Distinct NF-kB activation pathways engaged by T-cell receptor and co-receptor CD28 on T-cells

The transcription factor nuclear factor-kB (NF-kB) is critical for the induction of inflammatory responses in T-cells, survival and differentiation. Antigen receptor (TCR) and co-receptor CD28 are the central regulators of NF-kB activation in T-cells. Progress in understanding NF-kB activation in T-cells has occurred over the years with the identification of individual adapters such as ADAP and GRB-2 and enzymes such as PKC-θ that regulate NF-kB. However, little is known whether the engagement of distinct modules by the TCR and CD28 account for the cooperative effects of the two receptors in activating NF-kB. In this review, we discuss recent advances in our understanding of NF-kB regulation by TCR and CD28

Immune adaptor SKAP1 acts a scaffold for Polo-like kinase 1 (PLK1) for the optimal cell cycling of T-cells.

While the immune cell adaptor protein SKAP1 mediates LFA-1 activation induced by antigen-receptor (TCR/CD3) ligation on T-cells, it is unclear whether the adaptor interacts with other mediators of T-cell function. In this context, the serine/threonine kinase, polo-like kinase (PLK1) regulates multiple steps in the mitotic and cell cycle progression of mammalian cells. Here, we show that SKAP1 is phosphorylated by and binds to PLK1 for the optimal cycling of T-cells. PLK1 binds to the N-terminal residue serine 31 (S31) of SKAP1 and the interaction is needed for optimal PLK1 kinase activity. Further, siRNA knock-down of SKAP1 reduced the rate of T-cell division concurrent with a delay in the expression of PLK1, Cyclin A and pH3. Reconstitution of these KD cells with WT SKAP1, but not the SKAP1 S31 mutant, restored normal cell division. SKAP1-PLK1 binding is dynamically regulated during the cell cycle of T-cells. Our findings identify a novel role for SKAP1 in the regulation of PLK1 and optimal cell cycling needed for T-cell clonal expansion in response to antigenic activation

CTLA-4 and PD-1 Control of T-Cell Motility and Migration: Implications for Tumor Immunotherapy

CTLA-4 is a co-receptor on T-cells that controls peripheral tolerance and the development of autoimmunity. Immune check-point blockade (ICB) uses monoclonal antibodies (MAbs) to block the binding of inhibitory receptors (IRs) to their natural ligands. A humanized antibody to CTLA-4 was first approved clinically followed by the use of antibody blockade against PD-1 and its ligand PD-L1. Effective anti-tumor immunity requires the activation of tumor-specific effector T-cells, the blockade of regulatory cells and the migration of T-cells into the tumor. Here, we review data implicating CTLA-4 and PD-1 in the motility of T-cells with a specific reference to the potential exploitation of these pathways for more effective tumor infiltration and eradication

TCR and CD28 activate the transcription factor NF-κB in T-cells via distinct adaptor signaling complexes.

The transcription factor NF-κB is needed for the induction of inflammatory responses in T-cells. Whether its activation by the antigen-receptor and CD28 is mediated by the same or different intracellular signaling pathways has been unclear. Here, using T-cells from various knock-out (Cd28(-/-), adap(-/-)) and knock-in (i.e. Cd28 Y-170F) mice in conjunction with transfected Jurkat T-cells, we show that the TCR and CD28 use distinct pathways to activate NF-κB in T-cells. Anti-CD28 ligation alone activated NF-κB in primary and Jurkat T-cells as measured by NF-κB reporter and EMSA assays. Anti-CD28 also activated NF-κB normally in primary T-cells from adap(-/-) mice, while anti-CD3 stimulation required the adaptor ADAP. Over-expression of ADAP or its binding partner SKAP1 failed to enhance anti-CD28 activation of NF-κB, while ADAP greatly increased anti-CD3 induced NF-κB activity. By contrast, CD28 activation of NF-κB depended on GRB-2 binding to CD28 as seen in CD28 deficient Jurkat T-cells reconstituted with the CD28 YMN-FM mutant, and in primary T-cells from CD28 Y170F mutant knock-in mice. CD28 associated with GRB-2, and GRB-2 siRNA impaired CD28 NF-κB activation. GRB-2 binding partner and guanine nucleotide exchange factor, VAV1, greatly enhanced anti-CD28 driven activation of NF-κB. Further, unlike in the case of anti-CD28, NF-κB activation by anti-CD3 and its cooperation with ADAP was strictly dependent on LAT expression. Overall, we provide evidence that CD28 and the TCR complex regulate NF-κB via different signaling modules of GRB-2/VAV1 and LAT/ADAP pathways respectively.This work was supported by Wellcome Trust Progam Grant (PG) PKAG/504 to Principal Research Fellow (PRF) C.E. Rudd. We are grateful to Oreste Ocuto and Enzo Cerundolo from University of Oxford for providing CHC17 and 1G4 (Cd28−/−) Jurkat cells.This paper was originally published in Immunology Letters (Thaker YR, Schneider H, Rudd CE, Immunology Letters 2015, 163, 1, 113–119, doi:10.1016/j.imlet.2014.10.020)

