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The Journal of Immunology, 2006, 176: 5760-5771.
Copyright © 2006 by The American Association of Immunologists

CD16+ Monocyte-Derived Macrophages Activate Resting T Cells for HIV Infection by Producing CCR3 and CCR4 Ligands1

Petronela Ancuta*, Patrick Autissier{dagger}, Alysse Wurcel{ddagger}, Tauheed Zaman{ddagger}, David Stone{ddagger} and Dana Gabuzda2,*

* Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02115; {dagger} Beth Israel Deaconess Center, Boston, MA 02115; and {ddagger} Lemuel Shattuck Hospital, Jamaica Plain, MA 02130


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The CD16+ monocyte (Mo) subset produces proinflammatory cytokines and is expanded in peripheral blood during progression to AIDS, but its contribution to HIV pathogenesis is unclear. In this study, we investigate the capacity of human CD16+ and CD16– Mo subsets to render resting CD4+ T cells permissive for HIV replication. We demonstrate that CD16+ Mo preferentially differentiate into macrophages (M{phi}) that activate resting T cells for productive HIV infection by producing the CCR3 and CCR4 ligands CCL24, CCL2, CCL22, and CCL17. CD16+, but not CD16–, Mo-derived M{phi} from HIV-infected and -uninfected individuals constitutively produce CCL24 and CCL2. Furthermore, these chemokines stimulate HIV replication in CD16– Mo:T cell cocultures. Engagement of CCR3 and CCR4 by CCL24 and CCL2, respectively, along with stimulation via CD3/CD28, renders T cells highly permissive for productive HIV infection. Moreover, HIV replicates preferentially in CCR3+ and CCR4+ T cells. These findings reveal a new pathway of T cell costimulation for increased susceptibility to HIV infection via engagement of CCR3 and CCR4 by chemokines constitutively produced by CD16+ Mo/M{phi}. Thus, expansion of CD16+ Mo in peripheral blood of HIV-infected patients and their subsequent recruitment into tissues may contribute to chronic immune activation and establishment of viral reservoirs in resting T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Peripheral blood monocytes (Mo)3 consist of subsets with distinct phenotypic and functional characteristics (1, 2). The expression of CD16 (Fc{gamma}RIII) distinguishes two Mo subsets: a minor CD16+ subset and a major CD16– subset (1). CD16+ Mo represent a heterogeneous population, consisting of CD14highCD64high and CD14lowCD64low (2, 3) and M-DC8+ and M-DC8– Mo (4). Compared with CD16– Mo, CD16+ Mo produce higher levels of TNF and IL-1 (4, 5), express higher levels of HLA-DR, CD40, CD86, CD11a, and CD11c (6), and preferentially differentiate into dendritic cells (DC) upon transendothelial migration (7) or engagement of TLR2/1(8). Thus, CD16+ Mo may represent a pool of DC precursors and a source of proinflammatory cytokines. CD16+ are also distinguished from CD16– Mo by their pattern of chemokine receptor expression. CD16+ Mo express CX3CR1 and migrate in response to CX3CL1 (3, 9), whereas CD16– Mo express CCR1 and CCR2 and migrate in response to CCL2 and CCL3 (3, 10). Thus, CD16+ Mo may be preferentially recruited into anatomic sites expressing CX3CL1, such as lymph nodes (11), brain (12), and intestine (13).

CD16+ Mo represent 5–10% of total Mo in the blood of healthy individuals (1), but are dramatically expanded in HIV-infected patients (14, 15), particularly during progression to AIDS (16, 17). Highly active antiretroviral therapy induces only a modest decrease in the frequency of CD16+ Mo (15). CD16+ Mo express high levels of activation markers including HLA-DR, CD69, CD40, and CD23 (6, 14, 16, 18) and constitutively produce IL-1 and TNF (14), cytokines that promote HIV replication in T cells (19). Therefore, expansion of CD16+ Mo in HIV-infected patients may contribute to chronic immune activation and persistent HIV replication.

