM/ml lenalidomide prevented CXCL12-induced increase in p-S727-STAT3 and resulted in significant reduction in the IL-10 response by CLL cells. Lenalidomide also suppressed IL-10-induced Y705-STAT3 phosphorylation in healthy T cells, thus reversing CLL-induced T cell dysfunction. We next confirmed the in vivo relevance of our findings using PBMC cryopreserved from patients treated with lenalidomide monotherapy (NCT00535873). When compared to pretreatment samples, CLL cells from on-treatment samples produced less IL-10 and showed significantly improved T cell function. We thus conclude that the capacity of CLL to produce IL-10 is mediated by the CXCL12-CXCR4-STAT3 pathway and may contribute to immunodeficiency in patients. Lenalidomide can reverse CLL-induced immunosuppression through multiple mechanisms that involve abrogation of the CXCL12-CXCR4-S727STAT3-mediated IL-10 response by CLL B cells and prevention of IL-10-induced phosphorylation of Y705-STAT3 in T cells. Disclosures Estrov: incyte: Consultancy, Research Funding. Wierda:Glaxo-Smith-Kline Inc.: Research Funding; Celgene Corp.: Consultancy. Rezvani:Pharmacyclics: Research Funding.

scholarly journals Treatment with Acalibrutinib, Ibrutinib and CD19 CAR T Cells Restore the Number of Granulocytic Myeloid Derived Supressor Cells in CLL-Bearing Mice

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3032-3032
Author(s):  
Arantxa Romero-Toledo ◽  
Robin Sanderson ◽  
John G. Gribben
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Population ◽  
Research Funding ◽  
Cell Function ◽  
Car T Cells ◽  
Car T Cell ◽  
T Cell Function ◽  
Car T ◽  
Secondary Lymphoid Organs

The complex crosstalk between malignant chronic lymphocytic leukemia (CLL) cells and the tumor microenvironment (TME) is not fully understood. CLL is associated with an inflammatory TME and T cells exhibit exhaustion and multiple functional defects, fully recapitulated in Eµ-TCL1 (TCL1) mice and induced in healthy mice by adoptive transfer (AT) of murine CLL cells, making it an ideal model to test novel immunotherapies for this disease. Myeloid-derived suppressor cells (MDSCs), a non-leukemic cell type within the TME, are immature myeloid cells with the ability to suppress T cell function and promote Treg expansion. In humans, CLL cells can induce conversion of monocytes to MDSCs provoking their accumulation in peripheral blood (PB). MDSCs include two major subsets granulocytic (Gr) and monocytic (M)-MDSC. In mice, Gr-MDSCs are defined as CD11b+Ly6G+Ly6Clo and M-MDSC as CD11b+Ly6G-Ly6Chi. Both murine and human MDSCs express BTK. We observed that in CLL-bearing mice, MDSCs cells are lost in PB as disease progresses. Treatment with both BTK inhibitors (BTKi), ibrutinib (Ibr) and acalabrutinib (Acala), result in shift of T cell function from Th2 towards Th1 polarity and increase MDSC populations in vivo. We aimed to determine whether combination treatment with BTKi and chimeric antigen receptor (CAR) T cells renders recovery of the MDSC population in CLL-bearing mice. To address this question we designed a two-part experiment, aiming to mimic the clinically relevant scenario of pre-treatment of CLL with BTKi to improve CAR T cell function. Part 1 of our experiment consisted of 4 groups (n=12) of 2.5 month old C57/Bl6 mice. Three groups had AT with 30x106 TCL1 splenocytes. A fourth group of WT mice remained CLL-free as a positive control and donors for WT T cells. When PB CLL load reached >10% (day 14) animals were randomized to either Ibr or Acala at 0.15 mg/l in 2% HPBC or no treatment for 21 days. All animals from part 1 were culled at day 35 post-AT and splenic cells were isolated, analyzed and used to manufacture CAR T cells. WT, CLL, Ibr and Acala treated T cells were activated and transduced with a CD19-CD28 CAR to treat mice in part 2. Here, 50 WT mice were given AT with 20x106 TCL1 splenocytes for CLL engraftment. All mice were injected with lymphodepleting cyclophosphamide (100mg/kg IP) one day prior to IV CAR injection. At day 21 post-AT, mice were treated with WT CAR, CLL CAR, IbrCAR, AcalaCAR or untransduced T cells. MDSC sub-populations were monitored weekly in PB and SP were analysed by flow cytometry. As malignant CD19+CD5+ cells expands in PB, the overall myeloid (CD19-CD11b+) cell population was not affected, but MDSCs significantly decreased (p<0.0001). Treatment with Acala, but not Ibr restores total MDSCs. However, MDSC impairment occurs in the Gr- but not M- MDSC population and both Acala and Ibr restores this population (Figure 1a). When we examined the spleen, treatment with both Ibr (p<0.001) and Acala (p<0.001) reduced CD5+CD19+ cells, whereas neither BTKi affected the overall myeloid (CD19-CD11b+) cell population. Gr-MDSCs were restored by both treatments whilst M-MDSCs were only restored after Ibr treatment (p<0.001 in each case). In part 2 of this experiment we observed that treatment with all CAR-T cell groups provokes the clearance of all CD19+CD5+ cells. The overall CD19-CD11b+ population stays the same across all mice groups 35 days after treatment in PB with any group of CAR and untransduced T cells. Overall MDSC population is maintained following all CAR T cells compared to CLL-bearing mice (p<0.0001) and it is the Gr- but not the M- MDSC population which is recovered in PB (Figure 1b). These parts of the experiments can of course be influenced by treatment with cyclophosphamide. We conclude that novel therapies for CLL treatment have an effect not only in CLL cells but also in non-malignant cell components of the TME. In this animal model of CLL, the rapid expansion of CLL cells in PB and secondary lymphoid organs provokes loss of MDSC, particularly the Gr-MDSC subpopulation is affected. Treatment with BTKi and CAR T cells provokes clearance of CLL cells in PB and spleen allowing MDSC recovery; suggesting this may be BTK and ITK independent. We continue to explore secondary lymphoid organs to further characterize the shift of the CLL microenvironment from an immunosuppressive to an immune effective one and its impact on immune function in this model. Disclosures Sanderson: Kite/Gilead: Honoraria. Gribben:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding.

