Distinct Roles Of PD-1 Itsm and ITIM In Regulating Interactions With SHP-2, ZAP-70 and Lck, and PD-1-Mediated Inhibitory Function

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 191-191 ◽  
Author(s):  
Pranam Chatterjee ◽  
Nikolaos Patsoukis ◽  
Gordon J. Freeman ◽  
Vassiliki A. Boussiotis
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Fusion Proteins ◽  
Cell Activation ◽  
Cytoplasmic Tail ◽  
Inhibitory Function ◽  
Gst Fusion Proteins

Abstract Programmed death (PD)-1 plays a prominent role in the induction and maintenance of peripheral tolerance. The biochemical mechanisms via which PD-1 mediates its inhibitory function remain poorly understood. The cytoplamsic tail of PD-1 contains two structural motifs, an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). It has been reported that SHP-2 tyrosine phosphatase constitutively interacts with PD-1 ITSM and is involved in PD-1-mediated inhibitory function. We sought to identify the nature of PD-1: SHP-2 interaction and to determine whether other TCR-proximal signaling molecules might interact with PD-1 cytoplasmic tail. SHP-2 has two SH2 domains (N-SH2 and C-SH2) and one PTP domain. To identify the region of SHP-2 that interacts with PD-1 we generated five different GST-fusion proteins in which GST was fused with either SHP-2 full length (FL), SHP-2-N-SH2, SHP-2-C-SH2, SHP-2-ΔN-SH2 (lacking the N-terminus SH2 domain) or SHP-2-PTP. Pull down assays with each GST-fusion protein using lysates from naive and activated primary human T cells revealed that PD-1 interacted with GST-SHP-2 fusion protein only after T cell activation along with simultaneous PD-1 ligation. This interaction was mediated selectively via the SH2 domains of SHP-2, indicating that PD-1 requires prior tyrosine phosphorylation in order to undergo interaction with SHP-2. To identify the mechanism of PD-1 tyrosine phosphorylation governing PD-1: SHP-2 interaction, we used COS cells to express PD-1 along with either empty vector, the TCR proximal tyrosine kinase Fyn, or a kinase inactive mutant of Fyn, followed by pull down with each SHP-2-GST fusion protein. No interaction between PD-1 and SHP-2-GST fusion proteins was detected in lysates from COS cells expressing empty vector or kinase inactive Fyn mutant. In contrast, in the presence of active Fyn, PD-1 underwent tyrosine phosphorylation and was able to interact with GST fusion proteins of SHP-2-FL, SHP-2-N-SH2, SHP-2-C-SH2 and SHP-2-ΔN-SH2 but not SHP-2-PTP, providing evidence that PD-1: SHP-2 interaction requires tyrosine phosphorylation of PD-1 by Src family kinases for subsequent SH2-mediated recruitment of SHP-2. To determine the structural and functional role of each individual tyrosine in the ITIM and the ITSM of PD-1 cytoplasmic tail in PD-1: SHP-2 interaction in vivo, we used Jurkat T cells to express cDNA of either PD-1 wild type, PD-1 with the ITIM tyrosine mutated to phenylalanine (PD-1.Y223F), PD-1 with the ITSM tyrosine mutated to phenylalanine (PD-1.Y248F) or PD-1 with both ITIM and ITSM tyrosines mutated to phenylalanine (PD-1.Y223F/Y248F). After activation, PD-1 wild type underwent tyrosine phosphorylation and developed a robust interaction with SHP-2. PD-1.Y223F retained the ability to undergo interaction with SHP-2 after activation, whereas PD-1.Y248F and PD-1.Y223F/Y248F were unable to interact with SHP-2. We examined whether the PD-1 cytopasmic phosphotyrosines might interact with other SH2 domain containing proteins with critical role in T cell activation. We determined that after T cell activation, PD-1 displayed interaction with ZAP-70 and with activated Lck as determined by PD-1 immunoprecipitation followed by immunoblot with antibodies specific for ZAP-70 and for the activation-specific phospho-LckY394. These interactions remained unaffected in T cells expressing PD-1.Y223F but were abrogated in T cells expressing PD-1.Y248F or PD-1.Y223F/Y248F indicating a mandatory role of phosphorylated ITSM but not ITIM for these associations. However, despite their distinct ability to mediate interactions of PD-1 with SHP-2, Lck and ZAP-70, both phosphorylated ITSM and ITIM had a mandatory role in the inhibitory effect of PD-1 on T cell activation. In T cells expressing either PD-1.Y223F or PD-1.Y248F, PD-1-mediated inhibition of IL-2 production was diminished by 50%, but was almost abrogated in T cells expressing the double mutant PD-1.Y223F/Y248F. Our results indicate that the cytoplasmic tail of PD-1 requires tyrosine phosphorylation in order to mediate phosphorylation-dependent interactions and inhibition on T cell activation. Although phosphorylation-dependent interactions of PD-1 with SHP-2, ZAP-70 and Lck involve Y248 in the ITSM, yet unidentified interactions of Y223 in the ITIM are mandatory for PD-1-mediated inhibitory function on T cell activation. Disclosures: Freeman: Boehringer-Ingelheim: Patents & Royalties; Bristol-Myers-Squibb/Medarex: Patents & Royalties; Roche/Genentech: Patents & Royalties; Merck: Patents & Royalties; EMD-Serrono: Patents & Royalties; Amplimmune: Patents & Royalties; CoStim Pharmaceuticals: Patents & Royalties; Costim Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees.

