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Persistence of HIV in Gut-Associated Lymphoid Tissue despite Long-Term Antiretroviral Therapy

  1. Tae-Wook Chun1,a,
  2. David C. Nickle6,a,
  3. Jesse S. Justement1,
  4. Jennifer H. Meyers1,
  5. Gregg Roby1,
  6. Claire W. Hallahan2,
  7. Shyam Kottilil1,
  8. Susan Moir1,
  9. JoAnn M. Mican4,
  10. James I. Mullins6,
  11. Douglas J. Ward7,
  12. Kovacs Joseph A.5,
  13. Peter J. Mannon3 and
  14. Anthony S. Fauci1
  1. 1Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
  2. 2Biostatistical Research Branch, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
  3. 3Laboratory of Host Defense National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
  4. 4Division of Clinical Research, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
  5. 5Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, Maryland
  6. 6Department of Microbiology, University of Washington, Seattle
  7. 7Dupont Circle Physicians Group, Washington, DC
  1. Reprints or correspondence: Tae-Wook Chun, PhD, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10, Room 6A32, 9000 Rockville Pike, Bethesda, Maryland 20892 (twchun{at}nih.gov).
 
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Abstract

Human immunodeficiency virus (HIV) persists in peripheral blood mononuclear cells despite sustained, undetectable plasma viremia resulting from long-term antiretroviral therapy. However, the source of persistentHIVin such infected individuals remains unclear. Given recent data suggesting high levels of viral replication and profound depletion of CD4+ T cells in gut-associated lymphoid tissue (GALT) of animals infected with simian immunodeficiency virus and HIV-infected humans, we sought to determine the level of CD4+ T cell depletion as well as the degree and extent of HIV persistence in the GALT of infected individuals who had been receiving effective antiviral therapy for prolonged periods of time. We demonstrate incomplete recoveries of CD4+ T cells in the GALT of aviremic, HIV-infected individuals who had received up to 9.9 years of effective antiretroviral therapy. In addition, we demonstrate higher frequencies of HIV infection in GALT, compared with PBMCs, in these aviremic individuals and provide evidence for cross-infection between these 2 cellular compartments. Together, these data provide a possible mechanism for the maintenance of viral reservoirs revolving around theGALTof HIV-infected individuals despite long-term viral suppression and suggest that theGALTmayplay a major role in the persistence of HIV in such individuals.

Despite the development of successful strategies for the treatment of HIV-infected individuals, it has not been possible to eradicate HIV in patients who receive effective antiretroviral therapy, mainly due to the persistence of viral reservoirs [1]. Among these, a pool of latently infected, resting CD4+ T cells has long been recognized as one of the major impediments to the eradication of HIV because of its extremely long intrinsic half-life and the ineffectiveness of antiretroviral therapy in eliminating this viral reservoir [2–5].

In addition, a number of studies conducted over the past several years have demonstrated low, but persistent, levels of ongoing HIV replication, even in infected individuals who had been receiving uninterrupted antiretroviral therapy that rendered them consistently aviremic for prolonged periods of time [6–13]. Furthermore, we demonstrated recently that the level of HIV infection in activated CD4+ T cells was significantly higher than that in resting CD4+ T cells in the peripheral blood; we also found evidence for cross-infection between these 2 cellular populations in infected individuals who had been receiving effective antiretroviral therapy for extended periods of time [14]. These findings provided convincing evidence for continued HIV replication and raised the question of the contribution of viral replication in lymphoid tissues to the persistence of HIV in treated patients. In addition, a number of studies have demonstrated a profound degree of CD4+ T cell depletion in gut-associated lymphoid tissue (GALT) associated with high levels of viral infection in HIV-infected individuals and in rhesus macaques infected with simian immunodeficiency virus (SIV) [15–22]. In addition, other studies have shown the presence of HIV proviral DNA in the GALT of infected individuals who received antiretroviral therapy for short periods of time [21, 23]. However, research has thus far not addressed either the degree and extent of persistent viral replication in the GALT or the dynamics of cross-infection and genetic relationship of HIV in the peripheral blood versus the GALT of HIV-infected individuals who have been receiving continuous and effective antiretroviral therapy for extended periods of time. For this reason, we undertook the present study.

