Sex as a biological variable. Self-reported biological sex of study participants was included as a variable for the analysis of Vδ2 T cell expansion frequencies. Reported answers included male and female patients for both PWH and PWOH.

Study participants. PWH with over a year of viral suppression on ART were enrolled at the George Washington University Medical Faculty Associates, Washington DC, and the University of North Carolina, Chapel Hill. An additional 16 deidentified buffy coats from PWOH were purchased through the Gulf Coast Regional Blood Center (Houston, Texas, USA).

Flow cytometry immunophenotyping and cell sorting. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-gradient and viably stored in 20% DMSO in Liquid Nitrogen until further use. Cells were thawed and counted 3 times with a LUNA-FL cell counter (Logo Biosystems), stained for viability with fixable Zombie Aqua (BioLegend), and incubated with TruStain FcX (BioLegend) to prevent nonspecific binding. Following blocking, cells were stained with monoclonal antibodies (BioLegend) targeting CD3 (clone SK3), Vδ1 (clone REA117, Miltenyi), Vδ2 (clone B6), CD8 (clone SK1), CD56 (clone 5.1H11), CD16 (clone 3G8), NKG2D (clone 1D11), NKG2A (clone S19004C), FasL (clone NOK-1), and TRAIL (clone RIK-2) for 20 minutes in the dark at 4°C. Cells were then washed, fixed in 2% PFA, and acquired on a LSRFortessa X-20 (BD Biosciences) and analyzed using FlowJo software v10.10.0 (BD Biosciences). For LCAs, expanded Vδ2 T cells were sorted by FACS using a SH800 (Sony Biotechnology). Postsort purities of Vδ2 T cells were > 99.9% of live cells.

Ex vivo expansion of Vδ2 T cells. PBMCs were magnetically depleted of αβ T cells (StemCell Technologies) and cultured in complete RPMI (cRPMI) supplemented with 1% penicillin/streptomycin (Sigma-Aldrich) and 10% FBS (Atlas Biologics) plus 3 μM ALN (Sigma-Aldrich) and 500 U/mL IL-2 (Peprotech). Cells were cultured at a density of 1 × 106/mL replenishing half of the media with 500 U/mL IL-2 every 2–3 days for a total of 14 days. At the end of the culture period, cells were harvested and immunophenotyping of expanded Vδ2 T cells conducted as described above. Expansions with Vδ2 T cells representing > 95% of total live cells were used in subsequent cytotoxic cocultures with HIV-infected target cells. For LCAs, highly purified Vδ2 T cells were isolated by FACS as described above.

Generation of CD4+ T cells and MDM targets. Target CD4+ T cells and MDMs were generated from PWOH, as previously described (16). Briefly, CD4+ T cells were isolated by negative magnetic selection (StemCell Technologies) and activated with 3 μg/mL of plate-bound αCD3 (clone OKT3), 5 μg/mL αCD28 (clone 28.2), and 100 U/mL of IL-2. Cells were cultured at a density of 1 × 106/mL for 3 days, when the cells were washed and resuspended at 0.5 × 106/mL in cRPMI and 100 U/mL IL-2 before being transferred to a culture flasks to allow for expansion for an additional 4 days.

In parallel, monocytes were isolated from PBMCs by positive magnetic selection for CD14+ cells (Miltenyi) and differentiated in the presence of cRPMI supplemented with 50 ng/mL M-CSF and 50 ng/mL GM-CSF (Peprotech). Cells were cultured at a density of 0.5 × 106/mL in low attachment plates for a total of 7 days with media supplemented with 50 ng/mL M-CSF and 50 ng/mL GM-CSF. On day 7, maturation into macrophages was visually assessed for cell spreading on the surface of the plate.

Infection of targets. Freshly prepared target CD4+ T cells were washed twice and infected with strain HIVBAL by spinoculation at 2,000g for 2 hours. Cells were washed twice to remove unbound virus and resuspended in cRPMI and 100 U/mL IL-2. Cells were seeded at a density of 1 × 106/mL and cultured for 3 days to promote infection by cell-to-cell transmission. On day 7 of MDM maturation, half of the media was removed and replenished with fresh cRPMI supplemented with 50 ng/mL M-CSF and 50 ng/mL GM-CSF and HIVBAL (NIH HIV Reagent Program) then incubated for 6 hours at 37°C. After incubation, virus-containing media were removed and replenished with fresh cRPMI and cytokines, and cells were cultured for 3 days.

