Research ArticleHIV

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

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Science Translational Medicine  29 Jan 2020:
Vol. 12, Issue 528, eaax6795
DOI: 10.1126/scitranslmed.aax6795
  • Fig. 1 T cell subset sorting strategy and culture in the multiple stimulation viral outgrowth assay.

    (A) Resting CD4+ T cells were isolated from leukapheresis products from 10 HIV-1–infected individuals on ART by bead depletion. Purified resting T cells were then sorted into the respective T cell subsets indicated (APC, allophycocyanin; PE, phycoerythrin; PEcy7; phycoerythrin–cyanine7). (B) Timeline for the multiple stimulation viral outgrowth assay. A small aliquot of the sorted resting T cells was set aside for analysis by the intact proviral DNA assay (IPDA) and calculation of intact and defective HIV-1 provirus frequencies (Fig. 2B). The remaining cells from each T cell subset were seeded in the multiple stimulation viral outgrowth assay as previously described (14). Sorted T cells and irradiated allogeneic PBMCs were placed in the top chamber, and MOLT-4/CCR5 cells were placed in the bottom chamber of the assay plate. The MOLT-4/CCR5 cells were used to expand virus released from T cells after stimulation with phytohemagglutinin (PHA) (9). T cells were stimulated with PHA on day 0, and then both the top and bottom chambers of each culture were split in half on day 9. One set of split wells was incubated without further stimulation until day 21 and analyzed by ELISA for HIV-1 p24 protein. This analysis provided the viral outgrowth data presented in Fig. 4. The other set of split wells was stimulated again with PHA. This process was repeated every 9 days for a total of four rounds of T cell stimulation. Analysis of HIV-1 p24 antigen in the culture supernatants was carried out 21 days after each stimulation with PHA (Fig. 5). Flow cytometric analysis of T cell subset marker expression was done every 5 to 7 days of culture (Fig. 3).

  • Fig. 2 CD4+ T cell subset frequencies in peripheral blood and proviral DNA content in each subset.

    (A) Representative results from the intact proviral DNA assay of sorted resting CD4+ T cells from an HIV-1–infected individual on ART. Droplets from the assay in quadrant 1 (Q1) contain HIV-1 proviruses with a 3′ deletion in the env gene or APOBEC3G-induced hypermutation. Quadrant 4 (Q4) contains proviruses with deletions at the 5′ end of the genome encompassing the packaging signal (ψ). Quadrant 2 (Q2) contains intact proviruses. Most droplets in quadrant 3 (Q3) do not contain a provirus. (B) Results of the intact proviral DNA assay detecting HIV-1 proviral DNA in resting CD4+ T cell subsets from 10 HIV-1–infected individuals on ART. Geometric means ± SD are shown. For samples in which no provirus was detected (open symbols), the limit of detection (LOD; four copies per 106 cells) was used in calculations. All T cell subsets contained at least one type of provirus (intact or defective). Mann-Whitney tests were performed to compare copies per million between each T cell subset. (C) Frequency of each T cell subset in leukapheresis samples from 10 HIV-1–infected individuals on ART and one healthy donor (HD1). Bars show means ± SD. (D) Contribution of each T cell subset to the circulating pool of intact HIV-1 proviruses determined from the frequency of intact proviruses found in each T cell subset (B) and the frequency of T cell subsets in peripheral blood of 10 HIV-infected individuals on ART (C). P values were determined using Mann-Whitney two-tailed U tests. TN, naïve T cells; TCM, central memory T cells; TTM, transitional memory T cells; TEM, effector memory T cells.

  • Fig. 3 Changes in activation state, proliferative status, and phenotypic marker expression after repeated stimulation of CD4+ T cell subsets.

    (A) Activation status of CD4+ T cell subsets was measured by flow cytometry during the multiple stimulation viral outgrowth assay. Results are the average of samples from four representative HIV-1–infected individuals on ART and are shown as the percent of T cells expressing CD25 and CD69. (B) Dilution of carboxyfluorescein diacetate succinimidyl ester (CFSE) due to T cell proliferation after the first round of T cell stimulation with phytohemagglutinin (PHA). Cells from each T cell subset were stained with CFSE and stimulated with PHA on day 0 and then analyzed by flow cytometry on days 4 and 9 after activation. (C) Up-regulation of the memory T cell marker CD45RO on naïve T cells (TN) from a representative HIV-1–infected individual on ART. Naïve T cells (91.6%) expressed CD45RO by day 4 after stimulation by PHA (also see fig. S1A). APC, allophycocyanin; PE, phycoerythrin. (D) Percent of cells from each T cell subset expressing CCR7 and CD27 at the indicated time points for T cells from 10 HIV-infected individuals on ART and one healthy donor (HD1). Gray arrows denote time points for stimulation of T cells with PHA. TN, naïve T cells; TCM, central memory T cells; TTM, transitional memory T cells; TEM, effector memory T cells.

  • Fig. 4 Viral outgrowth and inducibility after one round of CD4+ T cell subset activation.

    (A) Frequency of T cells from each subset that gave rise to viral outgrowth after one round of stimulation in the multiple stimulation viral outgrowth assay. Results are shown for T cells from 10 HIV-infected individuals on ART and are expressed as infectious units per million (IUPM). Open circles denote samples from which no viral outgrowth was observed; IUPM values for these samples represent maximum likelihood estimates based on the total number of wells plated for that T cell subset (94). Geometric means ± SD are shown. (B) Contribution of T cell subsets to the total pool of replication-competent HIV-1 proviruses induced after one round of stimulation, calculated using frequencies of each T cell subset in peripheral blood of the same participant (see Fig. 2C). Means ± SD are shown. (C) Percentages of intact proviruses that gave rise to replication-competent virus after one round of T cell stimulation are indicated. The inducibility index was calculated by dividing IUPM values by the number of intact proviral copies per million cells (ICPM). For samples with no viral outgrowth or intact proviruses detected, estimates based on the number of input T cells and the limit of detection for the intact proviral DNA assay were used in the calculation of the inducibility index (open circles). Transitional memory T cells (TTM) from participant #2461 were not cultured because of an insufficient number of cells. Geometric means ± SD are shown. P values were calculated using Mann-Whitney two-tailed U tests (****P < 0.0001, ***P < 0.001). TN, naïve T cells; TCM, central memory T cells; TEM, effector memory T cells.

