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Observational Study
. 2020 Jan 29;12(528):eaax6795.
doi: 10.1126/scitranslmed.aax6795.

Different human resting memory CD4+ T cell subsets show similar low inducibility of latent HIV-1 proviruses

Kyungyoon J Kwon  1 , Andrew E Timmons  1 , Srona Sengupta  1 , Francesco R Simonetti  1 , Hao Zhang  2 , Rebecca Hoh  3 , Steven G Deeks  3 , Janet D Siliciano  1 , Robert F Siliciano  4   5
Affiliations
Observational Study

Different human resting memory CD4+ T cell subsets show similar low inducibility of latent HIV-1 proviruses

Kyungyoon J Kwon et al. Sci Transl Med. .

Abstract

The latent reservoir of HIV-1 in resting CD4+ T cells is a major barrier to cure. It is unclear whether the latent reservoir resides principally in particular subsets of CD4+ T cells, a finding that would have implications for understanding its stability and developing curative therapies. Recent work has shown that proliferation of HIV-1-infected CD4+ T cells is a major factor in the generation and persistence of the latent reservoir and that latently infected T cells that have clonally expanded in vivo can proliferate in vitro without producing virions. In certain CD4+ memory T cell subsets, the provirus may be in a deeper state of latency, allowing the cell to proliferate without producing viral proteins, thus permitting escape from immune clearance. To evaluate this possibility, we used a multiple stimulation viral outgrowth assay to culture resting naïve, central memory (TCM), transitional memory (TTM), and effector memory (TEM) CD4+ T cells from 10 HIV-1-infected individuals on antiretroviral therapy. On average, only 1.7% of intact proviruses across all T cell subsets were induced to transcribe viral genes and release replication-competent virus after stimulation of the cells. We found no consistent enrichment of intact or inducible proviruses in any T cell subset. Furthermore, we observed notable plasticity among the canonical memory T cell subsets after activation in vitro and saw substantial person-to-person variability in the inducibility of infectious virus release. This finding complicates the vision for a targeted approach for HIV-1 cure based on T cell memory subsets.

