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. 2021 May 20;16(5):e0251632. doi: 10.1371/journal.pone.0251632

Fig 5. Allopregnanolone inhibits human and mouse T-cell proliferation, which cannot be rescued with GABA(A)R inhibitors.

Fig 5

A, B) Allopregnanolone causes a dose dependent inhibition of T cell proliferation in mouse and humans. Splenocytes were isolated from the spleens of female wildtype BALB/c mice, and PBMCs were isolated from human whole blood samples. Cells were stained with 5 μM CFSE, before being treated with soluble α-CD3 antibody (33 ng/ml for splenocytes, 100 pg/ml for PBMCs), in addition to either 1 μM, 10 μM, 50 μM or 100 μM allopregnanolone. Splenocytes were harvested following 48 hours of treatment, while PBMCs were harvested following 96 hours of treatment. The percentage of proliferating T cells in each condition was determined by flow cytometry through assessing the reduction in CFSE fluorescence as compared to a positive control of α-CD3 treatment alone. A. Dose dependent inhibition of CD8+ and CD4+ T cell proliferation by allopregnanolone in treated splenocytes, in addition to total splenocyte death in response to allopregnanolone treatment. B. Dose dependent inhibition of CD8+ and CD4+ T cell proliferation by allopregnanolone in treated PBMCs, in addition to total PBMC death in response to allopregnanolone treatment. C, D) Treatment with a combination of BMI and TPMPA cannot rescue allopregnanolone induced inhibition of proliferation in mice or humans. Splenocytes and PBMCs were treated with α-CD3 (33 ng/ml for splenocytes, 100 pg/ml for PBMCs), 50 μM allopregnanolone, and either a cocktail of GABA(A)R inhibitors consisting of 100 μM BMI and 50 μM TPMPA, or each individual inhibitor alone. The reduction in proliferation under each condition as compared to a positive control of α-CD3 alone was assessed by flow cytometry following 48 hours of treatment in splenocytes, and 96 hours of treatment in PBMCs. C. No recovery of allopregnanolone-induced inhibition of proliferation in response to treatment with BMI, TPMPA, or a combination of both inhibitors in splenocytes. D. No recovery of allopregnanolone-induced inhibition of proliferation in response to treatment with BMI, TPMPA, or a combination of both inhibitors in PBMCs. Data shown are from at least 3 independent experiments, with error bars (SD). Differences between groups were assessed by one way ANOVA. * = p<0.05. ** = p<0.01. *** = p<0.001. **** = p<0.0001. UN = untreated. AP = allopregnanolone.