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

Fig 4. Alprazolam causes suppression of human and mouse T cell proliferation, which can be recovered by multiple GABA(A)R inhibitors.

Fig 4

A, B) 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 11 μM, 33 μM, 100 μM or 300 μM alprazolam. 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 alprazolam in treated splenocytes, in addition to total splenocyte death in response to alprazolam treatment. B. Dose dependent inhibition of CD8+ and CD4+ T cell proliferation by alprazolam in treated PBMCs, in addition to total PBMC death in response to alprazolam treatment. C, D) Splenocytes and PBMCs were treated with α-CD3 (33 ng/ml for splenocytes, 100 pg/ml for PBMCs), 100 μM (for mouse) or 33 μM (for humans) alprazolam, 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. Treatment with the GABA(A)R inhibitor cocktail is able to rescue CD8+ and CD4+ T cell proliferation in murine T cells. D. Treatment with the GABA(A)R inhibitor cocktail is able to rescue CD8+ and CD4+ T cell proliferation in human T cells. Data shown are from 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.