scholarly journals Modulation of KV1.3 channels by protein kinase A I in T lymphocytes is mediated by the disc large 1-tyrosine kinase Lck complex

2012 ◽  
Vol 302 (10) ◽  
pp. C1504-C1512 ◽  
Author(s):  
Zerrin Kuras ◽  
Vladimir Kucher ◽  
Scott M. Gordon ◽  
Lisa Neumeier ◽  
Ameet A. Chimote ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Tyrosine Kinase ◽  
Signaling Pathway ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Function ◽  
Scaffolding Protein ◽  
T Cell Function

The cAMP/PKA signaling system constitutes an inhibitory pathway in T cells and, although its biochemistry has been thoroughly investigated, its possible effects on ion channels are still not fully understood. KV1.3 channels play an important role in T-cell activation, and their inhibition suppresses T-cell function. It has been reported that PKA modulates KV1.3 activity. Two PKA isoforms are expressed in human T cells: PKAI and PKAII. PKAI has been shown to inhibit T-cell activation via suppression of the tyrosine kinase Lck. The aim of this study was to determine the PKA isoform modulating KV1.3 and the signaling pathway underneath. 8-Bromoadenosine 3′,5′-cyclic monophosphate (8-BrcAMP), a nonselective activator of PKA, inhibited KV1.3 currents both in primary human T and in Jurkat cells. This inhibition was prevented by the PKA blocker PKI6–22. Selective knockdown of PKAI, but not PKAII, with siRNAs abolished the response to 8-BrcAMP. Additional studies were performed to determine the signaling pathway mediating PKAI effect on KV1.3. Overexpression of a constitutively active mutant of Lck reduced the response of KV1.3 to 8-Br-cAMP. Moreover, knockdown of the scaffolding protein disc large 1 (Dlg1), which binds KV1.3 to Lck, abolished PKA modulation of KV1.3 channels. Immunohistochemistry studies showed that PKAI, but not PKAII, colocalizes with KV1.3 and Dlg1 indicating a close proximity between these proteins. These results indicate that PKAI selectively regulates KV1.3 channels in human T lymphocytes. This effect is mediated by Lck and Dlg1. We thus propose that the KV1.3/Dlg1/Lck complex is part of the membrane pathway that cAMP utilizes to regulate T-cell function.

scholarly journals Hypoxia is a dominant remodeler of the CD8+ T cell surface proteome relative to activation and regulatory T cell-mediated suppression

