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The Journal of Immunology, 2002, 169: 2313-2322.
Copyright © 2002 by The American Association of Immunologists

Follicular Dendritic Cell-Mediated Up-Regulation of CXCR4 Expression on CD4 T Cells and HIV Pathogenesis1

Jacob D. Estes*, Brandon F. Keele*, Klara Tenner-Racz{dagger}, Paul Racz{dagger}, Michael A. Redd*, Tyler C. Thacker*, Yongjun Jiang*, Michael J. Lloyd*, Suzanne Gartner{ddagger} and Gregory F. Burton2,*

* Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602; {dagger} Department of Pathology and Korber Laboratory for AIDS Research, Bernhard-Nocht Institute for Tropical Medicine, Hamburg, Germany; and {ddagger} Department of Neurology, Johns Hopkins Hospital, Baltimore, MD 21231


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Follicular dendritic cells (FDCs) represent a major reservoir of HIV, and active infection occurs surrounding these cells, suggesting that this microenvironment is highly conducive to virus transmission. Because CD4 T cells around FDCs in germinal centers express the HIV coreceptor, CXCR4, whereas CD4 lymphocytes in many other sites do not, it prompted the hypothesis that FDCs may increase CXCR4 expression on CD4 T cells, thereby facilitating infection. To test this, HIV receptor/coreceptor expression was determined on CD4 T cells cultured with or without FDCs, and its consequence to infection was assessed by measuring virus binding and entry. FDCs had little effect on CCR5 or CD4 expression but increased CXCR4 expression on CD4 T cells. FDC-mediated up-regulation of CXCR4 on CD4 T cells occurred by 24 h and was sustained for at least 96 h in vitro, and FDC-CD4 T cell contact was necessary. Importantly, increased CXCR4 expression directly correlated with increased binding and entry of HIV-1 X4 isolates. Furthermore, CD4+CD57+ germinal center T cells expressed high levels of CXCR4 and supported enhanced entry of X4 HIV compared with other CD4 T cells from the same tissue. Thus, in addition to serving as a reservoir of infectious virus, FDCs render surrounding germinal center T cells highly susceptible to infection with X4 isolates of HIV-1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Follicular dendritic cells (FDCs)3 are located in the germinal centers (GCs) of secondary lymphoid tissues where they trap and retain Ags in the form of immune complexes (1, 2). FDC-trapped Ags persist for many months in an unprocessed form and maintain long term, memory IgG and IgE responses to soluble protein Ags (1, 3, 4, 5, 6, 7). FDCs also provide Ag-independent signals to T and B lymphocytes that alter their state of activation/proliferation and render B cells responsive to chemoattractants (8, 9, 10). Thus, in normal physiology, FDCs are important to the GC reaction that generates and maintains T-dependent humoral immune responses.

Early after infection with HIV, the secondary lymphoid tissue becomes a major reservoir of virus with the vast majority of HIV particles found on the surface of FDCs (estimated to be 1.5 x 108 copies of viral RNA per gram of lymphoid tissue) (11, 12, 13). Importantly, HIV immune complexes on FDCs are highly infectious even in the presence of high levels of neutralizing Ab (14). Our recent studies have indicated that FDCs maintain HIV infectivity in vivo for many months in the complete absence of viral infection and/or replication (15). Additionally, FDCs appear to increase the production of virus in CD4 lymphocytes (16). Thus, secondary lymphoid tissues, in particular the GC microenvironment, bring together a reservoir of trapped, infectious virus on FDCs, CD4-bearing target cells, and an environment of high cellular activation creating an ideal site for HIV infection. The importance of this microenvironment is underscored by the observation that in asymptomatic HIV-infected subjects, the lymphoid follicles are the primary site of active viral infection (17, 18, 19, 20).

HIV-1 infection of target cells requires the presence of both the CD4 receptor and a chemokine coreceptor. Two chemokine receptors serve as major coreceptors for HIV: CCR5 binds HIV-1 R5 virus; whereas CXCR4 binds HIV-1 X4 isolates (21, 22, 23, 24, 25, 26). T lymphocytes and macrophages in both lymphoid and nonlymphoid tissues are the major cell populations expressing HIV coreceptors. In lymph nodes, CXCR4-bearing cells are found almost exclusively inside GCs and in the medulla, whereas CCR5-bearing cells exist primarily in the medulla and extrafollicular areas of the cortex (27). Thus, HIV coreceptor expression appears to be differentially regulated within secondary lymphoid tissues with a major population of CXCR4-bearing target cells surrounding the FDC reservoir of infectious virus.

HIV-1 coreceptor density also appears to play an important role in HIV-1 infection. Individuals who are heterozygous for the {Delta}32-ccr5 polymorphism have decreased CCR5 expression and slower progression of HIV disease (28, 29). CXCR4 expression also affects viral entry as evidenced by the observation that slight increases in CXCR4 expression, arising from CD28 signaling or from HIV-1 Tat expression, lead to increased viral entry by the X4 molecular clone, HXB2 (30, 31). Because HIV coreceptor density appears to be important in HIV infection and disease progression, we reasoned that the predominance of CXCR4 in GCs might contribute to transmission of infection in these sites. Because FDCs are unique to lymphoid follicles and are juxtaposed to CD4 T cells expressing CXCR4, we sought to determine whether FDCs contributed to the expression of this HIV coreceptor on CD4 T cells and thus to viral pathogenesis. We report here that FDCs up-regulated CXCR4 on CD4 T lymphocytes although having little effect on CD4 or CCR5 expression. Furthermore, we found that increased CXCR4 expression on CD4 T cells augmented HIV-1 X4, but not R5, virus binding and entry. Finally, we observed that freshly isolated CD4+CD57+ GC T cells also expressed high levels of CXCR4 and were readily infected by doses of HIV that did not appear to infect other CD4 T cells with lower CXCR4 expression. Collectively, these data indicate that FDCs increase CXCR4 expression on CD4 T cells and that this increase may make GC T cells especially susceptible to HIV infection as well as potentially contributing to virus tropism changes that occur in many infected subjects.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Flow cytometric analysis

