Monocyte-derived macrophages contain persistent latent HIV reservoirs

Abstract

The development of persistent cellular reservoirs of latent human immunodeficiency virus (HIV) is a critical obstacle to viral eradication since viral rebound takes place once anti-retroviral therapy (ART) is interrupted. Previous studies show that HIV persists in myeloid cells (monocytes and macrophages) in blood and tissues in virologically suppressed people with HIV (vsPWH). However, how myeloid cells contribute to the size of the HIV reservoir and what impact they have on rebound after treatment interruption remain unclear. Here we report the development of a human monocyte-derived macrophage quantitative viral outgrowth assay (MDM-QVOA) and highly sensitive T cell detection assays to confirm purity. We assess the frequency of latent HIV in monocytes using this assay in a longitudinal cohort of vsPWH (n = 10, 100% male, ART duration 5-14 yr) and find half of the participants showed latent HIV in monocytes. In some participants, these reservoirs could be detected over several years. Additionally, we assessed HIV genomes in monocytes from 30 vsPWH (27% male, ART duration 5-22 yr) utilizing a myeloid-adapted intact proviral DNA assay (IPDA) and demonstrate that intact genomes were present in 40% of the participants and higher total HIV DNA correlated with reactivatable latent reservoirs. The virus produced in the MDM-QVOA was capable of infecting bystander cells resulting in viral spread. These findings provide further evidence that myeloid cells meet the definition of a clinically relevant HIV reservoir and emphasize that myeloid reservoirs should be included in efforts towards an HIV cure.

Fig. 1. Development of the MDM-QVOA.

a, To determine the appropriate expander cell line, one viremic vPWH was activated with PMA in the context of MT-4, Molt-4-CCR5, CEMx174 and media only, dotted line indicates limit of detection (LOD). b, To determine the best activation condition, 7 participants (n = 7, 4 vPWH and 3 virally suppressed vsPWH) were activated with PMA, IL-4, TNF𝛼 and media with and without MT-4 expander cells; mean ± s.d. c, To determine whether macrophages derived from negatively selected monocytes could be reactivated similarly to macrophages derived from whole PBMCs, we compared 3 participants (n = 3, 1 vPWH and 2 vsPWH) activated with PMA and co-cultured with MT-4. d–f, To determine the appropriate assay to detect T cell contamination, in the well or via phagocytosis, we assessed CD3ε and TCRβ RNA expression in CD4 T cells isolated from healthy donors (HD). d, The CD4 T cells from 3 HD were serially diluted, lysed and RNA extracted to measure TCRβ and CDε expression; mean ± s.d. TCRβ (e) and CDε (f) showed similar variability across replicates, except at the low end of the assay. g, CD4 T cells were isolated from 8 HD; 1 × 10⁶ CD4s per donor were lysed and assessed for CD3ε and TCRβ expression to determine the copies of each per cell; bar indicates median value. h, Healthy MDMs were co-cultured with HIV+ CD4 T cells from two donors (CP11 and 21) with and without PMA activation to determine whether HIV+ CD4 T cells were able to transfer viral nucleic acids to MDM; n = 2. i, A schematic of the final MDM-QVOA experimental design. Source data

Fig. 2. MDMs from vsPWH have consistent levels of HIV DNA over time.

a, Ten vsPWH were assessed for HIV gag DNA, gag RNA and tat/rev RNA in isolated CD4 T cells (n = 10) and MDMs (n = 20), 6 donors were repeated 2–4 times. ****P < 0.0001, two-tailed unpaired t-test. b, HIV gag DNA in MDMs was assessed in 6 donors at multiple blood draws between 150–1,300 d apart. c, The number of CD4 T cells per million cells plated in MDM cultures, calculated using TCRβ RNA and CD4 percentages in whole blood; n = 15, 3 donors were repeated 2–3 times, line indicates median. d, In a subset of individuals, HIV gag DNA was assessed in monocytes and MDM from the same blood draw; n = 4. e, Monocytes and CD4 T cells were isolated from 30 vsPWH and assessed for HIV proviral DNA using IPDA. Intact, 3’ defective, 5’ defective and total proviral genome levels per million cells were compared between cell types; intact **P = 0.0014, 3’ del *P = 0.03, 5’ del ***P = 0.0005, total **P = 0.006, two-tailed unpaired t-test. f, Comparison of intact genome levels in a subset of participants that had detectable intact genomes in both CD4 and monocytes; n = 12, two-tailed paired t-test. NS, not significant. Each datapoint represents data from a specific participant, circles are CD4 data, squares are monocytes or MDM data and lines represent medians. Source data

Fig. 3. MDMs from vsPWH have reactivatable reservoirs that can be induced over time and stratify with HIV DNA burden.