2021 ◽  
Author(s):  
James Robert Byrnes ◽  
Amy M Weeks ◽  
Julia Carnevale ◽  
Eric Shifrut ◽  
Lisa Kirkemo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Function ◽  
Metabolic Reprogramming ◽  
T Cell Function ◽  
Human T Cells ◽  
Surface Proteome

Immunosuppressive factors in the tumor microenvironment (TME) impair T cell function and limit the anti-tumor immune response. T cell surface receptors that influence interactions and function in the TME are already proven targets for cancer immunotherapy. However, surface proteome remodeling of primary human T cells in response to suppressive forces in the TME has never been characterized systematically. Using a reductionist cell culture approach with primary human T cells and SILAC-based quantitative cell surface capture glycoproteomics, we examined how two immunosuppressive TME factors, regulatory T cells (Tregs) and hypoxia, globally affect the activated CD8+ surface proteome (surfaceome). Surprisingly, the CD8+/Treg co-culture only modestly affected the CD8+ surfaceome, but did reverse several activation-induced surfaceomic changes. In contrast, hypoxia dramatically altered the CD8+ surfaceome in a manner consistent with both metabolic reprogramming and induction of an immunosuppressed state. The CD4+ T cell surfaceome similarly responded to hypoxia, revealing a novel hypoxia-induced surface receptor program. Our findings are consistent with the premise that hypoxic environments create a metabolic challenge for T cell activation, which may underlie the difficulty encountered in treating solid tumors with immunotherapies. Together, the data presented here provide insight into how suppressive TME factors remodel the T cell surfaceome and represent a valuable resource to inform future therapeutic efforts to enhance T cell function in the TME.

Specificity of JAK-Kinase Inhibition Determines Impact on T-Cell Function

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1410-1410 ◽  
Author(s):  
Florian Perner ◽  
Felix C Saalfeld ◽  
Tina M Schnoeder ◽  
Denise Wolleschak ◽  
Corinna Fahldieck ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Function ◽  
T Cell Proliferation ◽  
Kinase Inhibition ◽  
T Cell Function ◽  
Jak Kinase ◽  
Cd69 Expression

Abstract Inhibitors of JAK2-kinase (Ruxolitinib, Momelotinib) are already approved or currently investigated in advanced clinical trials for treatment of myeloproliferative neoplasia (MPN). Besides their effect on mutated JAK2-kinase these compounds inhibit wildtype JAK and thereby impair JAK-STAT-signaling, which is an important pathway for proliferation and activation of other cell types such as human T-cells. Accumulating evidence suggests that they may also exert substantial immunosuppressive activity. Very recent reports highlighting hepatitis B reactivation complemented the series of severe infections in ruxolitinib-treated patients among which cryptococcus neoformans pneumonia, toxoplasmosis retinitis, disseminated tuberculosis, and progressive multifocal leukencephalopathy are the most alarming. We hypothesized that JAK-kinase inhibitors may act as immunosuppressant drugs by impairment of T-cell responses through inhibition of T-cell signaling (JAK-STAT pathway) and that specificity of JAK-kinase inhibition may be of major importance for the degree of T-cell inhibition. Therefore we investigated the effects of pharmacological JAK-kinase inhibition on healthy donor (HD-) and MPN patient T-cells. Selective inhibitors of JAK2-kinase (BSK805) and JAK3-kinase (BQM245) as well as clinically relevant inhibitors of JAK1/2-kinases (Ruxolitinib and Momelotinib) were used for pharmacologic inhibition. The SRC-kinase inhibitor Dasatinib served as a positive control for T-cell inhibition. Knockdown of specific JAK-kinases by RNAi was used to control for target specificity. In regard to T-cell receptor (TCR)-mediated signaling we investigated bona fide signaling molecules downstream of the TCR by Western Blotting. Besides SRC-kinases like LCK also ZAP70, PLCG1 and the MAPK/ERK pathway have been described to play a pivotal role in T-cell activation. In our data set, selectivity of JAK-kinase inhibition (JAK2, JAK3 or JAK1/2) influenced TCR-signaling in regard to overall tyrosine phosphorylation but also in regard to downstream effectors such as ERK. As activation and proliferation of primary T-cells is a critical step in immune responses against viral and tumor antigens we aimed to investigate the influence of JAK-kinase inhibition on activation and proliferation of human T-cells. T-cells from healthy donors were stimulated using either PHA 0.5% or CD3/CD28 beads to ensure a more T-cell receptor specific stimulation. CD69 expression was used as a marker for T-cell activation and CFSE staining was applied to assess for T-cell proliferation. Using CD3/CD28 stimulation, CD69 expression was almost abrogated following Dasatinib treatment and proliferation was significantly reduced. Applying relevant doses of specific JAK2 and JAK3 inhibitors to isolated T-cells did neither influence CD69 expression nor T-cell proliferation. These findings are confirmed by RNAi. In contrast, clinically relevant doses of JAK1/2 inhibitors Ruxolitinib and Momelotinib, respectively reduced CD69 expression and T-cell proliferation. Likewise, T-cells derived from MPN patients treated with Ruxolitinib revealed decreased CD69 expression and decreased proliferative capacity upon stimulation, compared to untreated patients or HD-controls. In order to investigate T-cell function, we assessed for allo-reactivity in a mixed lymphocyte culture. Human pan-T-cells were co-cultured with allogeneic antigen presenting cells. T-cell reactivity – as measured by 3H-thymidine incorporation – was significantly impaired by Ruxolitinib and Momelotinib. Specific inhibition of JAK2 or JAK3 kinase, however, did not affect T-cell reactivity. These effects could be confirmed using T-cells derived from Ruxolitinib-treated MPN patients. Investigation of leukemia- and virus-antigen-specific T-cell responses are currently under way to gain deeper insight regarding this clinically relevant scenario. Taken together, specificity of JAK-kinase inhibition influences the inhibitory potential on T-cell function. JAK1 kinase seems to play an important role in T-cell activation, as unspecific inhibitors of JAK1 & JAK2 Kinase inhibit T-cell function while selective inactivation of JAK2 kinase leaves T-cell function almost unaffected. Heterogeneity in T-cell function of Ruxolitinib-treated patients is an important finding that deserves detailed investigation. Disclosures Heidel: Novartis: Consultancy.

