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Primary HIV-1 Infection Is Associated with Preferential Depletion of CD4+ T Lymphocytes from Effector Sites in the Gastrointestinal Tract

Primary HIV-1 Infection Is Associated with Preferential Depletion of CD4+ T Lymphocytes from... Given its population of CCR5-expressing, immunologically activated CD4 T cells, the gas- trointestinal (GI) mucosa is uniquely susceptible to human immunodeficiency virus (HIV)-1 infection. We undertook this study to assess whether a preferential depletion of mucosal CD4 T cells would be observed in HIV-1–infected subjects during the primary infection period, to examine the anatomic subcompartment from which these cells are depleted, and to examine whether suppressive highly active antiretroviral therapy could result in complete immune re- constitution in the mucosal compartment. Our results demonstrate that a significant and prefer- ential depletion of mucosal CD4 T cells compared with peripheral blood CD4 T cells is seen during primary HIV-1 infection. CD4 T cell loss predominated in the effector subcompart- ment of the GI mucosa, in distinction to the inductive compartment, where HIV-1 RNA was present. Cross-sectional analysis of a cohort of primary HIV-1 infection subjects showed that although chronic suppression of HIV-1 permits near-complete immune recovery of the peripheral blood CD4 T cell population, a significantly greater CD4 T cell loss remains in the GI mucosa, despite up to 5 yr of fully suppressive therapy. Given the importance of the mucosal compartment in HIV-1 pathogenesis, further study to elucidate the significance of the changes observed here is critical. Key words: primary HIV-1 • GALT • CD4 T cells Introduction The gastrointestinal (GI) mucosa harbors the majority cytes express the CCR5 chemokine coreceptor (3–8). The of the body’s lymphocytes compared with the peripheral percentage of mucosal CD4 cells that express the CXCR4 blood, which contains only 2–5% of all lymphocytes (1, 2). chemokine coreceptor is similar to that seen in the blood In addition to representing the largest lymphoid organ, certain (8). Additionally, due to its proximity to the external envi- characteristics of the mucosal compartment render it ex- ronment and constant exposure to a myriad of food and tremely permissive to HIV-1 infection and supportive of microbial antigens, a predominant number of GI mucosal HIV-1 replication. Compared with circulating lympho- CD4 T cells are activated and well differentiated with a cytes, a greater percentage of these mucosal CD4 lympho- memory phenotype (9, 10). Lastly, the GI mucosa is main- tained in a state of physiological inflammation characterized by a high percentage of proinflammatory, HIV-1–stimulatory S. Mehandru and M.A. Poles contributed equally to this work. cytokines (11). Address correspondence to Martin Markowitz, Aaron Diamond AIDS Increasing evidence suggests that virologic and immuno- Research Center, The Rockefeller University, 455 First Ave., 7th Fl., logic events during acute and early HIV-1 infection have a New York, NY 10016. Phone: (212) 448-5020; Fax: (212) 725-1126; email: [email protected] A portion of this data was presented in abstract forms at Digestive Dis- ease Week, May 2004 (New Orleans, LA) and the second international Abbreviations used in this paper: GALT, gut-associated lymphoid tissue; GI, workshop for acute and early HIV-1 infection, May 2004 (Bethesda, gastrointestinal; HAART, highly active antiretroviral therapy; MMC, MD). No similar paper has been or will be submitted elsewhere. mucosal mononuclear cell; SIV, simian immunodeficiency. 761 J. Exp. Med. © The Rockefeller University Press • 0022-1007/2004/09/761/10 $8.00 Volume 200, Number 6, September 20, 2004 761–770 http://www.jem.org/cgi/doi/10.1084/jem.20041196 The Journal of Experimental Medicine crucial role in determining the rapidity of clinical progres- jects were treated with a multidrug regimen consisting of combi- nations of nucleoside (nucleotide) reverse transcriptase inhibitors sion in infected individuals (4, 12). Because the GI tract is with either a ritonavir-boosted protease inhibitor and/or a non- an important route for HIV-1 entry and a potentially dom- nucleoside reverse transcriptase inhibitor. inant site of HIV-1 replication, it is vital that early GI mu- All eight subjects, five women and three men, identified during cosal events be carefully examined to better understand the chronic HIV-1 infection were not receiving HAART at the time pathogenesis of acute and early HIV-1 infection. Owing to of biopsy. The mean age of this group was 38 yr. The risk factors the difficulty in identifying patients during acute HIV-1 in- for HIV acquisition in this group included heterosexual sex (n fection and the added complexity of obtaining GI biopsy 4), homosexual sex (n  2), and intravenous drug abuse (n  2). samples in these patients, study of the mucosal events dur- The mean CD4 T cell count in this group was 216 cells/mm , ing acute/early HIV-1 infection has to date been lacking, with a mean log plasma HIV-1 RNA level of 4.7 copies/ml. and the majority of our current understanding is derived The 10 HIV-1–uninfected subjects were recruited from a popula- tion undergoing routine screening colonoscopy. This group was from the simian immunodeficiency (SIV)-macaque model. comprised of six men and four women. None of the HIV-1– In the macaque model, acute SIV infection results in pro- infected or –uninfected subjects was found to have macroscopic found depletion of mucosal CD4 T cells (2, 13–17) with evidence of GI mucosal disease, nor were any concomitant patho- relative preservation of CD4 T cells in the peripheral logical processes found on histological examination. All enrolled blood and lymph nodes over the first 3 wk. Mucosal CD4 subjects signed an informed consent form that was approved by T cell depletion is seen whether the inoculum was deliv- the institutional review boards of The Rockefeller University, ered parenterally (2, 3, 14) or mucosally (3, 15). A subset of Bellevue Hospital Center, and Manhattan Veteran’s Administra- CD4 T cells that have a highly activated, memory pheno- tion Center. All clinical investigation was conducted according to type are found to be the most significantly depleted (18). the principles expressed in the Helsinki Declaration. Because mucosal sites abound in CD4 T cells with this Endoscopic biopsies were obtained from the colon from mac- roscopically normal mucosa at a standardized site in the rectosig- phenotype, the majority of CD4 T cell depletion in acute moid region 30–40 cm from the anal margin. This site was cho- SIV infection is confined to the mucosal compartment. Al- sen for all sampling to avoid potential regional variation and the though immunologic and virologic events during primary potential for confounding effects from infectious or traumatic SIV infection have been well described, mucosal events proctitis that would be expected to occur distal to this location. have been described in only two primary HIV-1–infected Biopsies were taken using large-cup endoscopic biopsy forceps human subjects. These patients, infected for 4 and 6 wk, re- (outside diameter, 3.3 mm; Microvasive Radial Jaw; Boston Sci- spectively, showed depletion of mucosal CD4 T cells (19). entific) and (a) immediately placed in tissue culture medium We undertook this study to characterize changes in the (RPMI 1640; Mediatech Inc.), (b) placed into 2-ml prelabeled GI tract in a cohort of patients with acute and early HIV-1 cryovials (Nalgene) and frozen in liquid nitrogen, or (c) placed in infection, and to understand the effects of treatment with formalin to preserve tissue architecture. Formalin-fixed tissues highly active antiretroviral therapy (HAART). were washed with PBS, transferred to 100% alcohol, and pro- cessed for immunohistochemistry and in situ hybridization. Phle- botomy was undertaken immediately before endoscopy. Materials and Methods Immediately after acquisition, mucosal mononuclear cells (MMCs) Patients and Sample Acquisition. Peripheral blood and rectosig- were enzymatically isolated from mucosal biopsies using a 30-min moid colonic mucosal tissue were collected from HIV-1–infected incubation in collagenase type II (Clostridiopeptidase A; Sigma- and –uninfected subjects. A total of 27 HIV-1–infected and 10 Aldrich) followed by mechanical separation through a blunt- HIV-1–uninfected subjects were studied. Of the HIV-1 infected, end, 16-gauge needle. The digested cell suspension was strained 19 subjects were identified during primary HIV-1 infection. Pa- through a 70-m disposable plastic strainer. Immediately after tients were all viremic and staged as per the National Institutes of isolation, cells were washed with PBS and resuspended in PBS Health (NIH)-sponsored Acute and Early Disease Research Pro- containing antibodies for flow cytometry. PBMCs were prepared gram (AIEDRP) as follows: enzyme-linked immunosorbent assay by centrifugation on a Ficoll-Hypaque density gradient (Media- negative (stage Ia), Western blot indeterminate (stage Ib), or a de- tech). PBMCs were stained for flow cytometry immediately after tuned ELISA (20) nonreactive (stage II) or a documented nega- isolation. tive within 6 mo of presentation (stage III). Eight HIV-1–infected Flow Cytometry. Cell surface expression of lymphocyte anti- subjects were studied during the chronic stage of HIV-1 infec- gens was identified by monoclonal antibody staining of freshly tion. The primary infection cohort was subgrouped as follows isolated MMCs and PBMCs, followed by flow cytometry using (see Table I): group 1: HAART naive, newly diagnosed subjects a FACSCalibur (Becton Dickinson Immunocytometry Systems (n  11), as well as two subjects who were treated with HAART [BDIS]) with analysis using CELLQuest software (BDIS). Mono- for 1 wk before biopsy; and group 2: HIV-1–infected subjects, clonal antibodies used in this study included anti–human CD3 identified during the primary infection stage and had received FITC (clone UCHT1; BDIS), anti–human CD3-PE (clone HAART for up to 5 yr (n  8). Group 2 is comprised of subjects SK-7; BDIS), anti–human CD3–peridinin chlorophyll-a protein who had received HAART for 6 mo (n  2), 1 yr (n  2), 2 yr (clone SK-7; BDIS), anti–human CD4-allophycocyanin (clone (n  2), 3 yr (n  1), and 5 yr (n  1). There was no difference RPA T4; BD Biosciences), anti–human CD8 PE (clone RPA between