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The Journal of Immunology, 2001, 166: 6437-6443.
Copyright © 2001 by The American Association of Immunologists

Nonproliferating Bystander CD4+ T Cells Lacking Activation Markers Support HIV Replication During Immune Activation1

David Scales*, Houping Ni*, Farida Shaheen{dagger}, John Capodici*, Georgetta Cannon* and Drew Weissman2,*

* Division of Infectious Diseases and {dagger} Center for AIDS Research, University of Pennsylvania, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
HIV replicates primarily in lymphoid tissue and immune activation is a major stimulus in vivo. To determine the cells responsible for HIV replication during Ag-driven T cell activation, we used a novel in vitro model employing dendritic cell presentation of superantigen to CD4+ T cells. Dendritic cells and CD4+ T cells are the major constituents of the paracortical region of lymphoid organs, the main site of Ag-specific activation and HIV replication. Unexpectedly, replication occurred in nonproliferating bystander CD4+ T cells that lacked activation markers. In contrast, activated Ag-specific cells were relatively protected from infection, which was associated with CCR5 and CXC chemokine receptor 4 down-regulation. The finding that HIV replication is not restricted to highly activated Ag-specific CD4+ T cells has implications for therapy, efforts to eradicate viral reservoirs, immune control of HIV, and Ag-specific immune defects.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human immunodeficiency virus infection of PBMC requires T cell activation to complete reverse transcription (1, 2, 3, 4, 5), while infection is aborted in quiescent peripheral blood CD4+ T cells in vitro. The majority of active HIV replication in vivo occurs in CD4+ T cells in the paracortical region of lymphoid tissue (6, 7). Model systems that use dendritic cell (DC)3 stimulation of CD4+ T cells to replicate lymphoid environments have been developed and have been shown to be useful in studying physiologic interactions that regulate HIV replication (8, 9, 10, 11, 12, 13, 14, 15). These systems differ in that they do not require exogenous stimulators or cytokines, including serum, and rely on the presentation of Ags by DC to activate CD4+ T cells. HIV replication in vivo is regulated by immune system activation. In HIV-infected individuals or SIV-infected macaques immunized against or infected with an infectious organism, immune system activation correlated with the induction of viral replication in vivo (16, 17, 18, 19, 20). The amount of viral replication observed after vaccination with influenza or tetanus toxoid or during active infection with Mycobacterium tuberculosis was associated with the stage of HIV disease (18, 19) and the ability to respond in vitro to the Ag (19, 21). These studies suggested that the level of increase in viral replication correlated with immune system activation against an antigenic challenge and have led to the hypothesis that antigenic activation in lymphoid organs drives HIV replication (reviewed in Ref. 22).

In contrast to mitogen activation, Ag-specific activation is complex. During a response to Ag, both Ag-specific and bystander T cells become activated. Bystander activation represents non-TCR-mediated activation as a result of mediator release or triggering of adhesion or costimulatory molecules (23, 24, 25). Earlier studies of bystander activation suggested that it was common in CD8+ T cells and more limited in CD4+ T cells. With the advent of newer techniques for measuring Ag-specific cells, it appears that bystander activation in CD8+ T cells is, in fact, minimal (26, 27, 28). On the other hand, CD4+ T cell bystander activation is being observed in an increasing number of experimental systems (29, 30). Bystander activation of CD4+ T cells has also been demonstrated to be relevant as part of the immunopathology of multiple viral diseases, including herpes simplex (31), hepatitis B and C (32), diabetes induced by coxsackie virus (33), and dengue (34).

In this report, we identify the populations of CD4+ T cells that replicate HIV during an Ag-specific immune response. As a model, we used DC presentation of superantigen (SA) to and stimulation of CD4+ T cells. SA are bacteria- or virus-derived proteins that bind to MHC class II molecules and a constant region of the variable {beta}-chain of the TCR. The ability of SA to activate a large population of T cells defined by V{beta} chain expression enables accurate identification of Ag-specific cells. SA stimulation of CD4+ T cells closely mimics the recognition of specific Ag (35). Although there may be subtle differences between Ag- and SA-activated CD4+ T cells depending on the system used (36, 37, 38), most studies find that the requirements of Ag and SA activation are similar. Efficient SA stimulation of T cells uses adhesion and coactivation molecule interactions shared by peptide Ag stimulation, including CD4 signaling through p56lck (39), CD28-B7 (40, 41), ICAM-1-LFA-1 (42), LFA-3-CD2 (43), CD40-CD40 ligand (44), CD49d subunit-bearing integrins (45), and CD69 (46) interactions. SA-stimulated cells show a dependence on IL-2 (47) and differential up-regulation of CD2, LFA-1, CD25, CD28, CD69, and HLA-DR on stimulated cells (48). Thus, a model system using DC presentation of SA to CD4+ T cells was employed as a physiologic representation of the site of the linked processes of HIV replication and Ag-specific activation (10, 12).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

