Interaction of T-cell-specific adapter protein with Src- and Tec-family kinases.

Adapter proteins are flexible and dynamic modulators of cellular signalling that are important for immune cell function. One of these, the T-cell-specific adapter protein (TSAd), interacts with the non-receptor tyrosine kinases Src and Lck of the Src family kinases (SFKs) and Itk of the Tec family kinases (TFKs). Three tyrosine residues in the TSAd C-terminus are phosphorylated by Lck and serve as docking sites for the Src homology 2 (SH2) domains of Src and Lck. The TSAd proline-rich region (PRR) binds to the Src homology 3 (SH3) domains found in Lck, Src and Itk. Despite known interactors, the role TSAd plays in cellular signalling remains largely unknown. TSAd's ability to bind both SFKs and TFKs may point to its function as a general scaffold for both kinase families. Using GST-pulldown as well as peptide array experiments, we found that both the SH2 and SH3 domains of the SFKs Fyn and Hck, as well as the TFKs Tec and Txk, interact with TSAd. This contrasts with Itk, which interacts with TSAd only through its SH3 domain. Although our analysis showed that TSAd is both co-expressed and may interact with Fyn, we were unable to co-precipitate Fyn with TSAd from Jurkat cells, as detected by Western blotting and affinity purification mass spectrometry. This may suggest that TSAd-Fyn interaction in intact cells may be limited by other factors, such as the subcellular localization of the two molecules or the co-expression of competing binding partners.

Exploring the role of the multiple sclerosis susceptibility gene CLEC16A in T cells.

C-type lectin-like domain family 16 member A (CLEC16A) is associated with autoimmune disorders, including multiple sclerosis (MS), but its functional relevance is not completely understood. CLEC16A is expressed in several immune cells, where it affects autophagic processes and receptor expression. Recently, we reported that the risk genotype of an MS-associated single nucleotide polymorphism in CLEC16A intron 19 is associated with higher expression of CLEC16A in CD4+ T cells. Here, we show that CLEC16A expression is induced in CD4+ T cells upon T cell activation. By the use of imaging flow cytometry and confocal microscopy, we demonstrate that CLEC16A is located in Rab4a-positive recycling endosomes in Jurkat TAg T cells. CLEC16A knock-down in Jurkat cells resulted in lower cell surface expression of the T cell receptor, however, this did not have a major impact on T cell activation response in vitro in Jurkat nor in human, primary CD4+ T cells.

Tyr192 Regulates Lymphocyte-Specific Tyrosine Kinase Activity in T Cells.

TCR signaling critically depends on the tyrosine kinase Lck (lymphocyte-specific protein tyrosine kinase). Two phosphotyrosines, the activating pTyr394 and the inhibitory pTyr505, control Lck activity. Recently, pTyr192 in the Lck SH2 domain emerged as a third regulator. How pTyr192 may affect Lck function remains unclear. In this study, we explored the role of Lck Tyr192 using CRISPR/Cas9-targeted knock-in mutations in the human Jurkat T cell line. Our data reveal that both Lck pTyr394 and pTyr505 are controlled by Lck Tyr192 Lck with a nonphosphorylated SH2 domain (Lck Phe192) displayed hyperactivity, possibly by promoting Lck Tyr394 transphosphorylation. Lck Glu192 mimicking stable Lck pTyr192 was inhibited by Tyr505 hyperphosphorylation. To overcome this effect, we further mutated Tyr505 The resulting Lck Glu192/Phe505 displayed strongly increased amounts of pTyr394 both in resting and activated T cells. Our results suggest that a fundamental role of Lck pTyr192 may be to protect Lck pTyr394 and/or pTyr505 to maintain a pool of already active Lck in resting T cells. This provides an additional mechanism for fine-tuning of Lck as well as T cell activity.

Adaptor proteins: Flexible and dynamic modulators of immune cell signalling.

