Role Of LAT In T Cells In The Pathogenesis Of Severe Aplastic Anemia

ObjectiveTo explore the level of LAT in peripheral blood (PB) CD3+T cells from patients with Severe Aplastic Anemia (SAA), the relationship between the level of LAT and function moleculs (perforin and granzymeB) in PB CD8+T cells and the role LAT played in T cells, and to prove the role of LAT in hyperfunction and hyperproliferation of T cells and to explore the importance of LAT on immunopathogenesis of SAA further.Method14untreated SAA patients,12remission patients, as well as12normal controls were enrolled in this study.First section LAT and related signaling molecules (ZAP-70and pLAT) of peripheral blood T cells from14SAA patients,12remission SAA patients and12healthy controls were detected by flow cytometry. The CD3+T cells were sorted by magnetic activated cell sorting system (MACS), and the mRNA expression of LAT were analyzed by Quantitative real-time PCR.Function molecules (perforin and granzymeB) of peripheral blood CD8+T cells from SAA patients, remission group and healthy controls were detected by flow cytometry. Correlation of LAT in CD8+T cells and function of CD8+T cells was analyzed.Second section The sorted peripheral blood CD3+T cells from SAA patients were nucleofected with LAT-siRNA and siRNA negative control plasmids The nucleofected T cells were activated with anti-CD3(1μg/ml) and anti-CD28(1μg/ml) and cultured for48h at37℃with5%CO2. Cells were harvested for flow cytometric analysis and the supernatants were collected and preserved at-20℃for ELISA.The purified peripheral blood T cells from normal controls were nucleofected with pCMV-myc-LAT and pCMV-myc plasmids. The nucleofected T cells were activated with anti-CD3(1μg/ml) and anti-CD28(1μg/ml) and cultured for48h at37℃with5%CO2. Cells were harvested for flow cytometric analysis and continuous culture, and the supernatants were collected and preserved at-20℃for ELISA. After counted,1×106harvested T cells nucleofected with pCMV-myc-LAT/pCMV-myc and1*106K562cells were cocultured for48h at37℃with5%CO2. Apoptosis of K562cells was detected by flow cytometry after staining with AnnexinV.ResultsFirst section The median expressions of LAT, ZAP-70and pLAT in T cells were49.55%,33%and8.31%in newly diagnosed SAA patients, and14.81%,4.06%and0.27%in normal controls. The expression of LAT, ZAP-70and Plat were higher in newly diagnosed cases than that in healthy controls (P<0.05). The relative LAT mRNA levels was evidently increased in the peripheral blood T cells from untreated cases compared to normal controls (P<0.05). After immune suppressive therapy (IST), the expression of LAT and its related molecules decreased dramatically. The expression of LAT, ZAP-70and pLAT were30.25%,19.23%and2.29%in T cells were lower in recovery patients than that in newly diagnosed SAA patients statistically (P<0.05). Meanwhile, the relative LAT mRNA levels was evidently decreased in the peripheral blood T cells from recovery cases compared to that from newly diagnosed cases (P<0.05). In SAA patients and recovery group, the expression of LAT in CD3+T cells is positively associated with percentage of CD3+T cells (r=0.1831, P<0.05). In SAA patients and controls, the expression of LAT in CD8+T cells is positively associated with expression of perforin in CD8+T cells (r=0.6503, P<0.05). Meanwhile, the expression of LAT in CD8+T cells is also positively associated with expression of granzymeB in CD8+T cells (r=0.1936, P<0.05)Second section After nucleofected with LAT-siRNA plasmid, expression of LAT in T cells from SAA patients was lower than that was nucleofected with si-RNA negative control plasmid (P<0.05). The expression of molecules (perforin and granzyme B) in CD8+T cells with inhibited expression of LAT (LAT-siRNA) were significantly lower than controls (si-RNA negative control)(P<0.05). The production cytokine IFN-y in supernatant were significantly reduced in peripheral blood T cells with inhibited expression of LAT (LAT-siRNA) compared to controls (si-RNA negative control)(P<0.05), but there was no difference of IL-4between these two groups. After nucleofected with Pcmv-myc-LAT plasmid, expression of LAT in T cells from healthy cases was higher than that was nucleofected with Pcmv-myc plasmid (P<0.05). The expression of molecules (perforin and granzyme B) in CD8+T cells with inhibited expression of LAT (pCMV-myc-LAT) were significantly higer than controls (pCMV-myc)(P<0.05). The production cytokine IFN-y in supernatant was significantly increased in peripheral blood T cells with overexpression of LAT (pCMV-myc-LAT) compared to controls (Pcmv-myc)(P<0.05), but there was no significant difference of IL-4between these two groups. After cocultured with peripheral blood T cells nuleofected with pCMV-myc-LAT and pCMV-myc plasmids for48h, the apoptosis rate of K562cells was determined by flow cytometry. The apoptosis rate of K562cells cocultured with LAT-overexpressed T cells (pCMV-myc-LAT) was significantly higher than that cocultured with controls (pCMV-myc)(P<0.05).Conclusion(1) By Quantitative real-time PCR and flow cytometry, we found that the expression of LAT in CD3+T cells and its related signaling molecules were higher in SAA patients, which means LAT may contribute to hyperproliferation of T cells in SAA patients.(2) In SAA patients and controls, the expression of LAT in peripheral blood CD8+T cells was positively associated with function molecules (perforin and granzyme B) of CD8+T cells, and inhibited expression of LAT could down-regulate the expression of function molecules in CD8+T cells while overexpression of LAT could up-regulated the expression of function molecules in CD8+T cells. LAT may lead to hyperfunction of CD8+T cells in SAA patients.(3) Over expression of LAT could enhance the production of IFN-y and inhibited the production of IL-4.