AIM V serum-free medium supplemented with glutamine (2 mM; Life Technologies, Rockville, MD); GM-CSF, IL-4, and TNF-{alpha} (R&D Systems, Minneapolis, MN); PGE3 (Cayman Chemical, Ann Arbor, MI); Toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin B SA (Sigma, St. Louis, MO); mAbs, CD16, and CD56 (Accurate Chemical and Scientific, Westbury, NY); HLA-DR-FITC (41) and HLA-DR-PE (Becton Dickinson, Franklin Lakes, NJ); CD83-PE (49) and Ki67-FITC (Immunotech, Fullerton, CA); 2D7-FITC or -PE, and 12G5-FITC or -PE (PharMingen, San Diego, CA); V{beta}2-biotin (Immunotech); and streptavidin-PerCP and streptavidin-FITC (Becton Dickinson) were used. CD16 and CD56 magnetic beads were prepared by incubating goat anti-mouse IgG beads (Dynal, Lake Success, NY) with anti-CD16 and anti-CD56 mAb according to the manufacturer’s instructions.

Culture system

DC were prepared as previously described (50) with minor modifications. Briefly, PBMC were isolated from leukapheresis packs obtained under an institutional review board-approved protocol from healthy volunteers by Ficoll-Hypaque density gradient centrifugation. Monocytes were purified from PBMC by discontinuous Percoll gradient centrifugation. The low density fraction was depleted of B, T, and, in certain experiments, NK cells using magnetic beads specific for CD2, CD16, CD19, and CD56 according to the manufacturer’s instructions (Dynal). The purified monocytes were cultured at 106 cells/ml in culture medium with GM-CSF (50 ng/ml) and IL-4 (100 ng/ml). TNF-{alpha} (1 ng/ml) and PGE3 (500 nM) were added 1 day before use to obtain mature, CD83+ (60–90%) DC. B cells were purified from PBMC using positive selection with CD19 beads (Dynal). Autologous HLA-DR-, CD4+ T cells were purified by negative selection first by incubating on human Ig (10 µg/ml; Sigma)-coated plates for 1 h and then with negative selection using magnetic beads specific for CD8, CD19, CD16, CD56, and HLA-DR (Dynal). DC and CD4+ T cells were cocultured, and TSST-1 SA (0.01–0.02 ng/ml) was added at the same time.

Analyses

HIV Ba-L, UGO24, SF162, IIIB, TH026 (obtained from AIDS Reference and Reagent Program), BL2 (51), 89.6 (52), and LL-7 (53) (obtained from Ronald Collman, University of Pennsylvania, Philadelphia, PA; 5–20 ng of p24) were added to 3-day-old DC/CD4+ T cell (1/10)/SA-TSST-1 (0.01 ng/ml) cocultures (10–20 x 106 cells). Sixteen to 48 h later, the populations of Ag-specific (expressing V{beta}2, which is bound by TSST-1) and bystander (expressing other V{beta} chains) activated cells were purified from the bulk culture using flow cytometric sorting (FACStarPlus; Becton Dickinson) with V{beta}2-biotin mAb followed by streptavidin-PE and HLA-DR-Fl staining. Ninety-seven to 99% purity of the V{beta}2+ cells was obtained; 94–98% purity of the V{beta}2- cells was achieved. DNA was isolated as described previously (21). RNA was isolated from cell pellets using Quantum Prep Master Blaster (Bio-Rad, Hercules, CA) and was reverse transcribed using the Superscript kit (Life Technologies). HIV infection of CD4+ T cells was determined by real-time quantitative PCR (Perkin-Elmer 7700; Perkin-Elmer, Norwalk, CT) for gag DNA and RNA and GAPDH DNA according to the manufacturer’s instructions. The PCR primer sequences for HIV gag were SK38/39 and a SK19 probe (54) modified with molecular beacon technology. The PCR primers for GAPDH were: forward, 5'-GGTGGTCTCCTCTGACTTCAACA-3'; reverse, 5'CCAGCCACATACCAGGAAATG-3'; and a molecular beacon-based probe with the sequence 5'-FAM-GCGAGCCTGGCATTGCCCTCAACGACCACGCTCGC-Dabcyl-3' (Integrated DNA Technologies, Coralville, IA).