Redox regulation of T-cell receptor signaling

2015 ◽  
Vol 396 (5) ◽  
pp. 555-569 ◽  
Author(s):  
Luca Simeoni ◽  
Ivan Bogeski
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Cell Activation ◽  
Redox Regulation ◽  
Molecular Mechanisms ◽  
Cell Function ◽  
Cell Receptor ◽  
T Cell Function

Abstract T-cell receptor (TCR) triggering by antigens activates a sophisticated intracellular signaling network leading to transcriptional activation, proliferation and differentiation of T cells. These events ultimately culminate in adaptive immune responses. Over recent years it has become evident that reactive oxygen species (ROS) play an important role in T-cell activation. It is now clear that ROS are involved in the regulation of T-cell mediated physiological and pathological processes. Upon TCR triggering, T cells produce oxidants, which originate from different cellular sources. In addition, within inflamed tissues, T cells are exposed to exocrine ROS produced by activated phagocytes or other ROS-producing cells. Oxidative modifications can have different effects on T-cell function. Indeed, they can stimulate T-cell activation but they can be also detrimental. These opposite effects of oxidation likely depend on different factors such as ROS concentration and source and also on the differentiation status of the T cells. Despite the well-stablished fact that ROS represent important modulators of T-cell activation, the precise molecular mechanisms of their action are far from clear. Here, we summarize the present knowledge on redox regulation of T-cell function with a particular emphasis on the redox regulation of TCR signaling.

scholarly journals Optimizing T Cell-Based Therapy for Glioblastoma

2021 ◽  
Vol 12 ◽  
Author(s):  
Aida Karachi ◽  
Farhad Dastmalchi ◽  
Saina Nazarian ◽  
Jianping Huang ◽  
Elias J. Sayour ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Treatment Strategies ◽  
Cell Dysfunction ◽  
Cell Based Therapy ◽  
Car T ◽  
Transfer Strategies ◽  
T Cell Dysfunction ◽  
T Cell Transfer

Evading T cell surveillance is a hallmark of cancer. Patients with solid tissue malignancy, such as glioblastoma (GBM), have multiple forms of immune dysfunction, including defective T cell function. T cell dysfunction is exacerbated by standard treatment strategies such as steroids, chemotherapy, and radiation. Reinvigoration of T cell responses can be achieved by utilizing adoptively transferred T cells, including CAR T cells. However, these cells are at risk for depletion and dysfunction as well. This review will discuss adoptive T cell transfer strategies and methods to avoid T cell dysfunction for the treatment of brain cancer.