Cell surface Ags were detected using the following mAbs: anti-human CD3-PE and PC5 (UCHT1), anti-human CD4-PC5 (13B8.2), anti-human CD45RO-PE (UCHL1), anti-human CD57-FITC or biotin (NC1), and anti-CD69-PE (TP1.55.3) (Immunotech, Westbrook, ME); anti-human CCR5-FITC (2D7), anti-human CXCR4-PE (12G5) (BD PharMingen, San Diego, CA); mouse IgM anti-human FDC (HJ2) (gift from Dr. M. Nahm, University of Alabama, Birmingham, AL); and donkey, F(ab')2 anti-mouse IgM-FITC (Jackson ImmunoResearch Laboratories, West Grove, PA). Mouse isotype-matched control IgG1 (679.1 Mc7) and IgG2a (U7.27) (Immunotech) were also used. Cells were incubated for 30 min on ice first with Fc-blocking reagent (Miltenyi Biotec, Auburn, CA) and then with specific mAbs. Cells were then washed in PBS, and 10,000 CD4 T cells were analyzed using an EPICS XL flow cytometer with EXPO32 ADC software (Beckman Coulter, Fullerton, CA) for immunofluorescence analysis. Positive gating was established using mouse isotype-matched control Abs to define background fluorescence. Propidium iodide (PI) (0.5 µg; Sigma-Aldrich, St. Louis, MO) labeling was used to exclude dead cells. Quantitation of Ab-binding sites (ABS) was performed using Quantum Simply Cellular Microbeads (Sigma-Aldrich) per the manufacturer’s instructions and as previously described (32).

FDC isolation

Human FDCs were isolated from tonsillar tissue as described (15). FDC-enriched preparations prepared by this procedure were examined by flow cytometry and typically contain 75–90% FDCs with residual cells consisting of T and B lymphocytes. Because FDCs are resistant to radiation, the preparations are subjected to 3000 rad of gamma-irradiation to inhibit the ability of contaminating lymphocytes to support HIV infection. The function of the FDCs was confirmed by their ability to provide costimulation to lymphocytes as previously described (9). In all experiments, an FDC:CD4 T cell ratio of 1:10 was used because we found this to result in optimal FDC-lymphocyte interactions. In some experiments, FDCs were specifically depleted by collecting the effluent from the MACS columns used to positively select FDCs, and these cells were then subjected to a further round of FDC depletion using HJ2 and magnetic beads (rat anti-mouse IgM Dyna-beads; Dynal Biotech, Great Neck, NY) at a concentration of 10 beads per target cell. This treatment removed >=90% of the FDCs (9).

T cell preparations

Human PBLs were isolated from whole blood using Ficoll-Paque (Amersham Pharmacia Biotech, Piscataway, NJ), and CD4 T lymphocytes were enriched by negative selection using a CD4+ T cell isolation kit (Miltenyi Biotec). The resulting preparations were >=95% CD4 T cells as assessed by flow cytometry. Human tonsillar CD4 T cells were purified after mechanical tissue homogenization or after enzymatic digestion of the tonsillar tissue used for FDC isolations. Isolated cells were then centrifuged (700 x g for 25 min) on preformed, 50% continuous Percoll gradients. T cells were then obtained from two fractions: the low density layer (1.050–1.060 g/ml), which also contains the FDCs and activated lymphocytes; and the high density layer (1.074–1.087 g/ml) which contains predominantly resting T cells. For CD4+CD57+ GC T cell isolation, human tonsillar tissue were cut into small sections and cells mechanically separated from tissue by repeat pipeting. RBCs were removed from the lymphocytes by lysis in RBC lysis buffer (155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA) for 5 min at room temperature. CD4 T lymphocytes were enriched by negative selection using a CD4+ T cell isolation kit (Miltenyi Biotec) followed by positive selection using anti-CD57-biotin and streptavidin microbeads by MACS (Miltenyi Biotec). The resulting CD4+CD57+ preparations were >=92% pure as assessed by flow cytometry.

FDC-CD4 T cell cocultures

FDCs and CD4 T cells isolated from PBLs or from autologous tonsillar tissue were cocultured at a density of 1 x 104 FDC per 1 x 105 CD4 T cells in complete tissue culture medium (CM) consisting of RPMI 1640 supplemented with HEPES buffer (20 mM), nonessential amino acid solution (1x), L-glutamine (2 mM), 10% heat-inactivated, defined FBS (all from HyClone Laboratories, Logan, UT) and gentamicin (50 µg/ml; Life Technologies, Gaithersburg, MD). Cocultures were performed in 5-ml round-bottom culture tubes (Falcon) or 24-well, tissue culture plates with 0.4 µm Transwell inserts (Corning Glass, Corning, NY) separating FDCs (top) from CD4 T cells (bottom) in CM. Where indicated, CD4 T cells were cultured with paraformaldehyde-fixed (4% w/v) FDCs in lieu of functional FDCs. CD4 T cells were cocultured with FDCs or FDC-depleted cells for the indicated times, and at each time point cells were removed, washed, and analyzed by quantitative, multicolor flow cytometry for surface receptor expression.