groups 1 and 2 with respect to CD4 T cell count on T8; BD Biosciences), anti–human CXCR4-PE (clone 12G5; BD presentation (P  0.5), log HIV-1 viral load on presentation (P  Biosciences), and anti–human CCR5-FITC (clone 2D7/CCR5; 0.7), or the estimated duration of infection on presentation (P  BD Biosciences). During flow cytometry, lymphocytes, initially 0.1). The mean age for primary infection subjects was 35 yr. All identified by their forward and side scatter characteristics, were subjects contracted HIV-1 sexually during same sex contact. Sub- subject to phenotypic analysis. Dead cells were excluded from Primary HIV-1 Infection CD4 T Cell Depletion Gastrointestinal Tract analysis using 7-aminoactinomycin D (Calbiochem). To deter- subjects using a two-sample, unequal variance t test. All reported mine the percentages of CD4 and CD8 cells in the T cell pop- p-values were two-sided at the 0.05 significance level using Win- ulation, gated lymphocytes were initially examined for the ex- dows software (SPSS 11.0; SPSS Inc.). pression of CD3. The CD3 lymphocytes were then analyzed for expression of CD4 and CD8 receptors. To evaluate the expres- sion of chemokine coreceptors, gated lymphocytes were initially Results examined for the expression of CD4 receptor. The CD4 lym- Primary HIV-1 Infection Is Associated with a Preferential phocytes were further examined for the expression of chemokine Depletion of mucosal CD4 T Cells. Isolated MMCs and coreceptors CCR5 and CXCR4. PBMCs from all subjects were examined using flow cy- Light Microscopy and Immunohistochemistry. For light micro- tometry to determine the percentage of CD4 T cells (Fig. scopic evaluation, tissues were fixed in 4% neutral-buffered forma- 1 A). The percentage of mucosal T cells expressing CD4 was lin and embedded in paraffin. 5-m thick sections were cut and significantly less than the percentage of CD4 T cells in the stained with hematoxylin and eosin and Giemsa stains. Immuno- histochemistry was also performed on paraffin-embedded sections peripheral blood in every patient in this group (Fig. 1 B). after high temperature antigen retrieval as described previously When cumulative data from group 1 was analyzed, the (21). The following antibodies were used according to the manu- mean mucosal CD4 T cell percentage was 15.7  3.6, and facturer’s instructions: NCL-CD4-IF6 (anti-CD4 antibody; No- was significantly less than the mean CD4 T cell percent- vocastra) and C8/144B (anti-CD8 antibody; Dakopatts). Binding of antibodies was visualized by the alkaline phosphatase/antialka- line phosphatase method using new fuchsin as a chromogen. In Situ Hybridization. The in situ hybridization was per- formed on paraffin sections as described previously (21). In brief, 5-m sections were cut onto slides coated with 3-amino-propyl- trietho-silane. Dewaxed paraffin-embedded sections were either boiled in a domestic pressure cooker in citrate buffer (pH 6.0) for 5 min or treated with 0.01 mg/ml proteinase K for 8 min at room temperature and subjected to in situ hybridization to detect viral RNA. We used a S-labeled, single stranded antisense RNA probe of HIV-1 (Lofstrand Labs). The probe was com- posed of fragments of 1.4–2.7 kb, which collectively represent 90% of the HIV-1 genome (22). The specific activity of the probe was 2  10 dpm of probe/milliliter. The hybridization was performed overnight at 45C in a moist chamber. The slides were washed, digested with RNase (Boehringer) at 37 C for 40 min, and washed again. The slides were then dipped into photo emulsion (NTB2; Eastman Kodak Co.), exposed for 7 d, devel- oped, counterstained with hematoxylin, and mounted. As a posi- tive control, paraffin-embedded sections from the spleen of an HIV-infected patient were used. As a negative control, sections were hybridized with a S-labeled sense probe. Quantitative Analysis of HIV RNA Cells, CD4 Cells, and CD8 Cells. The autoradiographs were examined with a mi- croscope equipped with epiluminescent illumination (Axiophot; Carl Zeiss MicroImaging, Inc.), a 3CD camera, and a PC-based image analysis system (KS 4000; Kontron). Cells were considered positive for viral gene expression if the grain count was more than six times the background. The positive cells were counted. The area occupied by lymphoid follicles was measured and the frequency of RNA-producing cells per mm of gut-associated lymphoid tissue (GALT) was calculated. To evaluate the number and distribution of T cell subsets, transmission light was used. Us- ing a 40 objective, a standard area was set by the image ana- Figure 1. CD4 T cells are preferentially depleted in the GI tract in lyzer. The number of positive cells within this unit area was de- acute and early HIV-1 infection. PBMCs and MMCs from the rectosigmoid termined by manual counting. For the lamina propria, a total of region from group 1, acute and early HIV-1 infection subjects (n  13), and 10–15 consecutive nonoverlapping fields was analyzed for each HIV-1–uninfected controls (n  10) were analyzed by flow cytometry. staining. For the GALT, two to five representative areas were CD3 gated lymphocytes were analyzed for the expression of CD4 and CD8. (A) A representative flow plot from subject 105 is depicted. CD8 chosen. The individual values obtained with each T cell marker T cells are shown on the x axis and CD4 T cells are shown on the y axis. were then pooled and the mean numbers of positive cells per unit (B) Comparison of CD4 T cells in the blood and GI tract of all 13 group area of lamina propria or GALT were determined separately. 1 primary infection subjects. The percent of CD4 T cells is shown in Statistical Methodology. Values are expressed as mean  stan- PBMCs (gray) and MMCs (white) per study subject. (C) Comparison of dard deviation. Statistical comparisons were made between the mean percent of CD4 T cells in the PBMCs of HIV-1–uninfected PBMCs and MMCs from individuals using a paired t test. Statisti- controls (black bar) and group1 subjects (gray bar), and MMCs of HIV-1– cal comparisons were made between HIV-1–infected and control uninfected controls (hatched bar) and group 1 subjects (white bar). Mehandru et al. age in the peripheral blood (42.3  14.7; P  0.001). The mean CD4/CD8 ratio in the MMCs (0.2  0.04) was also significantly lower than the mean CD4/CD8 ratio in the circulating PBMC compartment (0.9  0.6; P  0.002). In HIV-1–uninfected individuals, the percentage of CD4 T cells in the mucosa (56.4  8.8%) and blood (59.6 14.3%) were not significantly different (P  0.24), and the CD4/CD8 ratios in the mucosa and blood were 1.3 0.5% and 1.7  0.7%, respectively. Comparison of the percentages of CD4 T cells in the mucosa of HIV-1–uninfected and newly diagnosed subjects with primary HIV-1 infection (group 1) revealed highly statistically significant differences with marked depletion in the HIV-1–infected group (P  0.001; Fig. 1 C). As ex- pected, depletion of peripheral blood CD4 T cells was also observed when newly diagnosed, primary HIV-1–infected subjects were compared with HIV-1–uninfected subjects. However, the differences between the two groups were much less marked, though still significant (P  0.01). Primary HIV-1 Infection Is Associated with Preferential De- pletion of CCR5-expressing Mucosal CD4 T Cells. The Figure 2. CD4 T cells expressing CCR5 are preferentially depleted majority of GI MMCs express CCR5, either alone or along from the GI tract in acute and early HIV-1 infection. PBMCs (A) and with CXCR4 (4, 8). Given the high expression of CCR5 MMCs (B) from the rectosigmoid region from individuals with acute and in the mucosa and the possibility that the mucosal environ- early HIV-1 infection (group 1, n  13) and HIV-1–uninfected controls /CD4 gated lympho- (n  10) were analyzed by flow cytometry. CD3 ment plays an important role in the selection of the CCR5- cytes were analyzed for the expression of CXCR4, CCR5, or CCR5/ tropic strains of HIV-1 that predominate in the blood dur- CXCR4. HIV-1–uninfected PBMCs (black bars), group 1 PBMCs (gray ing the primary infection period, we sought to determine bars), HIV-1–uninfected MMCs (hatched bars), and group 1 MMCs the effect of primary HIV-1 infection on CCR5-expressing T cells expressing (white bars) are shown. Mean percent of CD4 chemokine receptors are depicted on the y axis and chemokine corecep- CD4 MMCs. Using four-color flow cytometry, we quan- tors CXCR4, CCR5, and CCR5/CXCR4 are grouped on the x axis. tified the percentage of CD3 /CD4 MMCs and PBMCs that expressed CCR5, CXCR4, or coexpressed CCR5 and CXCR4. Comparison was made between HIV-1–unin- and CXCR4. In the uninfected individuals, 56.2  13.9% fected subjects and the untreated primary HIV-1 infection of the CD4 mucosal T cells coexpressed CCR5 and CXCR4 subjects (group 1). In general there was no difference in lev- compared with just 21.1  14.3% in the group 1 subjects els of CD3 /CD4 cells expressing CCR5, CXCR4, or (P  0.001; Fig. 2 B). both in the peripheral blood of uninfected controls when CD4 T Cells Are Preferentially Depleted from the Effector compared with subjects in group 1 (Fig. 2 A). Mean levels Sites in the GI Mucosa. Having shown that the percentage of CD4 PBMC populations that expressed CXCR4 were of CD4 T cells is decreased in acute and early HIV-1 in- 64.3  15.2% in the HIV-1–uninfected subjects compared fection, we went on to examine the absolute number of with 63.7  11.8% in group 1 primary HIV-1 infection CD4 T cells in the GI biopsies. Using immunohistochem- subjects (P  0.6). Levels of CD3 /CD4 PBMCs express- istry, paraffin-embedded mucosal biopsies were compared ing CCR5 were 18.7  9.0% in HIV-1–uninfected subjects between HIV-1–uninfected individuals and subjects with compared with 14.8  11.0% in group 1 (P  0.4). Levels acute and early HIV-1 infection. To classify the anatomic of CD3 /CD4 PBMCs coexpressing CCR5 and CXCR4 subcompartment where CD4 T cell depletion occurred, were 9.6  5.8% in HIV-1–uninfected subjects versus 6.9 inductive and effector sites were examined separately. 