Phenotypic signatures of immune selection in HIV-1 reservoir cells

Human immunodeficiency virus 1 (HIV-1) reservoir cells persist lifelong despite antiretroviral treatment1,2 but may be vulnerable to host immune responses that could be exploited in strategies to cure HIV-1. Here we used a single-cell, next-generation sequencing approach for the direct ex vivo phenotypic profiling of individual HIV-1-infected memory CD4+ T cells from peripheral blood and lymph nodes of people living with HIV-1 and receiving antiretroviral treatment for approximately 10 years. We demonstrate that in peripheral blood, cells harbouring genome-intact proviruses and large clones of virally infected cells frequently express ensemble signatures of surface markers conferring increased resistance to immune-mediated killing by cytotoxic T and natural killer cells, paired with elevated levels of expression of immune checkpoint markers likely to limit proviral gene transcription; this phenotypic profile might reduce HIV-1 reservoir cell exposure to and killing by cellular host immune responses. Viral reservoir cells harbouring intact HIV-1 from lymph nodes exhibited a phenotypic signature primarily characterized by upregulation of surface markers promoting cell survival, including CD44, CD28, CD127 and the IL-21 receptor. Together, these results suggest compartmentalized phenotypic signatures of immune selection in HIV-1 reservoir cells, implying that only small subsets of infected cells with optimal adaptation to their anatomical immune microenvironment are able to survive during long-term antiretroviral treatment. The identification of phenotypic markers distinguishing viral reservoir cells may inform future approaches for strategies to cure and eradicate HIV-1.

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March 2021
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy Owing to study participant confidentiality concerns, viral sequencing data cannot be publicly released, but will be made available to investigators upon reasonable request and after signing a data sharing agreement. Correspondence and requests for materials should be addressed to Dr. Mathias Lichterfeld (mlichterfeld@partners.org).

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Life sciences study design
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Sample size
A total of n=4 ART-treated participants, and n=1 EC were analyzed in data described in Figure 1-4 (peripheral blood viral reservoir cells). Data from n=3 study participants are described in Figure 5 (lymph node reservoir cells). No computational approach was used to determine these sample sizes, testing was based on availability of more than 1 million memory CD4 T cells per study participant.
Data exclusions No data from the described individuals were excluded.

Replication
Multiple microfluidic cartridges were run for each patient sample. In selected cases (n=14), the proviral library from a given cartridge was sequenced twice, with similar results.
Randomization No randomization was performed, because we performed an analysis of study participants enrolled in an observational study.

Blinding
Coded samples from study participants were used throughout the study; laboratory personnel was not blinded with regard to the respective study subjects, since this was a non-interventional, observational study.
The following antibodies were used for cell sorting: CD3-PerCP-Cy5.  Figure 1A-B and the Extended Data Figure  9A-B

Recruitment
All study persons were recruited based on referral by HIV clinicians and infectious disease physicians. The enrollment protocols allowed recruited of men and women >18 years old, of any race or ethnicity. Patients were included in our prior studies and selected for this project according to the following criteria: availability of sufficient cells for experiments, availability of full-genome sequencing data and proviral integration site data, and relatively high frequency of genome-intact HIV-1 proviruses in CD4 T cells.

Ethics oversight
The MassGeneralBrigham Human Research Committee approved all sample collection at MGH and BWH; the IRB of the NIH supervised sample collection at the NIH Clinical Center.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
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Methodology Sample preparation
As described in the methods section of the manuscript. Cell population abundance Purity of sorted cell populations was >90%.

Gating strategy
The lymphocyte population was identified based on FSC/SSC characteristics, followed by identification of singlets on an FSC-Area vs FSC-Height plot. Viable cells were identified using live/dead viabiliy dye. The remaining gating strategy is shown in Extended Data Figure 6 and 7.
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