ABSTRACT
Follicular helper T (TFH) cells have been shown to support productive human immunodeficiency virus type 1 (HIV-1) replication and to serve as a key component of the latent viral reservoir. However, the viral characteristics of this latent reservoir and the clinical relevance of this reservoir remain unclear. In this study, we assessed the tropic composition of latent viruses from peripheral TFH (pTFH), non-TFH memory, and naive CD4+ T cells from individuals with HIV-1 infections on suppressive combined antiretroviral therapy (cART). X4-tropic latent HIV-1 was preferentially enriched in pTFH cells compared to levels in the other two subsets. Interestingly, the ratio of X4-tropic latent HIV-1 in pTFH cells not only was robustly and inversely correlated with blood CD4+ T cell counts across patients but also was prognostic of CD4+ T cell recovery in individuals on long-term cART. Moreover, patients with higher X4-tropic latent HIV-1 ratios in pTFH cells showed greater risks of opportunistic coinfections. These findings reveal the characteristics of latent HIV-1 in TFH cells and suggest that the ratio of X4-tropic latent HIV-1 in pTFH cells is a valuable indicator for disease progression and cART efficacy.
IMPORTANCE TFH cells have been shown to harbor a significant amount of latent HIV-1; however, the viral characteristics of this reservoir and its clinical relevance remain largely unknown. In this study, we demonstrate that X4-tropic latent HIV-1 is preferentially enriched in pTFH cells, which also accurately reflects the viral tropism shift. The ratio of X4-tropic proviruses in pTFH cells but not in other memory CD4+ T cell subsets is inversely and closely correlated with blood CD4+ T cell counts and CD4+ T cell recovery rates with cART. Our data suggest that the ratio of X4-tropic provirus in peripheral TFH cells can be easily measured and reflects disease progression and treatment outcomes during cART.
INTRODUCTION
The persistence of human immunodeficiency virus type 1 (HIV-1) despite long-term suppressive combined antiretroviral therapy (cART) has prevented curing this disease (1, 2). One of the major challenges for understanding HIV-1 persistence is the obscure nature of latent viral reservoirs, which remain only partially elucidated (1, 3). These latent reservoirs, which primarily reside in resting memory CD4+ T cells, have been shown to harbor integrated replication-competent HIV-1, which could cause viral rebound once cART is interrupted (4). Follicular helper T (TFH) cells have recently been identified as an important compartment for HIV-1 infection (5, 6) and could also serve as viral latent reservoirs (7). This lineage of CD4+ T cells is characterized by high expression levels of C-X-C chemokine receptor type 5 (CXCR5) and the transcription factor B cell lymphoma 6 (BCL6), as well as by the production of interleukin-21 (IL-21) (8, 9). In contrast to other CD4+ T cell populations that are depleted in infected subjects, TFH cells significantly accumulated in the lymph nodes (LNs) in individuals with chronic HIV-1 infections (5, 10, 11) and macaques with chronic simian immunodeficiency virus (SIV) infections (12). Instead of facilitating the generation of broadly neutralizing antibodies, this expansion of TFH cells has been linked to higher levels of viremia, impaired B cell immunity, and the onset of hypergammaglobulinemia in individuals with HIV-1 infections (10–12). These findings suggest that TFH cells not only are a significant cellular reservoir of replication-competent HIV-1 but also play crucial roles in the perturbation of B cell differentiation and the dysregulation of the antibody response in chronic HIV-1 infection. Although the role of TFH cells in latent HIV-1 infection has been the subject of intensive study in recent years, there is still little known regarding the characteristics of viruses that are harbored in TFH cells. In this study, we found that peripheral TFH (pTFH) cells were enriched for X4-tropic latent HIV-1. The ratio of X4-tropic latent HIV-1 in pTFH cells was significantly correlated with the number of peripheral CD4+ T cells and the recovery of peripheral CD4+ T cells during cART, as well as with disease progression.
RESULTS
X4-tropic HIV-1 is enriched in TFH cells.We collected peripheral blood from 41 individuals with chronic HIV-1 infections on suppressive cART. These individuals had documented diagnoses of HIV-1 infection and had been treated with cART for a period ranging from 20 to 144 months, and the plasma HIV-1 RNA had been below 20 copies per milliliter for each individual for at least 2 years (Table 1, patient IDs 1 to 41). The study flowchart is shown in Fig. 1A. We analyzed three subsets of CD4+ T cells in peripheral blood mononuclear cells (PBMCs) based on the expression of CXCR5 and CD45RO (Fig. 1B). These subsets included CXCR5+ pTFH cells, CXCR5− CD45RO− non-TFH naive CD4+ T (naive CD4) cells, and CXCR5− CD45RO+ non-TFH memory CD4+ T (mCD4) cells. pTFH cells had conventional TFH phenotypic and functional profiles (Fig. 1C).
