THE ROLE OF ZAP-70 KINASE IN THE FINE-TUNING OF TCR SIGNALLING : IMPLICATIONS FOR IMMUNOPATHOLOGY AND –THERAPY

ZAP-70 (zeta-chain associated 70 kDa) kinase is a key regulator of T cell receptor signaling. After ligand binding of the T cell receptor (TcR), Lck kinase phosphorylates tyrosine (Y) residues of the CD3 ζ chains and ZAP-70, which, in turn, phosphorylates a number of downstream target proteins (eg. LAT, SLP-76, PLCγ, Cbl). ZAP-70 itself contains a number of Y residues, which can be phosphorylated. Using an array of mutant cell lines where targeted Y-Phenylalanine (F) mutations were introduced into ZAP-70, we were able to characterize the fine details of TcR signaling. Our data confirmed the function of earlier described activator (Y315, Y493) and inhibitory (Y292, Y492) residues; moreover, we described the regulatory role of previously less-known (Y069, Y126, Y178) positions. Glucocorticoid treatment is widely used for suppressing the immune response, primarily through the inhibition of T cell functions. Our earlier work demonstrated, that ZAP-70 is also involved in non-genomic (rapid) GC signaling mechanisms. Using our Y-F mutant ZAP-70 expressing cell line array, we identified that Y315 and Y492 were phosphorylated upon short-term high dose GC analogue treatment. These results confirmed that ZAP-70 represents an important link between the non-genomic GC and TcR/CD3 signaling pathways. Moreover, potential role of ZAP-70 kinase was implicated in chronic lymphoid leukemia (CLL) and autoimmune arthritis. It has been shown in a subgroup of patients with CLL that the malignant B-lymphocytes express ZAP-70 kinase, which was associated with inferior clinical outcome and prognosis. Using two ZAP-70 specific antibodies recognizing different epitopes in the kinase, we performed intracellular staining of malignant B cells from CLL patients. Based on our preliminary experiments, it seems possible that the ZAP-70 molecule expressed in the tumorous B-cells is structurally different from that found in normal T-cells, as some patients showed positivity with either one or the other antibody, while the normal T-cells were positive with both antibodies, just as expected. A spontaneous single point mutation at 163 from Triptophane (W) to Cysteine (C) in the SH2 domain of ZAP-70 caused altered thymic selection and leads to the development of autoimmune arthritis in SKG mice. Another study has shown that targeted simultaneous mutation at positions Y315 and Y319 to Alanine led to similar defects in T cell development than in SKG mice, interestingly, however, these mice did not develop autoimmune arthritis despite the presence of rheuma factor in the sera, increased IL-17 production and impaired Treg development. These data clearly show, how our understanding about ZAP-70 kinase has emerged from being exclusively a T cell specific signaling molecule to an important therapeutic target and potential regulator of pathologies like CLL or autoimmune arthritis

Fine-tuning of proximal TCR signaling by ZAP-70 tyrosine residues in Jurkat cells

Zeta-chain-associated protein kinase of 70kDa (ZAP-70) kinase is a key regulator in the early steps of TCR signaling but some aspects of its fine regulation are still unclear. From its 31 tyrosine (Y) residues, 11 phosphorylation sites have been identified, some with activator (Y315 and Y493) or inhibitory (Y292 and Y492) and others with unknown function (Y069, Y126 and Y178). In our present work, we aimed to elucidate the role of different Y residues of ZAP-70, especially those with unknown function, in calcium signaling and the autoregulation of the kinase. ZAP-70-deficient Jurkat cells (P116) were stably reconstituted with point-mutated ZAP-70 constructs where tyrosine residues 069, 126, 178, 238, 292, 315, 492 or 493 were replaced with phenylalanine (F). The anti-CD3-elicited calcium signal increased in F069-, F292- and F492-ZAP-70-expressing cell lines but decreased in the F126-, F315- and F493-ZAP-70-expressing cell lines. ZAP-70 point mutations led to phosphorylation changes predominantly in SH2 domain containing leukocyte protein of 76kDa (SLP-76) but not linker of activated T cells (LAT) during CD3-activation; moreover, we detected basal hyperphosphorylation of SLP-76 Y128 in the F126-, F178- and F492-ZAP-70-expressing cell lines. In summary, Y069, Y178, Y292 and Y492 have inhibitory, while Y126, Y315 and Y493 activator role in anti-CD3-induced T-cell activation. Phosphorylation changes in LAT and SLP-76 suggest that fine regulation of ZAP-70 on calcium signaling is rather transmitted through SLP-76 not LAT. Additionally, negative or positive autoregulatory function of Y292 and Y493 or Y315, respectively, was revealed in ZAP-70. These data indicate that previously not characterized Y069, Y126 and Y178 in ZAP-70 participate in the fine regulation of TCR signaling

Effects of bowel cleansing on the composition of the gut microbiota in inflammatory bowel disease patients and healthy controls

Background: In patients with inflammatory bowel disease (IBD), Crohn’s disease (CD), and ulcerative colitis (UC), numerous cases of exacerbations could be observed after colonoscopy, raising the possible pathogenetic effect of colonic microbiota alterations in IBD flare. Objectives: We aimed to investigate the changes in the fecal microbiota composition in IBD patients influenced by the bowel preparation with sodium picosulfate. Design: We enrolled patients with IBD undergoing bowel preparation for colonoscopy in the prospective cohort study. The control group (Con) comprised non-IBD patients who underwent colonoscopy. Clinical data, blood, and stool samples were collected before colonoscopy (timepoint A), 3 days later (timepoint B), and 4 weeks later (timepoint C). Methods: Disease activity and gut microbiota changes were assessed at each timepoint. Fecal microbiota structure – at family level – was determined by sequencing the V4 region of the 16S rRNA gene. Statistical analysis included differential abundance analysis and Mann–Whitney tests. Results: Forty-one patients (9 CD, 13 UC, and 19 Con) were included. After bowel preparation, alpha diversity was lower in the CD group than in the UC (p = 0.01) and Con (p = 0.02) groups at timepoint B. Alpha diversity was significantly higher in the UC group than in the CD and Con (p = 0.03) groups at timepoint C. Beta diversity difference differed between the IBD and Con (p = 0.001) groups. Based on the differential abundance analysis, the Clostridiales family was increased, whereas the Bifidobacteriaceae family was decreased in CD patients compared to the Con at timepoint B. Conclusions: Bowel preparation may change the fecal microbial composition in IBD patients, which may have a potential role in disease exacerbation after bowel cleansing