Clone uchl1

PBMC were stained with monoclonal antibodies to CD4 (1:50, clone RPA-T4, Biolegend #300518), CD3 (1:50, clone OKT3, Biolegend #317332), CD45RO (1:40, clone UCHL1, Biolegend #304236) and CCR7 (1:40, clone G043H7, Biolegend #353216). Afterwards, cells were washed and CD45RO⁺ CCR7⁺ (central-memory) and CD45RO⁺ CCR7⁻ (effector-memory) and CD3⁺ CD4⁺ (total) CD4⁺ T-cells were sorted in a specifically designated biosafety cabinet (Baker Hood), using a FACS Aria cell sorter (BD Biosciences) at 70 pounds per square inch. Cell sorting was performed by the Ragon Institute Imaging Core Facility at MGH and resulted in isolation of lymphocytes with the defined phenotypic characteristics of >95% purity. Data were analyzed using FlowJo software (Treestar).

Resting CD4⁺ T cells from HIV-1-infected donors on suppressive ART were isolated as described above. To sort resting memory subsets, we incubated cells with FcgR block (BD Pharmingen) for 10 minutes before staining with a FITC-labeled antibody to CD3 (Biolegend; Clone HIT3a), phycoerythrin (PE)-Cy7-labeled antibody to CD4 (Biolegend; Clone RPA-T4), allophycocyanin (APC)-labeled antibody to CD45RO (Biolegend; Clone UCHL1), BV421-labeled antibody to CD27 (Biolegend; Clone O323) and PE-labeled antibody to CCR7 (Biolegend; Clone G043H7). Dead cells were excluded using propidium iodide. PE Mouse IgG2aκ, BV421 Mouse IgG1κ, and APC IgG2aκ isotype antibodies were used in fluorescence-minus-one controls to set sorting gates. Memory cells were distinguished from naive cells by the presence or absence of CD45RO staining, respectively. Central memory cells were distinguished from effector and transitional memory subsets by the presence of CCR7 as described by Sallusto et al³² . CCR7^- cells were subdivided into effector memory (Tem) (CD45RO+CCR7-) or transitional memory (Ttm) (CD45RO+CCR7-CD27+) as described by Chomont et al^{20 (link)}. Central, effector, and transitional resting memory subsets were sorted using a Beckman Coulter MoFlo XDT Cell sorter.