Fig. 4. SOCE defect in naive and memory CD4⁺ T cells from Orai1^KI/KI mice.

A, For measurements of SOCE by flow cytometry, mononuclear cells were isolated from lymph nodes of Orai1^KI/KI and Orai1^+/+ control mice, stained with antibodies to CD4, CD44 and CD62L, loaded with Fluo-4 and stimulated with thapsigargin (TG). Representative Ca²⁺ traces from naive CD4⁺ CD62L⁺ CD44^lo T cells (abbreviated as CD62L⁺) and memory CD4⁺ CD62L⁻ CD44^hi T cells (abbreviated as CD44^hi). * indicates the peak Ca²⁺ response; • indicates baseline [Ca²⁺]_i; dashed lines indicate initial rates of Ca²⁺ influx. B, Bar graphs show averages (±SEM) of peak Ca²⁺ responses (indicated by * in panel A) normalized to baseline (indicated by • in panel A) and initial rates of Ca²⁺ influx after readdition of 2 mM extracellular Ca²⁺ from n=13 (Orai1^+/+) and n=19 (Orai1^KI/KI) repeat experiments. p-values, Student’s t-test. C, Peak Ca²⁺ levels and influx rates in Orai1^KI/KI T cells in percent of values observed in wildtype T cells; derived from data in B. D, Comparable expression levels of ORAI1, ORAI2 and ORAI3 in naive and memory T cells. CD4⁺ and CD8⁺ naive CD62L⁺ CD44^lo and memory CD62L^– CD44^hi T cells from wildtype mice were sorted by FACS and analyzed by quantitative real-time PCR for mRNA expression of ORAI1, ORAI2 and ORAI3. Averages (±SEM) are from six repeat experiments (except for ORAI1 in CD8⁺ CD62L^–CD44^hi T cells, n=3) performed in triplicates. ΔC_T, threshhold cycle differential. p-values, Student’s t-test.