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

Altered cell cycle dynamics in schizophrenia

Background: The olfactory mucosa, the organ of smell in the nose, is a neural tissue that regenerates new sensory neurons throughout adult life. Based on this tissue, we previously demonstrated increased mitosis in olfactory biopsy cultures from schizophrenia patients compared with healthy control subjects. In addition, neural stem/progenitor cell cultures (neurosphere-derived cells) from nasal biopsies from individuals with schizophrenia show significantly altered gene and protein expression in key cell cycle control pathways

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

EdU, a new thymidine analogue for labelling proliferating cells in the nervous system

The standard method of labelling proliferating cells uses the thymidine analogue, bromodeoxyuridine (BrdU), which incorporates into the DNA during S-phase of the cell cycle. A disadvantage of this method is that the immunochemical processing requires pre-treatment of the cells and tissue with heat or acid to reveal the antigen. This pre-treatment reduces reliability of the method and degrades the specimen, reducing the ability for multiple immuno-fluorescence labelling at high resolution. We report here the utility of a novel thymidine analogue, ethynyl deoxyuridine (EdU), detected with a fluorescent azide via the “click” chemistry reaction (the Huisgen 1,3-dipolar cycloaddition reaction of an organic azide to a terminal acetylene). The detection of EdU requires no heat or acid treatment and the incorporated EdU is covalently conjugated to fluorescent probe. The reaction is quick and compatible with fluorescence immunochemistry and other fluorescent probes. We show here that EdU is non-toxic in vitro and in vivo and can be used in place of BrdU to label cells during neurogenesis and the progeny identified at least 30 days later. The fluorescent labelling of EdU, markedly improves the detection of proliferating cells and allows concurrent high resolution fluorescence immunochemistry

High-throughput analysis of T cell–monocyte interaction in human tuberculosis

The lack of efficient tools for identifying immunological correlates of tuberculosis (TB) protection or risk of disease progression impedes the development of improved control strategies. To more clearly understand the host response in TB, we recently established an imaging flow cytometer‐based in‐vitro assay, which assesses multiple aspects of T cell–monocyte interaction. Here, we extended our previous work and characterized communication between T cells and monocytes using clinical samples from individuals with different TB infection status and healthy controls from a TB endemic setting. To identify T cell–monocyte conjugates, peripheral blood mononuclear cells (PBMC) were stimulated with ds‐Red‐expressing Mycobacterium bovis bacille Calmette–Guérin or 6‐kDa early secreted antigenic target (ESAT 6) peptides for 6 h, and analyzed by imaging flow cytometer (IFC). We then enumerated T cell–monocyte conjugates using polarization of T cell receptor (TCR) and F‐actin as markers for synapse formation, and nuclear factor kappa B (NF‐κB) nuclear translocation in the T cells. We observed a reduced frequency of T cell–monocyte conjugates in cells from patients with active pulmonary tuberculosis (pTB) compared to latent TB‐infected (LTBI) and healthy controls. When we monitored NF‐κB nuclear translocation in T cells interacting with monocytes, the proportion of responding cells was significantly higher in active pTB compared with LTBI and controls. Overall, these data underscore the need to consider multiple immunological parameters against TB, where IFC could be a valuable tool

In vitro analysis of antigen induced T cell-monocyte conjugates by imaging flow cytometry

There is a lack of suitable correlates of immune protection against Mycobacterium tuberculosis (Mtb) infection. T cells and monocytes play key roles in host immunity against Mtb. Thus, a method that allows assessing their interaction would contribute to the understanding of immune regulation in tuberculosis (TB). We have established imaging flow cytometer (IFC) based in vitro assay for the analysis of early events in T cell-monocyte interaction, upstream of cytokine production and T cell proliferation. This was achieved through short term stimulation of peripheral blood mononuclear cells (PBMC) from healthy Norwegian blood donors with Mycobacterium bovis Bacille Calmette-Guérin (BCG). In our assay, we examined the kinetics of BCG uptake by monocytes using fluorescently labeled BCG and T cell-monocyte interaction based on synapse formation (CD3/TCR polarization). Our results showed that BCG stimulation induced a gradual increase in the proportion of conjugated T cells displaying NF-κB translocation to the nucleus in a time dependent manner, with the highest frequency observed at 6 h. We subsequently tested PBMC from a small cohort of active TB patients (n = 7) and observed a similar BCG induced NF-κB translocation in T cells conjugated with monocytes. The method allowed for simultaneous evaluation of T cell-monocyte conjugates and T cell activation as measured by NF-κB translocation, following short-term challenge of human PBMC with BCG. Whether this novel approach could serve as a diagnostic or prognostic marker needs to be investigated using a wide array of Mtb specific antigens in a larger cohort of patients with different TB infection status

In vitro analysis of antigen induced T cell-monocyte conjugates by imaging flow cytometry

There is a lack of suitable correlates of immune protection against Mycobacterium tuberculosis (Mtb) infection. T cells and monocytes play key roles in host immunity against Mtb. Thus, a method that allows assessing their interaction would contribute to the understanding of immune regulation in tuberculosis (TB). We have established imaging flow cytometer (IFC) based in vitro assay for the analysis of early events in T cell-monocyte interaction, upstream of cytokine production and T cell proliferation. This was achieved through short term stimulation of peripheral blood mononuclear cells (PBMC) from healthy Norwegian blood donors with Mycobacterium bovis Bacille Calmette-Guérin (BCG). In our assay, we examined the kinetics of BCG uptake by monocytes using fluorescently labeled BCG and T cell-monocyte interaction based on synapse formation (CD3/ TCR polarization). Our results showed that BCG stimulation induced a gradual increase in the proportion of conjugated T cells displaying NF-κB translocation to the nucleus in a time dependent manner, with the highest frequency observed at 6 h. We subsequently tested PBMC from a small cohort of active TB patients (n = 7) and observed a similar BCG induced NF-κB translocation in T cells conjugated with monocytes. The method allowed for simultaneous evaluation of T cell-monocyte conjugates and T cell activation as measured by NF-κB translocation, following short-term challenge of human PBMC with BCG. Whether this novel approach could serve as a diagnostic or prognostic marker needs to be investigated using a wide array of Mtb specific antigens in a larger cohort of patients with different TB infection status

Reduced MCMV Δm157 viral clearance in the absence of TSAd

The T cell specific adapter protein (TSAd) is expressed in activated T cells and NK cells. While TSAd is beginning to emerge as a critical regulator of Lck and Itk activity in T cells, its role in NK cells has not yet been explored. Here we have examined susceptibility to virus infections in a murine model using various viral infection models. We report that TSAd-deficient mice display reduced clearance of murine cytomegalovirus (MCMV) that lack the viral MHC class I homologue m157, which is critical for Ly49H-mediated NK cell recognition of infected cells. In this infection model, NK cells contribute in the early stages of the disease, whereas CD8+ T cells are critical for viral clearance. We found that mice infected with MCMV Δm157 displayed reduced viral clearance in the spleen as well as reduced proliferation in spleen NK cells and CD8+ T cells in the absence of TSAd. Though no other immunophenotype was detected in the infection models tested, these data suggests that in the absence of the Ly49H ligand activation, NK cell and CD8+ T cell responses may be compromised in TSAd-deficient mice

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