HIV binding and entry

CD4 T cells were incubated at 37°C in CM for 2 h (CXCR4low), 8 h (CXCR4int), or 18 h (CXCR4high) to induce cells with increasing levels of CXCR4 while allowing CD4 levels to remain constant. Where indicated, CD4 T cells were incubated with anti-human CXCR4 mAb (12G5; 10 µg) or anti-human CCR5 mAb (2D7; 10 µg) for 30 min on ice. For HIV-1 binding experiments using radiolabeled HIV-1, sucrose double-banded HIVIIIB (3.4 x 109 viral particles; Advanced Biotechnologies, Columbia, MD) was surface labeled with 125I (2 mCi) (Amersham Pharmacia Biotech) for 30 min on ice with Iodobeads (Pierce, Rockford, IL). Unbound 125I was removed by washing radiolabeled HIV through a Sephadex G-25 (Amersham Pharmacia Biotech) minicolumn. Western blot analysis coupled with autoradiography confirmed that the label was primarily on the surface of the virion as evidenced by radioactively labeled gp160, gp120 and gp41 but not p24. Triplicate cultures of CD4 T cells (1 x 106) expressing differential levels of CXCR4 were incubated with 125I surface labeled HIV-1IIIB having a specific activity of 0.5 µCi/µg (6.8 x 107 viral particles) for 2 h on ice, washed three times to remove unbound virus, and bound virus quantitated using a gamma counter. For nonradiolabeled HIV binding experiments, cells with differential CXCR4 expression were incubated with HIV blocking anti-CD4 Ab, SIM-4 (100 µl hybridoma supernatant; National Institutes of Health AIDS Research and Reference Reagent Program, Bethesda, MD), stromal cell-derived factor-1{alpha} (SDF-1{alpha} (2.5 µg; Upstate Biotechnology, Lake Placid, NY), macrophage-inhibitory protein-1{beta} (MIP-1{beta}) (2.5 µg; National Institutes of Health AIDS Research and Reference Reagent Program) or medium alone for 30 min on ice. Primary HIV-1 isolates 91US054 (X4) or 92US714 (R5) (National Institutes of Health AIDS Research and Reference Reagent Program) were then incubated with these cells at the indicated concentrations for 2 h on ice. Cells were washed extensively, and surface HIV was detected by measuring HIV gag p24 protein by ELISA according to the manufacturer’s instructions (Beckman Coulter). For HIV entry assays, cells with differential CXCR4 expression were incubated at 37°C with HIV-1IIIB at the indicated concentrations for 1 h, washed, and treated with trypsin (0.5% v/v; HyClone) for 20 min at 37°C to remove surface-bound virus. Cells were washed three times, and HIV entry was assessed by detection of intracellular HIV gag p24 by ELISA. In addition, HIV entry was performed by detection of reverse transcribed HIV gag products using DNA-PCR. In some experiments testing the specificity of the receptors involved, CD4 T cells (1 x 105) expressing differential CXCR4 expression or CD4 T cells (2.5 x 105) cultured with or without FDCs (2.5 x 104) were incubated with anti-CXCR4 mAb 12G5 (10 µg; BD PharMingen), anti-CCR5 mAb 2D7 (10 µg; BD PharMingen), SDF-1{alpha} (2.5 µg), or MIP-1{beta} (2.5 µg) or alone for 30 min on ice. These cells were incubated with HIV-1IIIB or X4 primary strain 91US054 for 1 h, washed three times, and incubated for an additional 12–13 h to allow reverse transcription to occur. Cells were washed three more times in PBS, lysed using 50 µl DNA lysis buffer (0.01% SDS, 0.001% Triton X-100, 0.42 µg/ml proteinase K in TE (10 mM THAM (Fisher, Pittsburgh, PA) and 1 mM EDTA). Cell lysis and protein digestion were performed at 55°C for 5 h, and the resulting DNA-containing cell lysate was simultaneously PCR amplified for both HIV gag and cellular {beta}-globin. PCR contained RNase-DNase-free H2O (Life Technologies), 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 200 µM dNTPs, 2.5 U/100 µl AmpliTaq DNA polymerase, 2 mM MgCl2 (all from PerkinElmer, Wellesley, MA), 0.5 µM concentrations each of HIV gag-SK38/39 primers (33) and 0.05 µM each of {beta}-globin-GH20/PC04 primers (34). Thirty cycles of DNA-PCR amplification were performed in a PTC-100 thermal cycler (MJ Research, Cambridge, MA) as follows: 94°C for 2 min, 55°C for 30 s, 72°C for 2 min; the initial cycle began with an additional 3-min denaturation step (94°C) and after the final cycle, an additional extension was performed for 10 min at 72°C). Amplicons were resolved on a 2% agarose gel by electrophoresis and transferred to HyBond-N+ nylon transfer membrane (Amersham Pharmacia Biotech), and Southern blot was performed using the HIV-1 gag-SK19 probe (33) with the ECL detection system (Amersham Pharmacia Biotech).

