Purity of isolated LMs. We first developed a protocol to purify LMs that limited the inadvertent presence of CD4+ T cells that could confound studies of HIV-1 reservoirs (8). To detect T cell contamination in LMs, we used a sensitive qPCR assay for CD3ε mRNA (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI121678DS1). The assay identified total T cell contamination in macrophages at a ratio of between 1:100 and 1:1,000, confirming 99% to 99.9% purity (Supplemental Figure 1B). The CD3ε qPCR assay circumvented the requirement of flow cytometry or FACS to ensure purity, which would have been challenging given limited cell numbers. To enhance LM purity, we progressively depleted T cells (Figure 1A), employing a strategy of positive selection of T cells using CD3 microbeads, plastic adherence, and deprivation of T cell mitogens over more than 30 days. T cell depletion concludedwith the use of Resimmune, a high-affinity anti-CD3 recombinant diphtheria toxin that has been used in patients to deplete T cells (9): we confirmed that Resimmune was specific for T cells, not macrophages (Supplemental Figure 1, C and D). In vitro we observed over 80% reduction in T cell viability using Resimmune (Supplemental Figure 1C) (10). The multistep protocol resulted in little evidence of T cell contamination in purified LMs (Figure 1B). In addition, purified LMs were cultured for more than 30 days in the presence of antiretrovirals (tenofovir disoproxil fumarate [TDF], emtricitabine [FTC], and raltegravir [RTG]) to fully suppress ex vivo infection of LMs by dying or phagocytosed CD4+ T cells that would have confounded our results.

Figure 1 Purification of liver macrophages yields minimal T cell contamination. (A) LMs were purified from fresh liver tissue using a multistep protocol that progressively depletes T cells: bulk liver tissue was disrupted, centrifuged on a Histodenz gradient (Sigma-Aldrich), and followed by depletion of T cells using CD3 microbeads. Mononuclear cell fractions were incubated on plastic to separate adherent LMs from nonadherent cells and then incubated with Resimmune, a high-affinity T cell toxin, to deplete remaining T cells. LMs were incubated for more than 30 days, deprived of T cell mitogens, and maintained in the presence of antiretrovirals (ARVs) to prevent ex vivo infection. (B) The purification protocol depletes T cells. In a sample isolation of LMs from an HIV-1 uninfected liver, a sensitive qPCR assay for CD3ε mRNA showed the absence of detectable T cells. LMs and hepatocytes were also detected by qPCR assays for CD68 and albumin mRNA, respectively. The fold increases were compared with GAPDH. However, when GAPDH was not detected, as shown by the open blue box (CD68), fold-change was calculated by assigning GAPDH the last cycle number of the qPCR. (C) LM purification resulted in minimal T cell contamination in tissues taken from HIV-1–infected people. LM purity and T cell contamination were measured in duplicate on LMs isolated from 9 HIV-1–infected people before Resimmune and compared with a dilution series of total T cells. The protocol resulted in no detectable T cell contamination of LMs from 8 of 9 participants. In LMs from LT02, 1% T cell contamination was detectable, which was removed by maintaining the LMs in culture devoid of T cell mitogens for more than 90 days and treatment with Resimmune. The T cell dilution series represents the standard curve derived by performing qPCR for CD3ε mRNA on total RNA extracted from 10-fold dilutions of unactivated CD3+ T cells isolated by MACS separation from PBMCs obtained from a healthy donor. Error bars indicate mean ± SD. *For N9, there appeared to be PCR inhibition in the sample as indicated by poor detection of a housekeeping gene by qPCR.

