Figure 4. Naïve CD4⁺ T cells induce B cell survival regulation via MIF secretion.

(A) Purified naïve wt B cells were cultured at a 1:1 ratio with 5×10⁶ wt CD4⁺ or CD8⁺ T cells. After 24 hrs, the cells were analyzed for T/B cell conjugates by flow cytometry by double positive staining for B220⁺ and CD3⁺. Results are shown as the percentage of conjugates. (B-C) Naïve wt B splenocytes were cultured alone or at a 1:1 ratio with 5×10⁶ wt or SAP^−/− CD4⁺ T or CD8⁺ T cells. After 24 hrs, cells were analyzed by flow cytometry for Annexin V/7AAD N=4. (B), or SLAMF6 surface expression (C) on B cells. N=4. (D) MIF expression was analyzed 5 hrs following Monensin treatment by FACS staining. Histograms show MIF expression by CD4⁺T cells, CD8⁺ T cells, and B cells compared to MIF expression on MIF^−/− splenocytes (background). (E) Naïve wt/SAP^−/−/SLAMF6^−/− T splenocytes were cultured alone or at a 1:1 ratio with 5×10⁶ wt B cells. After 24 hrs, expression of MIF in CD3⁺CD4⁺ was analyzed by flow cytometry. N=4 (F-H) Naïve wt B splenocytes were cultured alone or at a 1:1 ratio with 5×10⁶ wt or MIF^−/− T cells. After 24 hrs, cells were analyzed by flow cytometry for Annexin V/7AAD (F), CD74 (G) or SLAMF6 (H) surface expression on B cells. N=3. Results are shown as percentage (F) or MFI (G-H). In all experiments, each dot represents a biological repeat. N represents the number of experiments. Bars showing SEM; ns p≥ 0.05, * p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.