Local activator and T cell engager (LocATE) selectively blocks PD-L1 at the cytolytic synapse for deeper responses in multiple myeloma.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. 8045-8045 ◽  
Author(s):  
Christian Leisner ◽  
Leonardo Borras ◽  
Stephanie Jungmichel ◽  
Philipp Richle ◽  
Fabian Scheifele ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Fold Increase ◽  
Checkpoint Inhibition ◽  
Bone Marrow Biopsies ◽  
Immune Synapse

8045 Background: The BCMA-targeting bispecific T-cell engager AMG420 emerged as the first bispecific that achieved responses similar to CAR-T therapies in patients with relapsed/refractory (RR) multiple myeloma (MM). Despite improved ORR, the median duration until relapse is currently limited to approximately 12 months. Persistent minimal residual disease drives relapse and is characterized by increased expression of PD-1/PD-L1. Efficacy with checkpoint inhibitors is compromised by 1) their activity not been targeted specifically to the immune synapse between T cells and cancer cells, and 2) dose-limiting broadly distributed immune-related adverse events, which has halted several clinical trials. This underscores the need for localized checkpoint inhibition within the cytolytic synapse. We developed a Local Activator and T cell Engager (LocATE) antibody that combines binding to CD3 and BCMA with selective blockade of PD-L1 at the immune synapse in just one scaffold. Selectivity is achieved via low afffinity for PD-L1 and high affinity for BCMA. Methods: Antibody mediated Cytotoxicity (LDH assay) and T cell activation (IL-2 release) was measured in vitro using MM cell lines together with isolated human CD3+ T cells. Human ex vivo T cell activity and redirection was evaluated on fresh bone marrow biopsies from MM patients with different disease stages by automated microscopy (pharmacoscopy) and image analysis. Results: The LocATE antibody showed a 5-fold increase in T cell activation and MM cell killing in vitro compared to a BCMAxCD3 BiTE. Furthermore, patient-derived MM cells showed up to a 19-fold increase in T cell activation as compared to a BCMAxCD3 BiTE or a combination of BiTE and PD-L1 inhibitor, while no activity was observed on healthy cells. Conclusions: These results suggest that T cell redirection with simultaneous checkpoint inhibition in the synapse is highly potent while minimizing off-tumor toxicity, therefore, has high therapeutic potential for patients with relapsed MM.

Lenalidomide Decreases PD-1 Expression, Depletes Regulatory T-Cells and Improves Cellular Response to a Multiple Myeloma/Dendritic Cell Fusion Vaccine In Vitro

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 492-492 ◽  
Author(s):  
Katarina Luptakova ◽  
Brett Glotzbecker ◽  
Heidi Mills ◽  
Dina Stroopinsky ◽  
Baldev Vasir ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Fold Increase ◽  
Cellular Immunotherapy ◽  
Cd4 Cells

