(II) Evidence for altered expression of cell surface molecules on Teff that are important for engaging with Treg cells
Treg cells suppress via a variety of contact dependent and independent mechanisms (Figure 1). Some of the key molecules shown to be involved in promoting Treg suppression include CTLA-4, PD-1, CD39, CD73, PD-1, PD-L1, LAG3 etc. Several lines of data show some of these molecules, e.g. PD-1, PD-L1, CD39, CD73, LAG3 are altered in TB. PD-1 and PD-L1 expression is elevated in CD4+ T cells from TB subjects compared to healthy controls [97]; CD39 and CD73 are increased in lung parenchymal CD4+ T cells of Mtb infected mice [98] and LAG3 is increased in granuloma of macaques with active TB compared to those with latent TB [99]. However, the function of these molecules has not been specifically studied in the context of Treg suppression in TB. We provided some direct evidence for a role for PD-1/PD-L1 and β chemokine/CCR5 interactions. A comparative RNA seq analysis of Treg suppression sensitive (HLA-DR- Teff) and suppression resistant (comprising HLA-DR+ Teff + and HLA-DR- Teff) cells isolated from subjects with pulmonary TB showed elevated expression of PD-L1 and β-chemokines in the suppression sensitive HLA-DR- Teff fraction (Figure 2). Also, blockade of PD-1/PD-L1 and β-chemokine/CCR5 interactions resulted in loss of Treg suppression in these cells pointing to the importance of these pathways in maintaining Treg mediated homeostasis in TB (Figure 2). PD-1/PD-L1 interactions and β-chemokine/CCR5 interactions have been previously implicated to play a role in Treg mediated suppression [100, 101]. CCL3 and CCL4 secreted by Tregs serve as chemoattractants for Teff cells. CD4+ Teff cells from mice deficient in CCL3 and CCL4 fail to migrate and come in contact with Treg cells [101]. Moreover, Tregs from type-1 diabetes patients are deficient in CCL3 and CCL4 and this compromises their ability to suppress [101].
Apart from differences in PD-L1 and β-chemokine levels (Figure 2), our transcriptome analysis of Treg sensitive HLA-DR- and suppression resistant HLA-DR+ cells identified several additional cell surface markers (CD46, TRAIL, TRAF1, TRAF3, FASLG, CD30, SEMA7A) (Figure 3), some of which have previously been implicated in Treg function [102-105]. Engagement of complement receptor CD46 results in suppression of bystander CD4+ T cells via an IL-10 dependent mechanism [102, 103]. CD46 cross-linking also suppresses mycobacteria specific CD4+ T cell responses [106]. TRAIL is a regulator of T cell activation; its absence leads to autoimmunity and reduction in Treg frequencies while its presence dampens Th1 responses and boosts Tregs [104]. TRAF1 inhibits Th2 differentiation [107]; TRAF3 controls proximal T cell activation events and its absence in mice leads to elevated thymus derived Treg cell frequencies [105]; FASLG is a marker for T cell activation and is expressed on Th1 cells [108, 109]; Sema7a and CD30 have been implicated in Th1 and Th17 differentiation [110, 111]. However, a role for these pathways impacting T effector function in TB remains to be elucidated.