HLA-DR CD38 CD4 T Lymphocytes Have Elevated CCR5 Expression and Produce the Majority of R5-Tropic HIV-1 RNA In Vivo

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1 JOURNAL OF VIROLOGY, Oct. 2011, p Vol. 85, No X/11/$12.00 doi: /jvi Copyright 2011, American Society for Microbiology. All Rights Reserved. HLA-DR CD38 CD4 T Lymphocytes Have Elevated CCR5 Expression and Produce the Majority of R5-Tropic HIV-1 RNA In Vivo Amie L. Meditz, 1 Michelle K. Haas, 1 Joy M. Folkvord, 1 Kelsey Melander, 1 Russ Young, 1 Martin McCarter, 2 Samantha MaWhinney, 3 Thomas B. Campbell, 1 Yolanda Lie, 4 Eoin Coakley, 4 David N. Levy, 5 and Elizabeth Connick 1 * Division of Infectious Diseases, Department of Medicine, University of Colorado Denver, Aurora, Colorado 1 ; Department of Surgery, University of Colorado Denver, Aurora, Colorado 2 ; Department of Biostatistics and Informatics, University of Colorado Denver, Aurora, Colorado 3 ; Monogram Biosciences, South San Francisco, California 4 ; and New York University, College of Dentistry, New York, New York 5 Received 3 December 2010/Accepted 13 July 2011 Percentages of activated T cells correlate with HIV-1 disease progression, but the underlying mechanisms are not fully understood. We hypothesized that HLA-DR CD38 (DR 38 ) CD4 T cells produce the majority of HIV-1 due to elevated expression of CCR5 and CXCR4. In phytohemagglutinin (PHA)-stimulated CD8-depleted peripheral blood mononuclear cells (PBMC) infected with HIV-1 green fluorescent protein (GFP) reporter viruses, DR 38 T cells constituted the majority of CCR5 (R5)-tropic (median, 62%) and CXCR4 (X4)-tropic HIV-1-producing cells (median, 61%), although cell surface CCR5 and CXCR4 were not elevated in this subset of cells. In lymph nodes from untreated individuals infected with R5-tropic HIV-1, percentages of CCR5 cells were elevated in DR 38 CD4 T cells (median, 36.4%) compared to other CD4 T-cell subsets (median values of 5.7% for DR 38 cells, 19.4% for DR 38 cells, and 7.6% for DR 38 cells; n 18; P < 0.001). In sorted CD8 lymph node T cells, median HIV-1 RNA copies/10 5 cells was higher for DR 38 cells ( ) than for DR 38 ( ), DR 38 ( ), and DR 38 ( ) subsets (n 8; P < for all). After adjusting for percentages of subsets, a median of 87% of viral RNA was harbored by DR 38 cells. Percentages of CCR5 CD4 T cells and concentrations of CCR5 molecules among subsets predicted HIV-1 RNA levels among CD8 DR/38 subsets (P < for both). Median HIV-1 DNA copies/10 5 cells was higher in DR 38 cells (5,360) than in the DR 38 (906), DR 38 (814), and DR 38 (1,984) subsets (n 7; P < 0.031). Thus, DR 38 CD4 T cells in lymph nodes have elevated CCR5 expression, are highly susceptible to infection with R5-tropic virus, and produce the majority of R5-tropic HIV-1. PBMC assays failed to recapitulate in vivo findings, suggesting limited utility. Strategies to reduce numbers of DR 38 CD4 T cells may substantially inhibit HIV-1 replication. Activated T lymphocytes, identified by expression of CD38 (38) alone or in combination with HLA-DR (DR), are strongly implicated in the pathogenesis of HIV-1 infection. Susceptibility to HIV-1 infection has been linked to the percentages of activated CD4 T cells in peripheral blood (1, 24). Furthermore, the percentages of activated lymphocytes in peripheral blood (14, 21, 23) and lymph nodes (2, 35) are increased during HIV-1 infection, correlated with plasma HIV-1 RNA concentration (9, 19), and associated with disease progression (9, 16) and death (15, 30). The mechanisms underlying the strong association between activated lymphocytes and HIV-1 susceptibility and disease progression are not fully understood; both direct infection and replication of HIV-1 by activated CD4 lymphocytes and indirect effects of immune activation resulting * Corresponding author. Mailing address: Division of Infectious Diseases, Department of Medicine, University of Colorado Denver, E. 19th Avenue, Box B168, Aurora, CO Phone: (303) Fax: (303) liz.connick@ucdenver.edu. Present address: HIV Care and Support Program, Botswana- UPenn Partnership, Gaborone, Botswana. Present address: Abbott Laboratories, Abbott Park, IL. Published ahead of print on 3 August in CD4 T-cell depletion have been hypothesized to play a role (44). Knowledge of the proportion of virus replication that is supported by activated CD4 T cells could provide insight into the relative importance of direct infection of activated cells versus indirect effects of immune activation in HIV-1 immunopathogenesis. One study reported that HIV-1 gag DNA is elevated in activated peripheral blood memory CD4 T cells (defined as 38, DR, or Ki67 cells that also expressed CD45RO) than in other memory CD4 T cells (31), suggesting that activated cells may be preferentially infected in vivo. Nevertheless, neither the proportion of gag DNA nor the amount of HIV-1 RNA produced by activated memory CD4 T cells was determined in this study. Furthermore, it is unclear if peripheral blood measurements reflect those in lymphoid tissues, where the majority of HIV-1 replication occurs in vivo (12, 42, 45, 47). Most HIV-1 RNA in lymphoid tissues is produced by T lymphocytes (42, 47, 50). In situ analyses of lymph nodes from 5 HIV-1-infected humans in early HIV-1 infection revealed that approximately half of the HIV-1 RNA-producing cells expressed DR (59%) or Ki67 (43%), and in 10 subjects with AIDS, even higher proportions of virus-producing cells were found in DR (85%) and Ki67 (76%) cells (50). Nev