Mo do not support productive HIV infection in vitro (20, 21) and represent a minor viral reservoir in HIV-infected patients (22), whereas CD4+ T cells are the major targets for HIV replication in vivo (23). Resting T cells are resistant to HIV replication in vitro, with productive HIV infection requiring activation via TCR and costimulatory molecules (24, 25). Previous studies demonstrated preferential replication of HIV in Ag-specific T cells (26, 27), suggesting that APC such as DC and macrophages (M{phi}) play a critical role in disease pathogenesis. DC induce Ag-dependent T cell activation (28, 29) and mediate trans-HIV infection of T cells (30, 31). Additionally, DC and M{phi} produce soluble factors that promote HIV replication in T cells (32, 33, 34). Circulating CD16+ Mo share phenotypic and functional similarities with DC and M{phi} (3, 9, 10, 11, 12, 13). Furthermore, CD16+ Mo exposed to HIV in vitro promote highly efficient viral replication upon differentiation into M{phi} and interaction with PHA-stimulated autologous T cells, with conjugates formed between CD16+ Mo-derived M{phi} (Mo/M{phi}) and T cells being major sites of viral replication (35). However, mechanisms by which CD16+ Mo promote HIV replication in T cells are unclear.

In this study, we report that CD16+ Mo preferentially differentiate into M{phi} that activate resting T cells for productive HIV infection by producing the CCR3 and CCR4 ligands CCL24 (eotaxin-2), CCL2 (MCP-1), CCL22 (Mø-derived chemokine), and CCL17 (thymus and activation-regulated chemokine). CCL24 and CCL2, along with stimulation via CD3/CD28, render T cells highly permissive for productive HIV infection. Furthermore, CCR3+ and CCR4+ T cells are preferential targets for HIV replication. The ability to produce CCR3 and CCR4 ligands that activate resting T cells for HIV-1 infection endows CD16+ Mo/M{phi} with a novel previously unrecognized potential to contribute to chronic immune activation and HIV replication in resting T cells in vivo.


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

Recombinant IL-2, CCL2, and CCL24 were obtained from R&D Systems. Anti-CD3 and -CD28 Abs were obtained from BD Pharmingen. The following mAbs were used for FACS analysis: FITC anti-CD14, -CD16b, CD66b, PE anti-CD33, -CD19, and -CD56, PC5 anti-CD16 and -CD3 (Beckman Coulter), PE anti-HLA-DR, -CD4, -CD8, -CCR4, -CCR5, -CXCR4, -DC-SIGN, CD1a, -CD69, -CD25, -CD40 (BD Pharmingen), PE anti-CCR1, -CCR2, -CCR3 (R&D Systems), and FITC anti-CD1c (Miltenyi Biotec) mAbs. Matched isotype controls purchased from the same company were used as negative controls.

Cell sorting

PBMC were isolated from peripheral blood. The study protocol and informed consent forms were approved by the Dana-Farber Cancer Institute Institutional Review Board. Total Mo (tMo) and CD4+ T cells were isolated from PBMC by negative selection (Miltenyi Biotec) as described (35). The purity of the tMo fraction was >98%, as demonstrated by expression of Mo markers (CD14, HLA-DR, and CD33) and lack of expression of T cell (CD3), B cell (CD19), NK cell (CD56), neutrophil (CD16b and CD66b), and DC (CD1c) markers. The percentage of CD16+ Mo in tMo ranged between 5 and 15%. CD16+ and CD16– Mo were isolated from tMo by positive and negative selection, respectively, using CD16 beads (Miltenyi Biotec). Staining with anti-CD14 and -CD16 Abs showed >80% CD16+ cells in the CD16+ Mo fraction and >90% CD16– cells in the CD16– Mo fraction (86.1 ± 5.3% and 94 ± 3.3%, respectively, mean ± SD; n = 10). The total CD4+ T cell fraction consisted of >98% CD4+CD3+ cells and 5–10% cells expressing the activation markers HLA-DR, CD69, and/or CD25. Resting T cells were obtained by depleting the total T cell fraction of HLA-DR+CD69+CD25+ cells by FACS. CD16+ and CD16– Mo were isolated from PBMC of HIV-infected individuals by FACS sorting using a mixture of anti-CD14, and -CD16, -CD3, -CD19, -CD56, -CD16b, -CD66b, and -CD1c Abs.