Intracellular p24 gag Ag expression was determined 1–8 days postinfection by fixing cells in 2% paraformaldehyde for 30 min, permeabilizing with 0.1% saponin for 30 min, and including 0.05% saponin in the staining medium (PBS, 1% FCS, 1% normal mouse serum, and 0.2% NaN3) in all further staining steps. Cells were sequentially stained with p24 gag mAb (183-H12-5C from the AIDS Reference and Reagent Program) (55), goat anti-mouse IgG-FITC, 5% normal mouse serum (Sigma) to block further mouse Ig binding, V{beta}2-biotin, and streptavidin-PerCP plus HLA-DR-PE. Cells were then analyzed on a FACScan (Becton Dickinson).

Flow cytometry for chemokine receptor expression was performed by staining cells with V{beta}2-biotin in staining medium for 20 min on ice, followed by washing twice and incubation with streptavidin-PerCP, 2D7-FITC, and 12G5-PE for 20 min on ice, followed by washing and analysis on a FACScan.

Cell cycle analysis was performed on CD4+ T cells with or without DC and SA after 3 days of culture. Cells were stained with V{beta}2-biotin, streptavidin-FITC, and HLA-DR-PE, fixed in 70% ethanol for 30 min, and then incubated with actinomycin D (25 µg/ml; Sigma) for 30 min. Actinomycin D was used to allow three-color analysis. Cells were analyzed on a FACScan equipped with doublet discrimination mode to ensure analysis of single cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Indirect (bystander) activation occurs during a response to superantigen

Resting CD4+ T cells (HLA-DR-) were obtained by negative selection and cultured with autologous DC with or without the SA TSST-1. Since TSST-1 activates T cells bearing the V{beta}2 TCR chain, cells were stained for V{beta}2 to distinguish the Ag-specific (V{beta}2+) and bystander (V{beta}2-) populations. After 3 days of culture, HLA-DR-depleted CD4+ T cells demonstrated minimal activation within both the V{beta}2-positive and -negative populations (Fig. 1GoA). Coculture with autologous DC (Fig. 1GoB) increased activation of both populations. Based on acquisition of HLA-DR expression, the addition of TSST-1 resulted in near-complete activation of V{beta}2+ Ag-specific cells (upper right quadrant of Fig. 1GoC vs 1B) and a substantial increase in V{beta}2- bystander cell activation (upper left quadrant of Fig. 1GoC vs 1B). Similar bystander activation was observed for DC-T cell cocultures activated with staphylococcal enterotoxin B (data not shown). The level of bystander activation correlated in part with the ratio of DC to CD4+ T cells (Fig. 1GoD). This suggested that some component of cell-to-cell contact was necessary. If bystander activation of CD4+ T cells in this coculture system was independent of cell-to-cell contact, no decrease in bystander activation with low numbers of DC (1/50 CD4+ T cells) should be observed, since the V{beta}2+, CD4+ T cells remained almost completely activated (Fig. 1GoE).



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  		FIGURE 1. DC plus TSST-1 activation of V{beta}2+, CD4+ T cells results in bystander activation of V{beta}2- cells. Purified CD4+ T cells, depleted of activated (HLA-DR+) cells, alone (A), with DC (B), and with DC and TSST-1 (0.01 ng/ml; C) were cultured for 3 days and analyzed for HLA-DR expression in the V{beta}2+ (Ag-specific) and V{beta}2- (bystander) populations. The addition of SA to cocultures of DC and CD4+ T cells resulted in nearly complete activation of the V{beta}2-bearing CD4+ T cells (C, upper right quadrant) and a significant increase in activation of the V{beta}2- (bystander) population (C, upper left quadrant) compared with the absence of SA (B) or SA and DC (A). Decreasing the number of DC per CD4+ T cell resulted in a decrease in the level of activation of V{beta}2- cells in the presence of SA (D) but still activated the V{beta}2+, CD4+ T cells (E). The addition of SA to CD4+ T cells in the absence of DC resulted in no activation. Twenty thousand events were analyzed for each histogram. The data are representative of six experiments.