2 10190 MEDITZ ET AL. J. VIROL. ertheless, the magnitude of HIV-1 RNA and DNA harbored by DR 38 T cells, which are more strongly linked to viral load and disease progression than either DR cells or Ki67 cells alone, has never been quantified. Furthermore, mechanisms underlying infection and replication of HIV-1 by activated lymphocytes in lymphoid tissues, such as HIV-1 chemokine coreceptor expression, have not been evaluated. The purpose of the present study was to measure the amount of HIV-1 produced by DR 38 CD4 T cells in peripheral blood mononuclear cells (PBMC) in vitro and in lymphoid tissues in vivo and to investigate whether HIV-1 infection of these cells is related to the levels of HIV-1 chemokine coreceptors. We hypothesized that DR 38 CD4 T cells produce the majority of HIV-1 and that this is related to increased susceptibility of these cells to HIV-1 infection conferred by elevated levels of cell surface CCR5 and CXCR4. (Part of this research was presented at the 16th Conference for Retroviruses and Opportunistic Infections in 2009 [abstract 311].) MATERIALS AND METHODS Study subjects and clinical specimens. Individuals at low risk for HIV-1 infection donated peripheral blood specimens for in vitro HIV-1 replication assays. Inguinal lymph nodes were obtained as previously described (12) from individuals with documented HIV-1 infection for at least 6 months, who were not receiving antiretroviral therapy and had CD4 T-cell counts of 300/mm 3. None of these subjects had an opportunistic infection, malignancy, or acute illness at the time of lymph node excision. Inguinal lymph nodes were also obtained from HIV-1-seronegative individuals while they underwent a nonemergent surgical procedure in the groin area. Peripheral blood specimens were obtained at the same time as lymph node specimens. Informed consent was obtained from all subjects, and the study was approved by the Colorado Multiple Institutional Review Board. Peripheral blood mononuclear cells (PBMC) were isolated by density centrifugation using lymphocyte separation medium (Mediatech, Herndon, VA) according to the manufacturer s instructions. Inguinal lymph nodes were disaggregated as previously described (12), and cells were either used immediately in assays or cryopreserved for future studies. In HIV-1-seropositive subjects, peripheral blood CD4 T-cell counts were determined by flow cytometry, plasma HIV-1 RNA concentration was measured by Roche COBAS TaqMan 96 HIV-1 test (Indianapolis, IN), and HIV-1 coreceptor tropism was assayed by the Trofile test (Monogram Biosciences, South San Francisco, CA). For seronegative lymph node donors, HIV-1 serostatus was confirmed by an enzyme-linked immunosorbent assay (ELISA). In vitro infection with HIV-1 GFP reporter viruses. The HIV-1 NL4-3-based CXCR4 (X4)-tropic green fluorescent protein (GFP) reporter virus NLENG1- IRES has been described elsewhere (28). A CCR5 (R5)-tropic GFP reporter virus called NLYUV3-GFP was constructed from NLENG1-IRES by replacing the envelope V3 region with the V3 loops from the YU-2 HIV-1 molecular clone. CCR5 tropism was demonstrated by successful infection of CCR5-GHOST cells, but not CXCR4-GHOST cells (NIH AIDS Research and Reference Reagent Program) (33). Virus stocks were generated by transfection of 293T cells using Effectene (Qiagen, Valencia, CA), and p24 concentration was determined by ELISA (PerkinElmer, Shelton, CT). Freshly isolated PBMC from individuals at low risk for HIV-1 infection were depleted of CD8 cells using anti-cd8 antibody-coated magnetic beads (Invitrogen, Carlsbad, CA). The cells were stimulated with phytohemagglutinin (PHA) (5 g/ml, Sigma) in RPMI 1640 (Mediatech, Inc., Manassas, VA) with 10% fetal bovine serum (FBS) (Invitrogen Life Science, San Diego, CA) and 10 U/ml interleukin 2 (IL-2) (Roche, Indianapolis, IN) for 60 h, and then to cells were incubated with 0.5 ml to 1.5 ml of either CCR5-tropic reporter virus stock (ranging from 400 to 1,050 ng of p24 antigen/ml) or CXCR4-tropic reporter virus stock (ranging from 380 to 1,050 ng of p24 antigen/ml) for 2hat37 C. The cells were diluted to cells/ml in RPMI 1640 with 10% FBS and 10 U/ml IL-2 and incubated for 2 days at 37 C with 5% CO 2. The cells were then stained with monoclonal antibodies, CD3-PE (phycoerythrin [PE]-conjugated CD3), CD4-APC-H7 (allophycocyanin [APC] H7-conjugated CD4), HLA-DR-APC (APC-conjugated HLA-DR), CD38-PE- Cy5 (PE- and Cy5-conjugated CD38) (all obtained from BD Biosciences, San Diego, CA), and CD8-AF405 (Alexa Fluor 405 [AF405]-conjugated CD8) (Invitrogen Life Science). Data were acquired on an LSR II flow cytometer (BD Immunocytometry System, San Jose, CA) and analyzed using FlowJo (Tree Star, Ashland, OR). For a control, a well containing uninfected cells was included in each experiment. HIV-1 chemokine coreceptor flow cytometry analyses. Lymph node and whole blood cells were incubated with 1% normal goat serum to block nonspecific staining and then treated with antibodies to CD3-PE-Cy5 (BD Biosciences, San Jose, CA), CD4-APC-H7, CD8-AF405, CD38-fluorescein isothiocyanate (FITC) (Invitrogen Life Science), HLA-DR-APC, and CCR5-PE or CXCR4-PE (manufactured by BD Biosciences with known 1:1 PE/antibody ratio) for 30 min at room temperature. Red blood cells were lysed with fluorescence-activated cell sorting (FACS) lysis buffer (BD Biosciences) from whole-blood samples, and all cells were washed with phosphate-buffered saline (PBS) (Mediatech, Inc.) containing 1% bovine serum albumin (Sigma, St. Louis, MO) and fixed in 2% paraformaldehyde. Data were acquired using an LSR II flow cytometer (BD Immunocytometry Systems) and analyzed using FlowJo (Tree Star, Ashland, OR). QuantiBRITE beads (BD Biosciences, San Diego, CA) were used to determine the mean number of CCR5 or CXCR4 molecules on the surfaces of lymphocyte subsets as previously described (32). The linear range of these measurements was 474 to 69,045 PE molecules/cell. Sorting of lymph node cells into subsets. Cryopreserved lymph node cells were thawed, stained, and sorted on a FACSAria (BD Immunocytometry Systems, San Jose, CA). To determine the amount of HIV-1 RNA in CD4 and CD8 lymphocyte subsets, cells were stained with antibodies to CD3-PE-Cy5, CD4- APC-H7 (BD Biosciences) and CD8-AF405 (Invitrogen Life Science), gated on the CD3 population, and sorted into the following subsets: CD4 CD8, CD4 CD8, CD4 CD8, and CD4 CD8. To determine the amount of HIV-1 RNA and DNA harbored by subsets defined by HLA-DR and CD38, the cells were stained with antibodies to CD3-FITC, CD38 PE-Texas Red HLA-DR- APC (BD Biosciences) and CD8-PE (Invitrogen Life Science), gated on the CD3 CD8 population, and sorted into the following subsets: DR 38,DR 38,DR 38, and DR 38. Aliquots of 100,000 sorted cells were stored at 70 C as a dry pellet. Quantification of HIV-1 RNA within lymph node cell subsets. Cellular RNA was extracted using a RNA blood extraction kit (Qiagen, Gaithersburg, MD). HIV-1 RNA was quantified by a nested PCR amplification of the HIV-1 long terminal repeat (LTR) by previously described methods (8, 40). HIV-1 RNA standards were included in each PCR by dilution of known amounts of the HIV-1 BAL strain in a background of 10 5 HIV-1 uninfected tonsil cells ranging from 100 to 10 6 HIV copies/ml to obtain a range of 5 to 50,000 HIV copies per first-round PCR. A standard curve was constructed by plotting the cycle threshold in the second PCR (cutoff, fluorescence 10 times the background) versus RNA concentration of HIV-1 RNA standards. All HIV-1 RNA measurements were corrected for HIV-1 DNA contamination of the RNA preparations. HIV-1 DNA concentration in the RNA preparations was determined by PCR amplification without reverse transcription. The HIV-1 DNA concentration was subtracted from the measured HIV-1 RNA concentration to obtain a corrected HIV-1 RNA concentration. RNA recovery was measured by adding 50 to 500,000 copies of HIV-1 RNA (HIV-1 BAL strain) to tonsil cells from HIVseronegative donors at the time of RNA extraction. Mean HIV-1 RNA recovery was 74% (95% confidence interval [CI], 33 to 115%). Quantification of HIV-1 DNA within lymph node cell subsets. HIV-1 DNA was extracted using a DNeasy blood and tissue kit (Qiagen, Gaithersburg, MD). Cell-associated HIV-1 DNA was quantified by nested PCR amplification of the HIV-1 long terminal repeat. A total of 10 5 sorted lymph node cells were resuspended in 200 l of phosphate-buffered saline, and DNA was extracted and purified with the QIAamp blood kit (Qiagen, Inc., Chatsworth, CA). Purified cell DNA was quantitated by absorption spectroscopy at 260 nm and by real-time PCR quantification of human -actin DNA using TaqMan reagents according to the manufacturer s instructions (Applied Biosystems, Carlsbad, CA). HIV-1 DNA was quantified by a two-step PCR amplification by previously described methods (8, 40). HIV-1 DNA standards were included in each PCR by dilution of HIV-1-infected U1 cells (7) in a background of HIV-1 uninfected MT-2 cells (17) to obtain a range of HIV-1 DNA concentrations from 1.5 to 150,000 genome copies per first-round PCR. To determine the HIV-1 DNA concentration in samples, a standard curve was constructed by plotting the cycle threshold in the second PCR (cutoff, fluorescence of 10 times the background) versus DNA concentration of HIV-1 DNA standards. All PCRs were performed by a person who was blinded to the identity of the samples, and all measurements were the means of triplicate analyses. HIV-1 DNA recovery was measured by extracting total cellular DNA from 10 to 10 6 chronically HIV-1-infected U1 cells (2 copies

3 VOL. 85, 2011 ACTIVATED T CELLS HARBOR THE MAJORITY OF HIV RNA FIG. 1. Representative flow cytometry plot of CD8-depleted PHAstimulated PBMC 48 h after inoculation with R5-tropic HIV-1 GFP reporter virus. The cells were acquired from the lymphocyte gate of the forward-scatter versus side-scatter profile. The majority of GFP tonsil cells were CD4 negative. The cutoff between CD4 and CD4 cells is indicated by the broken line. CD4 dim cells were included in the CD4 gate. Data were analyzed using FlowJo software (Tree Star). pos, positive. of HIV-1 proviral DNA/cell) (11, 26). Mean HIV-1 DNA recovery was 50% (95% CI, 22 to 78%). Statistical analysis. All analyses assumed a two-sided test of hypothesis. Fisher s exact test and Wilcoxon rank sum tests were used for comparing demographic data between groups. Spearman correlations were used. Nonparametric Friedman tests were used to obtain overall P values for analysis of DR/38 subsets nested within subject. Assuming overall significance ( 0.05), Wilcoxon signedrank tests were used for subsequent pairwise comparisons under a Bonferroni s corrected significance level of (0.05/3). Analyses of log 10 HIV-1 RNA copies/ml as a function of subset percentage and density utilized the mixed procedure in SAS (SAS Institute, Inc., Cary, NC) with the repeated option for DR/38 subsets nested within subject. An unstructured variance/covariance matrix was chosen based on Akaike s information criterion (25). RESULTS HIV-1 replication is concentrated within DR 38 lymphocytes in cultured PBMC. To determine whether the majority of HIV-1 is produced by DR 38 CD4 T cells in vitro, phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) from 10 individuals at low risk for HIV-1 infection were infected with CCR5 (R5)- and CXCR4 (X4)- tropic HIV-1 green fluorescent protein (GFP) reporter viruses. It has previously been shown that replication of the X4-tropic reporter virus decreases CD4 expression (25), and we confirmed that this is also true for the R5-tropic reporter virus, as shown in a representative flow cytometry plot (Fig. 1). For this reason, PBMC were first depleted of CD8 cells, resulting in a median of 92% (range, 65% to 96%) of CD4 cells within the CD3 population. The cells were cultured with PHA for 2 days, then infected with R5- and X4-tropic HIV-1 GFP reporter viruses, and analyzed for GFP expression in the lymphocyte population 48 h later. The median percentages of GFP-positive (GFP ) lymphocytes were 0.07 (range, 0.03 to 1.25) for R5-tropic virus cultures and 0.19 (range, 0.06 to 1.82) for X4-tropic virus cultures (P 0.006). Medians of 25% and 21% of GFP cells in R5-tropic and X4-tropic cultures, respectively, expressed cell surface CD4 even when CD4 dim cells were included in the CD4 population (Fig. 1). The proportion of DR 38 lymphocytes was 2-fold higher in the GFP subset than in the GFP subset for both R5- tropic (median, 62.4% versus 31.6%, respectively) and X4- tropic virus cultures (median, 61.3% versus 31.6%, respectively) (Fig. 2A and B). There was no significant difference in the proportions of DR 38 cells in the GFP versus GFP lymphocyte populations (median values for R5-tropic virus cultures, 8.0% versus 7.7%, respectively; median value for X4- tropic virus cultures, 8.8% versus 8.2%, respectively). Significantly lower proportions of GFP compared to GFP lymphocytes were demonstrated in the DR 38 subset (median value for R5-tropic virus cultures, 1.4% versus 6.0%, respectively; median value for X4-tropic virus cultures, 1.7% versus 5.5%, respectively) and