HIV-1 infection

HIV 89.6-GFP is an HXBH10-based provirus expressing the R5X4 89.6 envelope and gfp in place of nef. HIV strains lacking GFP (e.g., YU2 and NDK) were used as negative controls for quantification of GFP expression. Virus stocks were prepared by transfection of 293 T cells with pHXBH10-89.6-GFP plasmids and reverse transcriptase (RT) activity was determined by [3H]thymidine incorporation (36). CD16+ and CD16– Mo were cocultured with autologous T cells (Mo:T cell ratio 1:2) in RPMI 1640 10% FBS medium in the absence of exogenous IL-2 for 3 days. Mo:T cocultures were incubated with HIV virus stock (40,000 [3H] cpm RT activity units/106 cells, corresponding to a multiplicity of infection of ~0.1/106 cells) for 3 h. Unbound virus was removed by washing and cells were cultured in RPMI 1640 10% FBS (2 x 106 cells/ml). In parallel, tMo were cocultured with T cells in the presence or absence of PHA (1 µg/ml) for 3 days and then exposed to HIV. Alternatively, T cells were pulsed with HIV for 3 h, washed, and cocultured with autologous CD16+ or CD16– Mo. Supernatants were recovered every 3–4 days and assessed for HIVp24 levels by ELISA (PerkinElmer). Mo:T cocultures were recovered at days 8–12 postinfection and stained with anti-CD3 and -CD14 or -CD33 Abs. Intracellular staining with anti-HIVp24 Ab (KC57; Beckman Coulter) was performed as described below. The percentage of GFP+CD3+CD33– T cells was determined by FACS. Cells were counted using Flow-Count fluorospheres (Beckman Coulter). In some experiments, Mo were cocultured with T cells in a Transwell system (0.1 µm pore size; Corning Costar).

Confocal microscopy

CD16+ Mo were cocultured with T cells for 3 days. Cocultures were harvested and the CD3+ fraction was isolated using CD3 immunobeads (Miltenyi Biotec). Mo:T conjugates were isolated from the CD3+ fraction by adherence to collagen-coated multichamber slides. Cells were stained with FITC CD14 mAb and 4',6-diamidino-2 phenylindole (Molecular Probes), and fixed using Cytofix/Cytoperm (BD Pharmingen). Conjugates were visualized by confocal microscopy.

BrdU incorporation

Mo subsets were cocultured with T cells in the presence or absence of PHA/IL-2 for 5 days. During the last 18 h, cells were incubated with BrdU (50 µM). Cocultures were harvested and stained with anti-CD33 and -CD3 Abs. Intracellular staining with anti-BrdU Ab was performed using the BrdU flow kit (BD Biosciences).

Cytokine Ab array

Two cytokine Ab arrays were used to detect expression of 120 cytokines/chemokines in Mo:T coculture supernatants (RayBiotech). The expression of each cytokine/chemokine was evaluated by measuring the OD of duplicate spots using Eagle Sight Software. The ratio between each cytokine spot and the positive control was calculated.

Chemokine detection

Mo were cocultured with T cells for 3 days and maintained in the presence of brefeldin A (10 µg/ml) for the last 18 h. Cocultures were harvested and stained with anti-CD33 and -CD3 Abs. Intracellular staining with anti-CCL2 (BD Biosciences) and -CCL24 Abs (PeproTech) was performed using Cytofix/Cytoperm. Intracellular expression of chemokines was analyzed in Mo/M{phi} (CD3–CD33+), Mo/M{phi}-T cell conjugates (CD3+CD33+), and T cells (CD3+CD33–) by FACS. CCL24, CCL2, CCL22, and CCL17 levels in Mo:T coculture supernatants were quantified by ELISA (R&D Systems).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD16+ Mo activate resting T cells for productive HIV infection

