Circulating HLA-DR+CD4+ effector memory T cells resistant to CCR5 and PD-L1 mediated suppression compromise regulatory T cell function in tuberculosis
Fig 8
PTB HLA-DR- but not HLA-DR+ total Teff are susceptible to Treg suppression via CCR5 and PD-L1.
(A, E) RNA was isolated from sorted PTB total and HLA-DR- Teff cells activated with anti-CD3/anti-CD28 for 2 hrs and converted to cDNA. Expression of CCL3L3, CCL4 and PD-L1 was measured by qRT-PCR. Expression is shown as relative copy number relative to GAPDH. (B) CCL3, CCL4 and CCL5 levels were measured in supernatants from cultures of PTB total and HLA-DR- cells activated with anti-CD3/anti-CD28 for 24 hrs by ELISA. CCR5 (C) and PD-L1 (F) expression was measured on unstimulated and anti-CD3/anti-CD28 activated PTB total and HLA-DR- Teff cells after 24 hrs and MFI of CCR5 (C) and PD-L1 (F) from multiple PTB donors was plotted for comparison between total and HLA-DR- Teff cells. CFSE labelled sorted PTB and IGRA-ve total and HLA-DR- Teff cells were co-cultured with autologous Treg cells at a ratio of 1:1 in the presence of either 5 μg/ml anti-CCR5 (D) or 5 μg/ml anti-PD-L1 (G). Appropriate 5 μg/ml isotype antibody was used as control in both cases and cells were activated with anti-CD3/anti-CD28 beads (beads:cell ratio of 1:1). Proliferation was measured by CFSE dilution after 4 days and percentage suppression was calculated (D and G). A total of 6–8 PTB and 6 IGRA-ve donors were used. Upper box shows data pertaining to CCR5 and lower box shows data pertaining to PD-L1. Paired Wilcoxon matched-pairs signed rank test with Bonferroni correction was used to determine P value. *p ≤ 0.03.