Clinical characteristics of the individuals with HIV-1 infections involved in this study
X4-tropic latent HIV-1 is enriched in TFH cells. (A) Flow chart for determining the tropism of HIV-1 residing in CD4+ T cells. (B) Representative phenotype, sorting strategy, and sorting purity of peripheral pTFH cells (CD3+ CD4+ CXCR5+), non-TFH memory (CD3+ CD4+ CXCR5− CD45RO+) CD4+ T cells (mCD4), and peripheral non-TFH naive (CD3+ CD4+ CXCR5− CD45RO−) CD4+ T cells. (C) Sorted CD4+ T cells were cocultured with B cells at a 1:1 ratio in the presence of 100 ng/ml staphylococcal enterotoxin B (SEB). Cytokine production was measured by ELISA after 5 days. (D) HIV-1 DNA load in sorted mCD4, pTFH, and naive CD4 cell populations from individuals with chronic HIV-1 infections (N = 41). (E) Frequencies of replication-competent latent HIV-1 in sorted CD4+ T cell populations (N = 29). (F) Frequencies of replication-competent latent HIV-1 per proviral DNA copy (N = 29). (G) The proportion of X4-tropic HIV-1 proviral DNA copies in sorted CD4+ T cell populations (N = 41). (H) The proportion of X4-tropic latent HIV-1 in sorted CD4+ T cell populations (N = 29). (I) Frequencies of X4-tropic replication-competent latent HIV-1 in sorted pTFH and mCD4 cells from individuals with chronic HIV-1 infections (N = 29). For panels C to I, the results are shown as the means. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) One-way ANOVA was used for this analysis. The data were from three experiments with cells from healthy donors. The Friedman test was used for the analysis shown in panel D. The Wilcoxon test was used for analyses shown in panels E to I. For panels E, F, H, and I, 29 of the 41 HIV-1 chronically infected individuals were tested with QVOA.
To measure latent HIV-1 in the aforementioned CD4+ T cell subsets, we performed quantitative real-time PCR (RT-qPCR) and quantitative viral outgrowth assay (QVOA). In the 41 enrolled subjects, the HIV-1 DNA level in pTFH cells was comparable to that in mCD4 cells, which is commonly considered an HIV-1 latent reservoir, and was significantly higher than that in naive CD4+ T cells (Fig. 1D). However, pTFH cells contained a larger pool of functionally inducible latent HIV-1, as shown by the higher levels of infectious virus outgrowth in the QVOA (Fig. 1E and F). These findings suggest that pTFH cells not only are important hosts for proviral HIV-1 DNA but also represent a major latent reservoir of replication-competent viruses.
Since pTFH cells characteristically expressed high levels of the HIV-1 coreceptor C-X-C chemokine receptor type 4 (CXCR4) during all phases of activation and in chronic HIV-1 infection (data not shown), we speculated that latent HIV-1 in pTFH cells has a distinct viral tropic preference. Therefore, we analyzed the tropism of both proviral DNA and outgrowth viruses from the QVOA in pTFH and mCD4 cells using deep sequencing. Indeed, we found that pTFH cells harbored a higher percentage of X4-tropic HIV-1 proviral DNA than mCD4 cells (Fig. 1G). Accordingly, the percentage of X4-tropic outgrowth HIV-1 in pTFH cells was also markedly higher than that in mCD4 cells (Fig. 1H). Considering both the levels of replication-competent viruses and the proportion of X4-tropic viruses, pTFH cells harbored a pool of X4-tropic latent HIV-1 that was twice as large as that in mCD4 cells (0.50 ± 0.18 and 0.24 ± 0.08 infectious units per million cells [IUPM] in pTFH and mCD4, respectively; means and standard errors of the means [SEM]; P < 0.05; Fig. 1I).
We also observed that the HIV-1 P24 protein in the QVOA culture supernatant of chronic aviremic HIV-1 individuals on cART was not detected until 15 days after activation and remained undetectable in the no-stimulation condition (Fig. 2A). In contrast, the HIV-1 P24 protein in the QVOA culture supernatant of viremic HIV-1 individuals pre-cART (patient IDs 42 to 44) became readily detectable at about day 3 with stimulation or after culture without stimulation for about 6 days (Fig. 2B). These findings suggest that the outgrowth viruses from individuals with chronic HIV-1 infections on long-term cART were released from the reactivated latent pool rather than from productive infected cells.
