Impacts Of Theileria Annulata-transfomation On The Cytokine Profiling Of B Lymphocytes And Endocytosis/Presentation Of Dendritic Cells Purified From Cattle

Theileria annulata?T.annulata?,an obligate intracellular protozoan parasite,deforms the monocytes/macrophages,dendritic cells and B-cells.The transformed cells obtained the ability for uncontrolled proliferation and the functions changed compared with the normal cells.The current study was conducted to find out the phenotypic and functional changes of transformed cells including surface marker expression,cytokine production,endocytosis rate and antigen presentation efficacy.During this study,the cell surface markers and antigen inside cells were analyzed by flow cytometry while expression levels of genes?cytokines,endocytosis regulatory proteins,small GTPase Rab family?were quantified by RT-qPCR.In this study,T.annuata sporozoites infected bovine B-cell line was established successfully.The immune response,particularly cytokine production?IL-1?,IL-1?,IL-4,IL-6,IL-8,IL-10,IL-16,TGF-?₁,TNF-?,LT-?/TNF-?,IFN-?and IFN-??from these transformed cells was measured.The established cell line was confirmed following identification of cluster differentiation 21?CD21?,immunoglobulin M?IgM?,and CD19-like?WC4?surface markers on normal and T.annulata transformed B-cells.The established T.annulata infected B-cell line has purity⁹9%?CD21⁺?while the other surface markers like IgM and WC4 were at the rate of 14.2⁶.5 and 2.46¹.3%,respectively at 6^thh and 10^th generations;compared with the normal purified B-cells?⁹9%pure?on which there identified IgM and WC4 with percentage presence of 96.6 and 85.9%,respectively.The IL-4 and IL-12?were significantly?p<0.01?up-regulated in transformed cells.Meanwhile,in buparvaquone?BW720c?treated transformed cells,IL-12?and IFN-?were significantly?p<0.01?up-regulated.Surprisingly,no significant?p>0.05?up-regulation of cytokines was found in LPS stimulated transformed cells.Moreover,IL-1?,IL-1?,IL-8 and IL-16 were significantly?p<0.01?up-regulated in LPS stimulated purified B cells compared with transformed cells.Furthermore,mechanism of endocytosis and antigen presentation ability for T lymphocytes proliferation by antigen presenting cells?APCs?like monocytes derieved dendritic cells?MoDCs?and T.annulata sporozoites transformed bovine dendritic cells?TaDCs?were assessed.The identified cell surface markers?CCR7,DEC-205 and CD209?were>70%in MoDCs,and<7%?CCR7 and CD209?and 78.5%?DEC-205?in TaDCs.The endocytosis of Ag in MoDCs was significantly?p<0.05?higher than TaDCs.However,intermittent shaking significantly?p<0.01?enhanced endocytosis rate in transformed cells while chemokine's ligand has non-significant?p>0.05?effect.The yeast derived mannan supplementation in cell culture,blocked mannan cell surface receptors?MRs?leading to reduced endocytosis rate ranged from 25.09⁴4.14 and 7.42³1.06%in MoDCs and TaDCs,respectively,depends on the duration of incubation period.We found that DMSO enhanced 128.9%endocytosis rate in TaDCs.Furthermore,the expression of endocytosis related surface receptors,ligand and Rac were down-regulated while Cdc42 and HS1 were up-regulated in TaDC cell line.The proliferation rate of T lymphocytes?CD4⁺and CD8⁺?were similar when co-cultured with TaDC-APC whereas it was higher for CD4⁺than CD8⁺after co-cultured with MoDCs-APC.On the other hand,the efficacy of TaDC for stimulation of T lymphocytes was found to be reduced with the increase in number of cell line generations.It was found that TaDC-APC and MoDC-APC have similar effect for intracellular antigen mobilization,no transfer of antigen from APCs to lymphocytes was found.Likewise,the transcription level of small GTPase of Rab family genes were significantly?P>0.001?down-regulated in progressive passages?>50?of TaDC cell line.The established B cell line can be used to investigate the further cytokine and immunoglobulin production that might be used as vaccine or prophylactic against the tropical theilerisosis.Furthermore,we can declare from this study that TaDC cell line at initial generation can be used as antigen presenting cells for T lymphocytes proliferation to produce cellular immune response against the presented antigen.The TaDC cell line can also be used to investigate some speicific antigen triggered immune response in vitro.