To determine whether CD16+ and CD16– Mo subsets differ in their ability to activate CD4+ T cells for productive HIV infection, we investigated HIV replication in T cells cocultured with autologous CD16+ or CD16– Mo. tMo were isolated from peripheral blood by negative selection, and CD16+ and CD16– Mo were isolated from tMo using CD16 beads (Fig. 1A). CD16+ and CD16– Mo exhibited similar morphology and viability in culture (data not shown) and lacked expression of DC-SIGN (35), a molecule involved in HIV capture and transinfection (31). Total T cells isolated by negative selection (35) (Fig. 1B, left panel) were cocultured with CD16+ and CD16– Mo in the absence of IL-2. In parallel, tMo were cocultured with T cells in the presence or absence of PHA. After 3 days, Mo:T cocultures were incubated with HIV. In initial experiments, several HIV viruses expressing GFP in place of the nef gene were tested for the ability to replicate in PHA/IL-2-stimulated PBMC. PBMC were exposed to CCR5 using (R5) NL4.3-BaL-GFP, CXCR4 using (X4) NL4.3-GFP, or dual tropic (R5X4) HXBH10-89.6-GFP (37). The R5X4 strain replicated to high levels, whereas the R5 and X4 strains replicated to intermediate and low levels, respectively (21,970 ± 2,170; 11,792 ± 1,714; and 4,537 ± 1,283 [3H] cpm RT activity units/ml, respectively, on day 9 postinfection, mean ± SD of duplicate wells). We also compared replication of NL4.3-BaL-GFP and HXBH10-89.6-GFP in T cells cocultured with Mo in the absence of IL-2. HXBH10-89.6-GFP replicated to higher levels in Mo:T cocultures than NL4.3-BaL-GFP, and also reached peak levels of HIV replication at earlier time points (12,487 ± 203 vs 2,245 ± 155 [3H] cpm RT activity units/ml on day 4 postinfection, mean ± SD of duplicate wells). Therefore, subsequent studies were performed with HXBH10-89.6-GFP, a virus that uses CXCR4 for entry into primary T cells and either CCR5 or CXCR4 for entry into Mo/M{phi} (37). Intracellular staining and FACS analysis demonstrated coexpression of GFP and HIVp24 (data not shown), allowing identification of HIV-infected cells as GFP+ cells. No GFP or HIVp24 expression was detected at the single-cell level in the absence of HIV. Significantly higher levels of HIV replication were observed in CD16+ compared with CD16– Mo:T cocultures (Fig. 1, C–F), with numerous GFP+ cells detected in CD16+ Mo:T cocultures as early as day 3 postinfection (Fig. 1C).


Figure 1
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  		FIGURE 1. CD16+ Mo/M{phi} render resting T cells permissive for productive HIV infection. A, CD16+ and CD16– Mo were analyzed by FACS for CD14 and CD16 expression. Results are representative of experiments performed with cells from 10 different donors. B, Total and resting CD4+ T cells isolated as described in Materials and Methods were analyzed by FACS for CD25, CD69, and HLA-DR expression. Results are representative of experiments performed with cells from three different donors. C–F, CD16+ and CD16– Mo were cocultured with autologous total T cells for 3 days. As controls, tMo were cocultured with total T cells in the presence or absence of PHA. Cocultures were incubated with HXBH10-89.6-GFP for 3 h and then maintained in RPMI 1640 10% FBS. C, Shown are fluorescent and phase contrast pictures at day 3 postinfection. D, Mo:T cocultures were stained with anti-CD33 and -CD3 mAbs, and the percentage of GFP+ T cell blasts gated as CD3+CD33– cells with high SSC/granularity characteristics was determined by FACS at day 9 postinfection. Results in C and D are representative of experiments performed with cells from eight different donors. E, The frequency of GFP+ T cell blasts was assessed by FACS in CD16+ and CD16– Mo:T cocultures, and (F) HIVp24 levels in supernatants were quantified by ELISA at days 8–12 postinfection. (Student’s t test, CD16+ vs CD16– Mo:T cocultures). G, CD16+ and CD16– Mo were cocultured with autologous resting T cells (total T cells depleted of CD25+CD69+HLA-DR+ cells) for 3 days and then pulsed with HXBH10-89.6-GFP. HIVp24 levels in supernatants were quantified by ELISA. Results (mean ± SD of duplicate wells) are representative of experiments performed with cells from three different donors. H, Total T cells were pulsed with HXBH10-89.6-GFP and then cocultured with autologous uninfected CD16+ and CD16– Mo. HIVp24 levels in supernatants were quantified by ELISA. Results are representative of experiments performed with cells from two different donors.