Levels of supernatant HIV-1 P24 in the QVOA. pTFH and mCD4 cell populations were sorted from patients with chronic (A; N = 3 [donors A, B, and C]) or viremic (B; N = 3 [donors D, E, and F]) HIV-1 infections. HIV-1 replication was assessed every 3 days with or without anti-CD3 plus anti-CD28 stimulation by measuring supernatant p24 levels using an ELISA kit. The limit of detection of this assay was 1 pg/ml, and the first time point (days in coculture) of detectable p24 in the supernatant is shown for each sample. Red, orange, and blue indicate different HIV-1 donors; circles indicate pTFH cells; triangles indicates mCD4 cells; solid lines indicate cells stimulated by anti-CD3 plus anti-CD28; and dotted lines indicate cells that were not stimulated.
To provide additional evidence regarding the tropism of latent HIV-1 in pTFH cells that complemented sequence-based algorithm predictions, we performed a series of functional experiments. In the present cohort, individuals were stratified into the X4-tropic high (X4high; N = 21) and the X4-tropic low (X4low; N = 20) groups according to the median percentage of X4-tropic proviruses in pTFH cells (Fig. 3A). We isolated outgrowth viruses from pTFH or mCD4 cells from both X4high (N = 6) and X4low (N = 6) individuals. The isolated viruses were then cocultured with PHA-activated CD4+ T cells derived from healthy donors in the presence of a CCR5 inhibitor (Maraviroc) and/or a CXCR4 inhibitor (AMD3100) (Fig. 3B and C). Infection with outgrowth viruses from X4high individuals was highly sensitive to AMD3100 (Fig. 3B), whereas outgrowth viruses from the X4low group were not significantly affected by this molecule (Fig. 3C). Moreover, in the X4high group, the combination of the two inhibitors did not yield an additional inhibitory effect on the infection of CD4+ T cells compared to applying AMD3100 alone, suggesting that the majority of outgrowth viruses released from the pTFH cells of X4high individuals were indeed X4 tropic. The coreceptor-dependent infectivity assays confirmed the X4 tropism of latent HIV-1 in pTFH cells.
Outgrowth HIV-1 released from the pTFH cells of X4high individuals are functionally X4 tropic. (A) According to the proportion of X4-tropic HIV-1 proviral DNA in pTFH cells, 41 individuals with chronic HIV-1 infections were divided into two groups. Patients with a proportion higher than the median were defined as X4high (N = 21), and the patients were otherwise defined as X4low (N = 20). (B and C) Outgrowth HIV-1 from pTFH or mCD4 cells of either X4high (B; N = 6) or X4low (C; N = 6) individuals were used to infect activated CD4+ T cells from healthy donors in the presence of the R5 and/or X4 inhibitor. These results were collected 7 days postinfection by flow cytometry. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Friedman test). The data are from three experiments with outgrowth viruses from six individuals with HIV-1 infections. R5 inhibitor, Maraviroc; X4 inhibitor, AMD3100.
To further demonstrate the capability of X4-tropic HIV-1 to establish latent infections in pTFH cells, we used a previously reported primary CD4+ T cell model of HIV-1 latency (13). In both freshly isolated samples from healthy donors and the Bcl-2-overexpressing primary CD4+ T cell model, pTFH cells were more permissive than mCD4 cells both to pseudotype and to wild-type X4-tropic HIV-1 infection (Fig. 4A and B). Upon stimulation by anti-CD3/CD28, suberoylanilide hydroxamic acid (SAHA), or bryostatin-1, pTFH cells exhibited significantly higher levels of latent HIV-1 reactivation than mCD4 cells (Fig. 4C), indicating that pTFH cells not only are capable of accommodating competent latent X4-tropic HIV-1 but also are superior to mCD4 cells at doing so.
pTFH cells support the establishment and reactivation of the X4-tropic HIV-1 latent reservoir in vitro. (A) pTFH and mCD4 cells were isolated from healthy donor PBMCs. Sorted cells were infected with wild-type X4-tropic HIV-1 NL4-3 or GFP-expressing CM6/X4-tropic-Env and were analyzed 5 days after infection by flow cytometry. (B) The infectivity of BCL-2-overexpressing mCD4 or pTFH cells by CM6/X4-tropic-Env. (C) Latently infected cells were activated by anti-CD3/CD28, SAHA, or bryostatin-1 and were analyzed by flow cytometry after 5 days of activation. Data are from five independent experiments. The results are shown as the means. **, P < 0.01; ***, P < 0.001 (paired t test).