8.1% in group 1 primary HIV-1 infection subjects (P  The inductive sites or lymphoid nodules consisted of a 0.4). In comparison, analysis of CD4 mucosal T cells re- centrally located B cell–dependent area with or without a vealed significant depletion in the HIV-1–infected subjects germinal center (Fig. 3 A). The follicle-associated epithe- that was most prominent in the CD3 /CD4 CCR5- lium and the dome area were noted in only a few of the sec- expressing populations. Although there was only a minor tions examined. The B cell follicle was surrounded by the degree of depletion of mucosal CD4 T cells expressing T-dependent zone, similar to that seen in the paracortex of a CXCR4 (76.3  14.4% in HIV-1–uninfected subjects vs. lymph node. In subjects with acute and early HIV-1 infec- 61.7  10.9 in group 1 subjects; P  0.03), the percentage tion (Fig. 3 B) and in HIV-1–uninfected controls (Fig. 3 C), of cells expressing CCR5 alone was 73.5  13.4% in HIV- the T cell zone was densely populated with CD4 T cells. In 1–uninfected subjects versus 42.5  18.9% in group 1 (P comparison, significantly more CD8 T cells were noted in 0.001). The most significant depletion was noted in the the subjects with acute and early HIV-1 infection (mean: population of CD4 mucosal T cells that coexpressed CCR5 130 cells/unit area; range: 95–137 cells/unit area) compared Primary HIV-1 Infection CD4 T Cell Depletion Gastrointestinal Tract 764 Figure 3. Effector sites (lamina pro- pria) of the GI tract show the most pro- nounced CD4 T cell depletion in acute and early HIV-1 infection. Immunohis- tochemical characterization of the effector and inductive sites in the rectal biopsies. Using a PC-based image analysis system (KS 4000; Kontron), a standard area was set by the image analyzer. For the lamina propria, a total of 10–15 consecutive nonoverlapping fields were analyzed for each staining. For the GALT, two to five representative areas were chosen. (A) At a magnification of 25, a Giemsa- stained section is shown depicting a lymphoid nodule (inductive site) where the germinal center (GC) and T cell zone (T) are indicated. The effector compartment represented by the lamina propria (LP) is seen in the lower part of the section. (B and C) The inductive sites are densely populated with CD4 T cells (stained red) in both group 1 (B) and HIV-1–uninfected (C) subjects. A magnification of 100. (D and E) Effector sites in acute and early HIV-1 infection (D) show significant depletion of CD4 T cells (stained red) compared with HIV-1–uninfected controls (E). A mag- nification of 100. (F) In the inductive and effector compartments, the mean of CD4 and CD8 T cells per unit area was determined and the CD4/CD8 ratio was calculated. A comparison of the mean CD4/CD8 ratio (represented on the y axis) between HIV-1–uninfected (gray bars) and group 1 subjects (white bars) is shown. with the HIV-1–uninfected controls (mean: 83 cells/unit situ hybridization was performed to localize HIV-1 RNA area; range: 81–140 cells/unit area). The mean CD4/CD8 within the effector and inductive subcompartments of the ratio in the inductive sites of HIV-1–uninfected controls was GI mucosa. Cells expressing HIV-1 RNA were predomi- 2.4 (range 1.6–3.1). In comparison, the mean CD4/CD8 ra- nantly localized to the organized lymphoid follicles or the tio in the inductive sites of subjects with acute and early inductive sites of the GI mucosa (Fig. 4, A and B), present HIV-1 infection was 1.3 (range 0.9–2.0). It is important to both in the T cell zone and the germinal centers. Even note that the lower CD4/CD8 ratio in HIV-1–infected sub- though CD4 T cells were depleted in the effector sites, of jects was due to an increase in the number of CD8 T cells the CD4 T cells that remained, productively infected cells and not due to a depletion of the CD4 T cells. were either absent or very rare. Interestingly, no viral trap- Significant CD4 T cell depletion was noted in the ef- ping by follicular dendritic cells was noted in the biopsies fector sites in subjects with acute and early HIV-1 infection of the group 1 primary infection subjects. (Fig. 3 D) compared with HIV-1–uninfected controls (Fig. Reconstitution of CD4 MMCs Is Incomplete Despite Sup- 3 E). In this site, the mean CD4/CD8 ratio was 0.4 (range pressive Antiretroviral Therapy. To examine whether appar- 0.1–0.7) in the HIV-1–infected subjects and 1.5 (1.1–2) in ently suppressive HAART would result in reconstitution the HIV-1–uninfected controls (Fig. 3 F). of CD4 MMCs, as has been described among CD4 HIV-1 RNA Is Located within the Inductive Sites of the GI PBMCs, subjects in whom HAART was initiated during Mucosa. Having shown that depletion of mucosal CD4 acute and early HIV-1 infection were examined. We T cells was primarily noted in the effector compartment, performed a cross-sectional analysis of primary HIV-1– we sought to determine whether this anatomic subcom- infected subjects who had been treated with regimens con- partment was also the principal site of viral replication. In taining three or four drug combinations for varying dura- Mehandru et al. 765 Figure 5. Prolonged HAART results in only partial reconstitution of the GI tract CD4 T cells. PBMCs and MMCs from the rectosigmoid region from HIV-1–uninfected individuals (n  10), subjects with acute and early HIV-1 infection (n  13), subjects with HIV-1 infection treated during the primary infection stage (n  8), and chronically infected HIV-1– untreated subjects (n  8) were analyzed by flow cytometry. The mean percent of CD4 T cells is shown in PBMCs (gray bars) and MMCs (white bars) per study group. than the mean CD4/CD8 ratio in the blood (1.7  0.6) Figure 4. HIV-1 RNA is localized in the organized lymphoid follicles and mucosa (1.3  0.4) of HIV-1–uninfected controls. It is of the GI tract in subjects with acute and early HIV-1 infection. In situ of interest that these two subjects had initiated HAART af- hybridization was performed using a S-labeled, single stranded antisense ter 28 and 26 d, respectively, of their estimated dates of in- RNA probe as described in Materials and Methods. Cells expressing fection (Table I). In subjects 106 and 923, the CD4/CD8 HIV-1 RNA (arrows) are present in the germinal center and T cell zone. A magnification of 40. ratio in the GI tract continued to be significantly lower compared with the blood, despite treatment with HAART for 3 and 5 yr, respectively. These two subjects had initi- tions (n  8), and eight untreated HIV-1–infected ated HAART after 168 and 204 d, respectively, of their es- subjects biopsied during chronic HIV-1 infection. The timated date of infection (Table I). Notably for subject 56, primary HIV-1 infection cohort included two subjects from the CD4/CD8 ratio in the blood normalized to 2.9 with group 1 who had been treated with suppressive HAART fully suppressive HAART for 2 yr. In comparison, the for 6 mo, two subjects who had been treated with suppres- CD4/CD8 ratio in the GI tract remained considerably sive HAART for 1 yr, two subjects who had been treated lower at 0.8. with suppressive HAART for 2 yr, and two subjects each As expected, in chronic, untreated HIV-1–infected sub- treated for 3 and 5 yr, respectively. The mean percentage jects, the mean CD4 T cell percentage continued to de- of CD4 T cells in the peripheral blood in this group was cline in both the blood (15.3  17%) and mucosal (12.5 51.3  11.3%, whereas in the GI mucosa, it was 35.4  11%) compartments (Fig. 5). 7.8% (P  0.008; Fig. 5). When the percentage of CD4 T cells of these subjects was compared with that from HIV- Discussion 1–uninfected subjects, there was a significant difference in the mucosal compartment (P  0.001), even though the Being that the GI tract is the largest lymphoid organ in difference in the blood was no longer statistically significant the body, it would follow that the potential for deleterious (P  0.06). The mean CD4/CD8 ratio in the mucosal effects of HIV-1 infection would be great at this site. Al- compartment (0.6  0.2) continued to be significantly though the effect of primary HIV-1 infection on lympho- lower than the CD4/CD8 ratio in the blood compartment cyte populations in the peripheral blood has been well (1.3  0.7) of HAART-treated primary infection subjects characterized, the events associated with primary HIV-1 (P  0.03). infection in GI mucosal lymphocyte populations in man To better understand the effect of HAART in this di- has to date remained largely unknown. Given the strong verse group, we analyzed each patient individually (Table selection pressure for CCR5 coreceptor using viruses dur- II). In the two subjects (100-6m and 336-6m) followed ing transmission of HIV-1 (23), it would be likely that the longitudinally for 6 mo, the CD4/CD8 ratio in the blood sizable population of CCR5-expressing, immunologically showed significant improvement over baseline with treat- activated CD4 T cells in the GI mucosa would be more ment (0.6 in the untreated stage to 1.1 after 6 mo of supportive of viral replication and susceptible to its delete- HAART in both subjects). However, the CD4/CD8 ratio rious effects as compared with the peripheral blood where in the GI tract continued to be low (0.4 and 0.5, respec- the T cells are predominantly CCR5 and naive (8). We tively). In subjects 78 and 132, the CD4/CD8 ratio in the undertook this study to assess whether a preferential deple- blood and GI tract equalized but continued to be lower tion of mucosal CD4 T cells would be observed in pri- Primary HIV-1 Infection CD4 T Cell Depletion Gastrointestinal Tract 766 Table I. Patient Characteristics CD4 T cell count Log plasma HIV-1 Estimated duration of infection (cells/mm ) (copies/ml) (days) Duration of HAART prior to biopsy Study Stage At presentation At biopsy At presentation At biopsy At presentation Prior to HAART initiation (days) Group 1 102 Ia 395 391 6.9 7.1 18 NA NA 100 II 610 595 5.8 5.5 45 NA NA 104 II 981 1,321 6.0 5.8 38 NA NA 107 Ia 456 426 6.1 5.6 24 NA NA 336 II 556 556 5.0 5.0 62 NA NA 109 II 340 340 6.3 6.3 19 NA NA 119 Ia 248 248 6.9 6.3 18 NA NA 122 III 285 400 5.1 4.9 72 NA NA 125 II 830 684 5.8 5.0 36 NA NA 127 Ia 390 350 4.8 4.9 23 NA NA 131 Ia 250 349 6.7 6.6 25 NA NA 105 Ia 374 462 7.1 4.7 27 7 7 106 Ia 317 368 6.8 5.4 26 7 7 Group 2 100-6m II 610 698 5.8 1.7 45 76 214 336-6m II 556 996 4.9 1.7 48 90 229 64 Ia 846 994 7.2 1.7 28 31 398 78 II 175 460 7.4 1.7 26 28 392 56 II 619 752 5.4 1.7 61 71 670 132 II 366 657 6.1 1.7 19 26 763 106 III 351 595 6.9 1.7 157 168 1,182 923 Ia 698 939 5.1 1.7 201 204 1,932 Subjects were staged as per the NIH-sponsored AIEDRP as follows: enzyme-linked immunosorbent assay negative (stage Ia), Western blot indeter- minate (stage Ib), or a detuned ELISA nonreactive (stage II) or a documented negative within 6 mo of presentation (Stage III). The approximate date of infection was calculated as follows: 2 wk before the onset of acute retroviral illness (97%); in cases where no acute retroviral illness symptoms were experienced, the midpoint between the last negative antibody result and first positive Western blot result was determined. mary HIV-1–infected subjects as has been observed in the Table II. Comparison of Blood and GI Tract Reconstitution SIV-macaque model. We went on to examine the ana- with HAART tomic subcompartment from which these cells are de- pleted and examine whether suppressive HAART therapy CD4/CD8 ratio CD4/CD8 ratio Duration of HAART Subject in the blood in the GI tract at the time of biopsy could result in complete immune reconstitution in the mucosal compartment. Previous studies of the effect of HIV-1 on the GI mu- 100-6m 1.1 0.4 6 mo cosal CD4 T cells during the primary infection period 336-6m 1.1 0.5 6 mo have been limited to a single report, with two subjects 64 1.6 0.6 1 yr studied during early HIV-1 infection. In both of these indi- 78 0.7 0.7 1 yr viduals, identified after 4 and 6 wk of the estimated date of 56 2.9 0.8 2 yr infection, mucosal CD4 T cell depletion was noted (19). 