 
At the peak of HIV replication, the majority of GFP+ cells were identified as T cell blasts based on CD3 (T cell marker) expression, lack of CD14 or CD33 (myeloid markers) expression, and forward scatter (FSC)/size and side scatter (SSC)/granularity characteristics (FSChighSSChigh) (Fig. 2A). The frequency of GFP+ T cells and HIVp24 levels in CD16+ Mo:T cocultures were similar to that in tMo:T/PHA cocultures, but significantly higher compared with that in CD16– Mo:T cocultures (Fig. 1, D–F, and data not shown). Conjugates formed between Mo/M{phi} and T cells were identified by simultaneous expression of CD3 and CD33 (Fig. 2B). GFP expression was detected in Mo/M{phi}:T conjugates, but not in Mo/M{phi} (CD3–CD33+), from CD16+ Mo:T cocultures (Fig. 2B). HIV replication was >2-fold higher in tMo:T compared with CD16– Mo:T cocultures (Fig. 1D and data not shown) and was similar in T cells cocultured with tMo preincubated or not with anti-CD16 beads (data not shown), indicating that CD16 beads used during the isolation protocol did not affect HIV replication. At the peak of HIV replication, the total number of T cells and T cell blasts was slightly higher in CD16+ Mo:T compared with CD16– Mo:T cocultures (Fig. 2, C and D). Trypan blue staining and FACS analysis of SSC/FSC characteristics indicated no significant loss of cell viability in uninfected CD16+ or CD16– Mo:T cocultures (data not shown). In contrast, the frequency of Mo/M{phi} and conjugates decreased dramatically in HIV-infected CD16+ compared with CD16– Mo:T cocultures (Fig. 2B, upper left and right panels, and data not shown) as a consequence of HIV-induced syncytia formation and cell lysis.


Figure 2
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  		FIGURE 2. CD16+ Mo/M{phi} activate T cells for HIV replication. CD16+ and CD16– Mo were cocultured with autologous total CD4+ T cells for 3 days and then incubated with HXBH10-89.6-GFP for 3 h. A, Mo:T cocultures harvested at the peak of HIV replication (days 8–12 postinfection) were double stained with CD33 and CD3 Abs and analyzed by FACS. Gate R1 excluded dead cells. T cells were identified in R1 as CD3+CD33– cells (gate R2) and consisted of small T cells (R3) and large T cell blasts (R4). The frequency of GFP+ cells was analyzed in total T cells, small T cells, and T cell blasts. B, Mo/M{phi} were identified in R1 as CD3–CD33+ cells (gate R3) and Mo/M{phi}:T conjugates as CD3+CD33+ events (gate R4). The frequency of GFP+ cells was analyzed in Mo/M{phi} and Mo/M{phi}:T conjugates from CD16+ and CD16– Mo:T cocultures (upper and lower panels, respectively). Results in A and B are representative of experiments performed with cells from three different donors. C and D, Mo:T cocultures harvested at the peak of HIV replication were stained with CD33 and CD3 Abs. C, Total T cells (CD3+CD33–) and (D) T cell blasts (SSChighFSChigh) were gated as described above and counted by flow cytometry using fluorescent beads (mean ± SEM; n = 6) (*, p < 0.05, Student’s t test; CD16+ vs CD16– Mo:T cocultures).