The ratio of X4-tropic proviral DNA in pTFH cells is associated with the level of replication-competent HIV-1 DNA.We next sought to investigate the potential clinical relevance of replication-competent latent X4-tropic HIV-1 in pTFH cells. To our surprise, the X4low and X4high groups showed similar levels of total HIV-1 DNA (Fig. 5A). However, the frequency of inducible replication-competent viruses was significantly higher in the X4high group (Fig. 5B). Furthermore, we assessed the tropism of outgrowth viruses from the QVOA by deep sequencing. In the X4low group, X4-tropic outgrowth viruses were almost undetectable in both pTFH and mCD4 cells, and the frequency of non-X4-tropic outgrowth viruses was not significantly different between pTFH and mCD4 cells (Fig. 5C). In contrast, the frequency of X4-tropic outgrowth viruses, but not that of non-X4-tropic outgrowth viruses, was significantly higher in X4high individuals than in X4low individuals (Fig. 5C and D). X4-tropic viruses were dominant in the pTFH cells of X4high individuals, suggesting that these X4-tropic replication-competent viruses accounted for the majority of the HIV-1 DNA content in pTFH cells (Fig. 1E and F). These data suggest that there is a close association between the ratio of X4-tropic proviruses in pTFH cells and the level of replication-competent HIV-1 proviral DNA.
X4-tropic HIV-1 accounts for a larger proportion of the replication-competent latent reservoir in individuals with chronic HIV-1 infections on cART. (A) Frequencies of replication-competent latent HIV-1 in sorted CD4+ T cell populations from individuals with chronic HIV-1 infections in both the X4high (N = 15) and X4low (N = 14) groups. (B) The proportion of X4- or non-X4-tropic replication-competent latent HIV-1 in sorted CD4+ T cell populations. The results are shown as the means and SEM. *, P < 0.05; ***, P < 0.001. Ordinary one-way ANOVA test was used for the analyses shown in panels A and B. (C) Analysis performed using the Wilcoxon test. (D) Three X4high individuals (D1 to D3) and three X4low individuals (D4 to D6) are presented. The phenotypic tropism of the lineages was identified based on the FPR for predicting the X4-tropic lineages using the Geno2Pheno algorithm. The lineages with FPR values of less than or equal to 10% were considered X4 tropic (red), whereas the values of more than 10% were non-X4 tropic (blue).
The ratio of X4-tropic proviral DNA in pTFH cells is correlated with treatment outcomes.We next examined whether the X4-tropic HIV-1 latent reservoir in pTFH cells was correlated with disease progression and treatment efficacy in individuals on cART. A schematic diagram of the analytic time points is shown in Fig. 6A. Pearson’s correlation analysis showed that there was no correlation between CD4+ T cell numbers at enrollment (pre-cART) and the ratio of X4-tropic proviral DNA (Fig. 6B and C). However, after at least 1 year of cART, the ratio of X4-tropic proviral DNA in pTFH cells, but not that in mCD4 cells, was negatively correlated with the number of total CD4+ T cells in the blood (pTFH cells, R = −0.8010 and P < 0.0001; mCD4 cells, R = −0.3116 and P = 0.0775) (Fig. 6D and E). Moreover, the number of CD4+ T cells was markedly higher in the X4low group than in the X4high group (Fig. 6F). Disease progression and treatment efficacy are commonly evaluated by the CD4+ T cell count recovery in the blood of patients. We found that the ratio of X4-tropic proviral DNA in pTFH cells from cART responders (absolute CD4+ T cell count of ≥200 CD4 cells/μl after at least 1 year of cART) was significantly lower than that in nonresponders (absolute CD4+ T cell count of <200 CD4 cells/μl after at least 1 year of cART) (Fig. 6G). In contrast, the ratio of X4-tropic proviral DNA in mCD4 cells was not significantly different between these two groups (Fig. 6H). More interestingly, when we regrouped the individuals by the ratio of X4-tropic proviral DNA in mCD4 cells (Fig. 6I), the number of CD4+ T cells also was not significantly different between the two groups (Fig. 6J).