DR 38 subset (median for R5-tropic virus cultures, 27.3% versus 54.7%, respectively; median for X4- tropic virus cultures, 22.9% versus 54.0%, respectively). Percentages of lymphocyte subsets defined by DR and 38 were not significantly different between infected cells and cells in uninfected control wells within each experiment (data not shown). In experiments using R5-tropic reporter virus, the mean fluorescence intensity (MFI) of GFP in DR 38 cells was significantly higher than in DR 38 cells (Fig. 2C) (P 0.014). No statistically significant differences in MFI were demonstrated between other subsets infected with either reporter virus (Fig. 2C and D). Further, when the fraction of GFP cells was evaluated within each subset, the DR 38 subset harbored significantly higher percentages of GFP lymphocytes than other subsets (P 0.03 for all pairwise comparisons to DR 38 ) (Fig. 2E and F). Thus, in contrast to our hypothesis, the majority of HIV-1-producing T cells were DR 38, whereas DR 38 cells constituted a minority of virus-producing cells after 48 h of in vitro culture. Heightened replication of HIV-1 by the DR 38 CD4 lymphocyte subset in vitro is not related to HIV-1 chemokine receptor levels. To evaluate whether preferential replication of HIV-1 by DR 38 T cells was related to HIV-1 chemokine receptor levels, we assessed CCR5 and CXCR4 expression on CD4 CD3 lymphocytes in additional experiments using peripheral blood samples from several of the same blood donors. Density centrifugation, as well as in vitro culture with PHA, are known to alter chemokine receptor levels (4, 32, 35). Because of this, we evaluated chemokine receptor expression on CD4 lymphocyte subsets defined by DR and 38 in whole blood, after density centrifugation, and after 2 days in culture with PHA (Fig. 3). Percentages of CCR5 and CXCR4 cells and concentrations of these receptors declined in most subsets after density centrifugation to isolate PBMC but recovered to levels similar to or higher than whole-blood levels in most subsets after 2 days in culture with PHA. At this time point, which corresponds to the time when virus was added to cultures in vitro and consequently when chemokine receptor levels would be of greatest relevance, there was no evidence that HIV-1 chemokine coreceptors were elevated on DR 38 CD4 cells compared to other subsets of cells. Indeed, the median proportion of CCR5 cells in PHA-stimulated PBMC tended to be lower among DR 38 CD4 cells (9.0%) than among DR 38 CD4 (11.1%; P 0.84), DR 38 CD4 (44.6%; P 0.09), and DR 38 CD4 (53.9%; P 0.031) cells (Fig.

4 10192 MEDITZ ET AL. J. VIROL. FIG. 2. Percentages of DR/38 lymphocyte subsets in non-virus-producing (GFP ) and virus-producing (GFP ) PHA-stimulated PBMC inoculated with R5-tropic (A) and X4-tropic (B) HIV-1 GFP reporter viruses. Mean fluorescence intensity (MFI) of GFP within virus-producing DR/38 subsets of cells inoculated with R5-tropic (C) and X4-tropic (D) HIV-1 reporter viruses. Percentages of GFP cells within DR/38 subsets inoculated with R5-tropic (E) and X4-tropic (F) HIV-1 reporter viruses. PBMC from 10 individuals at low risk for HIV-1 infection were depleted of CD8 cells, cultured for 2 days with PHA and IL-2, inoculated with HIV-1 GFP reporter viruses, and evaluated 48 h later by flow cytometry. For panels A through D, the percentages of DR/38 subsets of cells were determined after first gating on cells in the lymphocyte gate and then on GFP and GFP cells. For panels E and F, the percentages of GFP cells were determined after first gating on cells in the lymphocyte gate and then on each DR/38 subset. Lines link the values from an individual subject.

5 VOL. 85, 2011 ACTIVATED T CELLS HARBOR THE MAJORITY OF HIV RNA A). The numbers of cell surface CCR5 molecules per cell were higher on DR 38 cells (median, 4,094) than on DR 38 cells (median, 1,938; P 0.03) but did not differ significantly from those on DR 38 cells (median, 4,685; P 1.0) or DR 38 cells (median, 3,607; P 0.09) (Fig. 3B). No significant differences among subsets were observed in CXCR4 percentages (Fig. 3C; overall P 0.49) or concentrations (Fig. 3D; overall P 0.36). Thus, heightened replication of HIV-1 within the DR 38 lymphocyte subset in vitro was not related to HIV-1 chemokine receptor expression. DRⴚ 38ⴙ T cells predominate in culture conditions after 4 days of culture with PHA. To further investigate potential explanations for the concentration of virus replication within the DR 38 subset, data from the cells in control wells from the GFP reporter virus infection experiments were assessed to determine whether there was evidence that the proportions of subsets changed over time (Fig. 4). The median percentages of DR 38 cells increased from 37 at day 2 to 55 at day 4, the time of GFP analysis. The median percentages of DR 38 cells also increased from 12 at day 2 to 39 at day 4. In contrast, the percentages of both DR subsets declined between day 2 FIG. 4. Percentages of DR/38 CD4 T-cell subsets in PBMC after 2 and 4 days of culture with PHA. PBMC from 6 individuals at low risk for HIV-1 infection were depleted of CD8 cells and cultured with PHA. Using flow cytometry, the percentages of DR/38 subsets of cells were determined by first gating on cells in the lymphocyte gate and then on CD3 CD4 cells. Lines indicate paired values from one subject. Differences between day 2 and day 4 were significant within all subsets (P 0.03). FIG. 3. Percentages of CCR5 (A) and CXCR4 (C) and cell surface concentrations (B and D) of these receptors, respectively, on DR/38 CD4 T-cell subsets in whole-blood (WB) samples, PBMC isolated by density centrifugation, and CD8-depleted PHA-stimulated PBMC cultured for 2 days. Peripheral blood samples were obtained from 6 individuals at low risk for HIV-1 infection and stained with antibodies to cell surface markers at each time point, and results were determined by flow cytometry. Chemokine receptor expression on DR/38 subsets was determined after first gating on cells in the lymphocyte gate, then on CD3 CD4 cells, and finally on each DR/38 subset. Asterisks indicate the values for PHA-stimulated PBMC that were different from the values for whole blood (P 0.03 in all instances).