132 0.7 0.8 2 yr The remaining data suggesting the GI tract as the preferred 106 0.9 0.5 3 yr site for virus replication and CD4 T cell depletion are de- 923 1 0.5 5 yr rived from studies examining primary infection of macaques with SIV (2, 3, 13, 15, 17, 18, 24, 25). Mehandru et al. 767 Here we have described 13 subjects who are identified calized in the inductive compartment of the mucosa. These and studied extremely early in the course of HIV-1 infec- findings are consistent with the SIV-macaque model where tion. We have confirmed that indeed, significant mucosal during early infection, most of the SIV-infected cells are CD4 T cell depletion occurs in subjects during primary located in the inductive sites. In a recent study, Veazey et HIV-1 infection, before changes seen in the peripheral al. (26) noted that very early in the course of infection, i.e., blood. However, human mucosal CD4 T cell depletion from 7 to 14 d after infection, SIV-infected cells could be appears to be less marked than that described in primary localized in the effector compartment as well. However, SIV infection, where the percentage of CD4 T cells in the once effector CD4 T cells were depleted, SIV RNA was macaque small bowel was reduced to 10% by 3 wk after predominantly seen in the inductive compartment. Our infection (13). This is likely explained by a difference in the patients, although studied extremely early in the course of site sampled. All the biopsies in our studies were obtained HIV-1 infection, had already developed profound CD4 T from the rectal mucosa, which has both effector and induc- cell depletion in the effector sites. Thus, one possible ex- tive lymphoid tissue. In the macaque models described, the planation for the localization of HIV-1 RNA only in the biopsies are usually obtained from the proximal jejunum, inductive compartment is the loss of target cells from the which is devoid of immune inductive sites (26). As a result, effector sites by the time the biopsies were performed. samples obtained from the jejunum in macaque models Further studies are underway to better characterize the may demonstrate a much higher degree of CD4 T cell de- dynamics of CD4 T cell loss noted in the effector com- pletion compared with what is noted in this study. There partment. Some of the possibilities include decreased local may also be other factors responsible. SIV is introduced in proliferation, increased cell death in the effector compart- large inocula to attempt to guarantee infection. Simian ment due to apoptosis or activation-induced cell death AIDS is characterized by an accelerated natural history of (36), reduced homing of cells to the effector site from the the disease (27, 28) and is accompanied by higher peak periphery after antigen recognition due to either direct cy- plasma viral levels (29, 30) when compared with HIV-1 in- topathic effects of HIV-1 or immune activation-induced fection in man. Also, SIV is known to use alternative and cell death, or perhaps combinations of the above (37). additional coreceptors when compared with HIV-1 (31, By studying subjects in whom treatment was initiated 32), which may account for apparent differences in replica- during acute and early HIV-1 infection, we sought to un- tion dynamics. Yet unexplained differences may also exist derstand whether HAART would result in mucosal CD4 in host susceptibility to the two infections. T cell reconstitution. Guadalupe et al. (19) showed that in In addition to confirming the numerical decrease in mu- one subject, mucosal CD4 T cell reconstitution was at- cosal CD4 T cell percentage, we have gone on to charac- tained when HAART was initiated within 6 wk of the es- terize the relationship between CD4 T cell depletion and timated date of infection. In comparison, we did not see HIV-1 RNA localization within specific anatomic sub- immune reconstitution in the GI mucosa with HAART in compartments of the GI mucosa. Like the small intestine, our study population. It is to be noted that all eight patients the immune compartment in the rectal mucosa is also di- studied initiated therapy in the primary infection stage, re- vided into inductive and effector arms (24, 33). The orga- mained compliant with treatment, and continued to have nized lymphoid tissue with a high content of CD68 MHC undetectable plasma HIV-1 RNA levels for the entire du- class II macrophages and cells expressing costimulatory ration of the study period. The cohort remains small and molecules CD86 and CD40 serves as an antigen-presenting we plan to study more subjects longitudinally to better as- site (24). The majority of lymphocytes present in the in- sess the degree of immune reconstitution and the factors ductive compartment are antigen naive. However, once that may predispose to a greater or lesser response. activated and primed by antigen-presenting cells such as The consequence of early depletion and incomplete re- dendritic cells, GI mucosal lymphocytes can home back to constitution of mucosal CD4 T cells is unclear. Although the mucosa to perform effector functions (34). The lamina opportunistic infections remain rare until the peripheral propria lymphocytes, which are comprised of a high per- CD4 T cell count falls during the chronic infection pe- centage of differentiated effector cells (9, 10), serve as the riod, the long-term outcome of treated HIV-1 infection effector arm of the mucosal immune system (33). A limited remains unknown. Furthermore, the nature of the CD4 T number of studies have described the effects of SIV infec- cell loss requires additional study. Theoretically, the loss of tion (24–26) and chronic and advanced HIV-1 infection specific clones of CD4 T cells in the mucosa may predis- (35) on these distinct, anatomic subcompartments. Our re- pose to accelerated immune senescence, more rapid than sults suggest that as early as the primary infection stage, that seen as a consequence of aging, which in turn may re- there is a depletion of CD4 T cells from the effector com- sult in the increased incidence of malignancies, be they partment of the GI mucosa. Given the activated and differ- lymphomas or solid tumors such as adenocarcinomas. entiated phenotype of these cells, direct cytopathic effects Given the importance of the mucosal compartment in of the HIV-1 virus could be one mechanism explaining this HIV-1 pathogenesis, our findings may have important im- lesion. To assess this, we performed in situ hybridization plications with regard to treatment. Perhaps efforts to spe- for HIV-1 RNA to localize the sites of HIV-1 replication cifically inhibit T cell activation in the GI tract as well as in the GI mucosa. We observed that HIV-1 RNA was lo- drugs to more specifically interfere with HIV-1 entry (38, Primary HIV-1 Infection CD4 T Cell Depletion Gastrointestinal Tract 768 39) and replication in the GI mucosa during the primary 9. Kim, S.K., D.S. Reed, W.R. Heath, F. Carbone, and L. Le- francois. 1997. Activation and migration of CD8 T cells in infection period may result in lowering of the viral set the intestinal mucosa. J. Immunol. 159:4295–4306. point and in conjunction with HAART, result in improve- 10. Schieferdecker, H.L., R. Ullrich, H. Hirseland, and M. Zeitz. ment in clinical outcome. Such investigations are clearly 1992. T cell differentiation antigens on lymphocytes in the needed as the goal of optimized treatment of HIV-1 infec- human intestinal lamina propria. J. Immunol. 149:2816–2822. tion is yet to be attained. 11. McGowan, I.M., J. Elliott, M. Fuerst, P. Taing, J. Boscardin, M.A. Poles, and P.A. Anton. 2004. Increased HIV-1 mucosal We would like to thank the patients for their participation. We ac- replication is associated with generalized mucosal cytokine acti- knowledge the nursing staff at The Rockefeller University and vation. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. In press. Bellevue Hospital for their clinical assistance. We would like to 12. Lefrere, J.J., F. Roudot-Thoraval, M. Mariotti, M. Thauvin, thank Wendy Chen for help with the graphs and figures; Petra J. Lerable, J. Salpetrier, and L. Morand-Joubert. 1998. The Meyer, Birgit Raschdorff, and Gudrun Großschupff for their tech- risk of disease progression is determined during the first year nical assistance with immunohistochemistry and in situ hybridiza- of human immunodeficiency virus type 1 infection. J. Infect. tion; and Dr. Gerard Eberl for helpful discussion. Dis. 177:1541–1548. This work was supported in part by the following grants: The 13. Veazey, R.S., M. DeMaria, L.V. Chalifoux, D.E. Shvetz, AIEDRP (AI-41534) and the Virology Core of the Columbia-Rock- D.R. Pauley, H.L. Knight, M. Rosenzweig, R.P. Johnson, efeller Center for AIDS Research (P30 AI42848-05), AI-01668 and R.C. Desrosiers, and A.A. Lackner. 1998. Gastrointestinal AI-46964 from the National Institute of Allergy and Infectious Dis- tract as a major site of CD4 T cell depletion and viral repli- eases, the General Clinical Research Center grant from the National cation in SIV infection. Science. 280:427–431. Center for Research Resources at the NIH (M01-RR00102), and 14. Harouse, J.M., A. Gettie, R.C. Tan, J. Blanchard, and C. the German Ministry of Education and Research (BMBF) Contract Cheng-Mayer. 1999. Distinct pathogenic sequela in rhesus KompNet 01KI0211 (to P. Racz and K. Tenner-Racz). macaques infected with CCR5 or CXCR4 utilizing SHIVs. The authors have no conflicting financial interests. Science. 284:816–819. Submitted: 16 June 2004 15. Kewenig, S., T. Schneider, K. Hohloch, K. 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Primary HIV-1 Infection CD4 T Cell Depletion Gastrointestinal Tract http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Experimental Medicine Pubmed Central