Correlation between the ratio of X4-tropic proviral DNA in pTFH cells and CD4+ T cell numbers pre-/post-cART. (A) Schematic diagram of analytic time points. Thirty-three of the 41 HIV-1 donors participated in this investigation. (B and C) Correlations between the ratio of X4-tropic proviral DNA in pTFH cells (B) or mCD4 cells (C) and peripheral CD4+ T cell numbers at the point of enrollment (pre-cART) (N = 33; Pearson correlation). (D and E) Correlations between the ratio of X4-tropic proviral DNA in pTFH (D) or mCD4 cells (E) and peripheral CD4+ T cell numbers after at least 1 year of cART (N = 33; Pearson correlation). (F) The numbers of peripheral CD4+ T cells at the reference point in X4high (N = 20) and X4low individuals (N = 13). (G and H) The proportion of X4-tropic proviral DNA in pTFH (G) or mCD4 (H) cells from HIV-1 treatment responders (≥200 CD4 cells/μl; N = 19) or treatment nonresponders (<200 CD4 cells/μl; N = 14) at the reference point. (I) The distribution of the proportion of X4-tropic proviral DNA in mCD4 cells from 41 individuals with HIV-1 infections. The median is shown by the gray vertical dashed line. Individuals with HIV-1 infections were redivided into two groups based on the proportion of X4-tropic HIV-1 provirus in mCD4 cells. Patients with a proportion higher than the median were defined as mCD4-X4-tropic high (mCD4-X4high; N = 21), and patients were otherwise defined as mCD4-X4-tropic low (mCD4-X4low; N = 20). (J) The numbers of peripheral CD4+ T cells at the reference point in mCD4-X4-tropic high (N = 20) and low (N = 13) individuals. The results are shown as the means and SEM. ***, P < 0.001 (unpaired t test).
These findings revealed an inverse correlation between the blood CD4+ T cell number and the proportion of X4-tropic provirus in pTFH cells in cART-treated patients. We then assessed whether this X4-tropic provirus percentage is indicative of the dynamics of the blood CD4+ T cell number in individuals. Thus, we examined the blood CD4+ T cell counts of 20 chronically infected individuals for 15 to 30 months (median, 27 months), specifically for 6 to 12 months (median, 12 months) before and 6 to 18 months (median, 12 months) after the reference point when viral tropism was determined (month 0), as indicated in Fig. 6A. The peripheral CD4+ T cell counts and the viral loads in each individual were assessed every 3 months. All 20 individuals were on suppressive cART during the whole study period and had an undetectable viral load, as measured by typical clinical assays (plasma HIV-1 RNA copies of <20 per ml) for at least 2 years at month 0. The numbers of peripheral CD4+ T cells was not significantly different in the X4low group (N = 7) and the X4high group (N =13) at enrollment (pre-cART) (Fig. 7A). Interestingly, consistent with our previous findings, the numbers of peripheral CD4+ T cells were significantly higher in the X4low group (N = 7) than in the X4high group (N =13) at month 0 (post-cART) (Fig. 7B).
Ratio of X4-tropic proviral DNA in pTFH cells is correlated with CD4+ T cell recovery. Twenty of 41 HIV-1 donors participated in the long-term follow-up investigation. (A) CD4+ T cell numbers in X4low (N = 7) and X4high (N = 13) individuals at enrollment (pre-cART). (B) CD4+ T cell numbers in X4low (N = 7) and X4high (N = 13) individuals after 1 year of cART (after the reference point). (C) CD4+ T cell numbers in X4low (N = 7) and X4high (N = 13) individuals on cART. Month 0, reference point. (D) Correlations between the proportion of X4-tropic proviral DNA in pTFH cells at the reference point and peripheral CD4+ T cell numbers of different time points (measured every 3 months). CD4+ T cell numbers were detected in individuals with chronic HIV-1 infections on cART for 2 years (month 0, reference point; Pearson correlation). t tests were used for the analyses shown in panels A and B. The results are shown as the means and SEM. ***, P < 0.001.
To assess whether X4-tropic proviral DNA in pTFH cells reflected or was indicative of the trends in peripheral CD4+ T cell recovery during cART, we analyzed the correlation between the CD4+ T cell counts at different time points and the ratio of X4-tropic proviral DNA in pTFH cells at the reference point. The ratio of X4-tropic proviral DNA in pTFH cells was negatively correlated with peripheral CD4+ T cell numbers at least 6 months before the reference point and 12 months after the reference point (all P values of <0.05; Fig. 7C and D). Moreover, the increase in the CD4+ T cell count over time was remarkably larger in the X4low group than in the X4high group (Fig. 8A). The rate of growth (the difference between two adjacent detection points) was higher in the X4low group than in the X4high group (Fig. 8B). Similarly, three out of thirteen (23.1%) individuals of the X4high group had concurrent tuberculosis, and six (46.2%) had concurrent pneumonia during this study. Meanwhile, no cases of concurrent infection were reported in the X4low group (Fig. 8C). Together, our data suggest that even though there was no significant difference in the CD4+ T cell count pre-cART, the ratio of X4-tropic proviral DNA in pTFH cells post-cART was closely associated with the CD4+ T cell count, and more importantly, the ratio of X4-tropic proviral DNA reflected the recovery of CD4+ T cells and the risk of concurrent infection. Therefore, tropism analysis based on sequencing proviral DNA in pTFH cells may serve as a valuable indicator for treatment outcomes and has great clinical potential for the broad population of HIV-1-positive individuals on cART.