6 10194 MEDITZ ET AL. J. VIROL. TABLE 1. Demographic and clinical characteristics of HIV-1- seropositive and -seronegative subjects Group and subject a Age (yr) Sex b Race No. of CD4 T cells/ mm 3 No. of HIV- 1 RNA copies (log 10 copies/ml) HIV-1-seropositive subjects M Caucasian F Caucasian F Black M Black M Hispanic F Native American M Native American M Black M Caucasian M Black F Black F Hispanic F Black F Caucasian * 42 F Black * 36 M Black * 38 F Black F Black F Caucasian F Black F Black M Caucasian HIV-1-seronegative subjects F Caucasian M Caucasian M Caucasian M Caucasian F Caucasian F Hispanic a Asterisks denote the subjects who previously received antiretroviral therapy. At the time of enrollment in this study, they had been off therapy for 6 months or longer. b M, male; F, female. and day 4; in particular, DR 38 CD3 cells declined from a median of 36% at day 2 to a median of 3% at day 4 (P 0.031). Thus, significant fluctuations in lymphocyte subsets were observed between days 2 and 4 of in vitro culture, resulting in relative expansion of the DR 38 subset and profound reductions in the percentages of DR 38 cells. Clinical characteristics of human lymph node donors. To investigate the relationship between DR 38 CD4 T cells and HIV-1 replication in vivo, lymph nodes and peripheral blood samples from 22 HIV-1-seropositive individuals not receiving antiretroviral therapy and 6 HIV-1-seronegative individuals were evaluated. Demographic and clinical characteristics of these subjects are shown in Table 1. Overall, 55% of HIV-1-seropositive subjects and 45% of seronegative subjects were women (P 1.0). Compared to seronegative individuals, seropositive individuals were younger (median age, 34 versus 51 years; P 0.006) and more likely to be nonwhite (75% versus 17%; P 0.03). Among seropositive subjects, the median CD4 T-cell count was 621 (range, 256 to 1,117) cells/mm 3, and the median plasma viral load was 4.36 (range, 1.70 to 5.88) log 10 copies/ml. All HIV-1-infected subjects harbored only R5-tropic virus. The percentages of DR 38 CD4 T cells are elevated in lymph nodes of HIV-1-seropositive subjects and correlate with CD4 T-cell counts and plasma viral load. The percentages of lymph node DR 38 CD4 T cells were higher in HIV-1- seropositive subjects (median, 14.9; range, 2.1 to 32.8; n 18) than in seronegative subjects (median, 2.4; range, 1.3 to 10.5; n 6; P 0.004), consistent with previous studies (35, 49). In seropositive subjects, the percentages of DR 38 CD4 T cells were higher in lymph nodes (median, 20.6) than in whole blood (median, 6.9; n 11; P 0.001), as previously reported by others (35, 49). The percentages of lymph node DR 38 CD4 T cells correlated inversely with CD4 T cell counts (r 0.74; 95% CI, 0.9 to 0.41; P 0.002) and directly with plasma HIV-1 RNA (r 0.53; 95% CI, 0.08 to 0.8, P 0.03). Lymph node DR 38 CD4 T cells have elevated levels of cell surface HIV-1 chemokine coreceptors. To assess whether DR 38 CD4 T cells have increased expression of HIV-1 chemokine receptors compared to other CD4 T-cell subsets, the percentages of cells that expressed HIV-1 chemokine receptors and numbers of cell surface chemokine receptors were determined in lymph node cells from 18 HIV-1-seropositive and 6 HIV-1-seronegative individuals. Representative flow cytometry plots of the gating strategy used for stained lymph node cells are pictured in Fig. 5. Among HIV-1-seropositive subjects, the proportions of CCR5 cells were significantly higher in DR 38 lymph node CD4 T cells (median, 36.4%) than in all other subsets (median values of 5.7% DR 38 cells, 19.4% for DR 38 cells, and 7.6% for DR 38 cells) (Fig. 6A), whereas the percentages of CXCR4 cells did not significantly differ among subsets (Fig. 6B). The numbers of both CCR5 molecules and CXCR4 molecules per cell were also elevated on DR 38 cells (Fig. 6C and D). Similar patterns of HIV-1 chemokine receptor expression were observed in seronegative subjects, although differences among subsets were not statistically significant after adjusting for multiple comparisons. There was a trend for higher percentages of CCR5 cells in the DR 38 CD4 T-cell subset in HIV-1- seropositive versus HIV-1-seronegative subjects (median, 36.4 versus 22.5, respectively; P 0.06). However, there were no significant differences between seropositive and seronegative subjects in the proportions of CXCR4 cells (median value of 64.1 versus 78%, respectively; P 0.16), numbers of CCR5 molecules (median, 3,986 versus 4,364 mol/cell, respectively; P 0.67) or numbers of CXCR4 molecules (median, 2,933 versus 3,834 mol/cell, respectively; P 0.67) in the DR 38 CD4 T-cell subset. The majority of HIV-1-producing cells in vivo do not demonstrate cell surface CD4 expression. Our in vitro experiments demonstrated that CD4 is expressed on only a minority of virus-producing cells. To determine the extent to which CD4 is downregulated on virus-producing cells in vivo, we sorted CD3 lymph node cells from 6 untreated HIV-1-infected subjects into subsets defined by CD4 and CD8 expression. As shown in Fig. 7A, significantly more HIV-1 RNA copies/10 5 cells were found in CD4 CD8 T cells (median, ) than in CD4 CD8 (median, ), CD4 CD8 (median, ), and CD4 CD8 (median, ) (overall P 0.001; for all pairwise comparisons P 0.03) subsets. After adjusting for the proportion contributed by each subset (Fig. 7B), the CD4 CD8 fraction contributed a median of 48% of the total HIV-1 RNA copies compared to CD4 CD8 (median, 15%), CD4 CD8 (median, 29%), and CD4 CD8