Abstract Abstract 492 Introduction: We have developed a cancer vaccine for multiple myeloma in which patient derived tumor cells are fused with dendritic cells (DCs) such that a broad array of tumor antigens are presented in the context of DC mediated costimulation. In clinical studies, we have demonstrated that vaccination results in the induction of anti-tumor immunity and disease response in a subset of patients. A fundamental challenge limiting the efficacy of cellular immunotherapy is the immunosuppressive milieu that characterizes patients with myeloma. We have previously reported that the PD-1/PDL-1 pathway plays an important role in suppressing T cell immunity in patients with myeloma, PD-1 expression is upregulated on T cells isolated from patients with multiple myeloma, and PD-1 blockade is associated with enhancement of T-cell response to the vaccine. Lenalidomide is a potent anti-myeloma agent whose activity may be linked, in part, to its immunomodulatory properties. We hypothesized that lenalidomide would augment the capacity to elicit anti-myeloma immunity. In our current study, we examined the effect of lenalidomide on T-cell activation and polarization, PD-1 signaling, and vaccine-induced responses in vitro. Methods and results: Peripheral blood mononuclear cells were cultured in media containing IL-2 with and without 1μM lenalidomide. The expression of cell surface molecules and intracellular cytokines was assessed using flow cytometry. Exposure of unstimulated T cells to lenalidomide resulted in a decrease in the percentage of CD4+ T cells expressing PD-1 (from 8.0% to 5.6%, p=0.04) and a 2 fold increase in T-cell proliferation as measured by incorporation of tritiated thymidine. We then examined the effect of lenalidomide on T cell activation by ligation of the costimulatory complex using antibodies directed against CD3 and anti CD28. Most notably, the upregulation of PD-1 by CD3/CD28 ligation was markedly decreased in the presence of lenalidomide as measured in CD4+ cells (from 26% to 15%, p<0.0001) and in CD8+ cells (from 16% to 10% p<0.01). Ligation of CD3/CD28 in the presence of lenalidomide resulted in greater degree of Th1 polarization as manifested by a 2 fold increase in the percentage of CD8+ T cells expressing IFNγ (p=0.02) and a decrease in the percentage of regulatory T-cells (CD4+CD25+FoxP3+) from 6.88% to 3.13% (p=0.02). In addition, the percentage of NK cells (CD3-CD56+) expressing IFNγ following CD3/CD28 ligation was 5-fold greater (p=0.03) in the presence of lenalidomide. Lastly, we studied the effect of lenalidomide on T-cells stimulated in vitro by the DC/myeloma fusion vaccine. DC/myeloma fusions were generated as previously described. Fusion mediated stimulation of autologous T cells in the presence of lenalidomide resulted in an increase in the percentage CD4+ and CD8+ T cells expressing IFNγ, (5.35% to 8.79%, p=0.06; and 6.37% to 9.85%, p=0.03, respectively). The proportion of regulatory T-cells decreased from 9.57% to 4.43% in the presence of lenalidomide (p<0.01). As with non-specific stimulation, PD-1 expression on CD4+ cells in the presence of lenalidomide decreased from 24% to 19%. In concert with these findings, exposure to lenalidomide resulted in increased cytotoxic T lymphocyte mediated lysis of autologous tumor targets (from 25% to 36%). Conclusions: In vitro exposure to lenalidomide results in enhanced T-cell activation in response to direct ligation of the co-stimulatory complex and stimulation by the DC/myeloma fusion vaccine. Exposure to lenalidomide suppresses T cell expression of PD-1 and expansion of regulatory T cells, 2 critical pathways responsible for tumor mediated immune suppression. To our knowledge, this is the first demonstration of an interaction between lenalidomide and the PD-1/PDL-1 pathway. These findings support the development of cellular immunotherapy in conjunction with lenalidomide, including its use with the DC/myeloma fusion vaccine. Disclosures: No relevant conflicts of interest to declare.

Development of a fully human T cell engaging bispecific antibody for the treatment of multiple myeloma.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 8017-8017 ◽  
Author(s):  
Ben Buelow ◽  
Duy Pham ◽  
Starlynn Clarke ◽  
Shelley Force Aldred ◽  
Kevin Dang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Bispecific Antibody ◽  
Plasma Cells ◽  
Bispecific Antibodies ◽  
Human Pbmcs