Ratio of X4-tropic proviral DNA in pTFH cells is correlated with disease progression during cART. Twenty of 41 HIV-1 donors participated in the long-term follow-up investigation. (A) Change in the CD4+ T cell number (compared to month 0) in X4low (N = 7) and X4high (N = 13) individuals during cART. (B) After the reference point, the rate of CD4+ T cell incremental change (the CD4+ T cell count at each time point was compared to that at the last time point) in X4low (N = 7) and X4high (N = 13) individuals during cART. (C) Concurrent infections in X4low (N = 13) and X4high (N = 19) individuals. TB, tuberculosis.
DISCUSSION
X4-tropic viral variants appear in approximately 50% of individuals with chronic HIV-1 infections during the natural course of the disease (14). The emergence of plasma X4-tropic viruses is often associated with a decrease in the CD4+ T cell number and rapid disease progression (15). Although previous studies have indicated that the ratio of X4-tropic HIV-1 DNA increases during cART (15), the nature and the clinical relevance of the X4-tropic viral reservoir remain largely unknown. In this report, we showed that the level of X4-tropic proviral DNA was significantly higher in pTFH cells than in mCD4 cells. Moreover, QVOA indicated that both the level and the ratio of X4-tropic latent HIV-1 were markedly higher in pTFH cells than in mCD4 cells. The CXCR4-specific inhibitor AMD3100 effectively reduced the infectivity of outgrowth viruses from pTFH cells, supporting the X4 tropism of these viruses. Consistent with the enrichment of X4-tropic proviral DNA, we found that the frequency of replication-competent viruses was markedly higher in pTFH cells than in mCD4 cells. These findings suggested that pTFH serves as the major compartment for X4-tropic HIV-1 infection, replication, and latency.
Emerging evidence suggests that alterations in the TFH cell number and function are closely associated with the balance between HIV-1 and host immunity and serve as a marker for disease progression (16). To add to these findings, here we provide evidence supporting the potential prognostic values of the X4-tropic provirus ratio in pTFH cells. We found that this ratio was closely associated with the number of peripheral CD4+ T cells in cART-treated individuals. The ratio of X4-tropic proviral DNA in pTFH cells was inversely correlated with the circulating CD4+ T cell numbers at least 6 months before and 12 months after the reference point. Furthermore, the risk of concurrent opportunistic diseases was significantly increased in individuals whose pTFH cells harbored a higher ratio of X4-tropic viruses.
Thus, this study suggests that pTFH cells, which are selectively permissive to X4-tropic viruses, serve as a compartment for the accumulation and storage of X4-tropic proviruses and can accurately reflect the viral tropism shift. Interestingly, we found that the increase in the proportion of X4-tropic proviruses in pTFH also implicated a similar enrichment of X4-tropic HIV-1 proviruses in LN TFH cells (data not shown). Recent studies have demonstrated that germinal center (GC) TFH cells are enriched in lymph nodes of chronically HIV/SIV-infected individuals on ART (17, 18) and display a productive and latent infection viral reservoir. These TFH cells express a high level of PD-1, engagement of which with PD-L1 inhibits the IL-21 production by GC TFH cells and thereby impairs B-cell function and antibody production (10). Therefore, TFH cells play a dual role in chronic HIV/SIV infection (i) as a significant X4-tropic provirus reservoir and (ii) as a regulator of the host’s humoral immunity against the viruses (17). However, whether and how HIV-1 with different tropisms specifically affects TFH function and what role HIV-1 tropism shift may play in this process remain to be further illuminated. Although the detailed mechanism is still unclear, here we provide evidence showing close association between the enrichment of X4-tropic HIV-1 in pTFH and the disease risk progression on cART. Since viral outgrowth assay is time-consuming and costly, measurement of pTFH HIV-1 tropism may provide an alternative approach for measuring the characteristics of HIV-1 proviruses that reflects disease progression. With the experimental procedures mentioned above, the measurement of the X4-tropic ratio in pTFH is easily performed and has significant clinical potential.