Cytotoxic coculture. HIV-infected target cells were extensively washed and stained with 2.5 μM Cell Trace Violet (Thermo Fisher Scientific) in 1× PBS for 20 minutes at 37°C in the dark. The staining was then quenched with prewarmed cRPMI and incubated for an additional 5 minutes, washed, and resuspended in cRPMI at a concentration of 1 × 106 cells/mL. CD4+ T cell targets were cocultured with expanded allogeneic Vδ2 T cells at varying E:T ratios ranging from 1:5 to 50:1 and plated in triplicate wells. MDMs were cocultured at E:T ratios ranging from 1:1 to 10:1. Target cells cultured alone were used as controls and all conditions were incubated at 37°C for 18 hours. Following coculture, cells were harvested, washed, and stained for viability with fixable Zombie NIR (BioLegend) before being incubated with TruStain FcX (BioLegend) to prevent nonspecific binding. Following blocking, conditions with CD4+ T cells were stained with monoclonal antibodies (BioLegend) targeting CD3 (clone SK3), Vδ2 (clone B6), and CD4 (clone SK3) whereas MDM cocultures were stained with CD3 (clone SK3), Vδ2 (clone B6), CD14 (clone HCD14), CD16 (clone 3G8), and CD4 (clone SK3) for 20 minutes in the dark at 4°C. Cells were then washed, fixed, and permeabilized with Cytofix/Cytoperm (BD Biosciences) for 20 minutes at 4°C. Following permeabilization, cells were washed in 1× Perm/Wash Buffer then stained with a monoclonal antibody specific for HIVp24 (clone KC57, Beckman Coulter) for 20 minutes at 4°C. Samples were acquired on a LSR-Fortessa X-20 (BD Biosciences) and analyzed using FlowJo software v10.10.0 (BD Biosciences).

Cytotoxic coculture analyte detection. Supernatant was harvested following 18 hours from 10:1 cocultures as well as CD4+ T cell alone and Vδ2 T cell monoculture controls then stored at –80°C. Supernatants were then gently thawed, and 25 μL were used to detect cytotoxic analytes and cytokines using the Legendplex Human CD8/NK Panel Detection kit (BioLegend). Samples and standards were plated in technical duplicates. All samples were acquired on an LSR-Fortessa X-20 (BD Biosciences) and analyzed using the Legendplex Data Analysis Software Suite v2025.05.01. Results are represented as the comparison between HIV-infected CD4 cocultures with uninfected CD4 cells.

LCA. An adapted version of the LCA was performed, as previously described by our group (37). Briefly, PBMCs from ART-suppressed PWH were thawed and rested overnight in 10 nM Abacavir and 0.5 μM Nelfinavir (NIH HIV Reagent Program) to prevent new rounds of infection. The cells were washed to remove antiretrovirals, then CD4+ T cells were isolated by negative magnetic selection (StemCell Technologies) and viral replication was reactivated for 18 hours using 3 μg/mL phytohemagglutinin (PHA) and 100 U/mL IL-2 (Peprotech). Following reactivation, CD4+ T cells were washed twice and cocultured with FACS-purified expanded Vδ2 T cells at a 1:5 E:T ratio or alone for additional 18 hours. Vδ2 T cells were depleted from cultures by magnetic selection (Miltenyi), and remaining CD4+ T cells were resuspended in cRPMI and 20 U/mL IL-2 before 1 × 106 cells were plated per well of a 24-well plate for additional 19 days. Depletion of Vδ2 T cells was confirmed by flow cytometry showing a mean purity of 99% CD4+ T cells. On day 8, PHA-activated CD4+ T cells from HIV-seronegative donors were added to cultures to propagate outgrowth virus. Part of the media was replenished every 2–3 days, and supernatant was harvested on day 15 and 19 for HIVp24 ELISA quantification (R&D Systems). Results are reported as the number of positive wells compared between conditions.

Statistics. Statistical analyses were performed in GraphPad Prism or with the R programming language. Mann-Whitney U test was used to test for differences between ART-suppressed PWH and PWOH. Wilcoxon matched-pairs signed-rank test was used for paired analyses of phenotypes before and after expansion. Correlations between Vδ2 T cell phenotypes and participant clinical characteristics were performed using the Spearman’s ranked correlation test.

Study approval. Written informed consent was obtained from PWH at both the George Washington University Medical Faculty Associates and University of North Carolina, Chapel Hill, through IRB-approved protocols. Samples from PWOH purchased through the Gulf Coast Regional Blood Center were acquired without any associated personal identifiable information.

Data availability. Data values for all supporting figures within this study are available in the Supporting Data Values file.