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References
Descours, B. et al. CD32a is a marker of a CD4 T-cell HIV reservoir harbouring replication-competent proviruses. Nature 543, 564–567 (2017).
Boritz, E. A. et al. Multiple origins of virus persistence during natural control of HIV infection. Cell 166, 1004–1015 (2016).
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Data generation and analysis: L.P., J.A., T.W.S. and E.A.B. Study design and oversight: L.P., A.T.H., D.C.D., T.W.S. and E.A.B. Participant cohort and sample management: J.A., J.C., A.T., T.W.C., S.M. and T.W.S. Manuscript preparation: L.P., A.T.H., D.C.D., T.W.S. and E.A.B.
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Extended data figures and tables
Extended Data Fig. 1 Flow cytometry of CD32hi, CD32int and CD32lo CD4 T cell populations from PBMCs.
Single lymphocytes (first two columns) that were viable (third column), CD3+ (fourth column), CD4+ (fifth column), and CD32hi, CD32int or CD32lo (sixth column) were sorted as described in Descours et al.1.
Extended Data Fig. 2 Post-sort flow cytometry of CD3+CD4+ subsets that were CD32hi, CD32int or CD32lo.
Cells were sorted as in Extended Data Fig. 1. a, Overlay plots of CD32 and CD4 expression by cells in CD32hi, CD32int and CD32lo sorted populations. Note the heterogeneous pattern of cells from the CD32hi and CD32int populations. b, c, CD20, CD14 and CD3 staining in the CD32+ cells from the CD32hi (b) and the CD32int (c) subsets. Note the large proportions of all CD32+ cells bearing surface markers consistent with B cells (CD20+CD3−) or monocytes (CD14+CD3−) after sorting.
Extended Data Fig. 3 Flow cytometry of PBMCs sorted by alternative gating for CD32hi, CD32int and CD32lo CD4 T cell populations, as well as T cell populations bearing markers of B cells (T–B) or other non-CD4-T-cells (T–other).
Cells in an inclusive light scatter gate consistent with either small lymphocytes or larger cells (first column) were enriched for single cells (second column). Within these gates, viable CD3+ cells (third column) that were CD19− and CD20− (lower gate, fourth column), CD16− and CD14− (fifth column), CD123− (sixth column), CD4+ (seventh column), and CD32hi, CD32int or CD32lo were then collected. Cells that were CD3+ and bearing markers of B cells (T–B; upper gate, fourth column) or other non-CD4-T-cells (T–other; combined ungated events from fifth and sixth columns) were also collected in separate tubes.
Extended Data Fig. 4 Post-sort flow cytometry of CD32 and CD4 expression by CD32hi, CD32int, CD32lo, T–B and T–other cell subsets.
Cells were sorted as in Extended Data Fig. 3. Note the large proportions of all CD32+ cells that did not show high CD4 expression after sorting. Post-sort analyses of CD3+CD4+CD32hi populations were deferred in cases in which these populations were too small to permit both post-sort analysis and downstream HIV DNA quantification (that is, donors # 1, 4 and 6–8).
Extended Data Fig. 5 Flow cytometry, HIV DNA levels, and single-copy HIV DNA sequence analysis from CD32hi, CD32int and CD32lo CD4 T cell populations, and from T cells also bearing non-CD4-T-cell markers.
a, PBMCs from four additional study participants were collected from whole blood by venipuncture with immediate processing (without cryopreservation). The T–other population was collected as a combination of the ungated events from CD19/CD20, CD16/CD14 and γδ T cell receptor/CD123 exclusion plots (fourth, fifth and sixth columns). b, Left, copies of HIV DNA per million cells sorted from four additional study participants as in a. Right, percentages of all HIV DNA copies detected in blood cells deriving from CD32hi, CD32int, CD32lo and T–other subsets, calculated by adjusting values in the left panel for the relative proportions of these subsets determined using FACS data. c, Sequences of individual HIV DNA copies were determined by Sanger sequencing of products obtained by fluorescence-assisted clonal amplification, which amplifies a region of the HIV env gene. Phylogenetic trees were constructed as described in the Supplementary Methods. All Bonferroni-corrected Slatkin–Maddison P values for genetic compartmentalization between any two subsets were greater than 0.05 in all four participants.
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Pérez, L., Anderson, J., Chipman, J. et al. Conflicting evidence for HIV enrichment in CD32+ CD4 T cells. Nature 561, E9–E16 (2018). https://doi.org/10.1038/s41586-018-0493-4
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DOI: https://doi.org/10.1038/s41586-018-0493-4
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