scholarly journals Identification and Characterization of the Amphioxus Lck and Its Associated Tyrosine Phosphorylation-Dependent Inhibitory LRR Receptor

2021 ◽  
Vol 12 ◽  
Author(s):  
Jiatao Zhou ◽  
Zhihui Xiao ◽  
Yanli Zhan ◽  
Xuemei Qu ◽  
Sisi Mou ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Cell Activation ◽  
Spectrometric Analysis ◽  
Jurkat T Cells ◽  
Origin And Evolution ◽  
Inhibitory Function ◽  
Mutant Sequence

Amphioxus (e.g., Branchiostoma belcheri, Bb) has recently emerged as a new model for studying the origin and evolution of vertebrate immunity. Mammalian lymphocyte-specific tyrosine kinase (Lck) plays crucial roles in T cell activation, differentiation and homeostasis, and is reported to phosphorylate both the ITIM and ITSM of PD-1 to induce the recruitment of phosphatases and thus the inhibitory function of PD-1. Here, we identified and cloned the amphioxus homolog of human Lck. By generating and using an antibody against BbLck, we found that BbLck is expressed in the amphioxus gut and gill. Through overexpression of BbLck in Jurkat T cells, we found that upon TCR stimulation, BbLck was subjected to tyrosine phosphorylation and could partially rescue Lck-dependent tyrosine phosphorylation in Lck-knockdown T cells. Mass spectrometric analysis of BbLck immunoprecipitates from immunostimulants-treated amphioxus, revealed a BbLck-associated membrane-bound receptor LRR (BbLcLRR). By overexpressing BbLcLRR in Jurkat T cells, we demonstrated that BbLcLRR was tyrosine phosphorylated upon TCR stimulation, which was inhibited by Lck knockdown and was rescued by overexpression of BbLck. By mutating single tyrosine to phenylalanine (Y-F), we identified three tyrosine residues (Y539, Y655, and Y690) (3Y) of BbLcLRR as the major Lck phosphorylation sites. Reporter gene assays showed that overexpression of BbLcLRR but not the BbLcLRR-3YF mutant inhibited TCR-induced NF-κB activation. In Lck-knockdown T cells, the decline of TCR-induced IL-2 production was reversed by overexpression of BbLck, and this reversion was inhibited by co-expression of BbLcLRR but not the BbLcLRR-3YF mutant. Sequence analysis showed that the three tyrosine-containing sequences were conserved with the tyrosine-based inhibition motifs (ITIMs) or ITIM-like motifs. And TCR stimulation induced the association of BbLcLRR with tyrosine phosphatases SHIP1 and to a lesser extent with SHP1/2. Moreover, overexpression of wild-type BbLcLRR but not its 3YF mutant inhibited TCR-induced tyrosine phosphorylation of multiple signaling proteins probably via recruiting SHIP1. Thus, we identified a novel immunoreceptor BbLcLRR, which is phosphorylated by Lck and then exerts a phosphorylation-dependent inhibitory role in TCR-mediated T-cell activation, implying a mechanism for the maintenance of self-tolerance and homeostasis of amphioxus immune system and the evolutionary conservatism of Lck-regulated inhibitory receptor pathway.