 
To determine whether CD16+ Mo promote HIV replication in resting T cells, the total T cell fraction was depleted of cells expressing CD25, CD69, and HLA-DR (Fig. 1B), which are classical T cell activation markers (38). CD16+ and CD16– Mo were then cocultured with autologous total or resting T cells. The frequency of GFP+ T cells and HIVp24 levels was significantly higher when resting T cells were cocultured with CD16+ compared with CD16– Mo (Fig. 1G), similar to results obtained with total T cells.

The preceding results suggest that CD16+ Mo/M{phi} may promote HIV replication in T cells by inducing T cell activation and/or mediating HIV transinfection of T cells. To distinguish between these possibilities, total T cells were incubated with HXBH10-89.6-GFP and then cocultured with autologous CD16+ or CD16– Mo. HIV replication was detected when HIV-pulsed T cells were cocultured with CD16+ but not CD16– Mo (Fig. 1H). These findings imply that CD16+ Mo promote HIV replication in T cells by inducing T cell activation via soluble and/or membrane-bound molecules.

CD16+ Mo form conjugates with T cells and induce T cell activation

To determine whether CD16+ and CD16– Mo differ in their ability to activate T cells, the expression of T cell activation markers (CD25, CD69, CXCR3, and HLA-DR), HIV coreceptors (CCR5 and CXCR4), and M{phi}/DC markers (CD40, CD80, DC-SIGN, CD1a, and CD1c) was assessed in CD16+ and CD16– Mo:T cocultures before and after HIV infection. T cell (CD3) and myeloid (CD14 and CD33) markers were used to identify cells in Mo:T cocultures. Expression of CD14 is down-regulated during Mo differentiation into DC but not M{phi}, while CD33 is expressed on Mo, DC, and M{phi} (39). Before HIV exposure, CD16+ and CD16– Mo/M{phi} had a similar ability to form conjugates with T cells (Fig. 3A), with conjugate formation detected beginning at 2 h after coculture (data not shown). Conjugates typically consisted of one to two CD3+ T cells bound to a single CD14+ Mo/M{phi} (Fig. 3B). T cells cocultured with CD16+ and CD16– Mo expressed similar low levels of CCR5, high levels of CXCR4, moderate levels of CXCR3 and CD25, and low to undetectable levels of CD69 (Fig. 3C). HLA-DR expression was slightly higher on T cells cocultured with CD16+ compared with CD16– Mo (Fig. 3C). In the presence of T cells, CD16+ and CD16– Mo differentiated into M{phi} that lacked expression of the DC marker CD1a and expressed CD14, CD33, CD16, CCR5, CXCR4, CXCR3, HLA-DR, CD69, DC-SIGN, and CD40, with no significant differences between the two subsets (Fig. 3C and data not shown). We also investigated whether either Mo subset differentiated into DC after HIV exposure. CD16+ and CD16– Mo cocultured with T cells for 3 days and incubated in the presence or absence of HIV for 5 days exhibited a M{phi}-like phenotype, as indicated by the expression of CD14, DC-SIGN, and CD80 and lack of DC marker expression (CD1a and CD1c) (data not shown). Before HIV infection, CD16+ and CD16– Mo/M{phi}:T conjugates expressed similar levels of CCR5, CXCR4, HLA-DR, CD69, DC-SIGN, and CD40. However, CD16+ compared with CD16– Mo/M{phi}:T conjugates expressed significantly higher levels of CXCR3 and CD25 in all donors tested (Fig. 3C). T cell proliferation, measured by BrdU incorporation, was slightly higher in CD16+ compared with CD16– Mo:T cocultures maintained for 5 days in the absence of exogenous Ag (3.9 ± 1.7 vs 1.5 ± 0.5%; mean ± SD; n = 3; p < 0.05, Student’s t test) or in the presence of PHA (Fig. 3D). These results, together with those in Fig. 2, C and D, suggest that CD16+ Mo/M{phi} induce T cell activation and proliferation, particularly within Mo/M{phi}:T cell conjugates.