To maintain homeostasis, all cells respond to environmental cues via a multitude of surface receptors. In order to act appropriately in their environment, cells are dependent on the transduction of the incoming signal through tightly regulated and interconnected signalling pathways to the cell nucleus. In particular, cells implicated in the immune system greatly depend on such systems to respond in a flexible and dynamic manner to environmental challenges. One major group of intracellular proteins that are involved in these signalling pathways are adaptor proteins. Although adaptor proteins are essential for normal immune cell operation, the functional role of this group of signalling proteins remains to be fully appreciated. So far, research on adaptor proteins has revealed their unique potential in building transient complexes in a reversible, dynamic and inducible manner. In this review, we explore the roles of adaptor proteins - in space and time of intracellular signalling - and their associations with human disease. Examples of adaptor proteins expressed in hematopoietic cells highlight their crucial role in the immune system. Lastly, we present challenges faced in elucidating roles of adaptor proteins, as illustrated by the T cell-specific adaptor (TSAd) protein encoded by the SH2D2A gene.

A simple and efficient workflow for generation of knock-in mutations in Jurkat T cells using CRISPR/Cas9.

CRISPR/Cas9 is a powerful gene-editing tool allowing for specific gene manipulation at targeted sites in the genome. Here, we used CRISPR/Cas9-mediated gene editing to introduce single amino acid mutations into proteins involved in T cell receptor signalling pathways. Knock-in mutations were introduced in Jurkat T cells by homologous directed repair using single-stranded oligodeoxynucleotides. Specifically, we aimed to create targeted mutations at two loci within LCK, a constitutively expressed gene, and at three loci within SH2D2A, whose expression is induced upon T cell activation. Here, we present a simple workflow that can be applied by any laboratory equipped for cell culture work, utilizing basic flow cytometry, Western blotting and PCR techniques. Our data reveal that gene editing may be locus-dependent and can vary between target sites, also within a gene. In our two targeted genes, on average 2% of the clones harboured homozygous mutations as assessed by allele-specific PCR and subsequent sequencing. We highlight the importance of decreasing the clonal heterogeneity and developing robust screening methods to accurately select for correct knock-in mutations. Our workflow may be employed in other immune cell lines and acts as a useful approach for decoding functional mechanisms of proteins of interest.

The SH3 domains of the protein kinases ITK and LCK compete for adjacent sites on T cell-specific adapter protein.

T-cell activation requires stimulation of specific intracellular signaling pathways in which protein-tyrosine kinases, phosphatases, and adapter proteins interact to transmit signals from the T-cell receptor to the nucleus. Interactions of LCK proto-oncogene, SRC family tyrosine kinase (LCK), and the IL-2-inducible T cell kinase (ITK) with the T cell-specific adapter protein (TSAD) promotes LCK-mediated phosphorylation and thereby ITK activation. Both ITK and LCK interact with TSAD's proline-rich region (PRR) through their Src homology 3 (SH3) domains. Whereas LCK may also interact with TSAD through its SH2 domain, ITK interacts with TSAD only through its SH3 domain. To begin to understand on a molecular level how the LCK SH3 and ITK SH3 domains interact with TSAD in human HEK293T cells, here we combined biochemical analyses with NMR spectroscopy. We found that the ITK and LCK SH3 domains potentially have adjacent and overlapping binding sites within the TSAD PRR amino acids (aa) 239-274. Pulldown experiments and NMR spectroscopy revealed that both domains may bind to TSAD aa 239-256 and aa 257-274. Co-immunoprecipitation experiments further revealed that both domains may also bind simultaneously to TSAD aa 242-268. Accordingly, NMR spectroscopy indicated that the SH3 domains may compete for these two adjacent binding sites. We propose that once the associations of ITK and LCK with TSAD promote the ITK and LCK interaction, the interactions among TSAD, ITK, and LCK are dynamically altered by ITK phosphorylation status.

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.

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.

The endothelial adaptor molecule TSAd is required for VEGF-induced angiogenic sprouting through junctional c-Src activation.