Primary HIV-1 Infection Is Associated with Preferential Depletion of CD4+ T Lymphocytes from Effector Sites in the Gastrointestinal Tract

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Pubmed Central
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Copyright © 2004, The Rockefeller University Press
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0022-1007
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10.1084/jem.20041196
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Abstract

Given its population of CCR5-expressing, immunologically activated CD4 T cells, the gas- trointestinal (GI) mucosa is uniquely susceptible to human immunodeficiency virus (HIV)-1 infection. We undertook this study to assess whether a preferential depletion of mucosal CD4 T cells would be observed in HIV-1–infected subjects during the primary infection period, to examine the anatomic subcompartment from which these cells are depleted, and to examine whether suppressive highly active antiretroviral therapy could result in complete immune re- constitution in the mucosal compartment. Our results demonstrate that a significant and prefer- ential depletion of mucosal CD4 T cells compared with peripheral blood CD4 T cells is seen during primary HIV-1 infection. CD4 T cell loss predominated in the effector subcompart- ment of the GI mucosa, in distinction to the inductive compartment, where HIV-1 RNA was present. Cross-sectional analysis of a cohort of primary HIV-1 infection subjects showed that although chronic suppression of HIV-1 permits near-complete immune recovery of the peripheral blood CD4 T cell population, a significantly greater CD4 T cell loss remains in the GI mucosa, despite up to 5 yr of fully suppressive therapy. Given the importance of the mucosal compartment in HIV-1 pathogenesis, further study to elucidate the significance of the changes observed here is critical. Key words: primary HIV-1 • GALT • CD4 T cells Introduction The gastrointestinal (GI) mucosa harbors the majority cytes express the CCR5 chemokine coreceptor (3–8). The of the body’s lymphocytes compared with the peripheral percentage of mucosal CD4 cells that express the CXCR4 blood, which contains only 2–5% of all lymphocytes (1, 2). chemokine coreceptor is similar to that seen in the blood In addition to representing the largest lymphoid organ, certain (8). Additionally, due to its proximity to the external envi- characteristics of the mucosal compartment render it ex- ronment and constant exposure to a myriad of food and tremely permissive to HIV-1 infection and supportive of microbial antigens, a predominant number of GI mucosal HIV-1 replication. Compared with circulating lympho- CD4 T cells are activated and well differentiated with a cytes, a greater percentage of these mucosal CD4 lympho- memory phenotype (9, 10). Lastly, the GI mucosa is main- tained in a state of physiological inflammation characterized by a high percentage of proinflammatory, HIV-1–stimulatory S. Mehandru and M.A. Poles contributed equally to this work. cytokines (11). Address correspondence to Martin Markowitz, Aaron Diamond AIDS Increasing evidence suggests that virologic and immuno- Research Center, The Rockefeller University, 455 First Ave., 7th Fl., logic events during acute and early HIV-1 infection have a New York, NY 10016. Phone: (212) 448-5020; Fax: (212) 725-1126; email: [email protected] A portion of this data was presented in abstract forms at Digestive Dis- ease Week, May 2004 (New Orleans, LA) and the second international Abbreviations used in this paper: GALT, gut-associated lymphoid tissue; GI, workshop for acute and early HIV-1 infection, May 2004 (Bethesda, gastrointestinal; HAART, highly active antiretroviral therapy; MMC, MD). No similar paper has been or will be submitted elsewhere. mucosal mononuclear cell; SIV, simian immunodeficiency. 761 J. Exp. Med. © The Rockefeller University Press • 0022-1007/2004/09/761/10 $8.00 Volume 200, Number 6, September 20, 2004 761–770 http://www.jem.org/cgi/doi/10.1084/jem.20041196 The Journal of Experimental Medicine crucial role in determining the rapidity of clinical progres- jects were treated with a multidrug regimen consisting of combi- nations of nucleoside (nucleotide) reverse transcriptase inhibitors sion in infected individuals (4, 12). Because the GI tract is with either a ritonavir-boosted protease inhibitor and/or a non- an important route for HIV-1 entry and a potentially dom- nucleoside reverse transcriptase inhibitor. inant site of HIV-1 replication, it is vital that early GI mu- All eight subjects, five women and three men, identified during cosal events be carefully examined to better understand the chronic HIV-1 infection were not receiving HAART at the time pathogenesis of acute and early HIV-1 infection. Owing to of biopsy. The mean age of this group was 38 yr. The risk factors the difficulty in identifying patients during acute HIV-1 in- for HIV acquisition in this group included heterosexual sex (n fection and the added complexity of obtaining GI biopsy 4), homosexual sex (n  2), and intravenous drug abuse (n  2). samples in these patients, study of the mucosal events dur- The mean CD4 T cell count in this group was 216 cells/mm , ing acute/early HIV-1 infection has to date been lacking, with a mean log plasma HIV-1 RNA level of 4.7 copies/ml. and the majority of our current understanding is derived The 10 HIV-1–uninfected subjects were recruited from a popula- tion undergoing routine screening colonoscopy. This group was from the simian immunodeficiency (SIV)-macaque model. comprised of six men and four women. None of the HIV-1– In the macaque model, acute SIV infection results in pro- infected or –uninfected subjects was found to have macroscopic found depletion of mucosal CD4 T cells (2, 13–17) with evidence of GI mucosal disease, nor were any concomitant patho- relative preservation of CD4 T cells in the peripheral logical processes found on histological examination. All enrolled blood and lymph nodes over the first 3 wk. Mucosal CD4 subjects signed an informed consent form that was approved by T cell depletion is seen whether the inoculum was deliv- the institutional review boards of The Rockefeller University, ered parenterally (2, 3, 14) or mucosally (3, 15). A subset of Bellevue Hospital Center, and Manhattan Veteran’s Administra- CD4 T cells that have a highly activated, memory pheno- tion Center. All clinical investigation was conducted according to type are found to be the most significantly depleted (18). the principles expressed in the Helsinki Declaration. Because mucosal sites abound in CD4 T cells with this Endoscopic biopsies were obtained from the colon from mac- roscopically normal mucosa at a standardized site in the rectosig- phenotype, the majority of CD4 T cell depletion in acute moid region 30–40 cm from the anal margin. This site was cho- SIV infection is confined to the mucosal compartment. Al- sen for all sampling to avoid potential regional variation and the though immunologic and virologic events during primary potential for confounding effects from infectious or traumatic SIV infection have been well described, mucosal events proctitis that would be expected to occur distal to this location. have been described in only two primary HIV-1–infected Biopsies were taken using large-cup endoscopic biopsy forceps human subjects. These patients, infected for 4 and 6 wk, re- (outside diameter, 3.3 mm; Microvasive Radial Jaw; Boston Sci- spectively, showed depletion of mucosal CD4 T cells (19). entific) and (a) immediately placed in tissue culture medium We undertook this study to characterize changes in the (RPMI 1640; Mediatech Inc.), (b) placed into 2-ml prelabeled GI tract in a cohort of patients with acute and early HIV-1 cryovials (Nalgene) and frozen in liquid nitrogen, or (c) placed in infection, and to understand the effects of treatment with formalin to preserve tissue architecture. Formalin-fixed tissues highly active antiretroviral therapy (HAART). were washed with PBS, transferred to 100% alcohol, and pro- cessed for immunohistochemistry and in situ hybridization. Phle- botomy was undertaken immediately before endoscopy. Materials and Methods Immediately after acquisition, mucosal mononuclear cells (MMCs) Patients and Sample Acquisition. Peripheral blood and rectosig- were enzymatically isolated from mucosal biopsies using a 30-min moid colonic mucosal tissue were collected from HIV-1–infected incubation in collagenase type II (Clostridiopeptidase A; Sigma- and –uninfected subjects. A total of 27 HIV-1–infected and 10 Aldrich) followed by mechanical separation through a blunt- HIV-1–uninfected subjects were studied. Of the HIV-1 infected, end, 16-gauge needle. The digested cell suspension was strained 19 subjects were identified during primary HIV-1 infection. Pa- through a 70-m disposable plastic strainer. Immediately after tients were all viremic and staged as per the National Institutes of isolation, cells were washed with PBS and resuspended in PBS Health (NIH)-sponsored Acute and Early Disease Research Pro- containing antibodies for flow cytometry. PBMCs were prepared gram (AIEDRP) as follows: enzyme-linked immunosorbent assay by centrifugation on a Ficoll-Hypaque density gradient (Media- negative (stage Ia), Western blot indeterminate (stage Ib), or a de- tech). PBMCs were stained for flow cytometry immediately after tuned ELISA (20) nonreactive (stage II) or a documented nega- isolation. tive within 6 mo of presentation (stage III). Eight HIV-1–infected Flow Cytometry. Cell surface expression of lymphocyte anti- subjects were studied during the chronic stage of HIV-1 infec- gens was identified by monoclonal antibody staining of freshly tion. The primary infection cohort was subgrouped as follows isolated MMCs and PBMCs, followed by flow cytometry using (see Table I): group 1: HAART naive, newly diagnosed subjects a FACSCalibur (Becton Dickinson Immunocytometry Systems (n  11), as well as two subjects who were treated with HAART [BDIS]) with analysis using CELLQuest software (BDIS). Mono- for 1 wk before biopsy; and group 2: HIV-1–infected subjects, clonal antibodies used in this study included anti–human CD3 identified during the primary infection stage and had received FITC (clone UCHT1; BDIS), anti–human CD3-PE (clone HAART for up to 5 yr (n  8). Group 2 is comprised of subjects SK-7; BDIS), anti–human CD3–peridinin chlorophyll-a protein who had received HAART for 6 mo (n  2), 1 yr (n  2), 2 yr (clone SK-7; BDIS), anti–human CD4-allophycocyanin (clone (n  2), 3 yr (n  1), and 5 yr (n  1). There was no difference RPA T4; BD Biosciences), anti–human CD8 PE (clone RPA between groups 1 and 2 with respect to CD4 T cell count on T8; BD Biosciences), anti–human CXCR4-PE (clone 12G5; BD presentation (P  0.5), log HIV-1 viral load on presentation (P  Biosciences), and anti–human CCR5-FITC (clone 2D7/CCR5; 0.7), or the estimated duration of infection on presentation (P  BD Biosciences). During flow cytometry, lymphocytes, initially 0.1). The mean age for primary infection subjects was 35 yr. All identified by their forward and side scatter characteristics, were subjects contracted HIV-1 sexually during same sex contact. Sub- subject to phenotypic analysis. Dead cells were excluded from Primary HIV-1 Infection CD4 T Cell Depletion Gastrointestinal Tract analysis using 7-aminoactinomycin D (Calbiochem). To deter- subjects using a two-sample, unequal variance t test. All reported mine the percentages of CD4 and CD8 cells in the T cell pop- p-values were two-sided at the 0.05 significance level using Win- ulation, gated lymphocytes were initially examined for the ex- dows software (SPSS 11.0; SPSS Inc.). pression of CD3. The CD3 lymphocytes were then analyzed for expression of CD4 and CD8 receptors. To evaluate the expres- sion of chemokine coreceptors, gated lymphocytes were initially Results examined for the expression of CD4 receptor. The CD4 lym- Primary HIV-1 Infection Is Associated with a Preferential phocytes were further examined for the expression of chemokine Depletion of mucosal CD4 T Cells. Isolated MMCs and coreceptors CCR5 and CXCR4. PBMCs from all subjects were examined using flow cy- Light Microscopy and Immunohistochemistry. For light micro- tometry to determine the percentage of CD4 T cells (Fig. scopic evaluation, tissues were fixed in 4% neutral-buffered forma- 1 A). The percentage of mucosal T cells expressing CD4 was lin and embedded in paraffin. 5-m thick sections were cut and significantly less than the percentage of CD4 T cells in the stained with hematoxylin and eosin and Giemsa stains. Immuno- histochemistry was also performed on paraffin-embedded sections peripheral blood in every patient in this group (Fig. 1 B). after high temperature antigen retrieval as described previously When cumulative data from group 1 was analyzed, the (21). The following antibodies were used according to the manu- mean mucosal CD4 T cell percentage was 15.7  3.6, and facturer’s instructions: NCL-CD4-IF6 (anti-CD4 antibody; No- was significantly less than the mean CD4 T cell percent- vocastra) and C8/144B (anti-CD8 antibody; Dakopatts). Binding of antibodies was visualized by the alkaline phosphatase/antialka- line phosphatase method using new fuchsin as a chromogen. In Situ Hybridization. The in situ hybridization was per- formed on paraffin sections as described previously (21). In brief, 5-m sections were cut onto slides coated with 3-amino-propyl- trietho-silane. Dewaxed paraffin-embedded sections were either boiled in a domestic pressure cooker in citrate buffer (pH 6.0) for 5 min or treated with 0.01 mg/ml proteinase K for 8 min at room temperature and subjected to in situ hybridization to detect viral RNA. We used a S-labeled, single stranded antisense RNA probe of HIV-1 (Lofstrand Labs). The probe was com- posed of fragments of 1.4–2.7 kb, which collectively represent 90% of the HIV-1 genome (22). The specific activity of the probe was 2  10 dpm of probe/milliliter. The hybridization was performed overnight at 45C in a moist chamber. The slides were washed, digested with RNase (Boehringer) at 37 C for 40 min, and washed again. The slides were then dipped into photo emulsion (NTB2; Eastman Kodak Co.), exposed for 7 d, devel- oped, counterstained with hematoxylin, and mounted. As a posi- tive control, paraffin-embedded sections from the spleen of an HIV-infected patient were used. As a negative control, sections were hybridized with a S-labeled sense probe. Quantitative Analysis of HIV RNA Cells, CD4 Cells, and CD8 Cells. The autoradiographs were examined with a mi- croscope equipped with epiluminescent illumination (Axiophot; Carl Zeiss MicroImaging, Inc.), a 3CD camera, and a PC-based image analysis system (KS 4000; Kontron). Cells were considered positive for viral gene expression if the grain count was more than six times the background. The positive cells were counted. The area occupied by lymphoid follicles was measured and the frequency of RNA-producing cells per mm of gut-associated lymphoid tissue (GALT) was calculated. To evaluate the number and distribution of T cell subsets, transmission light was used. Us- ing a 40 objective, a standard area was set by the image ana- Figure 1. CD4 T cells are preferentially depleted in the GI tract in lyzer. The number of positive cells within this unit area was de- acute and early HIV-1 infection. PBMCs and MMCs from the rectosigmoid termined by manual counting. For the lamina propria, a total of region from group 1, acute and early HIV-1 infection subjects (n  13), and 10–15 consecutive nonoverlapping fields was analyzed for each HIV-1–uninfected controls (n  10) were analyzed by flow cytometry. staining. For the GALT, two to five representative areas were CD3 gated lymphocytes were analyzed for the expression of CD4 and CD8. (A) A representative flow plot from subject 105 is depicted. CD8 chosen. The individual values obtained with each T cell marker T cells are shown on the x axis and CD4 T cells are shown on the y axis. were then pooled and the mean numbers of positive cells per unit (B) Comparison of CD4 T cells in the blood and GI tract of all 13 group area of lamina propria or GALT were determined separately. 1 primary infection subjects. The percent of CD4 T cells is shown in Statistical Methodology. Values are expressed as mean  stan- PBMCs (gray) and MMCs (white) per study subject. (C) Comparison of dard deviation. Statistical comparisons were made between the mean percent of CD4 T cells in the PBMCs of HIV-1–uninfected PBMCs and MMCs from individuals using a paired t test. Statisti- controls (black bar) and group1 subjects (gray bar), and MMCs of HIV-1– cal comparisons were made between HIV-1–infected and control uninfected controls (hatched bar) and group 1 subjects (white bar). Mehandru et al. age in the peripheral blood (42.3  14.7; P  0.001). The mean CD4/CD8 ratio in the MMCs (0.2  0.04) was also significantly lower than the mean CD4/CD8 ratio in the circulating PBMC compartment (0.9  0.6; P  0.002). In HIV-1–uninfected individuals, the percentage of CD4 T cells in the mucosa (56.4  8.8%) and blood (59.6 14.3%) were not significantly different (P  0.24), and the CD4/CD8 ratios in the mucosa and blood were 1.3 0.5% and 1.7  0.7%, respectively. Comparison of the percentages of CD4 T cells in the mucosa of HIV-1–uninfected and newly diagnosed subjects with primary HIV-1 infection (group 1) revealed highly statistically significant differences with marked depletion in the HIV-1–infected group (P  0.001; Fig. 1 C). As ex- pected, depletion of peripheral blood CD4 T cells was also observed when newly diagnosed, primary HIV-1–infected subjects were compared with HIV-1–uninfected subjects. However, the differences between the two groups were much less marked, though still significant (P  0.01). Primary HIV-1 Infection Is Associated with Preferential De- pletion of CCR5-expressing Mucosal CD4 T Cells. The Figure 2. CD4 T cells expressing CCR5 are preferentially depleted majority of GI MMCs express CCR5, either alone or along from the GI tract in acute and early HIV-1 infection. PBMCs (A) and with CXCR4 (4, 8). Given the high expression of CCR5 MMCs (B) from the rectosigmoid region from individuals with acute and in the mucosa and the possibility that the mucosal environ- early HIV-1 infection (group 1, n  13) and HIV-1–uninfected controls /CD4 gated lympho- (n  10) were analyzed by flow cytometry. CD3 ment plays an important role in the selection of the CCR5- cytes were analyzed for the expression of CXCR4, CCR5, or CCR5/ tropic strains of HIV-1 that predominate in the blood dur- CXCR4. HIV-1–uninfected PBMCs (black bars), group 1 PBMCs (gray ing the primary infection period, we sought to determine bars), HIV-1–uninfected MMCs (hatched bars), and group 1 MMCs the effect of primary HIV-1 infection on CCR5-expressing T cells expressing (white bars) are shown. Mean percent of CD4 chemokine receptors are depicted on the y axis and chemokine corecep- CD4 MMCs. Using four-color flow cytometry, we quan- tors CXCR4, CCR5, and CCR5/CXCR4 are grouped on the x axis. tified the percentage of CD3 /CD4 MMCs and PBMCs that expressed CCR5, CXCR4, or coexpressed CCR5 and CXCR4. Comparison was made between HIV-1–unin- and CXCR4. In the uninfected individuals, 56.2  13.9% fected subjects and the untreated primary HIV-1 infection of the CD4 mucosal T cells coexpressed CCR5 and CXCR4 subjects (group 1). In general there was no difference in lev- compared with just 21.1  14.3% in the group 1 subjects els of CD3 /CD4 cells expressing CCR5, CXCR4, or (P  0.001; Fig. 2 B). both in the peripheral blood of uninfected controls when CD4 T Cells Are Preferentially Depleted from the Effector compared with subjects in group 1 (Fig. 2 A). Mean levels Sites in the GI Mucosa. Having shown that the percentage of CD4 PBMC populations that expressed CXCR4 were of CD4 T cells is decreased in acute and early HIV-1 in- 64.3  15.2% in the HIV-1–uninfected subjects compared fection, we went on to examine the absolute number of with 63.7  11.8% in group 1 primary HIV-1 infection CD4 T cells in the GI biopsies. Using immunohistochem- subjects (P  0.6). Levels of CD3 /CD4 PBMCs express- istry, paraffin-embedded mucosal biopsies were compared ing CCR5 were 18.7  9.0% in HIV-1–uninfected subjects between HIV-1–uninfected individuals and subjects with compared with 14.8  11.0% in group 1 (P  0.4). Levels acute and early HIV-1 infection. To classify the anatomic of CD3 /CD4 PBMCs coexpressing CCR5 and CXCR4 subcompartment where CD4 T cell depletion occurred, were 9.6  5.8% in HIV-1–uninfected subjects versus 6.9 inductive and effector sites were examined separately. 