scholarly journals 206 The development of ‘chimeric CD3e fusion protein’ and ‘anti-CD3-based bispecific T cell activating element’ engineered T (CAB-T) cells for the treatment of solid malignancies

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A217-A217
Author(s):  
Andy Tsun ◽  
Zhiyuan Li ◽  
Zhenqing Zhang ◽  
Weifeng Huang ◽  
Shaogang Peng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
T Cell Activation ◽  
Cell Activation ◽  
In Vivo Studies ◽  
Cytokine Release ◽  
Car T Cells ◽  
Car T

BackgroundCancer immunotherapy has achieved unprecedented success in the complete remission of hematological tumors. However, serious or even fatal clinical side-effects have been associated with CAR-T therapies to solid tumors, which mainly include cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), macrophage activation syndrome, etc. Furthermore, CAR-T therapies have not yet demonstrated significant clinical efficacy for the treatment of solid tumors. Here, we present a novel T cell therapeutic platform: a Chimeric CD3e fusion protein and anti-CD3-based bispecific T cell activating element (BiTA) engineered T (CAB-T) cells, which target tumor antigens via the secretion of BiTAs that act independently of MHC interactions. Upon BiTA secretion, CAB-T cells can simultaneously achieve anti-tumor cytotoxic effects from the CAB-T cells and simultaneously activate bystander T cells.MethodsCAB-T cells were generated by co-expressing a chimeric CD3e fusion protein and an anti-CD3-based bispecific T cell activating element. The chimeric CD3e contains the extracellular domain of CD3e, a CD8 transmembrane domain, 4-1BB costimulatory domain, CD3z T cell activation domain and a FLAG tag, while the BiTA element includes a tumor antigen targeting domain fused with an anti-CD3 scFv domain and a 6x His-tag. CAR-T cells were generated as a control. Cytokine release activity, T cell activation and exhaustion markers, T cell killing activity and T cell differentiation stages were analysed. We also tested their tumor growth inhibition activity, peripheral and tumor tissue distribution, and their safety-profiles in humanized mouse models.ResultsCAB-T cells have similar or better in vitro killing activity compared with their CAR-T counterparts, with lower levels of cytokine release (IL-2 and IFNγ). CAB-T cells also showed lower levels of exhaustion markers (PD-1, LAG-3 and TIM-3), and higher ratios of naive/Tscm and Tcm T cell populations, after co-culture with their target tumor cells (48h). In in vivo studies, CAIX CAB-T and HER2 CAB-T showed superior anti-tumor efficacy and tumor tissue infiltration activity over their corresponding CAR-T cells. For CLDN18.2 CAB-T cells, similar in vivo anti-tumor efficacy was observed compared to CAR-T after T cell infusion, but blood glucose reduction and animal mortality was observed in the mice administered with CAR-T cells.ConclusionsThe advantages of CAB-T in in vitro and in vivo studies may result from TCR signal activation of both the engineered CAB-T cells and the non-engineered bystander T cells via cross-bridging by the secreted BiTA molecules, thus offering superior anti-tumor efficacy with a potential better safety-profile compared to conventional CAR-T platforms.

scholarly journals PAG-Associated FynT Regulates Calcium Signaling and Promotes Anergy in T Lymphocytes