 
Since SA can lead to either activation and expansion of the specific V{beta}-bearing cells or anergy and T cell depletion depending on the culture conditions, we chose conditions designed to allow expansion without depletion of V{beta}2+, CD4+ T cells (40). To confirm this, we stained for V{beta}2 expression 2 wk after TSST stimulation and found no evidence of V{beta}2 depletion. In addition, restimulation with TSST-1 2 wk after initial stimulation again resulted in activation of V{beta}2+ T cells, demonstrating that anergy had not been induced. DC activation of CD4+ T cells in the absence of exogenous Ag can be substantial (3–15%) especially in the presence of FCS, which is known as the autologous MLR and can support HIV replication (10, 12). This phenomenon probably represents the presentation of Ags obtained by the DC before and during preparation. The autologous MLR can be substantially, although not completely, reduced by using serum-free medium for DC generation and T cell stimulation, which was employed in all experiments in this report.

HIV replication occurs in both Ag-specific and bystander CD4+ T cells

To determine the populations of CD4+ T cells responsible for HIV-1 replication during SA stimulation, DC-CD4+ T cell cocultures were infected and analyzed for infection by intracellular p24 gag expression over the subsequent 1–8 days. The HIV-1 strains analyzed included CCR5-using macrophage-tropic prototype (SF162, BAL) and primary isolates (LL-7, BL2, TH026); a dual-tropic strain that uses both CCR5 and CXCR4 (89.6); and CXCR4-using T-tropic prototype (IIIB) and primary (UGO24) isolates. Both V{beta}2+ Ag-specific and V{beta}2- bystander cells demonstrated p24 gag staining (Fig. 2Go and data not shown). The frequency of p24+ expression was typically 2- to 3-fold higher among V{beta}2+ cells than V{beta}2- bystander cells. However, as only 10–20% (11.2% in the experiment shown) of the CD4+ T cells in cultures with DC plus SA expressed V{beta}2, approximately two to four times as many bystander cells were infected as Ag-specific cells. In the absence of SA, DC-CD4+ T cell cocultures resulted in <1% p24 gag-positive cells, with equal frequencies in the V{beta}2+ and V{beta}2- populations.



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  		FIGURE 2. Ag-specific and bystander CD4+ T cells replicate HIV after stimulation by DC and SA. Three days after stimulation, cocultures were infected with HIV at 20 ng/20 million CD4+ T cells. Three days later, cells were stained with V{beta}2 and p24 gag mAb and analyzed. The histograms presented were gated on T cells and V{beta}2 expression. Values are the percentage of cells above control staining. Fifty thousand events were analyzed for each histogram. The data are representative of at least three separate experiments with each virus.

 
The relative mean fluorescence of p24 gag staining in Ag-specific cells was higher than that in bystander cells, suggesting more HIV p24 gag protein per cell. Since lower levels of p24 gag expression by infected bystander cells combined with relatively low sensitivity of p24 gag staining as a measure of infection could lead to underestimating the number of infected bystander cells, we used another approach to analyze the relative infection of these cell populations. Real-time quantitative DNA and RNA PCR was used on HIV-infected CD4+ T cells stimulated by DC-SA and sorted by V{beta}2 expression (Fig. 3Go). For all HIV strains, similar levels of HIV gag DNA per cell (Fig. 3GoA) were observed in the Ag-specific and bystander populations. When corrected for the percentage of each population in the culture, there were 2- to 8-fold more copies of HIV gag DNA within the bystander population. In contrast, HIV gag RNA quantitation demonstrated that the Ag-specific cells had more copies of HIV gag RNA per infected cell than did the bystander cells (Fig. 3GoB), in agreement with the higher levels of p24 gag staining (Fig. 2Go). Of note, variation between experiments was mainly due to the differences in the percentage of V{beta}2+ cells in the culture, while within each experiment, all strains demonstrated a similar distribution of infection in bystander vs Ag-specific cells. Thus, bystander cells represented the majority of infected cells, while Ag-specific activated cells were a more highly productive compartment.



