Fig. 3: d-serine inhibits IFN-γ production by CD8+ T cells.
a, Intracellular survival of H37Rv in BMDMs treated with or without d-serine (10 mM) (left) or Rv0884c_cKDTet treated with or without ATc in BMDMs (right) was assessed using c.f.u. assay. b, Bacterial viability of Rv0884c_cKDTet treated with or without ATc in BMDMs was assayed using the LIVE/DEAD BacLight bacterial viability kit. c,f, Naïve CD8+ T cells were activated and differentiated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2 and IL-12 p70 for 5 days and treated with PBS, d-serine or l-serine. Percentage of IFN-γ+ (c) and CD69+ (f) CD8+ T cells was assayed. APC, allophycocyanin. d,e, WT mice, Rag1−/− mice and Rag1−/− mice adoptively transferred with CD8+ T cells (Rag1−/−+CD8) were aerosol-infected with ~200 c.f.u. of indicated Mtb strains (d, Rv0884c_cKDTet with or without ATc, Rv0884c_cKDTet+Rv0884c (F108A) with ATc, and Rv0884c_cKDTet+Rv0884c with ATc; e, H37Rv) or treated with d-serine. Bacterial load in lung tissues at 4 weeks post infection was determined using c.f.u. assay. g, After co-culturing H37Rv-infected BMDMs (MOI = 5) and activated TB10Rg3 T cells treated with or without d-serine for 2 h, percentage of CD69+ TB10Rg3 T cells was measured in TB10Rg3 T cells. h, After co-culturing H37Rv-infected BMDMs (MOI = 5) and activated TB10Rg3 T cells treated with or without d-serine for 3 days, the expression of IFN-γ was measured using ELISA. i, After 1 day of infection with Mtb strains, BMDMs were co-incubated with activated TB10Rg3 T cells treated with or without d-serine for 3 days, and intracellular c.f.u. in BMDMs infected for 1 or 4 days were determined. No T, group of unpulsed BMDMs infected with H37Rv for 4 days without co-incubation with T cells; d1, group of unpulsed BMDMs infected with H37Rv for 1 day without co-incubation with T cells; d4, group of BMDMs infected with H37Rv for 4 days; Rg3, group of pulsed or unpulsed BMDMs infected with H37Rv for 4 days and co-incubated with TB10Rg3 T cells. j,k, C57BL/6 mice were aerosol-infected with ~200 c.f.u. per mouse of H37Rv and administered with d-serine for 4 weeks. Percentages of IFN-γ+ (j) and CD69+ (k) CD8+ T cells in lung tissues were measured; FMO was used as control. Data in a–k represent one experiment with at least three independent biological replicates. Results are shown as mean ± s.e.m. Two-way ANOVA with Tukey’s multiple comparisons test (i), one-way ANOVA with Tukey’s multiple comparisons test (c,d,f) and two-tailed unpaired Student’s t-test (a,b,e,g,h,j,k) were used for statistical analysis.