Next, we tested whether HIV-1–infected CD4+ T cells might falsely enhance the apparent abundance of macrophage infection if the 2 cell populations were cocultured to model the possibility of T cells contaminating LMs at the limit of our detection. We cocultured different combinations of HIV-1–infected versus uninfected CD4+ T cells with HIV-1–infected or uninfected monocyte-derived macrophages (MDMs) in the presence or absence of Resimmune, mimicking possible culture conditions (Supplemental Figure 2). Cells were infected separately and then combined, testing whether quantities of HIV-1 proviral DNA were different between test conditions after 30 days of coculture. CD4+ T cells were mixed with macrophages in a ratio of 1:100, conservatively reflecting the lower limit of detection of our CD3ε mRNA assay for T cell contamination. Upon mixing, cocultured cells were incubated with antiretrovirals to prevent transmission of infection from one cell population to the other. After 30 days of coculture of MDMs from 3 healthy donors with CD4+ T cells from the same donors, we did not find evidence that CD4+ T cell infection enhanced the measured abundance of HIV-1 proviral DNA in mixed cultures. Moreover, MDMs showed evidence of infection even in the presence of contaminating CD4+ T cells that were never infected. Since antiretrovirals were introduced into the wells at the time of coculture, the last result demonstrates that macrophage infection was sufficient on its own to yield proviral DNA 30 days after infection, irrespective of whether CD4+ T cells contaminated the cultures. Intriguingly, however, we found that only 2 of 3 donors showed evidence of HIV-1 infection in isolated MDMs that were never cocultured with CD4+ T cells after 30 days. In parallel, we characterized the donors’ CCR5Δ32 status: donor 1 had a copy of the CCR5Δ32 mutation, whereas donors 2 and 3 had only WT alleles. Taken together, these results exclude the possibility that CD4+ T cells could confound our findings in LMs from HIV-1–infected people. Furthermore, we were able to detect CD3ε mRNA in lysed cells more than 30 days after coculture (data not shown), indicating that our assays were sensitive enough to detect the presence of contaminating T cells if they were present.

Liver macrophage viral outgrowth assay. We used a variety of cell lines to identify which would be the most robust at replicating HIV-1 from LMs (Supplemental Figure 3). Curiously, although CEMx174 cells have been previously described to not contain CCR5 on their surface, their replication of R5-tropic HIV-1 was comparable to CCR5-expressing MOLT-4 cells that have been recently described (11). In addition, CEMx174 cells gave the most reproducible results when inoculated with R5-tropic HIV-1, as has been described recently for macrophage-derived SIV (12). Next, we infected LMs in vitro using a bicistronic GFP-expressing R5-tropic strain of HIV-1 that has been previously shown to replicate robustly in human MDMs (13). We continued to detect HIV-1 RNA in LM supernatants for more than 100 days after infection despite periodic media change, strongly supporting productive infection and indicating that these were viruses that were released by LMs. Although the amounts of HIV-1 RNA released from infected LMs were lower than has been described for CD4+ T cells, they are consistent with previous reports of macrophage infection (4, 5). The LM supernatants were then transferred to CEMx174 cells, which began to fluoresce green after incubation (Supplemental Figure 4). Moreover, HIV-1 RNA was detectable in CEMx174 supernatants for up to 15 days despite media changes (Supplemental Figure 5).

To mimic recovery of LM-derived HIV-1 from ART-suppressed individuals, HIV-1–infected LMs were treated with antiretrovirals that were sufficient to inhibit infection. After 21 days of antiretroviral exposure, LMs were stimulated with interferon gamma (IFNG) followed by recombinant Tat (rTat) and their supernatants were transferred to CEMx174, testing whether HIV-1 released from LMs that themselves could not complete the HIV-1 life cycle (because of antiretrovirals) was sufficient to propagate infection in a viral outgrowth assay (VOA). Upon lysis, CEMx174cells were found to contain HIV-1 proviral DNA using a sensitive qPCR assay (Supplemental Figure 6), whereas control CEMx174 cells that were inoculated with media from uninfected LMs failed to show HIV-1 DNA. Additionally, CEMx174 cells that were inoculated with LMs treated with antiretroviral for 12 and 20 days propagated HIV-1 for up to 15 days after incubation (Supplemental Figure 5). Taken together, these results support the assumption that if LMs are productively infected, CEMx174 target cells can be used to propagate infectious virus in an adapted VOA.

