Mapping the extent of heterogeneity of human CCR5⁺CD4⁺T cells in peripheral blood and lymph nodes

Background: CD4(+)T cells that express the chemokine receptor, CCR5, are the most important target of HIV-1 infection, but their functions, phenotypes and anatomical locations are poorly understood. We aimed to use multiparameter flow cytometry to better define the full breadth of these cells. Methods: High-parameter fluorescence flow and mass cytometry were optimized to analyse subsets of CCR5(+)memory CD4(+)T cells, including CD25(high)CD127(dim)Tregs, CXCR3(+)CCR6- Th1-like, CCR6(+)CD161(+)CXCR3- Th17-like, integrins alpha 4(+)ss7(+)gut-homing, CCR4(+)skin-homing, CD62L(+)lymph node-homing, CD38(+)HLA-DR(+)activated cells, and CD27-CD28- cytotoxic T lymphocytes, in a total of 22 samples of peripheral blood, ultrasound-guided fine needle biopsies of lymph nodes and excised tonsils. CCR5(+)antigen-specific CD4(+)T cells were studied using the OX40 flow-based assay. Results: 10-20% of CCR5(+)memory CD4(+)T cells were Tregs, 10-30% were gut-homing, 10-30% were skin-homing, 20-40% were lymph node-homing, 20-50% were Th1-like and 20-40% were Th17-like cells. Up to 30% were cytotoxic T lymphocytes in CMV-seropositive donors, including cells that were either CCR5(high)Granzyme K(+)or CCR5(dim)Granzyme B+. When all possible phenotypes were exhaustively analysed, more than 150 different functional and trafficking subsets of CCR5(+)CD4(+)T cells were seen. Moreover, a small population of resident CD69(+)Granzyme K(+)CCR5(+)CD4(+)T cells was found in lymphoid tissues. CMV- andMycobacterium tuberculosis-specific CD4(+)T cells were predominantly CCR5(+). Conclusion: These results reveal for the first time the prodigious heterogeneity of function and trafficking of CCR5(+)CD4(+)T cells in blood and in lymphoid tissue, with significant implications for rational approaches to prophylaxis for HIV-1 infection and for purging of the HIV-1 reservoir in those participants already infected.