a, Ten vsPWH were assessed for reactivatable reservoirs in CD4 T cells and MDMs isolated from the same blood draw using the cell-specific CD4 and MDM QVOAs. b, Four participants returned for a second visit 150–280 d after the first visit. All participants had repeat MDM-QVOA completed and one participant also had a repeat CD4-QVOA completed (CP36, orange circle). Two of the 4 participants returned for a third follow-up visit 1,174 and 1,502 d after their first visit to repeat the MDM-QVOA. c, Average HIV gag RNA copies per million cells plated in the CD4 and MDM QVOA; n = 9 CD4 and n = 10 MDM, not significant via unpaired t-test. d, Participants with detectable IUPM values in MDM-QVOA had higher levels of HIV gag DNA compared with those with undetectable IUPM values; n = 9 detectable and n = 5 undetectable, two-tailed unpaired Student’s t-test P = 0.0122. e, MDM DNA levels positively correlated with MDM IUPM values; simple linear regression R² = 0.48 and P = 0.04. f,g, Comparing immune cell percentages in blood from participants with detectable (n = 10) and undetectable (n = 5) IUPM values in MDM QVOA; total monocytes (TLR2+/CD3−) and CD4 T cells (f), and monocyte subsets (g). Source data

Fig. 4. Virus released in MDM-QVOAs can spread in an activated CD4 T cell line and are distinct.

a,b, QVOA culture supernatants were used to spinoculate MT-4s and determine whether viral isolates produced in the QVOAs are capable of spread. One representative positive CD4-QVOA well from 4 vsPWH (a) and one representative positive MDM-QVOA well from 5 vsPWH (b) are shown; viral input was normalized to 800 copies of HIV gag RNA per ml. c, The nef gene was sequenced at limiting dilution from positive QVOA wells in the CD4 and MDM assays. Nef sequences were aligned and a tree was generated using maximum likelihood estimation using the bootstrap method to test phylogeny (1,000 replications). Bootstrap outcomes are labelled at each participant node, >80 was considered significant. Each color represents a specific participant, circles indicate CD4 sequences, squares MDM sequences and diamond the reference sequence. Source data

Extended Data Fig. 1. Comparison of expander cell lines for MDM-QVOA development.

Cell lines, MT-4, CEMx174 and MOLT4-CCR5 were assessed for HIV entry receptor expression CCR5, CXCR4 and CD4. Cell were gated on singlets and then live cells, shaded histogram is unstained cells, black line marker of interest.

Extended Data Fig. 2. Comparison of whole blood, PBMC, negatively selected monocyte subsets.

Whole blood (A), PBMC (B) and negatively selected monocytes (C) from one representative subject used to show that negative selection results in less CD4 contamination in cells plated and similar monocyte subset percentages. Cells are first gated on TLR2 expression, monocytes (TLR2+) are further gated by CD14 and CD16 expression, classical (CD14 + CD16−), intermediate (CD14 + CD16+) and non-classical (CD14-CD16+). Lymphocytes (TLR2-) are gate based on CD3 and CD4 expression, CD4 T cells (CD3 + CD4+).

Extended Data Fig. 3. Viral replication of MDM and CD4 QVOA isolates.

Culture supernatants from CP11 (A), CP21 (B), CP25 (C), and CP36 (D) positive MDM and CD4 QVOA wells were used to infect MT-4 cells and viral kinetics were assessed by measuring HIV RNA in supernatant overtime. Source data

Extended Data Fig. 4. Highlighter plot comparing CP25 sequences with CP36 to rule out contamination.

CP25 CD4-A1 (m) was used as the master sequence to compare the other CP25-CD4 sequences and 2 representative CP36 CD4 and MDM sequences with CP25 MDM-A2 (yellow).

Extended Data Fig. 5

Nef sequences from positive MDM and CD4 QVOA wells.

Extended Data Fig. 6. Flow cytometry gating scheme for whole blood and T cell assessment in MDM with and without activation.

Post singlet gating samples are gated on TLR2 and side scatter to separate monocytes (TLR2+) from lymphocytes (TLR2−) (A). TLR2+ cells are then gated in monocytes subsets, classical (CD14 + CD16−), intermediate (CD14 + CD16+) and non-classical (CD14lo/-CD16+) (B). TLR2- cells are separated based on CD3 and CD159a expression (C) and then further gate on CD4 and CD8 expression (D). CD4 T cells are gated as (TLR2-CD3 + CD4 + CD8−). (E) MDM with and without activation for 12 days with PMA were removed from the plate with TrypLE and stained with Live/Dead Near IR and with or without CD3 for 30 minutes at 4C. Cells were permeabilized using Biolegend PermFast and stained with CD68 or matched IgG control. Cells were run immediately on a BD LSRFortessa. Cells were first gated to remove debris, then to remove doublets, and finally to remove dead cells.