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Materials and Methods

Patient population. A total of 8 HIV-infected individuals (mean CD4+ T cell count, 622 cells/µL blood; range, 348–1390 cells/µL; mean CD8+ T cell count, 996 cells/µL; range, 412–1854 cells/µL) who had been receiving effective antiviral therapy for an average of 8.4 years (range, 4.8–9.9 years) were studied (table 1). At time of the study, the participants had maintained consistently undetectable levels of plasma viremia (lower limit of detection, 50 copies HIV RNA/mL) for an average of 5.6 consecutive years (range, 3.0–7.3 years). Leukapheresis and endoscopic terminal ileum biopsies were conducted for each study participant in accordance with protocols approved by the institutional review board of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Isolation of resting and activated CD4+ T cells. Peripheral blood mononuclear cells (PBMCs) were obtained from leukapheresis by Ficoll-Hypaque density gradient centrifugation. CD4+ T cells were isolated from PBMCs obtained from HIV-infected individuals, by use of a column-based cell separation technique (StemCell Technologies), as described elsewhere [24]. To isolate resting and activated CD4+ T cells, total CD4+ T cells were labeled with anti-CD3 (APC), CD4 (FITC), CD25 (PE), CD69 (PE), and HLA-DR (PE) antibodies (BD Biosciences). Then, CD3+/CD4+/CD25−/CD69−/HLA-DR− (resting) and CD3+/CD4+/CD25+/CD69+/HLA-DR+ (activated) cells were separated, by use of a fluorescence-activated cell sorter (FACSAria; BD Biosciences), to very high purity (>99.0%).

Quantitative real-time polymerase chain reaction for measurements of HIV DNA. To determine the frequency of CD4+ T cells carrying HIV proviral DNA in infected individuals, realtime polymerase chain reaction (PCR) was carried out on genomic DNA isolated from 1–2 × 106 purified resting or activated CD4+ T cells using the Puregene DNA isolation kit (Gentra) in accordance with the manufacturer's specifications. 1 µg of DNA was then used as a template for PCR in an iCycler (Bio-Rad). The amplification reaction was carried out in triplicate using 0.5 µmol/L primers, 0.2 µmol/L fluorescent probe, 0.8 mmol/L dNTPs, 5 mmol/L MgCl2, and 2.5 U Platinum Taq Polymerase (Invitrogen) in 50 µL total volume. The following primers were used: 5′-GGTCTCTCTGGTTAGACCAGAT-3′ (5′ primer) and 5′-CTGCTAGAGATTTTCCACACTG-3′ (3′ primer) along with the fluorescent probe 5′-6FAM-AGTAGTGTGTGC CCGTCTGTT-TAMRA-3′. PCR conditions consisted of a denaturation step at 95°C for 3 min, followed by 45 cycles of 15 sec at 95°C and 1minat 59°C. Serially diluted ACH-2DNA (40,000, 8000, 1600, 320, 64, 12.8, 2.56, and 0.56 cell equivalents per well in triplicates) was also subjected to the PCR conditions above to obtain standard curves. The detection limit of the assay was 2.56 copies of HIV DNA. After endoscopic terminal ileum biopsies, tissue samples were incubated with 0.5 mg/mL collagenase (Type II-S; Sigma) in RPMI containing 5% fetal bovine serum, HEPES, and pen-strep at 37°C for 30 min. After frequent pipetting and vortexing, cells were washed and stored on ice, and the remaining undigested tissue was treated with 1.0 mg/mL collagenase for an additional 30 min. The frequency of CD3+CD4+ T cells was determined by fluorescence-activated cell sorter analysis (FACS) (FACSCanto;BD Biosciences). A fraction of the cells was subjected to CD8 depletion (Invitrogen-Dynal) and the frequency of the percentage CD3+CD4+ T cells was determined by FACS. Approximately 200,000 CD8-depleted cells were lysed in 10 mmol/L Tris-HCl at pH 8 that contained 100 µg/mL proteinase K (Roche Applied Science) for 1 hour at 56°C, followed by heat inactivation of the enzyme. PCR specific for human β-actin DNA (Applied Biosystems) was carried out on the cell lysates described above to determine the exact copy number of cells per µL of cell lysate. Serially diluted ACH-2cell lysates were prepared to obtain standard curves. Finally, PCR specific for HIV DNA was carried out as described above and the number of copies of HIV DNA per 1 × 106 CD4+ T cells was calculated on the basis of results obtained from the FACS and PCR experiments.