An essential role for SKAP-55 in LFA-1 clustering on T cells that cannot be substituted by SKAP-55R

Lymphocyte function-associated antigen (LFA)-1 clustering, which is needed for high avidity binding to intercellular adhesion molecule (ICAM)-1 and -2, regulates T cell motility and T cell–antigen-presenting cell (APC) conjugation. In this study, down-regulation of SKAP-55 by small interfering RNAs (siRNAs) identified an essential role for this adaptor molecule in the T cell receptor (TCR)–mediated ”inside-out signaling” that is needed for LFA-1 clustering and T cell–APC conjugation. In contrast, down-regulation of SKAP-55 had no effect on TCR–CD3 clustering. Furthermore, the expression of the related protein SKAP-55R failed to compensate for the loss of SKAP-55 in LFA-1 clustering, indicating that SKAP-55 has a unique function that cannot be replaced by this closely related protein. Our findings therefore indicate that SKAP-55, unlike SKAP-55R, is specifically tailored as an essential component of the inside-out signaling events that couple the TCR to LFA-1 clustering and T cell–APC conjugation

Glycogen synthase kinase 3 (GSK-3) controls T-cell motility and interactions with antigen presenting cells.

OBJECTIVE: The threonine/serine kinase glycogen synthase kinase 3 (GSK-3) targets multiple substrates in T-cells, regulating the expression of Tbet and PD-1 on T-cells. However, it has been unclear whether GSK-3 can affect the motility of T-cells and their interactions with antigen presenting cells. RESULTS: Here, we show that GSK-3 controls T-cell motility and interactions with other cells. Inhibition of GSK-3, using structurally distinct inhibitors, reduced T-cell motility in terms of distance and displacement. While SB415286 reduced the number of cell-cell contacts, the dwell times of cells that established contacts with other cells did not differ for T-cells treated with SB415286. Further, the increase in cytolytic T-cell (CTL) function in killing tumor targets was not affected by the inhibition of motility. This data shows that the inhibition of GSK-3 has differential effects on T-cell motility and CTL function where the negative effects on cell-cell interactions is overridden by the increased cytolytic potential of CTLs

GTPase-activating protein Rasal1 associates with ZAP-70 of the TCR and negatively regulates T-cell tumor immunity.

Immunotherapy involving checkpoint blockades of inhibitory co-receptors is effective in combating cancer. Despite this, the full range of mediators that inhibit T-cell activation and influence anti-tumor immunity is unclear. Here, we identify the GTPase-activating protein (GAP) Rasal1 as a novel TCR-ZAP-70 binding protein that negatively regulates T-cell activation and tumor immunity. Rasal1 inhibits via two pathways, the binding and inhibition of the kinase domain of ZAP-70, and GAP inhibition of the p21ras-ERK pathway. It is expressed in activated CD4 + and CD8 + T-cells, and inhibits CD4 + T-cell responses to antigenic peptides presented by dendritic cells as well as CD4 + T-cell responses to peptide antigens in vivo. Furthermore, siRNA reduction of Rasal1 expression in T-cells shrinks B16 melanoma and EL-4 lymphoma tumors, concurrent with an increase in CD8 + tumor-infiltrating T-cells expressing granzyme B and interferon γ-1. Our findings identify ZAP-70-associated Rasal1 as a new negative regulator of T-cell activation and tumor immunity

Glycogen synthase kinase 3 (GSK-3) controls T-cell motility and interactions with antigen presenting cells.

OBJECTIVE: The threonine/serine kinase glycogen synthase kinase 3 (GSK-3) targets multiple substrates in T-cells, regulating the expression of Tbet and PD-1 on T-cells. However, it has been unclear whether GSK-3 can affect the motility of T-cells and their interactions with antigen presenting cells. RESULTS: Here, we show that GSK-3 controls T-cell motility and interactions with other cells. Inhibition of GSK-3, using structurally distinct inhibitors, reduced T-cell motility in terms of distance and displacement. While SB415286 reduced the number of cell-cell contacts, the dwell times of cells that established contacts with other cells did not differ for T-cells treated with SB415286. Further, the increase in cytolytic T-cell (CTL) function in killing tumor targets was not affected by the inhibition of motility. This data shows that the inhibition of GSK-3 has differential effects on T-cell motility and CTL function where the negative effects on cell-cell interactions is overridden by the increased cytolytic potential of CTLs