Figure 6. Vitamin D₃ regulates T cell growth and EAE.

(a) Purified mouse CD3⁺ T cells were stained with CFSE, stimulated with plate-bound anti-CD3 antibody in the presence or absence of 1,25(OH)₂D₃ (20 nM) for 3 days and analysed by fluorescence-activated cell sorting (FACS) for CFSE dilution/proliferation in CD4⁺ T cells. Horizontal bars mark the population of proliferating cells, with the percentage of proliferating cells noted above. (b) Purified human CD3⁺ T cells (n=8 donors per group) were pre-stimulated with plate-bound anti-CD3 for 2 days (1 μg ml⁻¹) then treated with 100 nM 1,25(OH)₂D₃ for 3 days. CD25⁺CD4⁺ blasting T cells were analysed for surface CTLA-4 expression by FACS. Horizontal bar represents the average L-PHA MFI of each treatment group. P value determined by t-test. (c) Purified mouse CD3⁺ T cells were stimulated with plate-bound anti-CD3 (250 ng ml⁻¹) in the presence or absence of 1,25(OH)₂D₃ (150 nM) for 5 days and analysed by FACS for internalization rates of cell surface CTLA-4 in CD4⁺ T cells. (d) PL/J mice treated as indicated were immunized with MBP+CFA and examined daily for clinical EAE. 1,25(OH)₂D₃ of 100 ng was administered by intraperitoneal injection every other day starting at day −3. SW was provided in drinking water and reduced L-PHA staining ∼50%. P values by Fisher's exact t-test, ***P<0.0001. (e) Female mice were immunized with 100 μg MBP+CFA by intraperitoneal injection and after 10 days, splenocytes were collected and re-stimulated in vitro with MBP in the presence or absence of 1,25(OH)₂D₃ and/or SW as indicated. Proliferation was assessed by CFSE dilution and analysed by FACS. Error bars are standard error of triplicate or greater values in all panels.