Endocytosis Deficient Murine Xcl1-Fusion Vaccine Enhances Protective Antibody Responses in Mice

Targeting antigen to surface receptors on dendritic cells (DCs) can improve antibody response against subunit vaccines. We have previously observed that human XCL1-fusion vaccines target murine Xcr1+ DCs without actively inducing endocytosis of the antigen, resulting in enhanced antibody responses in mice. However, the use of foreign chemokines for targeting is undesirable when translating this observation to human or veterinary medicine due to potential cross-reactive responses against the endogenous chemokine. Here we have identified a mutant version of murine Xcl1, labeled Xcl1(Δ1) owing to removal of a conserved valine in position 1 of the mature chemokine, that retains specific binding to Xcr1+ DCs without inducing endocytosis of the receptor. DNA immunization with Xcl1(Δ1) conjugated to influenza hemagglutinin (HA) induced improved antibody responses, with higher end point titers of IgG compared to WT Xcl1-HA. The Xcl1(Δ1) fusion vaccine also resulted in an increased number of HA reactive germinal center B cells with higher avidity toward the antigen, and serum transfer experiments show that Xcl1(Δ1)-HA induced antibody responses provided better protection against influenza infection as compared to WT Xcl1-HA. In summary, our observations indicate that targeting antigen to Xcr1+ DCs in an endocytosis deficient manner enhances antibody responses. This effect was obtained by introducing a single mutation to Xcl1, suggesting our strategy may easily be translated to human or veterinary vaccine settings

Structure function analysis of SH2D2A isoforms expressed in T cells reveals a crucial role for the proline rich region encoded by SH2D2A exon 7

BACKGROUND: The activation induced T cell specific adapter protein (TSAd), encoded by SH2D2A, interacts with and modulates Lck activity. Several transcript variants of TSAd mRNA exist, but their biological significance remains unknown. Here we examined expression of SH2D2A transcripts in activated CD4+ T cells and used the SH2D2A variants as tools to identify functionally important regions of TSAd. RESULTS: TSAd was found to interact with Lck in human CD4+ T cells ex vivo. Three interaction modes of TSAd with Lck were identified. TSAd aa239–256 conferred binding to the Lck-SH3 domain, whereas one or more of the four tyrosines within aa239–334 encoded by SH2D2A exon 7 was found to confer interaction with the Lck-SH2-domain. Finally the TSAd-SH2 domain was found to interact with Lck. The SH2D2A exon 7 encoding TSAd aa 239–334 was found to harbour information essential not only for TSAd interaction with Lck, but also for TSAd modulation of Lck activity and translocation of TSAd to the nucleus. All five SH2D2A transcripts were found to be expressed in CD3 stimulated CD4+ T cells. CONCLUSION: These data show that TSAd and Lck may interact through several different domains and that Lck TSAd interaction occurs in CD4+ T cells ex vivo. Alternative splicing of exon 7 encoding aa239–334 results in loss of the majority of protein interaction motives of TSAd and yields truncated TSAd molecules with altered ability to modulate Lck activity. Whether TSAd is regulated through differential alternative splicing of the SH2D2A transcript remains to be determined

T Cell Specific Adapter Protein (TSAd) Interacts with Tec Kinase ITK to Promote CXCL12 Induced Migration of Human and Murine T Cells

The chemokine CXCL12/SDF-1α interacts with its G-protein coupled receptor CXCR4 to induce migration of lymphoid and endothelial cells. T cell specific adapter protein (TSAd) has been found to promote migration of Jurkat T cells through interaction with the G protein β subunit. However, the molecular mechanisms for how TSAd influences cellular migration have not been characterized in detail. We show that TSAd is required for tyrosine phosphorylation of the Lck substrate IL2-inducible T cell kinase (Itk). Presence of Itk Y511 was necessary to boost TSAd\u27s effect on CXCL12 induced migration of Jurkat T cells. In addition, TSAd\u27s ability to promote CXCL12-induced actin polymerization and migration of Jurkat T lymphocytes was dependent on the Itk-interaction site in the proline-rich region of TSAd. Furthermore, TSAd-deficient murine thymocytes failed to respond to CXCL12 with increased Itk phosphorylation, and displayed reduced actin polymerization and cell migration responses. We propose that TSAd, through its interaction with both Itk and Lck, primes Itk for Lck mediated phosphorylation and thereby regulates CXCL12 induced T cell migration and actin cytoskeleton rearrangements