2007 ◽  
Vol 27 (5) ◽  
pp. 1960-1973 ◽  
Author(s):  
Dominique Davidson ◽  
Burkhart Schraven ◽  
André Veillette
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Protein Tyrosine Kinase ◽  
Cell Activation ◽  
T Cell Anergy ◽  
Cell Anergy ◽  
Anergic T Cells ◽  
Selective Enhancement

ABSTRACT Phosphoprotein associated with glycolipid-enriched membranes (PAG), also named Csk-binding protein (Cbp), is a transmembrane adaptor associated with lipid rafts. It is phosphorylated on multiple tyrosines located in the cytoplasmic domain. One tyrosine, tyrosine 314 (Y314) in the mouse, interacts with Csk, a protein tyrosine kinase that negatively regulates Src kinases. This interaction enables PAG to inhibit T-cell antigen receptor (TCR)-mediated T-cell activation. PAG also associates with the Src-related kinase FynT. Genetic studies indicated that FynT was required for PAG tyrosine phosphorylation and binding of PAG to Csk in T cells. Herein, we investigated the function and regulation of PAG-associated FynT. Our data showed that PAG was constitutively associated with FynT in unstimulated T cells and that this association was rapidly lost in response to TCR stimulation. Dissociation of the PAG-FynT complex preceded PAG dephosphorylation and PAG-Csk dissociation after TCR engagement. Interestingly, in anergic T cells, the association of PAG with FynT, but not Csk, was increased. Analyses of PAG mutants provided evidence that PAG interacted with FynT by way of tyrosines other than Y314. Enforced expression of a PAG variant interacting with FynT, but not Csk, caused a selective enhancement of TCR-triggered calcium fluxes in normal T cells. Furthermore, it promoted T-cell anergy. Both effects were absent in mice lacking FynT, implying that the effects were mediated by PAG-associated FynT. Hence, besides enabling PAG tyrosine phosphorylation and the PAG-Csk interaction, PAG-associated FynT can stimulate calcium signals and favor T-cell anergy. These data improve our comprehension of the function of PAG in T cells. They also further implicate FynT in T-cell anergy.

Generation and biochemical analysis of human effector CD4 T cells: alterations in tyrosine phosphorylation and loss of CD3ζ expression

Blood ◽  
2001 ◽  
Vol 97 (12) ◽  
pp. 3851-3859 ◽  
Author(s):  
Sandeep Krishnan ◽  
Vishal G. Warke ◽  
Madhusoodana P. Nambiar ◽  
Henry K. Wong ◽  
George C. Tsokos ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Effector T Cells ◽  
T Cell Differentiation ◽  
Effector T Cell

Human effector T cells have been difficult to isolate and characterize due to their phenotypic and functional similarity to the memory subset. In this study, a biochemical approach was used to analyze human effector CD4 T cells generated in vitro by activation with anti-CD3 and autologous monocytes for 3 to 5 days. The resultant effector cells expressed the appropriate activation/differentiation markers and secreted high levels of interferon γ (IFN-γ) when restimulated. Biochemically, effector CD4 T cells exhibited increases in total intracellular tyrosine phosphorylation and effector-associated phosphorylated species. Paradoxically, these alterations in tyrosine phosphorylation were concomitant with greatly reduced expression of CD3ζ and CD3ε signaling subunits coincident with a reduction in surface T-cell receptor (TCR) expression. Because loss of CD3ζ has also been detected in T cells isolated ex vivo from individuals with cancer, chronic viral infection, and autoimmune diseases, the requirements and kinetics of CD3ζ down-regulation were examined. The loss of CD3ζ expression persisted throughout the course of effector T-cell differentiation, was reversible on removal from the activating stimulus, and was modulated by activation conditions. These biochemical changes occurred in effector T cells generated from naive or memory CD4 T-cell precursors and distinguished effector from memory T cells. The results suggest that human effector T-cell differentiation is accompanied by alterations in the TCR signal transduction and that loss of CD3ζ expression may be a feature of chronic T-cell activation and effector generation in vivo.

scholarly journals Partially Phosphorylated T Cell Receptor ζ Molecules Can Inhibit T Cell Activation