8017 Background: Although BCMA is a plasma cell specific surface molecule attractive as an antibody target in multiple myeloma, its scarcity on the cell surface may limit the efficacy of a conventional antibody. T-cell engaging bispecific antibody approaches are highly efficacious and are particularly well suited for a membrane target with limited expression, such as BCMA. Teneobio has developed a multivalent antibody platform based on modular human VH domains, which allowed us to build T cell engaging bispecific antibodies with low and high T cell agonistic activities. Methods: UniRats were immunized with either CD3 or BCMA antigens and antigen-specific UniAbs were identified by antibody repertoire sequencing and high-throughput gene assembly, expression, and screening. High affinity binding VH sequences were selected using recombinant proteins and cells. In vitro efficacy studies included T-cell activation by cytokine- and tumor cell kill by calcein-release assays. In vivo efficacy of the molecules was evaluated in NSG mice harboring myeloma cells and human PBMCs. Results: BCMA-specific UniAbs bound plasma cells with high affinities (100-700pM) and cross-reacted with cynomolgus plasma cells. Strong and weak T cell agonists were identified that bound human T cells with high and low affinities respectively and cross-reacted with cynomolgus T cells. T cell engaging bispecifics with a strong (H929 cytotoxicity:EC50=27pM) and a weak T cell activating arm (H929 cytotoxicity: EC50=1170pM) demonstrated T-cell activation and tumor-cell cytotoxicity in vitro; bispecifics with a weak CD3 engaging arm showed markedly reduced cytokine production even at doses saturating for cytotoxicity. In viv o, BCMAxCD3 bispecific antibodies reduced tumor load and increased survival when co-administered with human PBMCs as compared to controls. Conclusions: Our results suggest that T cell engaging bispecifics with low-affinity anti-CD3 arms could be preferred for the treatment of Multiple Myeloma.

scholarly journals Lenalidomide Treatment Restores In Vivo T Cell Activity in Relapsed/Refractory FL and DLBCL

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 729-729
Author(s):  
Cedric Menard ◽  
Joelle Dulong ◽  
Tien Tuan Nguyen ◽  
Nadège Bescher ◽  
Maelle Latour ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Proliferation ◽  
Immune Synapse ◽  
Anti Cd20

Abstract The immunostimulatory properties of lenalidomide have been mostly described in vitro while in vivo studies performed in multiple myeloma or chronic lymphocytic leukemia have reported a poorly characterized T-cell activation. A better understanding of lenalidomide kinetics and mechanisms of action is mandatory to optimize its combination with other immunotherapeutic agents in particular for the treatment of non Hodgkin lymphomas. We undertook a thorough immune monitoring of patients enrolled in the French multicenter clinical trial GALEN (ClinicalTrials.gov: NCT01582776) addressing the tolerance and efficacy of the association of lenalidomide and obinutuzumab, a glycoengineered type II anti-CD20 monoclonal antibody, in relapsed/refractory B-cell lymphomas. A 1-week interval between the start of lenalidomide and the first infusion of obinutuzumab was planned, allowing an assessment of the effect of lenalidomide alone on immune-related parameters separately from the combinatorial therapy. Serial blood samples were collected in 44 patients (16 DLBCL and 28 FL) to investigate T, B, NK, and myeloid subsets. In addition, in vitro functional assays were designed to address T cell functional features (proliferation, immune synapse, activity of regulatory T cells (Treg)). In the context of the association with obinutuzumab, we first checked that CD20 expression was not affected on circulating malignant and normal B cells (p=0.43 and 0.8; respectively). Interestingly, upon 1 week of lenalidomide treatment, normal B cells, unlike malignant B cells, upregulated MHC class II (p&lt;0.001 versus 0.16; respectively) while both increased the expression of the costimulatory molecule CD86 (p=0.001 and 0.002; respectively). More importantly, the T-cell capacity to mount a functional immune synapse with malignant B cells was restored in 5/6 relapsed/refractory patients (p&lt;0.001) and we confirmed that this stood true for 6 FL patients at diagnosis (p&lt;0.001). In addition, T cell proliferation was strongly increased in vivo as measured by Ki67 staining (p&lt;0.001) but also upon TCR stimulation ex vivo (p=0.002). This immunostimulatory effect could not be ascribed to a blockade of Treg inhibitory potential by lenalidomide as effector T-cell proliferation was similarly enhanced upon in vitro Treg depletion before and after lenalidomide treatment (p=0.02). In addition, T-cell activation was associated with a reshaping of memory T-cell distribution with the central memory subset dropping in favor of effector cells (p&lt;0.001 and 0.002 respectively). This restoration of T-cell functions was paralleled by the induction of activation markers on T cells such as HLA-DR, CD137, PD-1, and Tim-3 (p&lt;0.001 for all markers). Finally, immune stimulation was not confined to T cells as NK cells also upregulated CD137 (p&lt;0.001) but not PD-1 (p=0.53) expression. We also investigated the myeloid compartment including circulating MDSC and monocytes, both being putative precursors of tumor-associated macrophages. Within 1 week of lenalidomide, patients experienced a decrease of monocytes subsets count and an upregulation of the activation marker and Fcg receptor CD64 (p=0.006). Of note, preliminary experiments showed that, at least in some cases, in vitro exposure of macrophages to lenalidomide could enhance anti-CD20-mediated phagocytosis of tumor cells. Some of these immunological parameters were transiently modulated and returned to baseline levels upon lenalidomide washout but others were restored long term in particular the immune synapse score and memory T cell counts. We herein report for the first time early in vivo T cell activation by lenalidomide in relapse FL/DLBCL through a detailed phenotypic analysis strengthened by innovative functional assays. The study of T cells heterogeneity at the transcriptomic level is underway and the correlation of these immunomodulatory properties with clinical data is also currently being addressed. Our results will help build new and more relevant lenalidomide-based immunotherapeutic approaches. (This study was supported by research grants from Celgene and Roche companies) Disclosures Menard: Celgene: Consultancy; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy. Lamy: Roche: Consultancy, Honoraria. Morschhauser: Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Servier: Consultancy; Gilead: Consultancy. Tarte: Celgene: Consultancy, Research Funding; Novimmune: Research Funding; Roche: Consultancy.

