Effect of loss of PKC theta and p50+cRel on gene expression post T-cell stimulation

OT-1 Transgenic CD8 T-cells were isolated from spleens of WT, PKCθ theta KO, and p50 cRel DKO mice. The T-cells were either cultured with non-pulsed DC (WT only and signified as "WT - UN") or with BMDCs pulsed with the OVA peptide SIINFEKL (N4) (WT, PKCθ theta KO, and p50 cRel DKO and signified as 'genotype - N4') at a ratio of 1:10 (DC:T-cell) for 18 hours. DCs then were depleted from the culture and RNA was made from the T-cells to measure gene expression at the early / late stage of T-cell activation The T-cells were either cultured with non-pulsed DC (WT only and signified as "WT - UN") or with BMDCs pulsed with the OVA peptide SIINFEKL (N4) (WT, PKCθ theta KO, and p50 cRel DKO and signified as 'genotype - N4') at a ratio of 1:10 (DC:T-cell) for 18 hours.

OT-1转基因CD8阳性T细胞（OT-1 Transgenic CD8 T-cells）从野生型（Wild Type, WT）、PKCθ基因敲除（PKCθ KO）以及p50 cRel双基因敲除（p50 cRel DKO）小鼠的脾脏中分离得到。将分离得到的T细胞分为两种培养条件：一组与未负载抗原的树突状细胞（DC，仅野生型组，记为"WT - UN"）共培养；另一组与负载OVA肽段SIINFEKL（N4）的骨髓来源树突状细胞（Bone Marrow-derived Dendritic Cells, BMDC）按1:10的比例（DC:T细胞）共培养，涵盖野生型、PKCθ敲除及p50 cRel双敲除小鼠来源的T细胞，记为"基因型 - N4"，共培养时长为18小时。随后移除培养体系中的树突状细胞，提取T细胞的总RNA，以检测T细胞激活早期与晚期阶段的基因表达水平。