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  		FIGURE 3. Ag-specific compared with bystander CD4+ T cells contain a similar amount of HIV gag DNA (A) but more HIV gag RNA (B) per cell after stimulation by DC and SA and infection with macrophage (M), T cell line (T), and dual-tropic HIV. Cells were infected after 3 days of stimulation; 24 h later they were washed, stained, fixed, and sorted by V{beta}2 expression. The sorted cells were assayed for HIV gag DNA and RNA by real-time quantitative PCR. Copies of gag DNA or RNA were corrected for the number of cells in the samples determined by GAPDH DNA PCR. HIV was added at 5 ng/20 million CD4+ T cells. Similar results for HIV gag RNA were observed with all strains of HIV analyzed. The variation in individual experiments was <4-fold between V{beta}2+ and V{beta}2- cells for each virus, and individual viral differences in infection of subsets of CD4+ T cells were not present across experiments. Error bars represent the SEM. The data are representative of three experiments.

 
DC are the most efficient cell in presenting SA to CD4+ T cells and are the primary APC for naive and memory T cell responses, but other APC also participate in immune activation. Therefore, we conducted experiments using purified B cell presentation of SA to HLA-DR-, CD4+ T cells and PBMC stimulated with SA, which gave similar patterns of bystander activation and infection of both populations with the HIV strains Ba-L, 89.6, and IIIB. In addition, as an autologous MLR was not observed in B cell-CD4+ T cell cultures and PBMC, this demonstrated that bystander activation was responsible for HIV replication in V{beta}2-, CD4+ T cells.

Role of activation in HIV infection of bystander cells

An analysis of bystander cells from DC-SA cocultures demonstrated a portion of cells expressing HLA-DR, which was dramatically different from the Ag-specific cells that had high levels of HLA-DR (Fig. 1Go). Therefore, we determined whether infection was restricted to the HLA-DR+ subset of bystander activated CD4+ T cells. V{beta}2- bystander cells in cultures of DC, CD4+ T cells, and SA were analyzed for HLA-DR and p24 gag expression, which revealed that cells lacking the activation marker HLA-DR had significant p24 gag expression (Fig. 4Go).



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  		FIGURE 4. HLA-DR- bystander cells in cultures of DC, SA, and CD4+ T cells actively replicated HIV. CD4+ T cells infected with SF162 were stained for V{beta}2, HLA-DR, and isotype control (A) or p24 gag (B) mAb after 4 days. Histograms shown were gated on V{beta}-negative lymphocytes. Similar data were obtained with other viral strains. The data are representative of three experiments.

 
Cell cycle analysis of HLA-DR-positive and -negative bystander cells in DC-SA cocultures demonstrated 0.42% of HLA-DR- bystander cells in S phase, which was similar to unactivated CD4+ T cells, compared with 10.4% of Ag-specific cells and 2.02% of HLA-DR+ bystander activated cells (Fig. 5Go). Thus, since up to 14% of HLA-DR bystander cells were infected, and only 0.42% were in S phase, bystander CD4+ T cells present during an Ag-specific response were capable of replicating HIV in the absence of proliferation or detectable activation marker expression (HLA-DR, CD25, CD69, KI67).



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  		FIGURE 5. HLA-DR- bystander cells in SA-stimulated cultures do not proliferate. CD4+ T cells with or without DC and SA were stained for V{beta}2 and HLA-DR, fixed, stained with actinomycin D (7-AAD) to measure DNA content, and analyzed. Single-color histograms of DNA content for V{beta}2+ and V{beta}2- populations for each condition are shown along with V{beta}2- cells in DC-SA cocultures with and without HLA-DR expression. Different scales are used to normalize the histograms for the different percentages of each cell type in the culture. Values are the percentage of cells in S phase. The data represent two separate experiments.

 
Regulation of HIV coreceptor expression during Ag-specific activation

In addition to the ability of bystander cells to replicate HIV, a relative inability of Ag-specific cells to support HIV growth was also observed. In systems of mitogen-activated CD4+ T cells, high levels of both M- and T-tropic HIV infection and nearly complete depletion of CD4+ T cells with T-tropic HIV are typically observed. In contrast, we detected no substantial loss of V{beta}2+ T cells over time after infection. Furthermore, the percentage of Ag-specific cells expressing p24 gag was relatively low even though the Ag-specific cells in our culture system were highly activated (Fig. 1Go, C and E), proliferated with 10.4% of cells in S phase (Fig. 5Go), and contained the majority of cells incorporating [3H]thymidine (data not shown). The observation that highly activated and proliferating Ag-specific CD4+ T cells were only modestly infected was, thus, unexpected. To address a potential mechanism for the relative protection from HIV infection, we analyzed the expression of the chemokine receptor/HIV coreceptors CXCR4 and CCR5 on the two populations of CD4+ T cells during an Ag-specific response. Ag-specific cells demonstrated a significant, but not complete, down-regulation of CXCR4 expression (Fig. 6GoA). Over the next 10 days, CXCR4 expression on the V{beta}2+ cells never approached the level of expression on the V{beta}2- (Fig. 6GoB). CCR5 was similarly down-regulated on the V{beta}2+ cells, and its kinetics of reappearance were delayed compared with the V{beta}2- cells (Fig. 6Go, C and D). Thus, Ag-specific activation down-regulated the expression of CCR5 and CXCR4, which probably explained at least in part the relative resistance to infection.