Immunohistochemistry

CD4+ T cells in the GCs were characterized on frozen tonsil sections by two-color immunofluorescence. The sections were incubated with Abs Leu3a (CD4 IgG1; BD Biosciences, San Jose, CA) and Leu7 (CD57; BD Biosciences) for 45 min. The sections were rinsed in PBS and fixed in 4% (w/v) paraformaldehyde for 20 min. After a rinsing in PBS, goat anti-mouse IgG1-FITC (Southern Biotechnology Associates, Birmingham, AL) and then Alexa Fluor 488 conjugate (Molecular Probes, Eugene, OR) were applied for 30 min each. To visualize the binding of the CD57 mAb, the sections were incubated with an isotype-matched, biotin-conjugated secondary Ab (Dianova, Hamburg, Germany) and tetramethylrhodamine isothiocyanate-conjugated streptavidin (Jackson ImmunoResearch Laboratories). The sections were rinsed in PBS and examined by fluorescent microscopy.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FDC effects on CXCR4, CCR5 and CD4 expression on CD4 T cells

To begin to determine the ability of FDCs to affect HIV receptor expression on CD4 lymphocytes, we cultured resting CD4+ T cells with or without FDCs and examined CD4R, CCR5R, and CXCR4R expression after 24 and 72 h using quantitative flow cytometry (Fig. 1Go). FDCs had little effect on either CD4 or CCR5 expressed on T cells but increased the expression of CXCR4 at both the 24- and 72-h time intervals. Because FDCs did not appear to alter the expression of CD4 or CCR5 on T cells, we focused our efforts on CXCR4 and sought to establish the kinetics of FDC up-regulation of this receptor (Fig. 2GoA). Expression of CXCR4 on CD4 T cells cultured without FDCs increased during the first 24 h, as has been reported previously (35); however, this expression decreased thereafter whereas the presence of FDCs resulted in a rapid and sustained increase in CXCR4 expression. Examination of CD69 and CD25 activation markers failed to demonstrate any differences in cells incubated with or without FDCs. Because CXCR4 is found both at the cell surface and in intracellular stores (36), we examined intracellular CXCR4 in CD4 T cells and observed that the addition of FDCs had little effect on internal CXCR4 expression even though the surface expression of this receptor was increased over the same time period (data not shown). In addition to resting CD4+ T cells, FDCs increased CXCR4 receptor expression on stimulated (i.e., PHA, anti-CD3, anti-CD3/CD28, or IL-2) CD4+ lymphocytes with kinetics similar to resting CD4+ T cells (data not shown). Because our FDC-enriched preparations typically contain ~75–90% FDCs, with the remainder of the cells being lymphocytes, we sought to confirm that FDCs, and not residual T and B cells, mediated the observed increases in CD4 T cell expression of CXCR4. To test this, control tonsillar cell preparations specifically depleted of FDCs were cultured with CD4 T cells to determine their ability to increase CXCR4 expression (Fig. 2GoA, {blacktriangleup}). Cells depleted of FDCs had no ability to increase extracellular (or intracellular, data not shown) expression of CXCR4, thus indicating that FDCs and not other tonsillar cells were needed to induce CXCR4 up-regulation. Because CD4+ T cells cultured without FDCs demonstrated an initial rise in CXCR4 expression followed by a decrease by day 3 (see Fig. 2GoA, •), we next sought to determine whether FDCs could increase CXCR4 expression on these cells (i.e., that had recently down-regulated this receptor). We therefore cultured CD4 T cells for 3 days followed by an additional incubation with or without FDCs for 1 day and monitored CXCR4 expression (Fig. 2GoB). The addition of FDCs to these previously cultured T cells resulted in an increase of both the percentage of CD4+ T cells that expressed CXCR4 (from 14% to 72%) and the receptor density per cell (>200% increase). Thus, not only did FDCs increase CXCR4 expression on freshly isolated cells but also rescued expression on cells that had recently down-regulated this receptor.



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  		FIGURE 1. FDCs up-regulate CXCR4 but not CD4 or CCR5 expression on resting CD4 T cells. CD4 T cells (1 x 105) were cocultured with FDCs (1 x 104) in CM for 24 and 72 h followed by analysis of surface CD4, CCR5, and CXCR4 expression. After live gating, 10,000 CD4+ T cells were analyzed by quantitative flow cytometry with fluorescence intensity plotted on a log10 scale. CD4 expression was 33,303 vs 36,678 ABS on T cells alone and T cells cocultured with FDCs, respectively, at 24 h and 33,723 vs 31,625 at 72 h. CCR5 expression was 1037 vs 1060 ABS on CD4 T cells alone and CD4 T cells cocultured with FDCs, respectively, at 24 h and 714 vs 858 ABS at 72 h. CXCR4 expression was 2298 vs 3536 ABS on CD4 T cells alone and CD4 T cells cocultured with FDCs, respectively, at 24 h and 2401 vs 4603 at 72 h. FDCs increased CXCR4 expression on CD4 T cells at both 24 and 72 h (by 54 and 92%, respectively). Representative histograms from one of six independent experiments are shown. Although there was considerable donor-to-donor variability, FDCs always induced significant increases in CXCR4 expression on CD4 T cells.

 


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  		FIGURE 2. Kinetics of FDC-mediated up-regulation of CXCR4 expression on CD4 T cells. A, Freshly isolated CD4 T cells (2.5 x 105) were cultured alone (•), with FDCs ({blacksquare}), or with FDC-depleted cells ({blacktriangleup}) (10:1 CD4 T cells to FDC or FDC-depleted cells) for the indicated times. Cells were collected, washed, and labeled with Abs against CD4 and CXCR4. After live gating, 10,000 CD4+ T cells were analyzed by quantitative multicolor flow cytometry. Representative data from one of six independent experiments are shown. B, Freshly isolated CD4 T cells were cultured alone for 3 days followed by an additional 1-day incubation with or without FDCs. Cells were collected, washed, and labeled with Abs directed against CD4 and CXCR4 and analyzed as above. Analysis indicated that 632 and 2018 CXCR4 ABS were present on CD4 T cells cultured alone or with FDCs, respectively. C, Freshly isolated CD4 T cells (1 x 105) were cultured in 24-well tissue culture plates with or without FDCs or paraformaldehyde-fixed FDCs (1 x 104) for 24 h. Where indicated, CD4 T cells (bottom) were separated from FDCs (top) using 0.4-µm Transwell inserts. Representative data from one of three independent experiments are shown.