1999 ◽  
Vol 190 (11) ◽  
pp. 1627-1636 ◽  
Author(s):  
Ellen N. Kersh ◽  
Gilbert J. Kersh ◽  
Paul M. Allen
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Receptor ◽  
Inhibitory Function ◽  
Cell Clones ◽  
Agonist And Antagonist ◽  
Inhibitory Signaling

The T cell receptor complex (TCR) ζ chain is constitutively tyrosine phosphorylated specifically at two of the six ζ immunoreceptor tyrosine-based activation motif (ITAM) tyrosine residues in resting peripheral T cells. Further phosphorylation of ζ is induced by both agonist and antagonist ligands of the TCR, with agonists inducing complete phosphorylation of the ζ ITAM tyrosines. After antagonist stimulation, ζ phosphorylation is incomplete and generates discrete forms of partially phosphorylated ITAMs. Here, we mutate specific tyrosines in chimeric human CD8-ζ molecules to reflect phosphorylation in resting T cells as well as phosphorylation induced by agonist and antagonist ligands. We demonstrate that such partially phosphorylated TCR-ζ species can inhibit IL-2 production in T cell hybridomas and proliferation in T cell clones. This reveals a previously unrecognized, inhibitory function of partially phosphorylated ITAMs. These findings support the concept that TCR antagonism can arise through the generation of an inhibitory signal within the TCR complex and that constitutive ζ phosphorylation in resting T cells is an inhibitory signaling environment.

scholarly journals Co-Stimulatory Bispecific Antibodies Induce Enhanced T Cell Activation and Tumor Cell Killing in Breast Cancer Models

2021 ◽  
Vol 12 ◽  
Author(s):  
Karsten M. Warwas ◽  
Marten Meyer ◽  
Márcia Gonçalves ◽  
Gerhard Moldenhauer ◽  
Nadja Bulbuc ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
T Cell Activation ◽  
Fusion Proteins ◽  
Cell Activation ◽  
Tumor Antigens ◽  
Bispecific Antibodies ◽  
Tumoricidal Activity

Although T cell-recruiting CD3-binding bispecific antibodies (BiMAb) have been proven to be clinically effective for hematologic malignancies, the success of BiMAb targeting solid tumor-associated antigens (TAA) in carcinomas so far remains poor. We reasoned that provision of co-stimulatory BiMAb in combination with αTAA–αCD3 BiMAb would boost T cell activation and proliferative capacity, and thereby facilitate the targeting of weakly or heterogeneously expressed tumor antigens. Various αTAA–αCD3 and αTAA–αCD28 BiMAb in a tetravalent IgG1-Fc based format have been analyzed, targeting multiple breast cancer antigens including HER2, EGFR, CEA, and EpCAM. Moreover, bifunctional fusion proteins of αTAA–tumor necrosis factor ligand (TNFL) superfamily members including 4-1BBL, OX40L, CD70 and TL1A have been tested. The functional activity of BiMAb was assessed using co-cultures of tumor cell lines and purified T cells in monolayer and tumor spheroid models. Only in the presence of tumor cells, αTAA–αCD3 BiMAb activated T cells and induced cytotoxicity in vitro, indicating a strict dependence on cross-linking. Combination treatment of αTAA–αCD3 BiMAb and co-stimulatory αTAA–αCD28 or αTAA–TNFL fusion proteins drastically enhanced T cell activation in terms of proliferation, activation marker expression, cytokine secretion and tumor cytotoxicity. Furthermore, BiMAb providing co-stimulation were shown to reduce the minimally required dose to achieve T cell activation by at least tenfold. Immuno-suppressive effects of TGF-β and IL-10 on T cell activation and memory cell formation could be overcome by co-stimulation. BiMAb-mediated co-stimulation was further augmented by immune checkpoint-inhibiting antibodies. Effective co-stimulation could be achieved by targeting a second breast cancer antigen, or by targeting fibroblast activation protein (FAP) expressed on another target cell. In tumor spheroids derived from pleural effusions of breast cancer patients, co-stimulatory BiMAb were essential for the activation tumor-infiltrating lymphocytes and cytotoxic anti-tumor responses against breast cancer cells. Taken together we showed that co-stimulation significantly potentiated the tumoricidal activity of T cell-activating BiMAb while preserving the dependence on TAA recognition. This approach could provide for a more localized activation of the immune system with higher efficacy and reduced peripheral toxicities.