7 VOL. 85, 2011 ACTIVATED T CELLS HARBOR THE MAJORITY OF HIV RNA FIG. 5. Representative flow cytometry plots of lymph node cells. The cells were acquired from the lymphocyte gate of the forward- versus side-scatter profile. A dot plot was used to define CD3 CD4 cells (A) and gates for CD38 (B) and HLA-DR (C) set with fluorescence minus one (FMO) controls. CD4 CD3 lymphocytes were evaluated in a CD38 versus HLA-DR plot (D), and CCR5 expression was evaluated in four DR 38 subsets using histograms (E). Data were analyzed using FlowJo software (Tree Star). (median, 8%) (P 0.14 overall) subsets (Fig. 7C). Further analyses revealed that a strategy based on gating on CD4 T cells would include only 41% (range, 25 to 53%) of HIV-1 RNA, whereas a strategy based on gating on CD8 cells would include a median of 58% (range, 56 to 78%). Thus, on the basis of these data, we opted to sort CD8 rather than CD4 DR/38 T-cell subsets, as described below. HIV-1 RNA is concentrated in DR 38 T cells in lymph nodes. To evaluate whether DR 38 T cells produce the majority of HIV-1 in vivo, HIV-1 RNA was measured in lymph node CD8-T-cell subsets defined by DR and 38 from 8 HIV-1-seropositive subjects. As shown in Fig. 8A, significantly more HIV-1 RNA copies/10 5 cells were found in CD3 CD8 T cells that were DR 38 (median, ) than in DR 38 (median, ), DR 38 (median, ), and DR 38 subsets (median, ) (overall P 0.001; for all pairwise comparisons to DR 38, P 0.008). After adjusting for the proportion contributed by each subset (Fig. 8B), the DR 38 fraction contributed a median of 86% (range, 61% to 100%), of the total HIV-1 RNA copies (P overall; P for all pairwise comparisons to DR 38 ) (Fig. 8C). CCR5 expression on CD4 T cell DR/38 subsets predicts cell-associated HIV-1 RNA concentration within CD8 T-cell DR/38 subsets. We evaluated whether CCR5 expression on CD4 T cell DR/38 subsets predicted cell-associated HIV-1 RNA concentration within the sorted CD8 DR/38 subsets. The rationale for evaluating CCR5 expression on CD4 rather than CD8 subsets was that CD4 cells are the target cells for HIV-1, and both CD4 and CCR5 are downregulated on cells after infection with HIV-1 (6, 22, 48). The percentages of CCR5 CD4 T cells and concentrations of CCR5 receptors on the surfaces of CD4 T-cell subsets were significantly related to concentrations of virus within the subsets; log 10 HIV-1 RNA/10 5 cells increased 0.54 (95% CI, 0.33 to 0.74) for each 10% increase in the percentage of CCR5 (Fig. 9A) and 0.73 (95% CI, 0.38 to 1.11) for each increase of 1,000 in the number of CCR5 molecules per cell (Fig. 9B). CCR5 expression on CD8 T cells was less strongly related to HIV-1 RNA within CD8 DR/38 subsets; log 10 HIV-1 RNA/10 5 cells increased 0.85 (95% CI, 0.14 to 1.82; P 0.088) for each 10% increase in the number of CCR5 molecules per cell and 0.61 (95% CI, 0.25 to 1.48; P 0.16) for each increase of 1,000 in the number of CCR5 molecules per cell. HIV-1 DNA is concentrated in DR 38 T cells in lymph nodes. To evaluate whether elevated levels of HIV-1 RNA in DR 38 cells were related to increased infection of these cells, HIV-1 DNA measurements were performed in lymph node CD8 T-cell subsets from 7 subjects. Significantly more HIV-1 DNA copies/10 5 cells was found in lymph node DR 38 CD8 T cells (median, 5,360; n 7) than in DR 38 (median, 814; n 6; P 0.031), DR 38 (median, 1,984; n 6; P 0.031), and DR 38 (median, 906; n 7; P 0.016) subsets (Fig. 8D). After adjusting for the proportion of total cells contributed by each subset (Fig. 8B), DR 38 cells harbored a median of 64% (range, 19 to 87%) of CD8 T-cellassociated HIV-1 DNA in 5 subjects for whom DNA data were available for all 4 subsets (P 0.16) (Fig. 8E). DISCUSSION This is the first study to quantify HIV-1 RNA and chemokine coreceptor levels in activated T cells from human lym-

8 10196 MEDITZ ET AL. J. VIROL. FIG. 6. Percentages of CCR5 (A) and CXCR4 (B) and concentrations of these HIV-1 coreceptors (C and D), respectively, in DR/38 CD3 CD4 subsets of lymph node cells from HIV-1-seropositive individuals (n 18) and HIV-1-seronegative individuals (n 6). Each symbol represents the value for an individual. Short horizontal lines indicate median values for groups of individuals. P values indicate comparisons between DR 38 cells and other subsets in seropositive subjects. Asterisks denote P values that were no longer statistically significant after Bonferroni s correction for multiple comparisons (cutoff, P 0.017). Differences between DR 38 cells and other subsets in seronegative subjects and differences between seropositive and seronegative subjects were not statistically significant after adjusting for multiple comparisons. phoid tissues. While prior studies have suggested that activated lymphocytes are preferentially infected (31) and contribute to HIV-1 replication in vivo (51), the proportion of virus replication that occurs in these cells in vivo and the mechanisms underlying their facility to propagate HIV-1 are not known. The present study was designed to evaluate the hypothesis that activated CD4 T cells are the source of the majority of HIV-1 both in vitro and in vivo and to explore the role of HIV-1 chemokine coreceptors in promoting virus replication by these cells. In vitro, DR 38 T cells constituted a minority of virusproducing cells, and replication was unrelated to HIV-1 chemokine receptor expression. In vivo, the majority of R5- tropic HIV-1 RNA was found in DR 38 T cells, and in four of five subjects, the majority of HIV-1 DNA was found in these cells as well. The percentages of CCR5 cells were higher in the DR 38 CD4 T-cell subset than in other CD4 T-cell subsets in lymphoid tissues, and the percentages and density of CCR5 CD4 T cells predicted the amount of R5-tropic HIV-1 RNA among subsets. These data demonstrate that DR 38 CD4 T cells in lymphoid tissues are highly susceptible to infection with R5-tropic virus, possibly due to heightened expression of CCR5, and produce the majority of R5- tropic HIV-1 in vivo. In vitro studies in PBMC failed to recapitulate the in vivo observation that HIV-1 replication is concentrated within lymph node DR 38 lymphocytes. Instead, the majority of both R5- and X4-tropic HIV-1 replication in PBMC occurred within DR 38 lymphocytes and was unrelated to chemokine receptor expression. A dramatic decline in percentages of DR 38 cells was seen in culture, consistent with prior observations that activated T cells are predisposed to apoptosis (3, 18). In addition, some HIV-1-producing DR 38 cells may have been transformed into DR 38 cells because HIV-1 nef is known to downregulate major histocompatibility complex class II (MHC-II) by endocytosis (5, 43, 46). Thus, preferential replication of HIV-1 by DR 38 cells in vitro may have been due to relative resistance of these cells to HIV-1-induced apoptosis and/or downregulation of DR on HIV-1-producing DR 38 cells. Fundamental differences between peripheral blood and lymphoid tissue lymphocytes in their ability to support HIV-1 replication could explain the differences between cell types that propagated HIV-1 in vitro and in vivo. Tonsil cells