 
Because FDCs up-regulated CXCR4 expression on CD4 T cells, we next sought to establish the nature of the signal provided by FDCs. To determine whether an FDC-secreted soluble factor was responsible for CXCR4 up-regulation, we separated CD4 T cells from FDCs using a Transwell culture system (Fig. 2GoC). As before, FDCs up-regulated CXCR4 when cocultured directly with CD4 T cells; however, when FDCs were separated from CD4 T cells, CXCR4 up-regulation was abrogated. To further establish that a soluble factor was not necessary for CXCR4 up-regulation, we cultured CD4 T cells with paraformaldehyde-fixed FDCs and assessed CXCR4 expression after 24 h (Fig. 2GoC). Fixed FDCs were able to up-regulate CXCR4 on CD4 T cells as well as viable, functional FDCs, thus confirming the Transwell experiment and indicating that a membrane-bound molecule and not a soluble factor was necessary for CXCR4 up-regulation by FDCs.

Increased CXCR4 expression directly correlates with augmented HIV-1 binding and entry

Because FDCs increased CXCR4 expression on CD4 T cells, we sought to determine whether these cells demonstrated an increased susceptibility to HIV infection. To initially address this question, we examined the level of virus binding and subsequent entry into lymphocytes that expressed different levels of CXCR4 in a simplified system without FDCs. Because lymphocytes were found to increase their CXCR4 expression during the first 24 h of culture (Fig. 2GoA, •), we sought to determine whether these small increases could affect HIV binding and entry. CD4 lymphocytes were therefore incubated for 2, 8 or 18 h to induce differential expression of CXCR4. Examination of these cells indicated increased CXCR4 expression (915, 1,089, and 2,855 ABS at 2, 8, and 18 h, respectively), whereas the level of CD4 remained relatively unchanged (32,060, 31,837, and 32,284 ABS, respectively). On the basis of the amount of CXCR4 present, we arbitrarily designated these cell populations as low, intermediate, or high expressers and examined their respective ability to bind 125I-surface-labeled HIV-1IIIB. Binding of this radiolabeled X4 tropic virus to CD4 lymphocytes was directly proportional to the level of CXCR4 expressed on the cell surface (28,786 ± 3,974, 37,951 ± 5,481, and 67,424 ± 2,449 cpm of bound virus on CXCR4low, CXCR4int, and CXCR4high cells, respectively). We reasoned that if X4 virus binding depended primarily on HIV gp120-CD4/CXCR4 interactions, then virus isolates using CCR5 as a coreceptor would not demonstrate differential binding to the same cells. At the same time, we sought to confirm our results using radiolabeled virus with a procedure that did not require manipulation of the HIV surface. CD4 T cells with high or low CXCR4 expression were incubated with an R5 or X4 primary isolate of HIV-1, and surface-bound virus was assessed by quantitation of HIV-1 p24 (Fig. 3Go, A and B). As expected, only the binding of the X4 primary isolate correlated with the different amounts of CXCR4 expressed on the target cells. Furthermore, the addition of specific ligand for CXCR4 and CCR5 inhibited virus binding by the corresponding X4 and R5 virus, respectively. Thus, these data indicate that relatively small differences in cell surface expression of CXCR4 can have a strong effect on the amount of X4 tropic HIV-1 that associates with primary CD4 T cells.



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  		FIGURE 3. Increased CXCR4 expression results in augmented HIV-1 binding and entry into CD4 T cells. CD4+ T cells with low (Lo), intermediate, and high (Hi) CXCR4 expression were determined using quantitative flow cytometry with the individual ABS listed in the insets. A and B, CXCR4int and CXCR4high CD4 T cells (1 x 105) were incubated alone or with HIV-blocking anti-CD4 Ab, SIM-4 (100 µl hybridoma supernatant), SDF-1{alpha} (2.5 µg), or MIP-1{beta} (2.5 µg) for 30 min on ice and then incubated with either the X4 primary strain 91US054 (20 ng of p24) or the R5 primary strain 92US714 (20 ng of p24) for 2 h on ice to avoid virus internalization. Cells were washed three times to remove unbound virus and lysed, and bound p24 was analyzed by ELISA according to the manufacturer’s instructions. Data are represented as the amount of bound HIV p24 (picograms) per 1 x 105 CD4 T cells and are representative of three independent experiments. The amount of bound HIV was not statistically different between the CXCR4low-expressing cells and the CXCR4high-expressing cells treated with SDF-1{alpha} (p > 0.05). C, CXCR4high CD4 T cells were incubated alone or with anti-CXCR4 mAb 12G5 (10 µg), anti-CCR5 mAb 2D7 (10 µg), SDF-1{alpha} (2.5 µg), or MIP-1{beta} (2.5 µg) for 30 min on ice. HIV-1IIIB or X4 primary strain 91US054 (1 x 105 cpm reverse transcriptase activity) was added to CXCR4low and CXCR4high cells for 1 h at 37°C in CM. Cells were washed three times to remove the initial inoculum and incubated for an additional 12–13 h to allow efficient reverse transcription but not completion of the viral life cycle. Afterward, cells were lysed, and virus was detected by DNA-PCR amplification of HIV-1 gag. PCR amplification of the cellular {beta}-globin gene was also performed and used as a control for DNA loading and PCR amplification. D, Duplicate samples of CXCR4low, CXCR4int, or CXCR4high CD4 T cells were incubated with HIV-1IIIB (150 ng of p24) for 1 h at 37°C in CM. Cell samples were then subjected to treatment with trypsin (0.5% v/v) for 20 min to remove cell surface-associated HIV-1, washed three times, and lysed, and intracellular p24 was analyzed by ELISA according to manufacturer’s instructions. Data are presented as the amount of intracellular HIV p24 (picograms per 1 x 105 CD4 T cells) from three representative experiments.