Discovery of COM701, a therapeutic antibody targeting the novel immune checkpoint PVRIG, for the treatment of cancer.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 3074-3074 ◽  
Author(s):  
Spencer Liang ◽  
Ofer Levy ◽  
Sudipto Ganguly ◽  
Maya Kotturi ◽  
Ilan Vaknin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Function ◽  
Wild Type ◽  
High Affinity ◽  
T Cell Function

3074 Background: While inhibitors of CTLA4 and PD1 have emerged as effective cancer therapies, the majority of treated patients do not derive long term benefit. Employing our computational discovery platform, we discovered PVRIG as an immune suppressive molecule expressed on T and NK cells and identified COM701, an antibody (Ab) targeting human PVRIG that enhances T cell function and anti-tumor responses. Methods: Anti-human PVRIG Ab COM701 was identified as an antagonistic Ab that enhanced T cell function in multiple assays. Antagonistic anti-mouse PVRIG Abs and PVRIG deficient (PVRIG-/-) mice were generated and characterized using syngeneic tumor models. Results: PVRIG was induced upon T cell activation, with long term activation leading to the highest expression. PVRL2 was identified as the ligand for PVRIG, placing PVRIG in the DNAM/TIGIT immunoreceptor axis. Compared to normal adjacent tissues, PVRIG and PVRL2 were both induced in the tumor microenvironment of several human cancers. To target PVRIG for therapeutic intervention, we identified COM701, a high affinity Ab that disrupts the interaction of PVRIG with PVRL2. COM701 enhanced CD8 T cell proliferation and IFN-g production in vitro and had an additive or synergistic effect on T cell activation when further combined with an anti-PD1 or anti-TIGIT Ab. Consistent with a checkpoint function for human PVRIG, mouse PVRIG-/- T cells showed increased function compared to wild type T cells. A surrogate antagonistic anti-mPVRIG Ab reduced growth of CT26 and B16 tumors when combined with an anti-PDL1 Ab in vivo. MC38 tumors also grew slower in PVRIG-/- mice compared to wild type mice and ex vivo analysis pointed to functional differences in anti-cancer immunity. Conclusions: We demonstrated that targeting PVRIG with COM701, a high affinity antagonistic Ab, increased human T cell function. We further showed that PVRIG was induced in the tumor microenvironment and that disruption of PVRIG/PVRL2 interaction resulted in reduced tumor growth in preclinical models. These data demonstrate that PVRIG is a promising target for the treatment of cancer and provide the rationale for COM701 as a potential cancer immunotherapy.

scholarly journals Regulation of T-cell function by endogenously produced angiotensin II

2009 ◽  
Vol 296 (2) ◽  
pp. R208-R216 ◽  
Author(s):  
Nyssa E. Hoch ◽  
Tomasz J. Guzik ◽  
Wei Chen ◽  
Tenecia Deans ◽  
Samer A. Maalouf ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nadph Oxidase ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Function ◽  
Receptor Subtypes ◽  
Ang Ii ◽  
T Cell Function ◽  
Tnf Α

The adaptive immune response and, in particular, T cells have been shown to be important in the genesis of hypertension. In the present study, we sought to determine how the interplay between ANG II, NADPH oxidase, and reactive oxygen species modulates T cell activation and ultimately causes hypertension. We determined that T cells express angiotensinogen, the angiotensin I-converting enzyme, and renin and produce physiological levels of ANG II. AT1 receptors were primarily expressed intracellularly, and endogenously produced ANG II increased T-cell activation, expression of tissue homing markers, and production of the cytokine TNF-α. Inhibition of T-cell ACE reduced TNF-α production, indicating endogenously produced ANG II has a regulatory role in this process. Studies with specific antagonists and T cells from AT1R and AT2R-deficient mice indicated that both receptor subtypes contribute to TNF-α production. We found that superoxide was a critical mediator of T-cell TNF-α production, as this was significantly inhibited by polyethylene glycol (PEG)-SOD, but not PEG-catalase. Thus, T cells contain an endogenous renin-angiotensin system that modulates T-cell function, NADPH oxidase activity, and production of superoxide that, in turn, modulates TNF-α production. These findings contribute to our understanding of how ANG II and T cells enhance inflammation in cardiovascular disease.

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