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  		FIGURE 6. The Ag-specific (V{beta}2+), CD4+ T cells, stimulated with DC and SA, had reduced levels of CXCR4 and CCR5 compared with bystander activated (V{beta}2-) CD4+ T cells. A, CXCR4 expression was analyzed 3 days after stimulation of CD4+ T cells by DC and SA. B, Mean fluorescence of CXCR4 on V{beta}2+ and V{beta}2- populations over 10 days poststimulation demonstrated continued decreased expression on Ag-specific cells. C, DC and SA stimulation of CD4+ T cells led to down-regulation of CCR5 expression on the Ag-specific cells, but not the bystander cells, in cocultures. D, The percentages of CCR5-expressing cells in the V{beta}2+ and V{beta}2- populations over 10 days poststimulation show continued lower expression on Ag-specific cells. The data presented are representative of three individuals.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we demonstrate that HIV replication occurred in bystander cells in a lymphoid model of an Ag-specific immune response. Three novel findings relevant to understanding HIV replication in vivo and for potential therapeutic intervention are described. The first finding is that highly activated Ag-specific CD4+ T cells, although they are targets for HIV infection, are relatively protected compared with levels of infection induced by mitogen activation. This appears to be mediated by a down-regulation of the HIV coreceptors CCR5 and CXCR4. The second finding is that a much larger proportion of bystander cells are productively infected by HIV than Ag-specific cells during an Ag-specific response in a model of the lymphoid environment. Third, approximately two-thirds of the infected bystander cells do not express activation markers or proliferate. Although these bystander cells may not express activation Ags or cycle into S phase, they have received a signal from the products of the Ag-specific response that allows them to replicate HIV.

Model system for studying HIV replication in vitro

The initial identification of HIV as the causative agent of AIDS employed activated T cells (56). Further studies demonstrated that peripheral blood T cells, in the absence of activation, could not complete reverse transcription (1, 2, 3, 4, 5). The study of HIV replication in vitro requires the use of model systems. HIV replication (6, 7) and Ag-specific activation (reviewed in Ref. 57) primarily occur in the paracortical regions of lymphoid organs. Most systems used to study HIV replication in vitro employ PBMC. Unstimulated PBMC typically have <5% of CD4+ T cells expressing HLA-DR or other activation markers; after activation with mitogens or Abs against CD3 nearly all CD4+ T cells become activated. In a lymph node from an HIV-infected individual, up to 75% of the CD4+ T lymphocytes can express varying levels of HLA-DR, while T cells in lymphoid tissue from uninfected subjects have lower, but significant, levels of activation (reviewed in Ref. 58, 59). We and others have used DC activation of CD4+ T cells, with or without the addition of specific Ag, to model both Ag-specific activation and HIV replication based on the above observations (10, 11, 12, 13, 14, 50, 60, 61, 62).

Bystander cells during an Ag-specific response replicate HIV

During an Ag-specific immune response, multiple populations of cells are activated, only a portion of which recognize the initiating Ag and become highly activated. In this report we demonstrate that a population of cells responsible for most of the HIV-replicating cells were not directly activated by our model Ag. A minority portion of these bystander cells did become activated, as measured by expression of the activation markers HLA-DR, CD25, KI67, and CD69 (Fig. 1Go and data not shown), but did not produce higher levels of infection compared with the activation-negative cells.

It is likely that we are studying multiple populations of bystander CD4+ T cells. One population may be CD4+ T cells that are actively bound via their TCR to an APC that is presenting a peptide Ag bound to MHC class II, but the interaction is not "strong enough" (T cell cannot up-regulate APC function via CD40L, TCR-peptide/MHC interaction too weak, other defective or inefficient interactions between CD28 and B7, LFA-1 and ICAM-3, or CD2 and CD58) to activate the cell (63). When this T cell bound to an APC encounters an efficiently occurring APC-T cell interaction, soluble mediators may be released that allow the poorly interacting T cell to become activated. In addition, transcomplementation of costimulation has been described where another cell that is not presenting Ag to the T cell can supply the necessary B7 interaction and lead to activation (64). A second population of CD4+ T cells that is bound to the APC in an Ag-independent, non-TCR manner has been described that is bound via adhesion or coactivation molecule interactions (11). In the setting of a strong Ag-specific response, they become activated in the absence of TCR signaling. A third population of bystander CD4+ T lymphocytes that occurs without cell to cell contact, through mediator release, also may be present.