 
To determine whether increased binding of X4 virus led to increased entry, CD4 T cells expressing differential levels of CXCR4 were exposed to HIV-1IIIB or primary X4 HIV-1 and assessed for virus entry by DNA PCR amplification of the HIV gag gene (Fig. 3GoC). When entry using HIVIIIB was assessed, the signal obtained appeared much stronger in CXCR4high cells than in CXCR4low cells. Furthermore, when the HIV-1-binding sites of CXCR4 were inhibited by the addition of specific Ab (12G5) to CXCR4high cells, the intensity of the hybridization signal for the gag amplicon was reduced to a level similar to that obtained using CXCR4low CD4 T cells. In contrast to the results obtained with the CXCR4-specific Ab, no change was visualized when cells were treated with a CCR5-inhibiting Ab (2D7). Strikingly, HIV entry was much more robust in CXCR4high cells than in CXCR4low cells when a primary X4 strain of HIV-1 was used (Fig. 3GoC). The addition of either blocking CXCR4 Ab (12G5) or ligand (SDF-1{alpha}) inhibited virus entry into CXCR4high cells, whereas the addition of blocking CCR5 Ab (2D7) or ligand (MIP-1{beta}) had no effect. Finally, CD4 T cells were also examined for virus entry by measuring intracellular p24, which confirmed the PCR-based entry assays (Fig. 3GoD). These data were consistent with the hypothesis that HIV binding and entry were in large part dependent on CXCR4 expression levels.

Because our previous experiments assessing virus entry were performed in a simplified system where lymphocytes were induced to express CXCR4 in the absence of FDCs, we next sought to confirm that T cells cultured with FDCs also resulted in increased virus entry. CD4 T cells that had been incubated with or without FDCs for 1, 2, or 3 days (Fig. 2GoA) were exposed to HIV-1IIIB, after which viral entry was assessed by DNA PCR (Fig. 4Go). At the concentration of virus used, HIV-1 gag was detected only in CD4 T cells that were cocultured with FDCs, indicating that FDC-mediated increases in CXCR4 expression correlated with increased virus entry.



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  		FIGURE 4. FDC-induced up-regulation of CXCR4 results in augmented HIV-1 entry into CD4 T cells. CD4 T cells (1 x 105) were cultured alone or with FDCs (1 x 104) for the indicated times. Cells were collected, washed, and infected with HIV-1IIIB (2.5 x 104 cpm reverse transcriptase activity) for 1 h at 37°C. Cells were washed three times to remove unbound virus and incubated for an additional 12–13 h to allow reverse transcription to occur. Following incubation, DNA-containing cell lysates were PCR amplified for both HIV gag and cellular {beta}-globin genes. The amount of virus used in this experiment is reduced 4-fold from that used in Fig. 3GoD; with this lower concentration, only the cells expressing Hi CXCR4 demonstrated HIV DNA. ACH-2 cells (5 x 104), which contain one copy of the HIV-1 genome per cell, were used as a positive control.

 
GC T cells express high levels of CXCR4 and are highly susceptible to HIV entry

We reasoned that if FDCs contributed to HIV pathogenesis by increasing CD4 T cell expression of CXCR4 in vivo, then CD4 T cells surrounding FDCs within GCs of secondary lymphoid tissues should express high levels of CXCR4 and be more susceptible to virus entry than other CD4 T cells. To begin to test this hypothesis, we first sought to obtain GC CD4 T cells from tonsillar tissue. It has been reported that many GC T cells are CD4+CD45RO+ and that a majority also express CD57 (37, 38, 39, 40, 41). To determine whether isolation of CD4+CD57+ cells would represent true GC T cells, immunohistochemical analysis of tonsillar tissue was performed (Fig. 5Go). In agreement with previous data, CD4+CD57+ T cells were found exclusively in GCs and localized in the light zone of these structures, the same area in which FDCs reside. Depending on the plane of sectioning, 42–69% of the GC CD4+ T cells were CD57+. A few CD57+ lymphocytes were present in the extrafollicular lymphoid tissue, but they did not coexpress the CD4 Ag. To further characterize GC T cells, tonsillar CD4+CD57+ T cells were isolated and analyzed for both CD45RO and CD69 expression (Fig. 6Go, A and B). Consistent with previous reports, virtually all CD4+CD57+ GC T cells coexpressed both CD45RO and the early activation Ag CD69. Because CD4+CD57+ cells represented GC T cells, we examined these cells for CXCR4 expression (Fig. 6GoC). In six separate experiments, freshly isolated CD4+CD57+ T cells had higher CXCR4 expression (mean, 13,040 ± 3,528 ABS) than CD4+CD57- T cells (mean, 3,525 ± 1,029 ABS) from the same donor while at the same time expressing similar levels of CD4 (data not shown). Lastly, we sought to assess the susceptibility of GC CD4 T cells to HIV-1 entry compared with other CD4 T cells from the same tissue (Fig. 7Go). GC T cells appeared more susceptible to infection by HIV-1; this was particularly evident as the concentration of virus decreased. Thus, not only did FDCs increase CXCR4 expression on CD4 T cells in vitro but also T cells in GCs surrounding FDCs in vivo expressed higher levels of CXCR4 and demonstrated a corresponding increased susceptibility to infection ex vivo.