  • Fig. 5 Viral outgrowth and inducibility after multiple rounds of T cell stimulation.

    (A) Cumulative fractions of all wells positive for the HIV-1 p24 antigen 21 days after one, two, three, or four rounds of T cell stimulation with phytohemagglutinin (PHA). Data are graphed separately for T cells from the 10 HIV-1–infected individuals on ART. Transitional memory T cells (TTM) were not analyzed for participant #2461 because of an insufficient number of cells. (B) Cumulative fractions of all wells positive for HIV-1 p24 antigen 21 days after one to four rounds of T cell stimulation with PHA. Data are shown for four T cell subsets from 10 HIV-1–infected individuals on ART. (C) Frequency of T cells giving rise to replication-competent virus in the multiple stimulation viral outgrowth assay based on the initial number of T cells seeded. Geometric means ± SD are shown. P values were calculated using a Mann-Whitney U test (**P < 0.01). (D) Frequency of T cells giving rise to replication-competent virus in the multiple stimulation viral outgrowth assay based on the initial number of T cells seeded after one or four rounds of stimulation with PHA. Results are expressed as IUPM based on the initial number of T cells seeded and the number of wells that were positive for HIV-1 p24 antigen. P values were calculated using the Wilcoxon matched-pairs signed-rank test. (E) Contribution of each T cell subset to the total pool of replication-competent proviruses calculated from IUPM values (C) and the frequency of T cell subsets present in peripheral blood of HIV-infected individuals on ART (C). Means ± SD are shown. (F) The inducibility index was calculated from the ratio of IUPM values to intact copies per million cells for each T cell subset. Geometric means ± SD are shown. Mann-Whitney U tests showed no significant differences in inducibility indices across T cell subsets (P > 0.12). (G) Phenotype of cultured T cells at the time of each stimulation with PHA (left axis) overlaid with viral outgrowth results from that round of stimulation (right axis). Data are shown for two representative HIV-1–infected individuals on ART (top: #2026; bottom: #2669). TN, naïve T cells.

  • Fig. 6 Inducibility of proviruses from different T cell subsets.

    (A) Frequencies of T cells with intact HIV-1 proviruses for the four T cell subsets from 10 HIV-1–infected individuals on ART measured by the intact proviral DNA assay (squares). The frequencies of T cells from these same individuals that gave rise to viral outgrowth measured by the multiple stimulation viral outgrowth assay (triangles) are also indicated. Horizontal bars indicate geometric means ± SD. (B) Spearman’s rank correlations comparing IUPM values from the multiple stimulation viral outgrowth assay with ICPM values from the intact proviral DNA assay for each T cell subset from the 10 HIV-infected individuals on ART. Rho and P values are shown. (C) The fold change in IUPM values for each HIV-infected individual and for each T cell subset is indicated (n = 10 for TN, TCM, and TEM; n = 9 for TTM). TN, naïve T cells.

  • Fig. 7 Neighbor-joining env trees of replication-competent viral isolates.

    Neighbor-joining env trees of replication-competent viral isolates from the multiple stimulation viral outgrowth assay are shown. Sequences of the V3-V4 region of the env gene in replication-competent viruses from cultures 21 days after T cell stimulation (solid symbols) and proviral sequences from unfractionated resting CD4+ T cells (open circles) are shown for 10 HIV-infected individuals on ART.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/528/eaax6795/DC1

    Fig. S1. Representative flow cytometry plots of T cell subset phenotypes after stimulation.

    Fig. S2. Results of the multiple stimulation viral outgrowth assay and intact proviral DNA assay (excluding data points below the limit of detection).

    Fig. S3. Illustration of surface marker differences on resting versus activated CD4+ T cell subsets.

    Table S1. Characteristics of study participants.

    Table S2. Frequencies of proviral copies, infection frequencies, and assay input cell numbers for samples below the limit of detection.

    Table S3. Frequencies of subsets in peripheral blood, frequencies of proviral copies, infection frequencies, and inducibility indices for all T cell subsets from all 10 participants.

    Data file S1. Activation of T cell subsets in four representative participants.

    Data file S2. Average expression of CCR7 and CD27 for all T cell subsets from all participants.

  • The PDF file includes:

    • Fig. S1. Representative flow cytometry plots of T cell subset phenotypes after stimulation.
    • Fig. S2. Results of the multiple stimulation viral outgrowth assay and intact proviral DNA assay (excluding data points below the limit of detection).
    • Fig. S3. Illustration of surface marker differences on resting versus activated CD4+ T cell subsets.
    • Table S1. Characteristics of study participants.
    • Table S2. Frequencies of proviral copies, infection frequencies, and assay input cell numbers for samples below the limit of detection.
    • Table S3. Frequencies of subsets in peripheral blood, frequencies of proviral copies, infection frequencies, and inducibility indices for all T cell subsets from all 10 participants.

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    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Activation of T cell subsets in four representative participants.
    • Data file S2 (Microsoft Excel format). Average expression of CCR7 and CD27 for all T cell subsets from all participants.

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