9 VOL. 85, 2011 ACTIVATED T CELLS HARBOR THE MAJORITY OF HIV RNA FIG. 7. HIV-1 RNA measurements within subsets of CD4/CD8 CD3 lymph node cells (n 6). (A) Numbers of HIV-1 RNA copies/ 10 5 cells; (B) percentages of CD4/CD8 subsets within CD8 CD3 lymph node cells; (C) percentages of total RNA copies contributed by each CD4/CD8 subset after adjusting for percentages of subsets shown in panel B. For each box-and-whisker plot, the line indicates the median and the whiskers (error bars) indicate the range. Lymph node cells from untreated HIV-1-seropositive subjects were sorted into subsets on a cell sorter, and aliquots of 10 5 cells were frozen as pellets. HIV-1 RNA was then extracted and measured by PCR. have been demonstrated to be more permissive for HIV-1 than PBMC (10). In an ex vivo human tonsil tissue model, cells that expressed CD25 and/or DR were the major cell types that replicated R5 virus (3). In an ex vivo cervicovaginal tissue model, productive R5 infection occurred preferentially in CD38 T cells (41). Nevertheless, none of these studies examined whether cells that expressed both DR and 38 preferentially replicated HIV-1, and therefore, it is not possible to determine whether these model systems recapitulated the preferential propagation of HIV-1 by DR 38 cells that we observed in lymphoid tissues in vivo. Differences between acute HIV-1 infection of seronegative human cells using soluble extracellular virus compared to cell-to-cell transfer of virus in lymphoid tissues during chronic HIV-1 infection may also account for the failure of the vitro culture system to parallel HIV-1 replication in lymph node cells in vivo. Future studies could be directed at dissecting out reasons why PBMC assays failed to recapitulate in vivo findings from lymph node cells from untreated HIV-1-infected subjects. The discordance between our in vitro and in vivo findings illustrates the limitations of the PHA-stimulated PBMC model of HIV-1 replication and underscores the importance of validating in vitro findings with in vivo data. This is the first study to demonstrate that CCR5 is highly expressed on DR 38 CD4 T cells in lymph nodes and that differences in the amounts of HIV-1 RNA produced by DR/38 subsets in vivo are related to the percentages of CCR5 cells and density of CCR5 on these cells. One prior study that evaluated lymph nodes from HIV-1-infected individuals did not detect a correlation between CCR5 expression on CD4 T cells and lymph node cellular HIV-1 RNA (35). This study included individuals who were receiving antiretroviral therapy and individuals with X4-tropic HIV-1 in their analyses, however, which may have obscured an association. In vitro studies have suggested that the number of CCR5 molecules per cell is a critical determinant of HIV-1 susceptibility (27, 36, 38), and one group reported that the concentrations of CCR5 molecules on CD4 T cells, but not the percentages of CCR5 CD4 T cells in peripheral blood, correlate with viral load (39). In the present study, however, both the percentage of CCR5 cells and number of CCR5 molecules per cell correlated with HIV-1 RNA within lymphocyte subsets, suggesting that both may be major determinants of HIV-1 susceptibility in lymphoid tissues. Prior studies have demonstrated that HIV-1 infection itself does not alter CCR5 levels in peripheral blood CD4 T cells (29, 39), and we found similar percentages of CCR5 molecules in activated lymph node cells from seropositive and seronegative individuals in this study. Thus, the correlation between percentages and density of CCR5 activated T cells and HIV-1 RNA observed in this study is unlikely to be due to induction of CCR5 by HIV-1 replication. These findings provide circumstantial evidence that expression of CCR5 facilitates the preferential replication of R5-tropic HIV-1 by DR 38 T cells in vivo but do not eliminate the possibility that other factors may play a role. Further studies including studies of a larger number of subjects are warranted to confirm or refute the preliminary findings of the relationship between CCR5 expression and HIV-1 RNA from this study. Whether activated CD4 T cells propagate HIV-1 more efficiently than other CD4 T cells is unknown. In situ analyses of lymph nodes from 5 HIV-1-infected humans in early HIV-1 infection revealed that the amount of HIV-1 RNA per productively infected cell was approximately 4 times higher in HLA-DR or Ki67 cells than in other cells (50). Nevertheless, that study did not evaluate the amount of HIV-1 RNA in DR 38 cells. Two lines of evidence in the present study suggest that the amount of HIV-1 RNA produced per produc-

10 10198 MEDITZ ET AL. J. VIROL. Downloaded from FIG. 8. HIV-1 RNA and DNA measurements within subsets of DR/38 CD8 CD3 lymph node cells. (A) Numbers of HIV-1 RNA copies/10 5 cells; (B) percentages of DR/38 subsets within CD8 CD3 lymph node cells; (C) percentages of total RNA copies contributed by each DR/38 subset after adjusting for percentages of subsets shown in panel B; (D) numbers of HIV-1 DNA copies/10 5 cells; (E) percentages of total DNA copies contributed by each DR/38 subset after adjusting for percentages of subsets shown in panel B. Lymph node cells from untreated HIV-1-seropositive subjects were sorted into subsets on a cell sorter, and aliquots of 10 5 cells were frozen as pellets. HIV-1 RNA and DNA were then extracted and measured by PCR. on April 7, 2018 by guest tively infected DR 38 cell is not different from that produced by other cells. First, in vitro infection of PHA-stimulated PBMC with HIV-1 reporter viruses did not reveal consistent