Sequencing of HIV env isolated from infected CD4+ T cells. HIV DNA was amplified to single copy by limiting dilution PCR. First-round primers were ED5 (5′-ATGGG ATCAAAGCCTAAAGCCATGTG-3′, nucleotides 6556 to 6581, HIV-1 HXB2) and ED12 (5′-AGTGCTTCCTGCT GCTCCCAAGAACCCAAG-3′, nucleotides 7822 to 7792, HIV-1 HXB2). Second-round reactions consisted of 1 µL of the first-round product as template and primers DR7 (5′- TCAACTCAACTGCTGTTAAATGGCAGTCTAGC-3′, nucleotides 6989 to 7020, HIV-1 HXB2) and DR8 (5′-CACTTCT CCAATTGTCCCTCATATCTCCTCC-3′, nucleotides 7637 to 7667, HIV-1 HXB2) as previously described [14]. The final PCR products, (650 bp) spanning the C2 to V5 region of the HIV-1 env, were ligated into the sequencing vector pCR4-TOPO (Invitrogen), followed by transformation, amplification, and sequencing. The average number of HIV env sequences we examined in resting CD4+ T cells , activated CD4+ T cells, and GALT was 23, 24, and 27, respectively.

Phylogenetic trees and analysis of cross-infection. HIV env sequences isolated from blood-derived resting and activated CD4+ T cells and from the GALT of study participants were aligned with MUSCLE [25] and adjusted manually using MacClade (version 3) [26]. Any ambiguously aligned elements were removed before the phylogenetic trees were generated. A cladistic/parsimony-based approach was used to determine the number of cross-infection events among the 3 cellular compartments. First, a maximum likelihood tree was estimated with the algorithm implemented in PhyML and the HKY+G+I model of evolution was used to correct for multiple mutations at a single site [27]. The maximum likelihood tree above was used in MacClade [26] to calculate the minimum nonambiguous transitions of HIV from one cellular compartment to another on the phylogenetic tree. To calculate the level of diversity of the HIV env sequences in the blood and gut compartment, pairwise comparisons were measured to generate N(N-1)/2 values where N is the number of sequences. The null value was generated by jackknifing the pairwise distance of each population to the equal number of observed sequences (N) and then randomizing their assignment between the 2 populations followed by calculation of the differences of the mean values. The null value captures the variance of N observations and with an expectation of equal diversity between the 2 putative populations. The P values were then generated by comparing the observed difference in diversity with the distribution of chance differences in diversity estimated by 1000 random permutations (table 1).

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Results

Levels of CD4+ T cells and endoscopic analysis of GALT. We first determined the frequencies of CD3+CD4+ T cells in the peripheral blood and in tissue samples obtained from the GALT of HIV-infected individuals who had been receiving effective antiretroviral therapy for extended periods of time (table 1). The levels of CD3+CD4+ T cells in the GALT were significantly lower than that of the peripheral blood (P = .005). Although the percentage of CD4+ T cells in GALT is generally lower than that in peripheral blood of healthy individuals (∼40% vs. ∼65%, respectively) [16–18], the mean percentage of CD4+ T cells in the GALT of study participants was 11.3%, suggesting incomplete recovery of CD4+ T cells in that compartment, even after many years of effective antiretroviral therapy (figure 1A). The images obtained during endoscopy revealed a normal appearance in the terminal ileum of HIV-infected individuals who had been receiving effective antiretroviral therapy (figure 1B). Endoscopic findings included smooth villous surfaces and submucosal nodules of varying prominence consistent with typical secondary lymphoid follicles of GALT. Furthermore, the general appearance of the terminal ileum in our study subjects was indistinguishable from that of HIV-seronegative volunteers (figure 1B and data not shown).