The bystander cells in cultures of DC, CD4+ T cells, and SA contain the majority of infected cells as measured by p24 gag Ag expression and gag DNA content. The infected bystander cells express less HIV p24 gag protein and gag RNA per cell, demonstrating a lower production of HIV. The expression of p24 gag on bystander cells is independent of activation marker expression or proliferation, but is associated with some level of activation, because in the absence of DC and SA the cells are not infectable. The source of the activation includes both cytokines released and interactions between the bystander cells and DC, as preliminary studies separating bystander cells from DC-CD4+ T cell-SA cocultures by a membrane demonstrated reduced levels of HIV replication in the separated cells (D. Scales and D. Weissman, unpublished observations). Our data are in agreement with recent reports demonstrating that cytokines are capable of inducing HIV replication in unstimulated CD4+ T cells (65, 66). In addition, an in vivo correlate of our model has recently been reported where in situ examination for viral nucleic acids in tissue from HIV- and SIV-infected subjects has demonstrated low level infected CD4+ T cells lacking activation markers (67). As that study documented the presence of HIV in unactivated cells, but could not determine their activation state at infection, our data extend this observation by demonstrating the infection of nonreplicating CD4+ T cells.

Ag-specific cells are relatively protected from infection HIV

The regulation of the expression of CCR5 and CXCR4 expression on activated CD4+ T cells is complicated by the system used for analysis and the method of detection. Activation of PBMC with PHA was demonstrated to increase the expression of CXCR4. The expression of CXCR4 was found to be a balance between CD3 signaling, which increased CXCR4, and CD28 signaling, which decreased the expression (68). CCR5 was found to be present on a small subset of T cells, (CD26bright, CD45RO memory cells) and was down-regulated soon after PHA stimulation. Expression of CCR5 then slowly increased with the addition of IL-2 (69). Our current and previous studies of CXCR4 expression suggested that it was down-regulated with purification of PBMC and up-regulated after culture in the absence of stimulation (70). In this report Ag-specific activation led to a down-regulation of CCR5 and a significant, but incomplete, down-regulation of CXCR4 compared with bystander cells. Our results using an Ag-specific response in a model of the paracortical region of lymphoid organs are in agreement with other systems designed to mimic APC activation using anti-CD3 and anti-CD28 mAb, where a down-regulation of CCR5 and a protection from infection by R5 viruses was observed (71).

Current approaches to the treatment of HIV infection include the use of anti-viral agents, immune system modulators, and vaccines. With the improvement in virologic control of HIV, new therapies are being developed to target reservoirs of HIV infection. The findings presented in this report suggest that long-lived memory cell reservoirs (reviewed in Ref. 22) may be more dynamic and contain recently infected activation marker-negative CD4+ T cells. The data in this report support the finding of preserved common Ag-specific immune responses, but do not explain the early loss of HIV-specific responses. Further studies directed at defining the controls of bystander replication of HIV will be important as an alternate approach to inhibit viral production as well as for possibly enhancing Ag-specific and HIV-specific responses.


   Acknowledgments
 
We thank Drs. Ron Collman and Anthony S. Fauci for helpful discussions. We are grateful to the University of Pennsylvania Center for AIDS Research and the National Institute of Allergy and Infectious Disease AIDS Reference and Reagent Program.


   Footnotes
 
1 This work was supported by grants from the National Institute of Allergy and Infectious Disease (R21-45318) and National Heart, Lung, and Blood Institute (R01-62060). Back

2 Address correspondence and reprint requests to Dr. Drew Weissman, University of Pennsylvania, 536 Johnson Pavilion, Philadelphia, PA 19104. Back

3 Abbreviations used in this paper: DC, dendritic cell; SA, superantigen; CXCR4, CXC chemokine receptor 4; TSST-1; toxic shock syndrome toxin-1. Back

Received for publication October 2, 2000. Accepted for publication March 12, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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