Blocking ICOS-B7h Signal Can Inhibit the Function of Allogene T Lymphocytes and Decrease the Incidence and Severity of Acute Graft Versus Host Disease in Mouse Model

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4606-4606
Author(s):  
Xiaochen Bao ◽  
Ningxia Song ◽  
Bin Wang ◽  
Jianmin Wang
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Recipient Mouse ◽  
T Helper ◽  
Graft Versus Host ◽  
Ifn Γ

Abstract ICOS, a CD28 family member expressed on activated T cells, plays important roles in T cell activation and effector function. Here we report our results of biological activity of ICOS signal on allogeneic T lymphocytes and its effect on acute graft-versus-host disease in mouse model by blocking ICOS-B7h signal with ICOS-Ig fusion protein. Human ICOSIg fusion protein was harvested and purified from supernatant of CHO cells transfected with pSecTag2/Hygro A-ICOS-Ig in our lab. Spleen CD4+ cells from C57BL/6 mouse were stimulated with dendritic cells from BALB/C mouse, with different doses of ICOS-Ig or human-Ig (h-Ig) as controls. Allogeneic aGVHD model was established with lethally irradiated BALB/c recipients receiving allogeneic BM and spleen T cells from C57BL/6 mouse with 100ug ICOS-Ig or h-Ig intropenetoneally 4 times at day 0, day +2, +4 and +6 of transplantation. RESULTS: ICOS-Ig (10ug/mL) significantly inhibited proliferation of CD4+T cells ( P<0.01), decreased the level of TNF-α and elevated level of IL-4 in the supernatants of CD4+ T cells in response to allogeneic mature DCs but had no effect on IFN-γ production; ICOS-Ig blockade elevated apoptosis of splenic CD4+ T cells while had no effect on T cell activation (CD25 expression). ICOS-Ig blockade significantly attenuated the lethal GVHD that occurred in control recipient mice. The average survival time was 13.25±5.87 days for mice in h-Ig group, while 21.42±3.02 days for animals in ICOS-Ig group(p=0.0217). Pathologic evaluation revealed that the liver and intestine of animals in ICOS-Ig group has less lymphocyte infiltration and less architectural disruption than those in control h-Ig group; In vivo, ICOS-Ig had no effect on allogeneic T cells division (h-Ig :98.40±1.32, ICOS-Ig: 97.69±2.19 by FACS analysis of CFSE labeled lymphocyte at day 3 of transplantation) and no effect on the proportion of CD4+/CD8+ (h-Ig: 26.35±0.07, ICOS-Ig: 22.12±0.21), but increased apoptosis of allogeneic CD8+ T cells in GVHD model by FACS analysis of Annexin-V staining lymphocytes at day 10 of transplantation (h-Ig: 20.44±3.83, ICOS-Ig: 22.87±6.94 in CD4+ T cells; h-Ig: 18.73±7.43, ICOS-Ig: 24.03±5.4 in CD8+ T cells). Spleen T cells from mice after transplantation were stimulated by ConA ex vivo, ICOS-Ig group proliferated less than control h-Ig group through cell counting with CCK-8 (h-Ig: 0.86±0.04,ICOS-Ig: 0.69±0.12,P<0.05). (4) ICOS-Ig significantly reduced the secretion of IFN-γ and elevated IL-4 in the serum of recipient mouse. The IFN-γ (pg/mL) detected were 562.27±49.97 in h-Ig group, 49.79±2.81 in ICOS-Ig group; and the IL-4 (pg/mL) detected were 38.819±27.56 in h-Ig group,456.03±69.63 in ICOS-Ig group. (p<0.05). (5)ICOS-Ig significantly reduced the secretion of T-bet and elevated GATA-3 in the spleens of recipient mouse. The T-bet/GATA-3 detected were 1.87±0.65 in h-Ig group, 0.56±0.03 in ICOS-Ig (p=0.03). CONCLUSION: The ICOS-Ig fusion protein had bioactivity of inhibition of T cell proliferation and alternated the polarization of T helper cells; It promoted the apoptosis of allo-reactive T cells from donor animals but had no effect on the activation of allo-reactive CD4+T cells; ICOS-Ig blockade can prevent aGVHD through attenuating the function of the allo-reactive T cells, elevating apoptosis of allo-reactive T cells and alternating the polarization of T helper cells.