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  		FIGURE 5. GC CD4+ T cells express the CD57 Ag. Tonsillar sections from an HIV-seronegative individual showing CD4+ T cells (green) in the light zone of a GC. The CD57 Ag (red) is coexpressed on a subset of CD4+ lymphocytes (yellow) in these sites. Original magnification, x100.

 


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  		FIGURE 6. GC CD4+CD57+ T cells from human tonsillar tissue have increased CXCR4 expression compared with CD4+CD57- T cells from the same tissue. CD45RO (A) or CD69 expression (B) on CD4+CD57+ and CD4+CD57- human tonsillar T cells. Representative data from six donors are shown. C, CD4 T cells isolated from human tonsillar tissues were surface labeled with Abs against CD4, CD57, and CXCR4 followed by analysis using quantitative flow cytometry to determine the number of CXCR4 ABS. Inset, Representative overlay of CXCR4 expression. Data from six donors are shown. MFI, Mean fluorescence intensity. In every donor analyzed, the CD4+CD57+ T cells had significantly higher expression of CXCR4 than the CD4+CD57- cells from the same tissue.

 


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  		FIGURE 7. GC CD4+CD57+ T cells from human tonsillar tissue with increased CXCR4 expression are highly susceptible to HIV infection. Purified CD4+CD57+ or CD4+CD57- T cells (1.0 x 105) were incubated with decreasing concentrations of HIV-1IIIB (1 x 106, 5 x 105, and 5 x 104 cpm reverse transcriptase (RT) activity) for 1 h and incubated for an additional 12–13 h to allow reverse transcription to occur. After incubation, DNA-containing cell lysates were PCR amplified for both HIV gag and cellular {beta}-globin genes. CXCR4 ABS on CD4+CD57- and CD4+CD57+ cells were 5,372 and 17,439, respectively. HIV-1 entry into CD4+CD57+ T cells correlated with increased in vivo CXCR4 expression.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Throughout the course of disease, active HIV infection is found primarily in the lymphoid follicles of secondary lymphoid tissues where FDCs reside (12, 17, 18, 19, 20, 42, 43). Our previous work indicated that the microenvironment surrounding FDCs is ideally suited for the transmission of HIV infection because these cells maintain the virus in an infectious form for months in the absence of active infection and/or replication and can permit infection even in the presence of high levels of neutralizing Ab (14, 15, 16). In this study, we focused on expanding our understanding of FDC contributions to HIV pathogenesis. Specifically, we sought to determine whether FDCs altered the expression of receptors on CD4 T cells needed for HIV infection. We found that the presence of FDCs in cocultures led to increased expression of CXCR4 on CD4 T cells but had little effect on CCR5 or CD4 and that FDC-CD4 T cell contact was important for this observed CXCR4 up-regulation. Attachment of HIV to the surface of certain cell lines may occur through non-HIV receptor interactions (44); however, our data indicated that increased CXCR4 expression augmented both virus binding and entry by X4 (both laboratory and primary strains) but not primary R5 isolates of HIV-1 in CD4 T cells, suggesting that HIV coreceptor expression is a major factor in both HIV attachment and entry into primary lymphocytes. Importantly, examination of freshly isolated GC T cells that expressed the CD4+CD57+CD45RO+ phenotype revealed higher CXCR4 expression than other CD4 lymphocytes and indicated that these cells were more susceptible to infection by X4 HIV-1 isolates.

Our observation that FDCs increased CXCR4 expression, virus binding, and subsequent entry into CD4 lymphocytes supports the hypothesis that the microenvironment of the GC is highly conducive to HIV infection and that CXCR4high expressing CD57+ GC T cells may be particularly susceptible targets for this virus. Our findings add to those of Hufert et al. (45), who observed that CD57+ GC T cells were more susceptible than other cells to HIV infection by providing a potential mechanism for the increased receptor expression and showing that this correlated with susceptibility to infection. In this latter context, we also observed that increased CXCR4 became particularly important as the concentration of HIV was decreased, suggesting that as virus levels decrease in vivo, as would occur in subjects treated with current potent drug therapies, GC T cells with increased CXCR4 expression may be especially important targets for HIV. Our findings also strongly support the concept that relatively small differences in CXCR4 expression on primary target cells can have important consequences to HIV infection and suggest that in addition to serving as an important reservoir of replication-competent virus, FDCs may also alter the surrounding microenvironment to further support virus transmission.

The mechanism by which FDCs mediated increased CXCR4 levels on CD4 T cells has not been elucidated; however, data presented here indicate that the FDC effect was specific to CXCR4 expression because CD4 and CCR5 levels remained relatively unchanged (Fig. 1Go). Examination of intracellular stores of CXCR4 did not reveal any differences between cultures with or without FDCs even though cell surface expression was increased when FDCs were present. One potential explanation for this observation is a decrease in the turnover rate of CXCR4 in the presence of FDCs, but this must be formally tested.