Activation of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) by VEGF binding is critical for vascular morphogenesis. In addition, VEGF disrupts the endothelial barrier by triggering the phosphorylation and turnover of the junctional molecule VE-cadherin, a process mediated by the VEGFR2 downstream effectors T cell-specific adaptor (TSAd) and the tyrosine kinase c-Src. We investigated whether the VEGFR2-TSAd-c-Src pathway was required for angiogenic sprouting. Indeed, Tsad-deficient embryoid bodies failed to sprout in response to VEGF. Tsad-deficient mice displayed impaired angiogenesis specifically during tracheal vessel development, but not during retinal vasculogenesis, and in VEGF-loaded Matrigel plugs, but not in those loaded with FGF. The SH2 and proline-rich domains of TSAd bridged VEGFR2 and c-Src, and this bridging was critical for the localization of activated c-Src to endothelial junctions and elongation of the growing sprout, but not for selection of the tip cell. These results revealed that vascular sprouting and permeability are both controlled through the VEGFR2-TSAd-c-Src signaling pathway in a subset of tissues, which may be useful in developing strategies to control tissue-specific pathological angiogenesis.

Class II HLA interactions modulate genetic risk for multiple sclerosis.

Association studies have greatly refined the understanding of how variation within the human leukocyte antigen (HLA) genes influences risk of multiple sclerosis. However, the extent to which major effects are modulated by interactions is poorly characterized. We analyzed high-density SNP data on 17,465 cases and 30,385 controls from 11 cohorts of European ancestry, in combination with imputation of classical HLA alleles, to build a high-resolution map of HLA genetic risk and assess the evidence for interactions involving classical HLA alleles. Among new and previously identified class II risk alleles (HLA-DRB1*15:01, HLA-DRB1*13:03, HLA-DRB1*03:01, HLA-DRB1*08:01 and HLA-DQB1*03:02) and class I protective alleles (HLA-A*02:01, HLA-B*44:02, HLA-B*38:01 and HLA-B*55:01), we find evidence for two interactions involving pairs of class II alleles: HLA-DQA1*01:01-HLA-DRB1*15:01 and HLA-DQB1*03:01-HLA-DQB1*03:02. We find no evidence for interactions between classical HLA alleles and non-HLA risk-associated variants and estimate a minimal effect of polygenic epistasis in modulating major risk alleles.

Multiple Sclerosis Risk Allele in CLEC16A Acts as an Expression Quantitative Trait Locus for CLEC16A and SOCS1 in CD4+ T Cells.

For multiple sclerosis, genome wide association studies and follow up studies have identified susceptibility single nucleotide polymorphisms located in or near CLEC16A at chromosome 16p13.13, encompassing among others CIITA, DEXI and SOCS1 in addition to CLEC16A. These genetic variants are located in intronic or intergenic regions and display strong linkage disequilibrium with each other, complicating the understanding of their functional contribution and the identification of the direct causal variant(s). Previous studies have shown that multiple sclerosis-associated risk variants in CLEC16A act as expression quantitative trait loci for CLEC16A itself in human pancreatic ß-cells, for DEXI and SOCS1 in thymic tissue samples, and for DEXI in monocytes and lymphoblastoid cell lines. Since T cells are major players in multiple sclerosis pathogenesis, we have performed expression analyses of the CIITA-DEXI-CLEC16A-SOCS1 gene cluster in CD4+ and CD8+ T cells isolated from multiple sclerosis patients and healthy controls. We observed a higher expression of SOCS1 and CLEC16A in CD4+ T cells in samples homozygous for the risk allele of CLEC16A rs12927355. Pair-wise linear regression analysis revealed high correlation in gene expression in peripheral T cells of CIITA, DEXI, CLEC16A and SOCS1. Our data imply a possible regulatory role for the multiple sclerosis-associated rs12927355 in CLEC16A.

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 Tyr(280) (pTyr(280)) and pTyr(305). These predictions were confirmed by peptide array analysis, showing direct binding of recombinant Nck SH2 to both pTyr(280) and pTyr(305) 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.

Coordinated expression of DNAM-1 and LFA-1 in educated NK cells.