scholarly journals Immunomodulatory effects of different intravenous immunoglobulin preparations in chronic lymphocytic leukemia

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ana Colado ◽  
Esteban Enrique Elías ◽  
Valeria Judith Sarapura Martínez ◽  
Gregorio Cordini ◽  
Pablo Morande ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
T Cell Activation ◽  
Cell Activation ◽  
Lymphocytic Leukemia ◽  
Immunomodulatory Effects ◽  
Immunoglobulin Preparations

AbstractHypogammaglobulinemia is the most frequently observed immune defect in chronic lymphocytic leukemia (CLL). Although CLL patients usually have low serum levels of all isotypes (IgG, IgM and IgA), standard immunoglobulin (Ig) preparations for replacement therapy administrated to these patients contain more than 95% of IgG. Pentaglobin is an Ig preparation of intravenous application (IVIg) enriched with IgM and IgA (IVIgGMA), with the potential benefit to restore the Ig levels of all isotypes. Because IVIg preparations at high doses have well-documented anti-inflammatory and immunomodulatory effects, we aimed to evaluate the capacity of Pentaglobin and a standard IVIg preparation to affect leukemic and T cells from CLL patients. In contrast to standard IVIg, we found that IVIgGMA did not modify T cell activation and had a lower inhibitory effect on T cell proliferation. Regarding the activation of leukemic B cells through BCR, it was similarly reduced by both IVIgGMA and IVIgG. None of these IVIg preparations modified spontaneous apoptosis of T or leukemic B cells. However, the addition of IVIgGMA on in vitro cultures decreased the apoptosis of T cells induced by the BCL-2 inhibitor, venetoclax. Importantly, IVIgGMA did not impair venetoclax-induced apoptosis of leukemic B cells. Overall, our results add new data on the effects of different preparations of IVIg in CLL, and show that the IgM/IgA enriched preparation not only affects relevant mechanisms involved in CLL pathogenesis but also has a particular profile of immunomodulatory effects on T cells that deserves further investigation.

