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. 2020 Aug 17;11:4113. doi: 10.1038/s41467-020-17756-7

Fig. 4. T-cell effector functions are inhibited at acidic pH.

Fig. 4

a Interferon γ (IFNγ) production from C57BL/6 (B6) T-cells is reduced at low pHe, as determined by ELISA; n = 3, p = 0.00013. b INFγ production, measured over a range of pHe in T-cells from B6 mice as well as three antigen-specific strains. n = 3. IFNγ levels were compared with those at pHe 7.4 within each strain. B6 (pHe 6.8, p = 0.0034; pHe 6.6, p = 0.00013), OT-I (pHe 6.6, p = 0.0013), Pmel-1 (pHe 6.8, p = 0.017; pHe 6.6, p = 6.25E-6), OT-II (pHe 6.6, p = 0.046). c Time course of IFNγ levels in media following pH-manoeuvres that demonstrate the reversal of acid inhibition upon subsequent exposure to alkaline pH (rescue experiment); n = 3. d Interleukin-2 (IL-2) release, measured by ELISA in Pmel-1 and OT-II T-cells and a Jurkat leukaemia cell line, is reduced at low pHe; n = 3. Pmel-1, p = 8.77E-6; OT-II, p = 6.85E-9; Jurkat, p = 5.64E-8. e Relationship between cytokine levels at low and high pH, determined in paired experiments by the Cytokine Beads Array (CBA) assay. For most cytokines, with the exception of those highlighted in red (IP-10, MIG, MDC), acidic conditions evoked a reduction in release. f Rate of B6 cell proliferation measured by CellTrace Violet assay. g IFNγ production was measured, by ELISA, at the end of a 24 h preconditioning period (no OVA added) at either pHe 6.6 or 7.4, and then at the end of a consecutive 24 h period in the presence of antigen (OVA) at pHe 7.4. IFNγ production can be activated irrespective of whether cells had been preconditioned at pHe 6.6 or 7.4; n = 3. pHe 6.6 precondition, p = 2.82E-5; pHe 7.4 precondition, p = 4.25E-7. Asterisks (****) represent p < 0.0001. h IFNγ production by T-cells activated with dendritic cells (DC) and antigen (OVA) measured after 24 h at pHe 6.6 or 7.4, followed by measurements at the end of a subsequent 24 h period without stimulation at pHe 7.4 (rest). T-cells can become activated by DC/OVA at acidic or alkaline pHe, and fully retain the capacity to produce cytokines when transferred to alkaline media; n = 3. pHe 6.6 activation, p = 3.22E-7; pHe 7.4 activation, p = 0.29. Asterisks (****) represent p < 0.0001. i Flow cytometry. Intracellular IFNγ staining of T-cells activated with DC and antigen (OVA) measured after 24 h of treatment in either pHe 6.6 or 7.4 (top panels). Cells were then transferred to pHe 7.4 to rest in the absence of DC and OVA, and measurements were performed after 3 h of resting. All the experiments were repeated at least twice and expressed as mean ± SD and analyzed by two-tailed, unpaired t-test unless indicated otherwise. Significance level: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.