LMs from HIV-1–infected people. To test the contribution of LMs to HIV-1 infection in vivo, we purified LMs from liver tissue of HIV-1–infected individuals. Fresh liver tissue was obtained from a deceased HIV-1–infected patient (N7) who had not been taking ART for more than 6 months and who had a plasma HIV-1 RNA level greater than 500,000 cp/ml. Following isolation and culture of LMs that contained no detectable T cells, we detected polyadenylated HIV-1 RNA that is found only in spliced and genomic RNA (14) in supernatants 18 days after purification, demonstrating that LMs can be infected with HIV-1 in vivo. We next examined whether infectious viruses were recoverable from LMs in HIV-1–infected people who were virologically suppressed on ART. Fresh liver explant tissues were obtained intraoperatively from 7 HIV-1–infected individuals undergoing liver transplantation and from 1 individual at the time of death (N9). These individuals were taking ART at the time of liver recovery and had undetectable plasma HIV-1 RNA levels. Total HIV-1 proviral DNA was detectable in low levels in bulk liver tissue from 7 of 8 participants (data not shown). A median of 5.5 × 105 LMs (range 4.8 × 105–1.4 × 106) were purified from all participants and T cell exclusion was confirmed in all but 1 subject (LT02) who had approximately 1 T cell in 100 LMs (99% pure) (Figure 1C).

IFNG+rTat was used to stimulate LMs from HIV-1–infected people more than 30 days after LM purification (Figure 2). In one instance (N9), bacterial contamination was noted early after LM purification; therefore, LMs were stimulated only after 6 days after isolation. Following stimulation of LMs, supernatants were filtered and transferred twice over a 15-day interval to CEMx174 cells. Proviral DNA was detected in target cells incubated with LM supernatants from 6 of 8 participants and never in negative control target cells that were incubated with media alone (Figure 3A and Table 1). However, with the exception of LT02, target cells from other isolated LMs did not show evidence of HIV-1 propagation by way of HIV-1 RNA (Table 1). We did not observe high-level replication, as has been seen from resting CD4+ T cells (11). LT02 was also distinct in having taken ART for the shortest duration prior to liver transplantation (Figure 3B and Table 1). Taken together, these results demonstrate that LMs can harbor HIV-1 for prolonged periods, but these are inert with respect to propagation unless sampling occurred very early after ART-mediated suppression. We confirmed the presence of replication-competent HIV-1 in resting CD4+ T cells in this cohort. Using the conventional quantitative VOA that has been previously described and used extensively to quantify the latent reservoir (15), we estimated a similarly sized reservoir in resting CD4+ T cells from 2 of the people who also had contemporaneous LM VOA performed (Supplemental Table 1).

Figure 2 Schema for VOA performed on purified LMs. After purification of primary human LMs from HIV-1–infected subjects, cells were maintained ex vivo for more than 30 days. The VOA was performed on LMs from individuals taking ART. Isolated LMs were treated with Resimmune over 48 hours to deplete any remaining T cells. Following stimulation with IFNG and HIV-1 rTat, LM supernatants were filtered and transferred to target cells twice over 15 days and incubated. In addition, target cells were added to LMs. Cell-associated and supernatant HIV-1 DNA and RNA levels were measured on day 11 in target cells.

