Extended Data Figure 2: Resting CD4 T cells from tonsil include both naive and memory subsets.

a, CD4 T lymphocytes in lymphoid tissues contain a large population of central memory cells. To identify the sub-populations of CD4 T cells in human tonsil histocultures, we examined the expression pattern of CCR5, CD45RA, CD45RO, CD62L and CD27. Central memory CD4 T cells (T_CM) are characterized by expression of CD45RO⁺/CD62L⁺ or CD45RO⁺/CD27^{+ 29,31,55,56}. T_CM lack effector function and constantly travel through the lymph nodes in large quantities for antigen sampling, whereas effector memory cell (T_EM) mainly migrate to peripheral tissues^29,30,31. Analysis of these surface markers revealed at least three distinct maturation phenotypes. The majority of CD4 T lymphocytes exhibit a memory phenotype as determined by surface expression of CD45RO, among them more than two-thirds were found to be central memory cells (CD45RO⁺/CD62L⁺ and CD45RO⁺/CD27⁺). Similarly, a large population of CCR5-expressing CD4 T cells was found to have central memory phenotype (CCR5⁺/CD62L⁺ and CCR5⁺/CD27⁺). These findings are in accordance with previous studies in primary human lymphoid cultures^12,57,58. b, c, Memory lymphoid CD4 T cells represent preferential targets for productive infection by both the R5- and X4-tropic strains of HIV-1. To determine whether cell maturation influences susceptibility for productive infection, we measured the levels of productive infection using GFP reporter viruses harbouring either an X4-tropic or R5-tropic Env of HIV-1. Except for their select V3 loop envelope determinants, both reporters were derived from the same bicistronic Nef-IRES-GFP clone which produces fully replication-competent viruses¹⁶. Interestingly, productive infection of both X4-tropic or R5-tropic viral strains was detected in CXCR4-expressing cells, indicating that the CXCR4 co-receptor is equally present on CCR5-expressing cells, as was previously shown^12,57,58,59. Memory CD4 T cells (CD45RO⁺) were selectively productively infected in cultures infected with either X4-tropic or R5-tropic reporter virus. Similar findings were found in infected cultures activated with CD3/CD38 beads to achieve higher rates of infection. Among the memory CD4 T cells, T_EM cells became productively infected in higher quantities than T_CM (not shown). These data are the representative results of six independent analyses performed in tonsil cells isolated from six different donors.