MATERIALS AND METHODS
Human subjects.Peripheral blood was obtained from 44 individuals with chronic HIV-1 infections and 3 viremic HIV-1 individuals, and primary pTFH (CD3+ CD4+ CXCR5+), mCD4 (CD3+ CD4+ CXCR5− CD45RO+), or naive CD4 (CD3+ CD4+ CXCR5− CD45RO−) cells were isolated (Table 1). The 41 individuals with chronic HIV-1 infections had received cART for at least 20 months and had maintained undetectable HIV-1 viremia (<50 HIV-1 RNA copies per ml of plasma) for at least 12 months before this study. Cells from 29 of the 41 HIV-1 chronically infected individuals were tested with the QVOA. We successfully obtained blood CD4+ T cell numbers before cART at the enrollment point and at the reference point after at least 1 year of cART, as well as concurrent infection information, from 33 of the 41 HIV-1 chronically infected individuals. However, the complete follow-up records of CD4+ T cell numbers every 3 months from the enrollment point were obtained from only 20 of these 33 patients (Table 1). All of these individuals were recruited from Guangzhou Eighth People’s Hospital. Buffy coats were derived from the blood of healthy donors and were used for in vitro experiments. All human samples were anonymously coded in accordance with the local ethical guidelines (as stipulated by the Declaration of Helsinki). Written informed consent was provided by all study participants, and the protocol was approved by the institutional review board (IRB) of Guangzhou Eighth People’s Hospital (Guangzhou, China).
Isolation and culture of primary CD4+ T cell populations.The indicated CD4+ T cell populations were isolated by flow cytometry, and the culture conditions were previously described (19). Details on the antibodies used for flow cytometry are provided in Table 2. Data were acquired on an LSR II Fortessa or a FACSAria II flow cytometer (BD Biosciences) and were analyzed with FlowJo software (Tree Star).
Fluorochrome-conjugated antibodies used for flow cytometry
QVOA.The QVOA was carried out as previously described (20). Briefly, CD4+ T cell populations from individuals with HIV-1 infections were isolated as described above. Sorted cells were plated in replicate-limiting dilutions of 1 × 106, 2 × 105, 4 × 104, 8 × 103, and 1,600 cells per well and were stimulated with 1 μg/ml anti-CD3/CD28, 100 U/ml IL-2, and 2 × 106 allogeneic irradiated PBMCs from healthy donors. After 24 h of coculture, anti-CD3/CD28-activated CD4+ T cells from healthy donors were added to each well. Cultures were then incubated for 24 days, and supernatants were harvested and replaced every 3 days. HIV-1 production was measured by a P24 antigen capture enzyme-linked immunosorbent assay (ELISA; Perkin Elmer). The infectious units per million cells (IUPM) was estimated by a maximum likelihood method (21).
Measurement of HIV-1 proviral DNA.Genomic DNA was extracted from sorted CD4+ T cell subsets using a Quick-gDNA MicroPrep kit (Zymo Research). Real-time quantitative PCR was applied with the TaqMan (Life Technologies) gene expression assay using gag primers and probes (22). The sequences of the primers and probes are shown in Table 3.
Gene-specific primers and probes
Deep sequencing and the prediction of viral tropism.Genomic DNA from sorted CD4+ T cell populations was extracted as described above. Proviral DNA was extracted with a Quick-gDNA MicroPrep kit (Zymo Research). The sample preparation for deep sequencing was performed as previously described (22–24). Briefly, the env gene was amplified by two rounds of nested PCR. The sequences of primers used in this process are included in Table 3. The products of nested PCR were then purified using Agencourt AMPure XP beads (Beckman Coulter) and quantified using a Qubit fluorometric quantitation double-stranded DNA HS assay (Life Sciences, Invitrogen). DNA libraries were constructed using a NEBNext Ultra DNA library prep kit and were analyzed on an Agilent 2100 Bioanalyzer with a high-sensitivity DNA chip (Agilent Technologies) for size confirmation. The barcoded sequencing libraries were quantified by quantitative PCR using a KAPA library quantification kit (KAPA Biosystems). Finally, the sequencing libraries were prepared by following the MiSeq reagent kit preparation guide (Illumina) and were loaded onto a MiSeq sequencer (Illumina) for paired-end 250-bp reads. The raw base call files of each sample were first demultiplexed into a FASTQ format using bcl2fastq conversion software (Illumina). The quality control of FASTQ data was performed by FastQC software (www.bioinformatics.babraham.ac.uk), and the data were aligned to the Nucleotide Sequence Database (www.ncbi.nlm.nih.gov/genbank) using the basic local alignment search tool (BLAST) to avoid data distortion caused by experimental contamination. To ensure sequence quality, sequences with low quality were further trimmed or removed using Btrim software (http://graphics.med.yale.edu/trim). Sequences were trimmed if the average quality score of 5 continuous bases was less than 20, and sequences of less than 100 bases or that contained undetermined bases were removed. For demultiplexing, Cutadapt software was used to discard sequences without barcodes and to sort the sequences into the corresponding tagged samples based on their barcodes (https://cutadapt.readthedocs.io/en/stable). Paired-end reads with an overlap of ≥20 bases were merged into full-length sequences by FLASH software, and other reads that could not be joined were removed (http://www.cbcb.umd.edu/software/flash).