It appeared that the FDC-signaling that increased CXCR4 expression was mediated by a cell-bound and not a soluble molecule because FDC-T cell contact was necessary for modulation of CXCR4 by FDCs as evidenced by the failure to up-regulate the receptor when the FDCs were physically separated from the lymphocytes and because fixed FDCs were fully capable of inducing increased CXCR4 expression on CD4 T cells (Fig. 2GoC). That the needed FDC-CD4 T cell contact can occur in vivo seems likely because Yuda et al. (46) showed that CD4 T cells in the light zone of secondary lymphoid follicles appear to be in direct contact with FDCs. This contact may facilitate both B and T cell contributions to GCs because FDCs provide both Ag-dependent and Ag-independent signals to lymphocytes that are believed to be important to induction of optimal T-dependent humoral immune responses (10, 16, 47, 48). FDC-mediated up-regulation of CXCR4 on CD4 T cells is both Ag independent and primary as evidenced by the observation that both autologous (data not shown) and allogeneic FDCs (Figs. 1Go and 2Go) up-regulated this receptor equally well and that both unstimulated (Figs. 1Go and 2Go) and activated CD4 T cells underwent receptor up-regulation. Other FDC signals also function in an Ag-independent manner, including induction of B cell responsiveness to chemoattractants (also a primary signal), sparing of B lymphocytes from Fas-mediated apoptosis, and providing costimulatory signals to B lymphocytes (9, 10, 49). These signals, as well as those mediated by FDC-retained Ags, are thought to be important for the optimal development of GCs and the maintenance of memory Ab responses (47, 50, 51, 52, 53).

Although the importance of B and T lymphocytes in the GC reaction is appreciated, the role of FDCs in lymphocyte recruitment into these sites is not well defined. However, it is known that FDCs produce chemoattractant signals for both B and T cells (54) and induce a state of chemotactic responsiveness in B cells (10). Recently, a specialized Th cell, referred to as a follicular Th cell (TFH), has been identified (55, 56). These cells express CXCR5, and preferentially migrate to lymphoid follicles where they provide support for B cell differentiation (56, 57, 58). Kim et al. (41) recently observed that not all CXCR5+ CD4 T cells are TFH but that CD57+CXCR5+ tonsillar cells represent true TFH cells and preferentially migrate to B lymphocyte chemoattractant (CXCL13) which is found in the follicular mantle and on FDCs. In addition to expressing CXCR5, we found that CD4+CD57+ GC T cells also express high levels of CXCR4 (Fig. 5GoC); however, the role of this latter chemokine receptor in TFH migration or in the GC reaction has not been elucidated. Further clarification is needed to understand follicular localization of CD4+ lymphocytes and contributions of CXCR4, SDF-1{alpha}, and FDCs in this process.

In addition to FDC contributions to CXCR4 up-regulation, other signals present in the GC microenvironment have been shown to influence CXCR4 expression. The Th2 cytokine IL-4 has been shown to increase CXCR4 expression while decreasing CCR5 on CD4 T cells (59, 60, 61, 62). Interestingly, increased production of IL-4 has been demonstrated in HIV-1-infected individuals (59, 63, 64, 65, 66, 67); freshly isolated GC T cells (CD4+CD57+) consistently express IL-4 mRNA, whereas non-CD57-bearing Th cells are often negative for IL-4 mRNA (38). In addition to IL-4, TNF-{alpha}, important in FDC development and function (68), has also been shown to enhance CXCR4 expression on CD4 T cells (69). Thus, the GC microenvironment contributed by both FDCs and lymphocytes appears to be ideally suited for augmenting CXCR4 expression and maintaining HIV infection.

Because HIV infection of CD4 T cells with either X4 or R5 strains likely depends on threshold concentrations of CD4 and coreceptors (70, 71), FDCs and the GC microenvironment may be important in tropism shifts from R5 to X4 strains. Shortly after infection, most primary isolates from infected individuals use CCR5 as coreceptors even when the transmitting partner carries both R5- and X4-tropic viruses (72). However, host factors that regulate HIV-1 coreceptors, may be important in infected individuals, because later in the course of disease viruses that use CXCR4 often evolve and become dominant. This adaptation to CXCR4 specificity in vivo accelerates HIV-1 disease progression (73, 74, 75, 76). We reason that contributions from FDCs and the cytokine milieu present in the GC strongly favor CXCR4 expression on CD4 T cells and thus may be important over the course of disease in establishing an environment that favors the propagation of X4 virus quasi-species. In support of this reasoning, Berger et al (28). hypothesized that HIV replication in specialized tissue compartments, which predominate in expression of a coreceptor, might select for viral variants. The data reported here do not demonstrate that virus tropism shifts occur in GCs, but they do provide evidence that relatively small differences in HIV coreceptor expression can influence viral infection in primary cells and suggest that this may contribute to selection of X4 variants over time. Because HIV is trapped on the FDC network early after infection and remains infectious for long periods of time (15, 42), an understanding of the contributions of FDCs that lead to increased CXCR4 expression and heightened susceptibility to infection by X4 isolates of virus may be important to our ability to devise intervention strategies that effectively target this important HIV reservoir and microenvironment.


   Acknowledgments
 
We thank Dr. Kipp M. Robins and his associates at the Utah Valley Regional Medical Center, Health South Provo Surgical Center, and the Central Utah Surgical Center for providing tissues. We also thank the National Institutes of Health, AIDS Research and Reference Reagent Program (Rockville, MD), for supplying reagents.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI39963 (to G.F.B.). J.D.E. was supported in part by an American Society for Microbiology Undergraduate Research Fellowship and the Vanice, Glen W. and Keith Reid Endowment for Scientific Research at Brigham Young University. Back

2 Address correspondence and reprint requests to Dr. Gregory F. Burton, Department of Microbiology and Molecular Biology, Room 851 John A. Widtsoe Building, Brigham Young University, Provo, UT 84602. Back

3 Abbreviations used in this paper: FDC, follicular dendritic cell; ABS, Ab binding site; GC, germinal center; SDF-1{alpha}, stromal cell-derived factor-1{alpha}; TFH, follicular Th cell; PI, propidium iodide; MIP-1{beta}, macrophage-inhibitory protein-1{beta}. Back

Received for publication September 24, 2001. Accepted for publication June 26, 2002.


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