8.1% in group 1 primary HIV-1 infection subjects (P  The inductive sites or lymphoid nodules consisted of a 0.4). In comparison, analysis of CD4 mucosal T cells re- centrally located B cell–dependent area with or without a vealed significant depletion in the HIV-1–infected subjects germinal center (Fig. 3 A). The follicle-associated epithe- that was most prominent in the CD3 /CD4 CCR5- lium and the dome area were noted in only a few of the sec- expressing populations. Although there was only a minor tions examined. The B cell follicle was surrounded by the degree of depletion of mucosal CD4 T cells expressing T-dependent zone, similar to that seen in the paracortex of a CXCR4 (76.3  14.4% in HIV-1–uninfected subjects vs. lymph node. In subjects with acute and early HIV-1 infec- 61.7  10.9 in group 1 subjects; P  0.03), the percentage tion (Fig. 3 B) and in HIV-1–uninfected controls (Fig. 3 C), of cells expressing CCR5 alone was 73.5  13.4% in HIV- the T cell zone was densely populated with CD4 T cells. In 1–uninfected subjects versus 42.5  18.9% in group 1 (P comparison, significantly more CD8 T cells were noted in 0.001). The most significant depletion was noted in the the subjects with acute and early HIV-1 infection (mean: population of CD4 mucosal T cells that coexpressed CCR5 130 cells/unit area; range: 95–137 cells/unit area) compared Primary HIV-1 Infection CD4 T Cell Depletion Gastrointestinal Tract 764 Figure 3. Effector sites (lamina pro- pria) of the GI tract show the most pro- nounced CD4 T cell depletion in acute and early HIV-1 infection. Immunohis- tochemical characterization of the effector and inductive sites in the rectal biopsies. Using a PC-based image analysis system (KS 4000; Kontron), a standard area was set by the image analyzer. For the lamina propria, a total of 10–15 consecutive nonoverlapping fields were analyzed for each staining. For the GALT, two to five representative areas were chosen. (A) At a magnification of 25, a Giemsa- stained section is shown depicting a lymphoid nodule (inductive site) where the germinal center (GC) and T cell zone (T) are indicated. The effector compartment represented by the lamina propria (LP) is seen in the lower part of the section. (B and C) The inductive sites are densely populated with CD4 T cells (stained red) in both group 1 (B) and HIV-1–uninfected (C) subjects. A magnification of 100. (D and E) Effector sites in acute and early HIV-1 infection (D) show significant depletion of CD4 T cells (stained red) compared with HIV-1–uninfected controls (E). A mag- nification of 100. (F) In the inductive and effector compartments, the mean of CD4 and CD8 T cells per unit area was determined and the CD4/CD8 ratio was calculated. A comparison of the mean CD4/CD8 ratio (represented on the y axis) between HIV-1–uninfected (gray bars) and group 1 subjects (white bars) is shown. with the HIV-1–uninfected controls (mean: 83 cells/unit situ hybridization was performed to localize HIV-1 RNA area; range: 81–140 cells/unit area). The mean CD4/CD8 within the effector and inductive subcompartments of the ratio in the inductive sites of HIV-1–uninfected controls was GI mucosa. Cells expressing HIV-1 RNA were predomi- 2.4 (range 1.6–3.1). In comparison, the mean CD4/CD8 ra- nantly localized to the organized lymphoid follicles or the tio in the inductive sites of subjects with acute and early inductive sites of the GI mucosa (Fig. 4, A and B), present HIV-1 infection was 1.3 (range 0.9–2.0). It is important to both in the T cell zone and the germinal centers. Even note that the lower CD4/CD8 ratio in HIV-1–infected sub- though CD4 T cells were depleted in the effector sites, of jects was due to an increase in the number of CD8 T cells the CD4 T cells that remained, productively infected cells and not due to a depletion of the CD4 T cells. were either absent or very rare. Interestingly, no viral trap- Significant CD4 T cell depletion was noted in the ef- ping by follicular dendritic cells was noted in the biopsies fector sites in subjects with acute and early HIV-1 infection of the group 1 primary infection subjects. (Fig. 3 D) compared with HIV-1–uninfected controls (Fig. Reconstitution of CD4 MMCs Is Incomplete Despite Sup- 3 E). In this site, the mean CD4/CD8 ratio was 0.4 (range pressive Antiretroviral Therapy. To examine whether appar- 0.1–0.7) in the HIV-1–infected subjects and 1.5 (1.1–2) in ently suppressive HAART would result in reconstitution the HIV-1–uninfected controls (Fig. 3 F). of CD4 MMCs, as has been described among CD4 HIV-1 RNA Is Located within the Inductive Sites of the GI PBMCs, subjects in whom HAART was initiated during Mucosa. Having shown that depletion of mucosal CD4 acute and early HIV-1 infection were examined. We T cells was primarily noted in the effector compartment, performed a cross-sectional analysis of primary HIV-1– we sought to determine whether this anatomic subcom- infected subjects who had been treated with regimens con- partment was also the principal site of viral replication. In taining three or four drug combinations for varying dura- Mehandru et al. 765 Figure 5. Prolonged HAART results in only partial reconstitution of the GI tract CD4 T cells. PBMCs and MMCs from the rectosigmoid region from HIV-1–uninfected individuals (n  10), subjects with acute and early HIV-1 infection (n  13), subjects with HIV-1 infection treated during the primary infection stage (n  8), and chronically infected HIV-1– untreated subjects (n  8) were analyzed by flow cytometry. The mean percent of CD4 T cells is shown in PBMCs (gray bars) and MMCs (white bars) per study group. than the mean CD4/CD8 ratio in the blood (1.7  0.6) Figure 4. HIV-1 RNA is localized in the organized lymphoid follicles and mucosa (1.3  0.4) of HIV-1–uninfected controls. It is of the GI tract in subjects with acute and early HIV-1 infection. In situ of interest that these two subjects had initiated HAART af- hybridization was performed using a S-labeled, single stranded antisense ter 28 and 26 d, respectively, of their estimated dates of in- RNA probe as described in Materials and Methods. Cells expressing fection (Table I). In subjects 106 and 923, the CD4/CD8 HIV-1 RNA (arrows) are present in the germinal center and T cell zone. A magnification of 40. ratio in the GI tract continued to be significantly lower compared with the blood, despite treatment with HAART for 3 and 5 yr, respectively. These two subjects had initi- tions (n  8), and eight untreated HIV-1–infected ated HAART after 168 and 204 d, respectively, of their es- subjects biopsied during chronic HIV-1 infection. The timated date of infection (Table I). Notably for subject 56, primary HIV-1 infection cohort included two subjects from the CD4/CD8 ratio in the blood normalized to 2.9 with group 1 who had been treated with suppressive HAART fully suppressive HAART for 2 yr. In comparison, the for 6 mo, two subjects who had been treated with suppres- CD4/CD8 ratio in the GI tract remained considerably sive HAART for 1 yr, two subjects who had been treated lower at 0.8. with suppressive HAART for 2 yr, and two subjects each As expected, in chronic, untreated HIV-1–infected sub- treated for 3 and 5 yr, respectively. The mean percentage jects, the mean CD4 T cell percentage continued to de- of CD4 T cells in the peripheral blood in this group was cline in both the blood (15.3  17%) and mucosal (12.5 51.3  11.3%, whereas in the GI mucosa, it was 35.4  11%) compartments (Fig. 5). 7.8% (P  0.008; Fig. 5). When the percentage of CD4 T cells of these subjects was compared with that from HIV- Discussion 1–uninfected subjects, there was a significant difference in the mucosal compartment (P  0.001), even though the Being that the GI tract is the largest lymphoid organ in difference in the blood was no longer statistically significant the body, it would follow that the potential for deleterious (P  0.06). The mean CD4/CD8 ratio in the mucosal effects of HIV-1 infection would be great at this site. Al- compartment (0.6  0.2) continued to be significantly though the effect of primary HIV-1 infection on lympho- lower than the CD4/CD8 ratio in the blood compartment cyte populations in the peripheral blood has been well (1.3  0.7) of HAART-treated primary infection subjects characterized, the events associated with primary HIV-1 (P  0.03). infection in GI mucosal lymphocyte populations in man To better understand the effect of HAART in this di- has to date remained largely unknown. Given the strong verse group, we analyzed each patient individually (Table selection pressure for CCR5 coreceptor using viruses dur- II). In the two subjects (100-6m and 336-6m) followed ing transmission of HIV-1 (23), it would be likely that the longitudinally for 6 mo, the CD4/CD8 ratio in the blood sizable population of CCR5-expressing, immunologically showed significant improvement over baseline with treat- activated CD4 T cells in the GI mucosa would be more ment (0.6 in the untreated stage to 1.1 after 6 mo of supportive of viral replication and susceptible to its delete- HAART in both subjects). However, the CD4/CD8 ratio rious effects as compared with the peripheral blood where in the GI tract continued to be low (0.4 and 0.5, respec- the T cells are predominantly CCR5 and naive (8). We tively). In subjects 78 and 132, the CD4/CD8 ratio in the undertook this study to assess whether a preferential deple- blood and GI tract equalized but continued to be lower tion of mucosal CD4 T cells would be observed in pri- Primary HIV-1 Infection CD4 T Cell Depletion Gastrointestinal Tract 766 Table I. Patient Characteristics CD4 T cell count Log plasma HIV-1 Estimated duration of infection (cells/mm ) (copies/ml) (days) Duration of HAART prior to biopsy Study Stage At presentation At biopsy At presentation At biopsy At presentation Prior to HAART initiation (days) Group 1 102 Ia 395 391 6.9 7.1 18 NA NA 100 II 610 595 5.8 5.5 45 NA NA 104 II 981 1,321 6.0 5.8 38 NA NA 107 Ia 456 426 6.1 5.6 24 NA NA 336 II 556 556 5.0 5.0 62 NA NA 109 II 340 340 6.3 6.3 19 NA NA 119 Ia 248 248 6.9 6.3 18 NA NA 122 III 285 400 5.1 4.9 72 NA NA 125 II 830 684 5.8 5.0 36 NA NA 127 Ia 390 350 4.8 4.9 23 NA NA 131 Ia 250 349 6.7 6.6 25 NA NA 105 Ia 374 462 7.1 4.7 27 7 7 106 Ia 317 368 6.8 5.4 26 7 7 Group 2 100-6m