Polarity of CD4+ T cells towards the antigen presenting cell is regulated by the Lck adapter TSAd

Polarization of T cells towards the antigen presenting cell (APC) is critically important for appropriate activation and differentiation of the naïve T cell. Here we used imaging flow cytometry (IFC) and show that the activation induced Lck and Itk adapter T cell specific adapter protein (TSAd), encoded by SH2D2A, modulates polarization of T cells towards the APC. Upon exposure to APC presenting the cognate antigen Id, Sh2d2a−/− CD4+ T cells expressing Id-specific transgenic T cell receptor (TCR), displayed impaired polarization of F-actin and TCR to the immunological synapse (IS). Sh2d2a−/− T-cells that did polarize F-actin and TCR still displayed impaired polarization of PKCξ, PAR3 and the microtubule-organizing center (MTOC). In vitro differentiation of activated Sh2d2a−/− T cells was skewed towards an effector memory (Tem) rather than a central memory (Tcm) phenotype. A similar trend was observed for Id-specific TCR Sh2d2a−/− T cells stimulated with APC and cognate antigen. Taken together our data suggest that TSAd modulates differentiation of experienced T cells possibly through polarization of CD4+ T cells towards the APC

T cell specific adaptor protein (TSAd) promotes interaction of Nck with Lck and SLP-76 in T cells

Background The Lck and Src binding adaptor protein TSAd (T cell specific adaptor) regulates actin polymerization in T cells and endothelial cells. The molecular details as to how TSAd regulates this process remain to be elucidated. Results To identify novel interaction partners for TSAd, we used a scoring matrix-assisted ligand algorithm (SMALI), and found that the Src homology 2 (SH2) domain of the actin regulator Non-catalytic region of tyrosine kinase adaptor protein (Nck) potentially binds to TSAd phosphorylated on Tyr280 (pTyr280) and pTyr305. These predictions were confirmed by peptide array analysis, showing direct binding of recombinant Nck SH2 to both pTyr280 and pTyr305 on TSAd. In addition, the SH3 domains of Nck interacted with the proline rich region (PRR) of TSAd. Pull-down and immunoprecipitation experiments further confirmed the Nck-TSAd interactions through Nck SH2 and SH3 domains. In line with this Nck and TSAd co-localized in Jurkat cells as assessed by confocal microscopy and imaging flow cytometry. Co-immunoprecipitation experiments in Jurkat TAg cells lacking TSAd revealed that TSAd promotes interaction of Nck with Lck and SLP-76, but not Vav1. TSAd expressing Jurkat cells contained more polymerized actin, an effect dependent on TSAd exon 7, which includes interactions sites for both Nck and Lck. Conclusions TSAd binds to and co-localizes with Nck. Expression of TSAd increases both Nck-Lck and Nck-SLP-76 interaction in T cells. Recruitment of Lck and SLP-76 to Nck by TSAd could be one mechanism by which TSAd promotes actin polymerization in activated T cells

SH2D2A modulates T cell mediated protection to a B cell derived tumor in transgenic mice.