Modulation of T-Cell Functions in KIR2DL3 (CD158b) Transgenic Mice

Blood ◽  
1999 ◽  
Vol 94 (7) ◽  
pp. 2396-2402 ◽  
Author(s):  
Anna Cambiaggi ◽  
Sylvie Darche ◽  
Sophie Guia ◽  
Philippe Kourilsky ◽  
Jean-Pierre Abastado ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Transgenic Mice ◽  
T Cell Activation ◽  
Cell Activation ◽  
Killer Cell ◽  
In Vitro Proliferation ◽  
Cell Functions ◽  
Double Transgenic Mice

In humans, a minor subset of T cells express killer cell Ig-like receptors (KIRs) at their surface. In vitro data obtained with KIR+ β and γδ T-cell clones showed that engagement of KIR molecules can extinguish T-cell activation signals induced via the CD3/T-cell receptor (TCR) complex. We analyzed the T-cell compartment in mice transgenic for KIR2DL3 (Tg-KIR2DL3), an inhibitory receptor for HLA-Cw3. As expected, mixed lymphocyte reaction and anti-CD3 monoclonal antibody (MoAb)-redirected cytotoxicity exerted by freshly isolated splenocytes can be inhibited by engagement of transgenic KIR2DL3 molecules. In contrast, antigen and anti-CD3 MoAb-induced cytotoxicity exerted by alloreactive cytotoxic T lymphocytes cannot be inhibited by KIR2DL3 engagement. In double transgenic mice, Tg-KIR2DL3 × Tg-HLA-Cw3, no alteration of thymic differentiation could be documented. Immunization of double transgenic mice with Hen egg white lysozime (HEL) or Pigeon Cytochrome-C (PCC) was indistinguishable from immunization of control mice, as judged by recall antigen-induced in vitro proliferation and TCR repertoire analysis. These results indicate that KIR effect on T cells varies upon cell activation stage and show unexpected complexity in the biological function of KIRs in vivo.

scholarly journals T cell receptor zeta/CD3-p59fyn(T)-associated p120/130 binds to the SH2 domain of p59fyn(T).

1993 ◽  
Vol 178 (6) ◽  
pp. 2107-2113 ◽  
Author(s):  
A J da Silva ◽  
O Janssen ◽  
C E Rudd
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Intracellular Signaling ◽  
Cell Activation ◽  
Cell Receptor ◽  
Sh2 Domain ◽  
T Complex

Intracellular signaling from the T cell receptor (TCR)zeta/CD3 complex is likely to be mediated by associated protein tyrosine kinases such as p59fyn(T), ZAP-70, and the CD4:p56lck and CD8:p56lck coreceptors. The nature of the signaling cascade initiated by these kinases, their specificities, and downstream targets remain to be elucidated. The TCR-zeta/CD3:p59fyn(T) complex has previously been noted to coprecipitate a 120/130-kD doublet (p120/130). This intracellular protein of unknown identity associates directly with p59fyn(T) within the receptor complex. In this study, we have shown that this interaction with p120/130 is specifically mediated by the SH2 domain (not the fyn-SH3 domain) of p59fyn(T). Further, based on the results of in vitro kinase assays, p120/130 appears to be preferentially associated with p59fyn(T) in T cells, and not with p56lck. Antibody reprecipitation studies identified p120/130 as a previously described 130-kD substrate of pp60v-src whose function and structure is unknown. TCR-zeta/CD3 induced activation of T cells augmented the tyrosine phosphorylation of p120/130 in vivo as detected by antibody and GST:fyn-SH2 fusion proteins. p120/130 represents the first identified p59fyn(T):SH2 binding substrate in T cells, and as such is likely to play a key role in the early events of T cell activation.

scholarly journals CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac-dependent pathway

Blood ◽  
2009 ◽  
Vol 114 (3) ◽  
pp. 580-588 ◽  
Author(s):  
Kathrin Gollmer ◽  
François Asperti-Boursin ◽  
Yoshihiko Tanaka ◽  
Klaus Okkenhaug ◽  
Bart Vanhaesebroeck ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Early Time ◽  
Cell Receptor ◽  
Tcr Signaling ◽  
Activation Markers