PD-1 Inhibits TCR Proximal Signaling By Sequestering SHP-2 Phosphatase and Facilitating Csk-Mediated Inhibitory Phosphorylation of Lck

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 283-283 ◽  
Author(s):  
Kankana Bardhan ◽  
Nikolaos Patsoukis ◽  
Duygu Sari ◽  
Theodore Anagnostou ◽  
Pranam Chatterjee ◽  
...  
Keyword(s):  
T Cells ◽  
Plasma Membrane ◽  
T Cell ◽  
Fusion Protein ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cos Cells ◽  
Empty Vector ◽  
Human T Cells ◽  
Stimulation Of

Abstract Programmed death (PD)-1 is a negative regulator of T cell responses, which controls peripheral tolerance but also suppresses anti-viral and anti-tumor immunity. The biochemical mechanisms via which PD-1 inhibits T cell activation remain poorly understood. The cytoplamsic tail of PD-1 contains two structural motifs, an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). It has been reported that SHP-2 tyrosine phosphatase constitutively interacts with the ITSM and has a critical role in PD-1-mediated inhibitory function but the mechanism remains unclear. Although phosphatases are generally considered negative regulators of activation, somatic mutations of SHP-2 leading to gain of function have been identified in MDS and AML, where they induce activation of Ras-MAPK cascade. In contrast, fibroblasts and T cells from SHP-2 deficient mice display impaired activation of MAPK in response to growth factor-mediated activation and TCR/CD3-mediated activation, respectively. These studies suggest that SHP-2 has a role in activating some signaling pathways. In the present study we sought to determine the mechanism via which PD-1: SHP-2 interaction leads to inhibition of T cell activation. SHP-2 has two src homology domains, (N-SH2 and C-SH2) and one PTP domain. To identify the region of SHP-2 that interacts with PD-1, we generated five different GST-fusion proteins in which GST was fused with either SHP-2 full length (FL), SHP-2-N-SH2, SHP-2-C-SH2, SHP-2-ΔN-SH2 (lacking the N-terminus SH2 domain) or SHP-2-PTP. Pull-down assays using lysates from naive or activated primary human T cells revealed that PD-1 interacted with full length GST-SHP-2 fusion protein only after TCR/CD3-mediated activation and simultaneous PD-1 ligation. Pull-down assays with each GST-fusion protein showed that this interaction was mediated selectively via the SH2 domains of SHP-2, indicating that PD-1 requires prior tyrosine phosphorylation for interaction with SHP-2. The TCR itself lacks kinase activity but constitutively interacts with Fyn, the most TCR-proximal tyrosine kinase, which induces TCR phosphorylation upon oligomerization of the TCR/CD3 chains. To determine whether Fyn can also induce PD-1 phosphorylation during simultaneous ligation with TCR/CD3, we used COS cells to express PD-1 together with empty vector, Fyn, or a kinase inactive mutant of Fyn, followed by pull-down with GST-SHP-2 fusion protein. No interaction between PD-1 and GST-SHP-2 fusion protein was detected in lysates from COS cells expressing empty vector or kinase inactive Fyn mutant. In contrast, in the presence of active Fyn, PD-1 underwent phosphorylation and association with SHP-2. A key event required for the activation of the TCR/CD3 downstream signaling cascade is the downregulation of Csk-mediated inactivating C-terminal phosphorylation of Lck on Tyr-505. In epithelial cells, upon growth factor stimulation, SHP-2 is recruited to the plasma membrane and abrogates Csk recruitment and Csk-mediated inactivating C-terminal phosphorylation of Src kinase. We examined whether SHP-2 might be recruited to the plasma membrane during TCR/CD3-mediated stimulation and whether this event might be affected by PD-1: SHP-2 interaction. Using nitrogen cavitation to fractionate cytoplasmic and membranous compartments, we determined that stimulation of primary human T cells by TCR/CD3 resulted in robust membrane translocation of SHP-2. This event correlated with downregulation of the inactivating phosphorylation of Lck on Tyr-505. In contrast, stimulation of T cells with simultaneous ligation of PD-1 resulted in PD-1: SHP-2 association and sequestration, and prevented the translocation of SHP-2 to the membranous fraction and the downregulation of the inactivating Lck phosphorylation on Tyr-505. Moreover, phosphorylation of Lck substrates including PLC-γ1 was impaired. Consistent with the key role of PLC-γ1 in the activation of Ras/MAPK via RasGRP1, activation of Ras and MAPK was also defective. Our results unravel a previously unidentified mechanism via which PD-1 inhibits T cell activation by sequestering SHP-2 and preventing its translocation to the plasma membrane where SHP-2 plays a key role in activating the TCR signaling cascade by reversing Csk-mediated inactivating phosphorylation of Lck. Disclosures No relevant conflicts of interest to declare.