II 610 698 5.8 1.7 45 76 214 336-6m II 556 996 4.9 1.7 48 90 229 64 Ia 846 994 7.2 1.7 28 31 398 78 II 175 460 7.4 1.7 26 28 392 56 II 619 752 5.4 1.7 61 71 670 132 II 366 657 6.1 1.7 19 26 763 106 III 351 595 6.9 1.7 157 168 1,182 923 Ia 698 939 5.1 1.7 201 204 1,932 Subjects were staged as per the NIH-sponsored AIEDRP as follows: enzyme-linked immunosorbent assay negative (stage Ia), Western blot indeter- minate (stage Ib), or a detuned ELISA nonreactive (stage II) or a documented negative within 6 mo of presentation (Stage III). The approximate date of infection was calculated as follows: 2 wk before the onset of acute retroviral illness (97%); in cases where no acute retroviral illness symptoms were experienced, the midpoint between the last negative antibody result and first positive Western blot result was determined. mary HIV-1–infected subjects as has been observed in the Table II. Comparison of Blood and GI Tract Reconstitution SIV-macaque model. We went on to examine the ana- with HAART tomic subcompartment from which these cells are de- pleted and examine whether suppressive HAART therapy CD4/CD8 ratio CD4/CD8 ratio Duration of HAART Subject in the blood in the GI tract at the time of biopsy could result in complete immune reconstitution in the mucosal compartment. Previous studies of the effect of HIV-1 on the GI mu- 100-6m 1.1 0.4 6 mo cosal CD4 T cells during the primary infection period 336-6m 1.1 0.5 6 mo have been limited to a single report, with two subjects 64 1.6 0.6 1 yr studied during early HIV-1 infection. In both of these indi- 78 0.7 0.7 1 yr viduals, identified after 4 and 6 wk of the estimated date of 56 2.9 0.8 2 yr infection, mucosal CD4 T cell depletion was noted (19). 132 0.7 0.8 2 yr The remaining data suggesting the GI tract as the preferred 106 0.9 0.5 3 yr site for virus replication and CD4 T cell depletion are de- 923 1 0.5 5 yr rived from studies examining primary infection of macaques with SIV (2, 3, 13, 15, 17, 18, 24, 25). Mehandru et al. 767 Here we have described 13 subjects who are identified calized in the inductive compartment of the mucosa. These and studied extremely early in the course of HIV-1 infec- findings are consistent with the SIV-macaque model where tion. We have confirmed that indeed, significant mucosal during early infection, most of the SIV-infected cells are CD4 T cell depletion occurs in subjects during primary located in the inductive sites. In a recent study, Veazey et HIV-1 infection, before changes seen in the peripheral al. (26) noted that very early in the course of infection, i.e., blood. However, human mucosal CD4 T cell depletion from 7 to 14 d after infection, SIV-infected cells could be appears to be less marked than that described in primary localized in the effector compartment as well. However, SIV infection, where the percentage of CD4 T cells in the once effector CD4 T cells were depleted, SIV RNA was macaque small bowel was reduced to 10% by 3 wk after predominantly seen in the inductive compartment. Our infection (13). This is likely explained by a difference in the patients, although studied extremely early in the course of site sampled. All the biopsies in our studies were obtained HIV-1 infection, had already developed profound CD4 T from the rectal mucosa, which has both effector and induc- cell depletion in the effector sites. Thus, one possible ex- tive lymphoid tissue. In the macaque models described, the planation for the localization of HIV-1 RNA only in the biopsies are usually obtained from the proximal jejunum, inductive compartment is the loss of target cells from the which is devoid of immune inductive sites (26). As a result, effector sites by the time the biopsies were performed. samples obtained from the jejunum in macaque models Further studies are underway to better characterize the may demonstrate a much higher degree of CD4 T cell de- dynamics of CD4 T cell loss noted in the effector com- pletion compared with what is noted in this study. There partment. Some of the possibilities include decreased local may also be other factors responsible. SIV is introduced in proliferation, increased cell death in the effector compart- large inocula to attempt to guarantee infection. Simian ment due to apoptosis or activation-induced cell death AIDS is characterized by an accelerated natural history of (36), reduced homing of cells to the effector site from the the disease (27, 28) and is accompanied by higher peak periphery after antigen recognition due to either direct cy- plasma viral levels (29, 30) when compared with HIV-1 in- topathic effects of HIV-1 or immune activation-induced fection in man. Also, SIV is known to use alternative and cell death, or perhaps combinations of the above (37). additional coreceptors when compared with HIV-1 (31, By studying subjects in whom treatment was initiated 32), which may account for apparent differences in replica- during acute and early HIV-1 infection, we sought to un- tion dynamics. Yet unexplained differences may also exist derstand whether HAART would result in mucosal CD4 in host susceptibility to the two infections. T cell reconstitution. Guadalupe et al. (19) showed that in In addition to confirming the numerical decrease in mu- one subject, mucosal CD4 T cell reconstitution was at- cosal CD4 T cell percentage, we have gone on to charac- tained when HAART was initiated within 6 wk of the es- terize the relationship between CD4 T cell depletion and timated date of infection. In comparison, we did not see HIV-1 RNA localization within specific anatomic sub- immune reconstitution in the GI mucosa with HAART in compartments of the GI mucosa. Like the small intestine, our study population. It is to be noted that all eight patients the immune compartment in the rectal mucosa is also di- studied initiated therapy in the primary infection stage, re- vided into inductive and effector arms (24, 33). The orga- mained compliant with treatment, and continued to have nized lymphoid tissue with a high content of CD68 MHC undetectable plasma HIV-1 RNA levels for the entire du- class II macrophages and cells expressing costimulatory ration of the study period. The cohort remains small and molecules CD86 and CD40 serves as an antigen-presenting we plan to study more subjects longitudinally to better as- site (24). The majority of lymphocytes present in the in- sess the degree of immune reconstitution and the factors ductive compartment are antigen naive. However, once that may predispose to a greater or lesser response. activated and primed by antigen-presenting cells such as The consequence of early depletion and incomplete re- dendritic cells, GI mucosal lymphocytes can home back to constitution of mucosal CD4 T cells is unclear. Although the mucosa to perform effector functions (34). The lamina opportunistic infections remain rare until the peripheral propria lymphocytes, which are comprised of a high per- CD4 T cell count falls during the chronic infection pe- centage of differentiated effector cells (9, 10), serve as the riod, the long-term outcome of treated HIV-1 infection effector arm of the mucosal immune system (33). A limited remains unknown. Furthermore, the nature of the CD4 T number of studies have described the effects of SIV infec- cell loss requires additional study. Theoretically, the loss of tion (24–26) and chronic and advanced HIV-1 infection specific clones of CD4 T cells in the mucosa may predis- (35) on these distinct, anatomic subcompartments. Our re- pose to accelerated immune senescence, more rapid than sults suggest that as early as the primary infection stage, that seen as a consequence of aging, which in turn may re- there is a depletion of CD4 T cells from the effector com- sult in the increased incidence of malignancies, be they partment of the GI mucosa. Given the activated and differ- lymphomas or solid tumors such as adenocarcinomas. entiated phenotype of these cells, direct cytopathic effects Given the importance of the mucosal compartment in of the HIV-1 virus could be one mechanism explaining this HIV-1 pathogenesis, our findings may have important im- lesion. To assess this, we performed in situ hybridization plications with regard to treatment. Perhaps efforts to spe- for HIV-1 RNA to localize the sites of HIV-1 replication cifically inhibit T cell activation in the GI tract as well as in the GI mucosa. We observed that HIV-1 RNA was lo- drugs to more specifically interfere with HIV-1 entry (38, Primary HIV-1 Infection CD4 T Cell Depletion Gastrointestinal Tract 768 39) and replication in the GI mucosa during the primary 9. Kim, S.K., D.S. Reed, W.R. Heath, F. Carbone, and L. Le- francois. 1997. Activation and migration of CD8 T cells in infection period may result in lowering of the viral set the intestinal mucosa. J. Immunol. 159:4295–4306. point and in conjunction with HAART, result in improve- 10. Schieferdecker, H.L., R. Ullrich, H. Hirseland, and M. Zeitz. ment in clinical outcome. Such investigations are clearly 1992. T cell differentiation antigens on lymphocytes in the needed as the goal of optimized treatment of HIV-1 infec- human intestinal lamina propria. J. Immunol. 149:2816–2822. tion is yet to be attained. 11. McGowan, I.M., J. Elliott, M. Fuerst, P. Taing, J. Boscardin, M.A. Poles, and P.A. Anton. 2004. Increased HIV-1 mucosal We would like to thank the patients for their participation. We ac- replication is associated with generalized mucosal cytokine acti- knowledge the nursing staff at The Rockefeller University and vation. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. In press. Bellevue Hospital for their clinical assistance. We would like to 12. Lefrere, J.J., F. Roudot-Thoraval, M. Mariotti, M. Thauvin, thank Wendy Chen for help with the graphs and figures; Petra J. Lerable, J. Salpetrier, and L. Morand-Joubert. 1998. The Meyer, Birgit Raschdorff, and Gudrun Großschupff for their tech- risk of disease progression is determined during the first year nical assistance with immunohistochemistry and in situ hybridiza- of human immunodeficiency virus type 1 infection. J. Infect. tion; and Dr. Gerard Eberl for helpful discussion. Dis. 177:1541–1548. This work was supported in part by the following grants: The 13. Veazey, R.S., M. DeMaria, L.V. Chalifoux, D.E. 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The Journal of Experimental Medicine – Pubmed Central

Published: Sep 20, 2004

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