Abstract CD4+ T cells use the chemokine receptor CCR7 to home to and migrate within lymphoid tissue, where T-cell activation takes place. Using primary T-cell receptor (TCR)–transgenic (tg) CD4+ T cells, we explored the effect of CCR7 ligands, in particular CCL21, on T-cell activation. We found that the presence of CCL21 during early time points strongly increased in vitro T-cell proliferation after TCR stimulation, correlating with increased expression of early activation markers. CCL21 costimulation resulted in increased Ras- and Rac-GTP formation and enhanced phosphorylation of Akt, MEK, and ERK but not p38 or JNK. Kinase-dead PI3KδD910A/D910A or PI3Kγ-deficient TCR-tg CD4+ T cells showed similar responsiveness to CCL21 costimulation as control CD4+ T cells. Conversely, deficiency in the Rac guanine exchange factor DOCK2 significantly impaired CCL21-mediated costimulation in TCR-tg CD4+ T cells, concomitant with impaired Rac- but not Ras-GTP formation. Using lymph node slices for live monitoring of T-cell behavior and activation, we found that G protein-coupled receptor signaling was required for early CD69 expression but not for Ca2+ signaling. Our data suggest that the presence of CCL21 during early TCR signaling lowers the activation threshold through Ras- and Rac-dependent pathways leading to increased ERK phosphorylation.

107 Effects of IL-2 and IL-15 on the proliferative and antitumor capacities of allogeneic CD20 CAR-engineered γδ T cells in a 3D B cell lymphoma spheroid assay

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A119-A119
Author(s):  
Lu Bai ◽  
Kevin Nishimoto ◽  
Mustafa Turkoz ◽  
Marissa Herrman ◽  
Jason Romero ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cytokine Production ◽  
Γδ T Cells ◽  
Γδ T Cell ◽  
Car T

BackgroundAutologous chimeric antigen receptor (CAR) T cells have been shown to be efficacious for the treatment of B cell malignancies; however, widespread adoption and application of CAR T cell products still face a number of challenges. To overcome these challenges, Adicet Bio is developing an allogeneic γδ T cell-based CAR T cell platform, which capitalizes on the intrinsic abilities of Vδ1 γδ T cells to recognize and kill transformed cells in an MHC-unrestricted manner, to migrate to epithelial tissues, and to function in hypoxic conditions. To gain a better understanding of the requirements for optimal intratumoral CAR Vδ1 γδ T cell activation, proliferation, and differentiation, we developed a three-dimensional (3D) tumor spheroid assay, in which tumor cells acquire the structural organization of a solid tumor and establish a microenvironment that has oxygen and nutrient gradients. Moreover, through the addition of cytokines and/or tumor stromal cell types, the spheroid microenvironment can be modified to reflect hot or cold tumors. Here, we report on the use of a 3D CD20+ Raji lymphoma spheroid assay to evaluate the effects of IL-2 and IL-15, positive regulators of T cell homeostasis and differentiation, on the proliferative and antitumor capacities of CD20 CAR Vδ1 γδ T cells.MethodsMolecular, phenotypic, and functional profiling were performed to characterize the in vitro dynamics of the intraspheroid CD20 CAR Vδ1 γδ T cell response to target antigen in the presence of IL-2, IL-15, or no added cytokine.ResultsWhen compared to no added cytokine, the addition of IL-2 or IL-15 enhanced CD20 CAR Vδ1 γδ T cell activation, proliferation, survival, and cytokine production in a dose-dependent manner but were only able to alter the kinetics of Raji cell killing at low effector to target ratios. Notably, differential gene expression analysis using NanoString nCounter® Technology confirmed the positive effects of IL-2 or IL-15 on CAR-activated Vδ1 γδ T cells as evidenced by the upregulation of genes involved in activation, cell cycle, mitochondrial biogenesis, cytotoxicity, and cytokine production.ConclusionsTogether, these results not only show that the addition of IL-2 or IL-15 can potentiate CD20 CAR Vδ1 γδ T cell activation, proliferation, survival, and differentiation into antitumor effectors but also highlight the utility of the 3D spheroid assay as a high throughput in vitro method for assessing and predicting CAR Vδ1 γδ T cell activation, proliferation, survival, and differentiation in hot and cold tumors.