The functional capacity of NK cells is dynamically tuned by integrated signals from inhibitory and activating cell surface receptors in a process termed NK cell education. However, the understanding of the cellular and molecular mechanisms behind this functional tuning is limited. In this study, we show that the expression of the adhesion molecule and activation receptor DNAX accessory molecule 1 (DNAM-1) correlates with the quantity and quality of the inhibitory input by HLA class I-specific killer cell Ig-like receptors and CD94/NKG2A as well as with the magnitude of functional responses. Upon target cell recognition, the conformational state of LFA-1 changed in educated NK cells, associated with rapid colocalization of both active LFA-1 and DNAM-1 at the immune synapse. Thus, the coordinated expression of LFA-1 and DNAM-1 is a central component of NK cell education and provides a potential mechanism for controlling cytotoxicity by functionally mature NK cells.

Retinoic acid-induced IgG production in TLR-activated human primary B cells involves ULK1-mediated autophagy.

In the present study we have established a vital role of autophagy in retinoic acid (RA)-induced differentiation of toll-like receptor (TLR)-stimulated human B cells into Ig-secreting cells. Thus, RA enhanced autophagy in TLR9- and CD180-stimulated peripheral blood B cells, as revealed by increased levels of the autophagosomal marker LC3B-II, enhanced colocalization between LC3B and the lysosomal marker Lyso-ID, by a larger percentage of cells with more than 5 characteristic LC3B puncta, and by the concomitant reduction in the level of SQSTM1/p62. Furthermore, RA induced expression of the autophagy-inducing protein ULK1 at the transcriptional level, in a process that required the retinoic acid receptor RAR. By inhibiting autophagy with specific inhibitors or by knocking down ULK1 by siRNA, the RA-stimulated IgG production in TLR9- and CD180-mediated cells was markedly reduced. We propose that the identified prominent role of autophagy in RA-mediated IgG-production in normal human B cells provides a novel mechanism whereby vitamin A exerts its important functions in the immune system.

Retinoic acid enhances the levels of IL-10 in TLR-stimulated B cells from patients with relapsing-remitting multiple sclerosis.

We have explored the beneficial effects of retinoic acid (RA) on B cells from multiple sclerosis (MS) patients. When co-stimulated via the toll-like receptors (TLRs) TLR9 and RP105, MS B cells secreted less of the anti-inflammatory cytokine interleukin 10 (IL-10) compared to B cells from healthy controls. Importantly, RA enhanced the secretion of IL-10 by MS-derived B cells without affecting the levels of the pro-inflammatory cytokine TNF-α. RA revealed the same ability to induce IL-10 as did interferon-ß-1b (IFN-ß-1b), and B-cells from patients treated with glatiramer acetate or IFN-ß-1b still displayed the beneficial effects of RA on the IL-10/TNF-α ratio.

The kinase Itk and the adaptor TSAd change the specificity of the kinase Lck in T cells by promoting the phosphorylation of Tyr192.

The substrate specificity of Src family kinases (SFKs) is partly determined by their Src homology 2 (SH2) domains. Thus, transient alterations in the SH2 domain of SFKs might change their binding partners and affect intracellular signaling pathways. Lck is an SFK that is central to the initiation of T cell activation in response to ligand binding to the T cell receptor (TCR) and is also critical for later signaling processes. The kinase activity of Lck requires both the phosphorylation of an activating tyrosine residue and the dephosphorylation of an inhibitory tyrosine residue. We found that a third conserved tyrosine phosphorylation site (Tyr(192)) within the SH2 domain of Lck was required for proper T cell activation and formation of cell-cell conjugates between T cells and antigen-presenting cells. Through phosphopeptide arrays and biochemical assays, we identified several regulators of the actin cytoskeleton that preferentially bound to Lck phosphorylated at Tyr(192) compared to Lck that was not phosphorylated at this site. Two of these phosphorylation-dependent binding partners, the kinase Itk (interleukin-2-inducible Tec kinase) and the adaptor protein TSAd (T cell-specific adaptor), promoted the TCR-dependent phosphorylation of Lck at Tyr(192). Our data suggest that phosphorylation transiently alters SH2 domain specificity and provide a potential mechanism whereby SFKs may be rewired from one signaling program to another to enable appropriate cell activation.