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Text: Transcription factors T - bet and Blimp-1 are reported to play a crucial role in promoting cytotoxic CD8 + T cell differentiation ( 51,52 ) ; therefore , we tested what is the effect of NFAT2 absence in CTLs on the expression of T - bet and Blimp-1 . Purified CD8 + T cells from WT and NFAT2 fl / fl CD4 - Cre mice were differentiated in vitro into cytotoxic CD8 + T lymphocytes as described . ( B ) Total RNA isolated from naive CD8 + T cells ( NAIVE ) or from in vitro differentiated cytotoxic CD8 + T lymphocytes ( CTLs ) were analyzed for NFAT2 mRNA levels by real - time RT - PCR assay using SYBR green master mix . The data are normalized to the β - actin levels . ( c ) Total number of live WT and NFAT2 - deficient CD8 + T lymphocytes was recorded daily during in vitro differentiation using Trypan blue exclusion method . ( D ) Flow cytometric analysis of CD44 and CD25 expression on differentiated cytotoxic WT and NFAT2 - deficient CD8 + T cells at day 5 . The analysis of MFI from three independent experiments on the right . ( e ) At day 5 , differentiated cytotoxic CD8 + T cells were tested in cytotoxicity assay against P815 cells as described at indicated efector : target ratios or restimulated with PMA plus ionomycin for 6 h , and intracellular granzime B and IFN - γ production was analyzed by flow cytometry . The analysis of MFI from three independent experiments on the right ( F ) . ( g ) Total RNA was isolated from differentiated in vitro WT and NFAT2 - deficient cytotoxic CD8 + T lymphocytes , and T - bet and Blimp-1 mRNA levels were analyzed by real - time RT - PCR assay using Taqman probes . The data are normalized to the HPRT RNA levels . All data are shown as mean ± SD of three independent experiments . The ns indicates not significant and * indicates p < 0.05 compared to WT mice . NFAT2 - deficient CTLs had lower expression of both T - bet and Blimp-1 , although in both cases , the difference remained insignificant ( Figure 7 G ) .<|eot_id|><|start_header_id|>assistant<|end_header_id|>

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