The costimulatory activity of Tim-3 requires Akt and MAPK signaling and its recruitment to the immune synapse

2021 ◽  
Vol 14 (687) ◽  
pp. eaba0717
Author(s):  
Shunsuke Kataoka ◽  
Priyanka Manandhar ◽  
Judong Lee ◽  
Creg J. Workman ◽  
Hridesh Banerjee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Cell Receptor ◽  
Mapk Signaling ◽  
Inhibitory Protein ◽  
Immune Synapse

Expression of the transmembrane protein Tim-3 is increased on dysregulated T cells undergoing chronic activation, including during chronic infection and in solid tumors. Thus, Tim-3 is generally thought of as an inhibitory protein. We and others previously reported that under some circumstances, Tim-3 exerts paradoxical costimulatory activity in T cells (and other cells), including enhancement of the phosphorylation of ribosomal S6 protein. Here, we examined the upstream signaling pathways that control Tim-3–mediated increases in phosphorylated S6 in T cells. We also defined the localization of Tim-3 relative to the T cell immune synapse and its effects on downstream signaling. Recruitment of Tim-3 to the immune synapse was mediated exclusively by the transmembrane domain, replacement of which impaired the ability of Tim-3 to costimulate T cell receptor (TCR)–dependent S6 phosphorylation. Furthermore, enforced localization of the Tim-3 cytoplasmic domain to the immune synapse in a chimeric antigen receptor still enabled T cell activation. Together, our findings are consistent with a model whereby Tim-3 enhances TCR-proximal signaling under acute conditions.

scholarly journals Immune function of the blood-brain barrier: incomplete presentation of protein (auto-)antigens by rat brain microvascular endothelium in vitro.

1990 ◽  
Vol 110 (5) ◽  
pp. 1757-1766 ◽  
Author(s):  
W Risau ◽  
B Engelhardt ◽  
H Wekerle
Keyword(s):  
T Cells ◽  
Endothelial Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Rat Brain ◽  
Blood Brain Barrier ◽  
T Cell Activation ◽  
Cell Activation ◽  
Ifn Gamma

The endothelial blood-brain barrier (BBB) has a critical role in controlling lymphocyte traffic into the central nervous system (CNS), both in physiological immunosurveillance, and in its pathological aberrations. The intercellular signals that possibly could induce lymphocytes to cross the BBB include immunogenic presentation of protein (auto-)antigens by BBB endothelia to circulating T lymphocytes. This concept has raised much, though controversial, attention. We approached this problem by analyzing in vitro immunospecific interactions between clonal rat T lymphocyte lines with syngeneic, stringently purified endothelial monolayer cultures from adult brain micro-vessels. The rat brain endothelia (RBE) were established from rat brain capillaries using double collagenase digestion, density gradient fractionation and selective cytolysis of contaminating pericytes by anti-Thy 1.1 antibodies and complement. Incubation with interferon-gamma in most of the brain-derived endothelial cells induced Ia-antigens in the cytoplasm and on the cell surface in some of the cells. Before the treatment, the cells were completely Ia-negative. Pericytes were unresponsive to IFN-gamma treatment. When confronted with syngeneic T cell lines specific for protein (auto-)antigens (e.g., ovalbumin and myelin basic protein, MBP), RBE were completely unable to induce antigen-specific proliferation of syngeneic T lymphocytes irrespective of pretreatment with IFN-gamma and of cell density. RBE were inert towards the T cells, and did not suppress T cell activation induced by other "professional" antigen presenting cells (APC) such as thymus-derived dendritic cells or macrophages. IFN-gamma-treated RBE were, however, susceptible to immunospecific T cell killing. They were lysed by MBP-specific T cells in the presence of the specific antigen or Con A. Antigen dependent lysis was restricted by the appropriate (MHC) class II product. We conclude that the interaction of brain endothelial cells with encephalitogenic T lymphocytes may involve recognition of antigen in the molecular context of relevant MHC products, but that this interaction per se is insufficient to initiate the full T cell activation program.

Sign in / Sign up

Export Citation Format

Share Document