Analysis of HIV Reservoirs in Cellular Conjugates from Peripheral Blood

Defining distinctive attributes of HIV-infected cells will inform development of HIV cure-directed therapies. Prior ex vivo studies of blood and tissue have suggested that some HIV-infected CD4 T cells are found in conjugates with other cell types. Here, we analyzed levels and sequences of HIV nucleic acids in sorted cellular conjugates from PBMC. Compared to single CD4 T cells, conjugates containing CD4 T cells showed no enrichment for HIV DNA or RNA. However, in several HIV controllers, HIV DNA sequences from sorted conjugates were enriched for sequences closely related to plasma viruses. In ART-treated people, although subgenomic HIV DNA sequences in sorted conjugates and single cells were genetically intermingled, intact proviruses were more frequent in whole blood cells than in magnetically-purified CD4 T cells. We conclude that some HIV-infected cells have attributes that predict preferential loss during sample processing, and that may also reflect vulnerability to therapeutic targeting in vivo.


Introduction
7 cells and other cell types. These conjugates occurred at frequencies that were much higher than

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PBMC isolated from leukapheresis or whole blood that were CD3 + and viable were separated by 6 (1) CD32 expression and forward light scatter. CD32cells were divided into two populations: (2) 7 single CD4 T cells showing a low forward light scatter (FSC lo ) and (3) high forward light scatter 8 (FSC hi ). CD32 + cells were gated based on their expression of (4) ab TCR and CD14. Cells that 9 were only positive for TCR ab were further divided into (5) CD14and (6) CD14 + . CD4and CD4 + 10 T cells were sorted from these four cell populations: FSC lo , FSC hi , CD14 -, and CD14 + . 1 In a previous study, we found that the HIV-infected CD4 T cell pool in blood from HIV 2 controllers consisted predominantly of expanded cellular clones harboring archival proviruses, 3 with a much smaller population of cells harboring viruses genetically similar to plasma viruses 4 (Boritz et al., 2016). To determine in the present study whether such recent sequences might be 5 preferentially found in conjugate populations, we performed Sanger sequencing and phylogenetic 6 analysis on single-copy env PCR products from conjugates, single cells, and plasma virions in 7 HIV controllers. In two of three participants, we found extensive HIV genetic intermingling both 8 among sorted cell subsets and between each subset and plasma virions ( Figure 3A; participants 9 #1 and #2). In these two participants, there was no clear segregation between HIV DNA 10 sequences in conjugates and those in single CD4 T cells. In the third participant, however, we 11 found that while most HIV DNA sequences showed a clustered, archival pattern, rare sequences to single CD4 T cells, we found enrichment for very rare cells with markers of an actively 20 replicating pool in conjugates from one HIV controller, as well as evidence that recovery of this 21 cell population was influenced by upstream PBMC processing steps.

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We also performed Sanger sequencing and phylogenetic analysis on single-copy env 23 PCR products from conjugates and single cells in ART-treated participants. As in the three HIV activated platelets in HIV infection (Green et al., 2015;Real et al., 2020;Simpson et al., 2020), 3 we expanded the focus of these studies to include CD4-T-cell-platelet conjugates. We detected 4 CD4 T cells bearing platelet markers in freshly-acquired PBMC by labeling for CD42b +/-CD62P,

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and employed a gating strategy in which CD4 T cell conjugates with other cells (Non-CD4 + ) were 6 isolated first, followed by collection of CD4-T-cell-platelet conjugates (Plt + ) and single CD4 T 7 cells ( Figure 5). Using this strategy, we found that CD4-T-cell-platelet conjugates accounted for 8 2.5-11.6% of all cellular events in PBMC from HIV+ participants ( Table II)

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Cells that were CD19 + , CD20 + , CD14 + , CD16 + , gd TCR + , and CD123 + were (7) combined into one 7 population and (8) ab TCR + cells were gated (Non-CD4 + ). CD4and CD4 + T cells were sorted 8 from these three cell populations: CD4, Plt + , and Non-CD4 + . 9 10 11 region analyzed ( Figure 8A). Clusters of matching sequences representing possible expanded 1 cellular clones again included sequences both from single cells and from conjugates ( Figure  . Subgenomic sequence analysis from CD4, Plt + , and Non-CD4 + cells, sorted from whole genome using multiplexed digital-droplet PCR (Bruner et al., 2019). As shown in Figure 9B, HIV 1 env was detected by limiting dilution PCR in both whole blood and magnetically-purified CD4 T 2 cell DNA. As expected, given the lack of enrichment for CD4 T cells in whole blood DNA, levels 3 of HIV were lower in whole blood DNA samples. Single-copy sequence analysis showed 4 evidence neither for sample contamination nor for distinct HIV genetic subpopulations in whole 5 blood compared to CD4 T cells (Figure 9-figure supplement 4). Nonetheless, percentages of 6 intact proviruses were significantly higher in whole blood cell DNA than in purified CD4 T cell 7 DNA. This was true for all 8/10 participants in whom IPDA detected intact proviruses ( Figure 9C).

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Overall, therefore, using a distinct experimental approach in samples from ART-treated 9 individuals, we found evidence that a subset of cells containing intact HIV proviruses may be lost 10 in standard CD4 T cell isolation procedures.

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PBMC were then sorted without doublet exclusion into three populations: 1) single CD4 T cells

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In order to compare differences during sample preparation, whole blood samples obtained 7 by phlebotomy were processed using two different methodologies. Whole blood was lysed using