The high-quality sequence data sets were subsequently evaluated with the coreceptor tropism prediction algorithm Geno2pheno[coreceptor] (https://coreceptor.geno2pheno.org), which predicts HIV-1 coreceptor usage based on a database containing the typical motifs of the V3 loop of known phenotypic tropism based on a support vector machine. The false-positive rate (FPR) cutoff used to discriminate between CCR5 and CXCR4 was set to 10%, which corresponded to an X4 virus with a value of ≤10 and a non-X4 virus with a value of ≥10 (based on recommendations from the European Consensus Group on the Clinical Management of HIV-1 Tropism Testing) (25).
In vitro HIV-1 infection.CD4+ T cells from healthy donors were activated by 1 μg/ml anti-CD3/CD28 monoclonal antibodies (MAbs) and 100 U/ml IL-2 for 3 days, followed by infection with wild-type HIV-1NL4-3 and HIV-1 reporter viruses. HIV-1 reporter viruses were generated by cotransfecting 293T cells with pNL4-3-Δ6-drEGFP (CM6) and an X4 env expression vector (pX4-tropic-Env) or pVSV-G. Productive infection was determined by evaluating green fluorescent protein (GFP) or P24 expression using flow cytometry 5 days postinfection.
Generation of a primary cell model of HIV-1 latency.Primary pTFH and mCD4 cells were isolated from the PBMCs of healthy donors. Bcl-2-expressing CD4+ primary cells were established as described in a previous report (13). These cells were then infected by spinoculation with HIV-1 reporter viruses, such as HIV-1 CM6/X4-tropic-Env or HIV-1 vesicular stomatitis virus glycoprotein (VSV-G). After 4 to 6 weeks, allowing the infected cells to return to a resting state, GFP− cells were sorted by flow cytometry and were reactivated by various reagents.
Statistics.Statistical analysis was performed with GraphPad Prism 7. For data with a normal distribution, we used a Student's t test, and a nonparametric exact Wilcoxon signed-rank test was used to compare data that were not normally distributed. For multiple comparisons (including multiple two-group comparisons shown in the same panel), a one-way or two-way analysis of variance (ANOVA; for parametric data) followed by Bonferroni’s correction (only two groups were compared), Dunnett’s test (all groups were compared to one control group), Tukey’s multiple-comparison test (all groups were compared to each other), or a Kruskal-Wallis test (for nonparametric data) followed by Dunn’s multiple-comparison test was used. Correlation was estimated by Pearson correlation coefficients (for parametric data). P values of <0.05 were considered statistically significant.
ACKNOWLEDGMENTS
This work was supported by grants from the National Special Research Program of China for Important Infectious Diseases (2018ZX10302103 and 2017ZX10202102), the Important Key Program of Natural Science Foundation of China (81730060), and the Joint-Innovation Program in Healthcare for Special Scientific Research Projects of Guangzhou (201803040002), which were awarded to H.Z. This study was also supported by grants from the National Natural Science Foundation of China (81672024), Natural Science Foundation of Guangdong Province of China (2017A030306005 and 2016A030313325), and Pearl River Scholar Program of Guangdong, Guangdong Innovative and Entrepreneurial Research Team Program (2016ZT06S638), which were awarded to K.D., and by a grant from the Natural Science Foundation of Guangdong (2016A030313826), which was awarded to H.L.
F.Y., K.D., and H.Z. designed experiments. F.Y., Q.L., X.C., B.L., J.Z., X.Z., Z.L., and H.L. performed the experiments and analyzed and interpreted the data. J.L. and F.Y. performed the bioinformatics analysis. L.L., B.L., X.T., and W.C. provided clinical resources and technical support. K.D. and H.Z. supported and supervised the research. F.Y., K.D., and H.Z. wrote the manuscript, and all authors contributed to manuscript editing.
We have no competing interests to declare.
FOOTNOTES
- Received 24 July 2019.
- Accepted 18 October 2019.
- Accepted manuscript posted online 30 October 2019.
- Copyright © 2020 American Society for Microbiology.