Figure 1
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Figure 1

Levels of CD4+ T cells and endoscopic analysis of gut-associated lymphoid tissue (GALT) of infected individuals receiving effective antiretroviral therapy for prolonged periods of time. A, Levels of CD4+ T cells in peripheral blood and GALT (terminal ileum). Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll-Hypaque density gradient centrifugation. Single cell suspensions were prepared from the terminal ileum by treating tissue samples with collagenase Type II-S. Gray bars, mean values. The means of the percentage of CD3+CD4+ T cells were compared by the Student paired t test. B, Endoscopic analysis of the terminal ileum in study participants. The study participants were prepared for colonoscopy with Bisacodyl (Boehringer Ingelheim Pharmaceuticals) and standard oral gut lavage (Fleet Phospho-Soda; C. B. Fleet). Endoscopic biopsies were taken from the terminal ileum using a standard oval cup biopsy forceps.

Frequency of HIV infection in GALT. FACS-enriched resting (CD25−/CD69−/HLA-DR−) and activated (CD25+/CD69+/HLA-DR+) CD4+ T cells in blood, as well as CD8-depleted cells obtained from GALT, were subjected to quantitative PCR specific for the detection of HIV DNA. As shown in figure 2, HIV proviral DNA was readily detected in all cellular compartments tested, and the frequency of cells carrying HIV proviral DNA was highest in the GALT compartment (geometric mean, 4887 cells per million CD4+ T cells; range, 2572–8798 per million CD4+ T cells) when compared with resting CD4+ T cells (geometric mean, 1083 cells per million CD4+ T cells; range 535-2490 cells per million CD4+ T cells; P < .001) and activated CD4+ T cells (geometric mean, 1796 cells per million CD4+ T cells; range, 644–3275 cells per million CD4+ T cells; P = .001) in the PBMC compartment, suggesting that CD4+ T cells in GALT, even when present in low numbers, may support low but readily detectable levels of HIV replication in infected individuals, despite their having received years of effective antiretroviral therapy that resulted in sustained, undetectable levels of plasma viremia. Of note, the level of HIV proviral DNA in activated CD4+ T cells was positively correlated with that in GALT (r = 0.94; P = .01) (data not shown).

Figure 2
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Figure 2

Frequency of HIV proviral DNA in resting and activated CD4+ T cells sorted by fluorescence-activated cell sorter analysis (FACS) and CD8-depleted single cell suspensions from gut-associated lymphoid tissue (GALT) (terminal ileum). The HIV proviral loads in GALT were normalized based on the number of cells (β-actin) present in each sample following real-time polymerase chain reaction and FACS analysis. The values of HIV proviral DNA were logged; gray bars, geometric mean values. The geometric means of the levels of HIV proviral DNA were compared by the Student paired t test. The P values were adjusted for multiple comparisons by the Bonferroni method.

Phylogenetic analysis of HIV env DNA and evidence for cross-infection among resting and activated CD4+ T cells from peripheral blood and GALT. Finally, phylogenetic analysis of HIV env DNA(C2-V5) isolated from resting and activated CD4+ T cells in the PBMC compartment and CD8-depleted cells from the GALT compartment of the study participants was conducted in order to evaluate levels of cross-infection, including migration of virus and/or infected cells, between these cellular compartments. Based on phylogenetic and cladistic analyses of the history of migration mapped onto the branches of maximum-likelihood trees [14], our data provide evidence for HIV cross-infection among all 3 cellular compartments, suggesting that CD4+ T cells in the blood are subjected to continuous infection while trafficking through the GALT and that CD4+ T cells in the GALT may themselves be subjected to infection from infected CD4+ T cells trafficking through the gut (table 1 and figure 3A). This notion is further supported by the fact that the level of viral diversity among the HIV env sequences in the blood and GALT compartments in the majority of the study subjects examined in this study was not significantly different in ways that would suggest distinct reservoir-like characteristics in either population (table 1 and figure 3B). Thus, our data suggest that relatively low but detectable levels of cross-infection events involving virus and/or infected cells occur throughout the body, rather than being restricted to certain anatomical sites.

