Fig 1.
Expression of GABA(A)R subunits in mouse and human T cells.
A. Representative gel of PCR to amplify GABA(A)R subunits from mouse CD8+ cells. DNA ladder is GeneRuler 100 bp DNA Ladder (ThermoFisher, Cat. No. SM0244) B. Representative gel of PCR to amplify GABA(A)R subunits from human T cells. DNA ladder is GeneRuler 100 bp DNA Ladder (ThermoFisher, Cat. No. SM0244) C. Staining of mouse splenocytes for Gabrr2, Gabra1 and Gabrp subunits. Red–staining of the subunits, blue–DAPI. D. Staining of human PBMCs for the GABRR2, GABRA1 and GABRP (red), anti-CD3 (green) and DAPI (blue). Scale bars 10μm.
Table 1.
Expression levels of GABA(A)R subunits in various types of immune cells.
Fig 2.
Diazepam can inhibit T cell proliferation in a dose dependent manner.
Splenocytes were isolated from the spleens of female wildtype BALB/c mice, and PBMC 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 50 μM GABA alone, or 50 μM GABA plus 1 μM, 10 μM or 100 μM diazepam. Splenocytes were harvested following 48 hours of treatment, while PBMC 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. A. Representative flow cytometry plots depicting the gating strategy used to identify cell populations of interest from PBMC and splenocyte preparations. Lymphocytes were gated according to size (forward scatter; FSC) and granularity (side scatter; SSC). Within the lymphocyte gate, CD8+ and CD4+ T cells were further gated using established lineage markers for these cells (CD8 and CD4). Histograms were used to determine the percentage of proliferating cells present under each condition, compared to an untreated control. B. Exemplar flow cytometry plots (from human CD8+ T cells) depicting the dose dependent inhibition of α-CD3 stimulated proliferation caused by treatment of cells with 50 μM GABA and varying concentrations of diazepam. C. Dose dependent inhibition of α-CD3 stimulated proliferation in CD8+ and CD4+ T cells following treatment of splenocytes with 50 μM GABA and varying concentrations of diazepam. In addition, splenocyte death in response to diazepam treatment is shown. D. Dose dependent inhibition of α-CD3 stimulated proliferation in CD8+ and CD4+ T cell following treatment of PBMC with 50 μM GABA and varying concentrations of diazepam In addition, PBMC death in response to diazepam treatment is shown. 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.
Fig 3.
Diazepam inhibits mouse and human T cell proliferation via different pathways.
Splenocytes and PBMC were treated with α-CD3, 50 μM GABA, 100 μM diazepam, and a cocktail of GABA(A)R inhibitors consisting of 100 μM BMI and 50 μM TPMPA. 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 for splenocytes, and 96 hours of treatment in PBMC. A. Treatment with the GABA(A)R inhibitor cocktail is able to rescue CD8+ and CD4+ T cell proliferation in murine T cells. B. Treatment cannot rescue proliferation in humans T cells. Data shown is from one representative experiment, 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.
Fig 4.
Alprazolam causes suppression of human and mouse T cell proliferation, which can be recovered by multiple GABA(A)R inhibitors.
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.
Fig 5.
Allopregnanolone inhibits human and mouse T-cell proliferation, which cannot be rescued with GABA(A)R inhibitors.
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.