differences in the mean fluorescence intensity (MFI) of GFP between DR 38 cells and other subsets (Fig. 2). Second, the pattern of concentration of HIV-1 RNA and DNA among the 4 subsets in vivo (Fig. 8) was similar in most subjects, suggesting that the amount of DNA, rather than the cell type, was the major determinant of how much RNA was produced. Nevertheless, these observations do not exclude the possibility that DR 38 cells produce more virions per infected cell than other subsets. This question could be addressed definitively by limiting dilution assays and measurement of HIV-1 RNA among purified cells from each subset. A shortcoming of this study is that virus replication and chemokine receptor levels were determined only in lymph nodes, whereas HIV-1 is propagated within a variety of other secondary lymphoid tissues in vivo, including spleen and mucosal lymphoid tissues. The percentage of total body lymphocytes that are harbored within lymph nodes is controversial, but it has been estimated that approximately 41% of lymphocytes are found in lymph nodes (13). It would be important to determine in future studies whether R5-tropic HIV-1 replication in other secondary lymphoid tissues is concentrated within

11 VOL. 85, 2011 ACTIVATED T CELLS HARBOR THE MAJORITY OF HIV RNA FIG. 9. Percentage of CCR5 (A) and number of CCR5 molecules (B) on CD4 T cells within lymph node cell DR/38 subsets and predicted log 10 HIV-1 RNA within the corresponding DR/38 CD8 CD3 subset. activated lymphocytes and whether these cells also have elevated CCR5 expression. Another limitation of the present study is that none of the study subjects harbored X4-tropic virus, and consequently, conclusions concerning HIV-1 replication in vivo are limited to R5-tropic virus. Interestingly, unlike CCR5, the percentages of CXCR4 cells were not elevated in DR 38 CD4 T-cell subsets in lymph node (Fig. 6). Measurement of HIV-1 RNA in T-cell subsets from lymphoid tissues of individuals infected with X4-tropic virus or with mixed tropic viruses could potentially provide important insight regarding the immunopathogenesis of X4-tropic virus. A final limitation of this study is that although our gating strategy on CD8 T cells included the majority of HIV-1 RNA-producing cells, it excluded the CD4 CD8 subset, which proportionally accounted for a median of 29% of total RNA copies (Fig. 7C). One prior study characterized the HIV-1- specific functional properties of these cells in peripheral blood samples from HIV-1-infected individuals and concluded that they shared effector profiles of both CD4 and CD8 T cells (20). Virtually nothing is known, however, about the CD4 CD8 T-cell population in lymph nodes. Because of the low frequency of CD4 CD8 T cells in the lymph node cell population, we were unable to perform parallel sorting experiments to measure HIV-1 RNA in DR/38 subsets. Nevertheless, further study of this subset, which harbored the second highest burden of HIV-1 RNA, is warranted. The present study significantly advances understanding of the link between immune activation and HIV-1 pathogenesis, as it demonstrates that activated T cells are central to the propagation of HIV-1 in vivo. It has been hypothesized that immune activation may also contribute to CD4 T-cell depletion indirectly through a variety of mechanisms, including activation-induced death, immune senescence, and immune suppression (44). Results of the present study do not exclude a potential role for these mechanisms, although the need to invoke indirect mechanisms seems less clear in light of the pivotal role of activated lymphocytes in HIV-1 replication. The findings from this study suggest that therapeutic strategies to reduce the number of DR 38 CD4 T cells could limit HIV-1 replication and consequently disease progression. The efficacy and feasibility of such interventions are supported by the fact that HIV-1-infected individuals with remarkably low levels of immune activation and very low viral loads have been reported (7); these individuals seem to be otherwise healthy, suggesting that low levels of DR 38 CD4 T cells do not confer an immune deficit. Although therapies to suppress immune activation have not resulted in dramatic improvements in virologic control in HIV-1-infected individuals so far (34, 37), it is unclear whether these therapies truly reduced fractions of DR 38 T cells in lymphoid tissues where the majority of HIV-1 is produced. A better understanding of the ontogeny of DR 38 CD4 T cells in lymphoid tissues and identification of enzymes that are specific to their development could reveal potential targets for intervention. Development of in vitro models that accurately recapitulate HIV-1 replication in vivo could facilitate studies to develop targeted therapies to interrupt immune activation-associated HIV-1 replication in vivo. Ultimately, strategies aimed at reducing the number of DR 38 CD4 T cells could represent a new class of hostdirected therapies and further add to the armamentarium of antiretroviral therapies for HIV-1 infection. ACKNOWLEDGMENTS This work was supported by Public Health Services grant R21 HD from the National Institute of Child Health and Human Development and the University of Colorado Center for AIDS Research grants P30 AI and K08 AI from the National Institute of Allergy and Infectious Diseases, National Institutes of Health. We express our gratitude to the subjects who participated in this study. We also thank John Koeppe, Patty Caraway, Graham Ray, Cathi Basler, and Beverly Putnam for assistance in recruiting study subjects; Karen Whalen for assistance in lymph node surgical procedures; Mark Nehler and David Fullerton for assistance in procuring HIV-1-seronegative nodes; the NIH AIDS Research and Reference Reagent Program and Vineet N. Kewal Ramani and Dan R. Littman for provision of GHOST cells; and Elizabeth Kelly and Brent Palmer for assistance with cell sorting. REFERENCES 1. Begaud, E., et al Reduced CD4 T cell activation and in vitro susceptibility to HIV-1 infection in exposed uninfected Central Africans. Retrovirology 3:35.