scholarly journals The role of p21ras in CD28 signal transduction: triggering of CD28 with antibodies, but not the ligand B7-1, activates p21ras.

1994 ◽  
Vol 180 (3) ◽  
pp. 1067-1076 ◽  
Author(s):  
J A Nunès ◽  
Y Collette ◽  
A Truneh ◽  
D Olive ◽  
D A Cantrell
Keyword(s):  
Signal Transduction ◽  
T Cells ◽  
T Cell ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Tyrosine Kinases ◽  
Cell Activation ◽  
Signal Transduction Pathway ◽  
Biochemical Basis ◽  
Ras Activation

CD28 is a 44-kD homodimer expressed on the surface of the majority of human T cells that provides an important costimulus for T cell activation. The biochemical basis of the CD28 accessory signals is poorly understood. Triggering of the T cell antigen receptor (TCR) activates the p21ras proteins. Here we show that ligation of CD28 by a monoclonal antibody (mAb) also stimulates p21ras and induces Ras-dependent events such as stimulation of the microtubule-associated protein (MAP) kinase ERK2 and hyperphosphorylation of Raf-1. One physiological ligand for CD28 is the molecule B7-1. In contrast to the effect of CD28 mAb, the present studies show that interactions between CD28 and B7-1 do not stimulate p21ras signaling pathways. Two substrates for TCR-regulated protein tyrosine kinases (PTKs) have been implicated in p21ras activation in T cells: p95vav and a 36-kD protein that associates with a complex of Grb2 and the Ras exchange protein Sos. Triggering CD28 with both antibodies and B7-1 activates cellular PTKs, and we have exploited the differences between antibodies and B7-1 for p21ras activation in an attempt to identify critical PTK-controlled events for Ras activation in T cells. The data show that antibodies against TCR or CD28 induce tyrosine phosphorylation of both Vav and p36. B7-1 also induces Vav tyrosine phosphorylation but has no apparent effect on tyrosine phosphorylation of the Grb2-associated p36 protein. The intensity of the Vav tyrosine phosphorylation is greater in B7-1 than in TCR-stimulated cells. Moreover the kinetics of Vav tyrosine phosphorylation is prolonged in the B7-1-stimulated cells. These studies show that for CD28 signaling, the activation of p21ras correlates more closely with p36 tyrosine phosphorylation than with Vav tyrosine phosphorylation. However, the experiments demonstrate that Vav is a major substrate for B7-activated PTKs and hence could be important in CD28 signal transduction pathway.

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