Figure 3
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Figure 3

Phylogenetic analysis of HIV env DNA and evidence for cross-infection among resting and activated CD4+ T cells from peripheral blood and GALT in patients receiving effective antiretroviral therapy. A, Phylogenetic trees of HIV env sequences in resting (blue circles) and activated (red circles) CD4+ T cells in peripheral blood and in CD8-depleted cells from gut-associated lymphoid tissue (GALT) (green triangles) of 2 representative patients are shown. The outgroup sequences were obtained from unrelated HIV-infected patients. Phylogenetic and cladistic analyses were conducted as previously described [14]. The bars indicate genetic distance. The direction of HIV cross-infection is shown as arrows where “R,” “A,” and “G” indicate resting CD4+ T cells, activated CD4+ T cells, and GALT, respectively. HIV env sequences from the sigmoid colon were also included for phylogenetic and cladistic analyses. B, Diversity of HIV env sequences in the GALT and blood compartments (pairwise comparisons). Dots, genetic distance between pairs of sequences; red bars, means of the distributions.

Table 1
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Table 1

Profiles of HIV-infected individuals and genetic analysis of HIV env sequences.

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Discussion

The importance of GALT in the pathogenesis of HIV has been highlighted recently in various studies in which profound levels of CD4+ T cell depletion were demonstrated in HIV-infected individuals and in SIV-infected rhesus macaques, presumably as a consequence of high frequencies of viral infection [15–22]. Considering that the GALT comprises the largest component of the human lymphoid organ system and contains the highest number of CD4+ T cells in humans [28] that are susceptible to HIV infection (high levels of activation and expression of HIV coreceptors), it is thought to be a potentially important viral reservoir [29]. It is thus of considerable interest to investigate the degree and extent of viral replication in the GALT of infected individuals who have been receiving effective antiretroviral therapy for prolonged periods of time and who have maintained consistently undetectable levels of plasma viremia. Although peripheral lymphoid tissue may also play an important role in the maintenance of HIV reservoirs, our data indicate that CD4+ T cells in the GALT (and possibly tissue macrophages) of infected individuals who maintained long-term suppression of plasma viremia while receiving antiretroviral therapy continued to support the persistence of HIV. Although HIV DNA is mainly replication defective, the presence of viral DNA in various CD4+ T cell compartments in patients who have been receiving longterm treatment may serve as a useful marker for ongoing viral replication, especially in activated CD4+ T cells in the blood and GALT. In addition, our data also provide evidence for crossinfection between the blood and GALT compartments in patients who are receiving antiretroviral therapy, which may explain, in part, the persistence of HIV in peripheral blood CD4+ T cells, possibly as a consequence of new rounds of infection in the tissue compartment. The present study has implications for a more precise understanding of the scope of HIV persistence in treated individuals and for the design of therapeutic strategies aimed at attenuating and conceivably eliminating viral reservoirs. Considering the persistence of HIV in the GALT and in the peripheral blood of patients who receive effective antiviral therapy, intensification of existing drug regimens by the addition of new classes of antiretroviral drugs, such as HIV entry or integrase inhibitors, may be necessary to abrogate the low levels of ongoing viral replication that originate from the GALT. In addition, given the substantially higher levels of HIV infection in CD4+ T cells in the GALT, compared with the peripheral blood compartment, studies designed to measure the efficacy of newly developed therapeutic strategies may require sampling of the GALT in HIV-infected individuals for more accurate characterization of the size and persistence of the viral reservoir.

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Acknowledgments

We thank the patients for their participation in this study. We also thank Richard Kwan and the staff of the National Institute of Allergy and Infectious Diseases HIV Clinic for their invaluable assistance in the execution of this study.

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Footnotes

  • ↵a T-.W.C. and D.C.N. contributed equally to this work.

  • Potential conflicts of interest: none declared.

  • Financial support: National Institutes of Health (grant P30 AI27757 to J.I.M) and the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

  • Received June 27, 2007.
  • Accepted September 6, 2007.
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  1. J Infect Dis. (2008) 197 (5): 714-720. doi: 10.1086/527324
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