Figure 3 HIV-1 transmission from LMs of HIV-1–infected individuals. (A) LM VOA results for all HIV-1–infected individuals. Using qPCR, HIV-1 proviral DNA was measured in lysed target cells that were incubated with LM supernatants from each donor (red diamonds). Error bars represent mean ± SEM for some with multiple positive values. Grey triangles indicate negative control target cells that were inoculated with media alone before lysis. N7 is not included in this figure because HIV-1 RNA was detectable in LM supernatants before initiation of the LM VOA. (B) LM VOA yields HIV-1 RNA only in people with ART exposure less than 1 year. HIV-1 RNA was measured in target cell supernatants in LM VOA from all people, and in N7 LM supernatants directly. HIV-1 RNA was only detectable from LMs in 2 people who were also those who had the least amount of exposure to ART before liver explantation. N9 is not included in this figure because the duration of ART prior to liver explantation was not available. LOD, limit of detection; ND, not done.

Table 1 Liver macrophage viral outgrowth assay results

We were not able to differentiate between unintegrated and integrated DNA present in the LMs before VOA because of the limited number of LMs available for sequencing. We were only able to amplify a 160-bp portion of the HIV-1 pol gene from LM VOA of 5 individuals. These sequences closely matched bulk liver sequences, sharing a median p distance of 0.012 (range 0–0.031) with their respective bulk liver tissue HIV-1 sequences; in contrast, the median interindividual p distance was 0.037 (range 0.023–0.068, P < 0.005), despite limited variability in this region of the pol gene (nucleotide position 4900-5061 in reference HXB2 genome) (Figure 4).

Figure 4 HIV-1 pol sequence analysis reveals that liver macrophages and bulk liver contain genetically similar viruses. p distances between HIV-1 pol amplicons (160 bp) from purified LMs after culture or from bulk liver tissue were calculated. All comparisons were of 160 nucleotide amplicons, except comparisons including subject LT01, which used 44 nucleotide amplicons as that was the largest obtainable sequence. The LT02 LM sequence included in the analysis was amplified from RNA. (A) Pairwise p distances. Dark red indicates lower values (i.e., more genetically similar), and dark blue indicates higher values (i.e., more genetically distinct). (B) Comparison of inter- and intraindividual p distances between LMs and bulk liver amplicons from the same donor to p distances between LMs and bulk liver amplicons from different individuals. Error bars represent mean ± SD. P value was calculated by 2-tailed Mann Whitney U test, **P < 0.005.

HIV-1–infected LMs persist variably in vitro. Previous estimates of the t 1/2 of HIV-1–infected macrophages (14 days) have been relatively short compared with resting CD4+ T cells (44 months) (16). We attempted to measure the longevity of HIV-1–infected LMs using the LMs that we had previously infected in vitro. We observed minimal cell death within the first 4 weeks of infection. HIV-1 RNA remained detectable in LM supernatants from all 3 participants for greater than 30 days of infection (doi), and thereafter gradually decayed (Figure 5, A–C). Our findings were similar in LMs infected with the R5-tropic BaL HIV-1 strain (Supplemental Figure 7). LMs that were lysed on 181 doi revealed intracellular HIV-1 RNA. HIV-1 RNA from donor 1 LMs was largely undetectable in supernatants by 100 doi but showed occasional blips, whereas HIV-1 RNA was still detectable in LM supernatants from donors 2 and 3. HIV-1 RNA release continued even when antiretrovirals (TDF/FTC/RTG) were added to LM media in concentrations that were sufficient to inhibit replication in CD4+ T cells (Figure 5D). We observed an inconstant pattern with regard to HIV-1 dynamics in LMs during the 24 days of antiretrovirals, whereas GFP+ cells were visible throughout antiretroviral incubation (data not shown). Based on the viral kinetics during ART, the median of the infected LMs t 1/2 was estimated at 6.8 days (range 3.8–55 days) (Figure 5, A–C), although in some wells persistent HIV-1 RNA release prevented accurate calculation of t 1/2 . It should be noted that the t 1/2 was calculated based on decay kinetics of released RNA and this could possibly be longer because of the existence of virus-containing compartments in LMs (17). Indeed, we observed abundant intracellular HIV-1 RNA in LMs even in the absence of concomitant release of HIV-1 RNA in the supernatant (Supplemental Figure 7), consistent with this phenomenon.