BACKGROUND: T cell specific adapter protein (TSAd), encoded by the SH2D2A gene, modulates signaling downstream of the T cell receptor (TCR). Young, unchallenged SH2D2A-deficient C57BL/6 mice exhibit a relatively normal immune phenotype. To address whether SH2D2A regulates physiologic immune responses, SH2D2A-deficient TCR-transgenic BALB/c mice were generated. The transgenic TCR recognizes a myeloma-derived idiotypic (Id) peptide in the context of the major histocompatibility complex (MHC) class II molecule I-E(d), and confers T cell mediated resistance to transplanted multiple myeloma development in vivo. PRINCIPAL FINDINGS: The immune phenotype of SH2D2A-deficient C57BL/6 and BALB/c mice did not reveal major differences compared to the corresponding wild type mice. When challenged with myeloma cells, Id-specific TCR-transgenic BALB/c mice lacking SH2D2A displayed increased resistance towards tumor development. Tumor free TCR-transgenic SH2D2A-deficient mice had higher numbers of Id-specific single positive CD4+ thymocytes compared to TCR-transgenic wild-type mice. CONCLUSION: Our results suggest a modulatory role for SH2D2A in T cell mediated immune surveillance of cancer. However, it remains to be established whether its effect is T-cell intrinsic. Further studies are required to determine whether targeting SH2D2A function in T cells may be a potential adjuvant in cancer immunotherapy

T Cell Specific Adapter Protein (TSAd) Interacts with Tec Kinase ITK to Promote CXCL12 Induced Migration of Human and Murine T Cells

The chemokine CXCL12/SDF-1α interacts with its G-protein coupled receptor CXCR4 to induce migration of lymphoid and endothelial cells. T cell specific adapter protein (TSAd) has been found to promote migration of Jurkat T cells through interaction with the G protein β subunit. However, the molecular mechanisms for how TSAd influences cellular migration have not been characterized in detail. We show that TSAd is required for tyrosine phosphorylation of the Lck substrate IL2-inducible T cell kinase (Itk). Presence of Itk Y511 was necessary to boost TSAd's effect on CXCL12 induced migration of Jurkat T cells. In addition, TSAd's ability to promote CXCL12-induced actin polymerization and migration of Jurkat T lymphocytes was dependent on the Itk-interaction site in the proline-rich region of TSAd. Furthermore, TSAd-deficient murine thymocytes failed to respond to CXCL12 with increased Itk phosphorylation, and displayed reduced actin polymerization and cell migration responses. We propose that TSAd, through its interaction with both Itk and Lck, primes Itk for Lck mediated phosphorylation and thereby regulates CXCL12 induced T cell migration and actin cytoskeleton rearrangements.This article is from PLoS ONE 5 (2010): e9761, doi:10.1371/journal.pone.0009761. Posted with permission.</p

Targeting Influenza Virus Hemagglutinin to Xcr1(+) Dendritic Cells in the Absence of Receptor-Mediated Endocytosis Enhances Protective Antibody Responses

Targeting Ags to conventional dendritic cells can enhance Ag-specific immune responses. Although most studies have focused on the induction of T cell responses, the mechanisms by which targeting improves Ab responses are poorly understood. In this study we present data on the use of human XCL1 (hXCL1) and hXCL2 fusion vaccines in a murine model. We show that the human chemokines bound type 1 conventional dendritic cells (cDC1), and that immunization with influenza virus hemagglutinin fused to hXCL1 or hXCL2 induced full protection against influenza challenge. Surprisingly, the hXCL1- and hXCL2- fusion vaccines induced better long-term protection associated with stronger induction of neutralizing Abs, and more Ab-secreting cells in bone marrow. In contrast, murine Xcl1 fusion vaccines induced stronger CD8(+) T cell responses compared with hXCL1. Further analysis revealed that although murine Xcl1 fusion vaccines induced chemotaxis and were rapidly endocytosed by cDC1, hXCL1 and hXCL2 fusion vaccines did not induce chemotaxis, were less efficiently endocytosed, and consequently, remained on the surface. This difference may explain the enhanced induction of Abs when targeting Ag to cDC1 using hXCL1 and hXCL2, and suggests that immune responses can be manipulated in directing Abs or T cells based on how efficiently the targeted Ag is endocytosed by the DC