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Figures

Figure 1.
Figure 1.. Subset sorting strategy and MSVOA culture schematic.
(A) Resting CD4+ T-cells were isolated from leukapheresis products from 10 individuals on ART as described in Methods. Purified resting cells were sorted into the respective subsets as shown. (B) Timeline for MSVOA. A small aliquot of the sorted resting cells was set aside for IPDA analysis and calculation of intact and defective provirus frequencies (Fig. 2B). The remaining cells from each subset were seeded in the MSVOA as previously described (14). Cells were activated with PHA on day 0, then both top and bottom chambers of each cultures were split in half on day 9. One set of split wells was incubated without further stimulation until day 21 for p24 ELISA. These results provided the viral outgrowth data in Fig. 4. The other set of split wells was activated again with PHA. This process was repeated every 9 days for 4 total stimulations. Analysis of p24 in the supernatants was carried out 21 days after each PHA stimulation (Fig. 5). Flow cytometric analysis of subset marker expression was done every 5-7 days (Fig. 3).
Fig. 2.
Fig. 2.. Frequencies and proviral DNA content of CD4+ T-cell subsets in peripheral blood.
(A) Representative IPDA results from sorted resting CD4+ T-cells. Droplets in quadrant 1 (Q1) contain proviruses with a 3΄ deletion in the env gene and/or APOBEC3G-induced hypermutation. Q4 contains proviruses with deletions at the 5՛ end of the genome encompassing the packaging signal (ψ). Q2 contains intact provirus. Most droplets (Q3) do not contain a provirus. (B) IPDA analysis on resting CD4+ T-cells from each subset. Geometric means ± SD are shown. For samples in which no provirus was detected (open symbols), the limit of detection (LOD, 4 copies/106 cells) was used in calculations. All subsets contained at least one type of provirus (intact or defective). Mann-Whitney tests were performed to compare ICPM between each subset. P-values are two-tailed. (C) Frequency of each subset in leukapheresis samples from 10 individuals and 1 healthy donor (HD1). Bars show mean ± SD. (D) Contribution of each subset to the circulating pool of intact proviruses determined from the frequency of intact proviruses found in each subset (Fig. 2B) and subset frequency in peripheral blood (Fig. 2C).
Fig. 3.
Fig. 3.. Changes in activation state, proliferative status, and phenotypic marker expression following repeated activation of CD4+ T-cell subsets.
(A) Activation status of CD4+ T-cells throughout the MSVOA averaged across 4 representative individuals as percent of cells expressing CD25 and/or CD69. (B) CFSE dilution after first stimulation. Cells from each subset were stained with CFSE and activated on day 0, then analyzed by flow cytometry at days 4 and 9. (C) CD45RO expression on TN from representative individual. 91.6% of TN became CD45RO+ by day 4 after initial stimulation (Fig. S1A). (D) Percent of cells from each subset culture expressing CCR7 and CD27 at the indicated timepoints. Arrows denote stimulation timepoints.
Fig. 4.
Fig. 4.. Viral outgrowth and inducibility of CD4+ T-cell subsets after one stimulation.
(A) Frequency of cells giving rise to viral outgrowth after one stimulation. Results expressed as IUPM. Open circles denote samples from which no outgrowth was observed; IUPM values for these represent maximum likelihood estimates based on the total number of wells plated for that subset (97). Geometric means ± SD are shown. (B) Contribution of subsets to total pool of replication-competent proviruses induced after one stimulation, calculated using frequencies of each subset in peripheral blood (Fig. 2C). Means ± SD are shown. (C) Percentage of intact proviruses that gave rise to replication-competent virus after one stimulation. Inducibility index was calculated by dividing IUPM values by ICPM values. For samples with no outgrowth or intact proviruses detected, estimates based on the number of input cells and LOD for IPDA are used in the calculation of inducibility (open circles, see Methods). TTM from participant 2461 were not cultured due to insufficient number of cells. Geometric means ± SD are shown. Two-tailed P-values were calculated using Mann-Whitney U-tests (****P <0.0001, ***P <0.001).
Fig. 5.
Fig. 5.. Viral outgrowth and inducibility for T-cell subsets after multiple stimulations.
(A) Cumulative fraction of all positive wells that had positive p24 values 21 days after the indicated stimulation. Data are graphed separately for each individual. TTM were not analyzed for participant 2461 due to insufficient number of cells. (B) Cumulative fraction of all positive wells that had positive p24 values 21 days after the indicated stimulation. Data are graphed separately for each subset. (C) Frequency of cells giving rise to infectious virus in the MSVOA based on initial number of cells seeded. Geometric means ± SD are shown. P-values were calculated from Mann-Whitney U-tests (** P < 0.01). (D) Frequency of cells giving rise to infectious virus after one stimulation or after all 4 stimulations. Results are expressed as IUPM based on initial number of cells seeded and number of wells turned p24+. P-values between IUPM values after the indicated numbers of stimulations were calculated using Wilcoxon matched-pairs signed rank tests. (E) Contribution of each subset to total pool of replication-competent proviruses calculated from IUPM values (Fig. 5C) and the frequency of subsets present in peripheral blood (Fig. 2C). Means ± SD are shown. (F) Inducibility index calculated from ratio of MSVOA IUPM to ICPM from each subset (geometric means ± SD shown). On average, only 1.7% of intact proviruses measured across all subsets were induced to replicate in culture. Mann-Whitney tests showed no significant differences in inducibility indices across subsets (P>0.12). (G) Phenotype of cultured cells at the time of each stimulation (left axis) overlaid with outgrowth results from that stimulation (right axis) from representative individuals (top: 2026, bottom: 2669).
Fig. 6.
Fig. 6.. Inducibility of viruses from different subsets.
(A) Comparison of frequencies of cells with intact proviruses as measured by IPDA (squares, gray bars) and frequencies of cells giving rise to viral outgrowth as measured by MSVOA (triangles, black bars). Bars indicate geometric mean ± SD. (B) Spearman’s rank correlations between IPDA and MSVOA values within each subset. Rho and p-values are shown. (C) Fold-change in IUPM by individual and by subset (n = 10 for TN, TCM, TEM; n = 9 for TTM).
Fig. 7.
Fig. 7.. Neighbor-joining env trees of replication-competent viral isolates from the MSVOA.
Sequences of V3-V4 region of env from outgrowth viruses at 4 timepoints (solid symbols) and proviral sequences (open circles) from unfractionated resting CD4+ T-cells.
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References

    1. Siliciano JD, Kajdas J, Finzi D, Quinn TC, Chadwick K, Margolick JB, Kovacs C, Gange SJ, Siliciano RF, Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells, Nat. Med 9, 727–728 (2003). - PubMed
    1. Finzi D, Blankson J, Siliciano JD, Margolick JB, Chadwick K, Pierson T, Smith K, Lisziewicz J, Lori F, Flexner C, Quinn TC, Chaisson RE, Rosenberg E, Walker B, Gange S, Gallant J, Siliciano RF, Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy, Nat. Med 5, 512–517 (1999). - PubMed
    1. Chun TW, Finzi D, Margolick J, Chadwick K, Schwartz D, Siliciano RF, In vivo fate of HIV-1-infected T cells: quantitative analysis of the transition to stable latency, Nat. Med 1, 1284–1290 (1995). - PubMed
    1. Chun TW, Carruth L, Finzi D, Shen X, DiGiuseppe JA, Taylor H, Hermankova M, Chadwick K, Margolick J, Quinn TC, Kuo YH, Brookmeyer R, Zeiger MA, Barditch-Crovo P, Siliciano RF, Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection, Nature 387, 183–188 (1997). - PubMed
    1. Chun TW, Stuyver L, Mizell SB, Ehler LA, Mican JA, Baseler M, Lloyd AL, Nowak MA, Fauci AS, Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy, Proc. Natl. Acad. Sci. U. S. A 94, 13193–13197 (1997). - PMC - PubMed

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