Figure 3
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  		FIGURE 3. CD16+ Mo/M{phi} form conjugates with T cells and promote T cell activation. CD16+ and CD16– Mo were cocultured with T cells for 3 days. A, FACS analysis of Mo:T cocultures distinguished three subsets: T cells (CD14–CD3+), Mo/M{phi} (CD14+CD3–), and Mo:T conjugates (CD14+CD3+). The percentage of cells in each quadrant is shown. Results are representative of experiments performed with cells from 10 different donors. B, CD16+ Mo:T conjugates were isolated as described in Materials and Methods, stained with anti-CD14 and -CD3 Abs, and visualized by confocal microscopy. C, The phenotype of T cells, Mo/M{phi}, and Mo/M{phi}:T conjugates (gated as indicated in Fig. 2, A and B) from CD16+ and CD16– Mo:T cocultures was analyzed by FACS (n = 4–10) before HIV infection. (**, p < 0.05, *, p < 0.1, sign-rank test; CD16+ vs CD16– Mo:T cocultures). D, CD16+ and CD16– Mo were cocultured with autologous T cells in the presence or absence of PHA for 5 days. During the last 18 h, cells were incubated with BrdU (50 µM). Cells were recovered and stained with anti-CD33 and -CD3 Abs. Incorporation of BrdU in T cells was quantified by intracellular staining with anti-BrdU Ab and FACS analysis. Results are representative of experiments performed with cells from two different donors.

 
Differential expression of cytokines/chemokines in CD16+ and CD16– Mo:T cocultures

To investigate mechanisms involved in CD16+ Mo-induced T cell activation, we used a cytokine/chemokine Ab array to screen for 120 soluble factors in Mo:T cocultures. Coculture of CD16+ Mo with total T cells in the absence of Ag for 3 days resulted in significantly higher expression (1.5-fold cutoff) of angiogenin, CCL24, CCL2, IL-1ra, CCL22, TGF-beta3, and CCL4 in culture supernatants compared with CD16– Mo:T cocultures (Fig. 4, A and B, and data not shown). Incubation of Mo:T cocultures with HIV induced expression of a large number of cytokines and chemokines including IL-10, CCL5, IL-13, CCL8, CCL17, GM-CSF, CCL1, IFN-{gamma}, IL-6, CXCL1–3, and CCL3 (Fig. 4, C and D, and data not shown). At the peak of HIV replication, higher levels of HIVp24 in CD16+ compared with CD16– Mo:T cocultures (Fig. 1) were associated with significantly higher expression of CCL24, CCL5, IL-13, CCL17, CCL22, GM-CSF, CCL1, and IFN-{gamma}, whereas the expression of IL-10, CCL2, CCL8, IL-6, CXCL1–3, IL-8, CCL3, and CCL4 was similarly high in CD16+ and CD16– Mo:T cocultures (Fig. 4, C and D, and data not shown).


Figure 4
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  		FIGURE 4. Expression of cytokines/chemokines in CD16+ and CD16– Mo:T cocultures. Supernatants from Mo:T cocultures were harvested (A and B) before HIV infection (day 3 of coculture) or (C and D) at the peak of HIV replication (days 5–9 postinfection) and then (A and C) blotted against a membrane spotted with anti-cytokine Abs (Ab array C1000 VI). B and D, The OD of each spot was determined, and the ratio between the OD of cytokine and positive control spots was calculated. Results (mean ± SD of duplicate spots) are representative of experiments performed using cells from three different donors. E and F, CCL24, CCL2, CCL22, and CCL17 levels in Mo:T cell supernatants (E) before HIV infection (n = 4) and (F) at the peak of HIV replication (n = 5) were quantified by ELISA (Student’s t test; CD16+ vs CD16– Mo:T cocultures).

 
Among chemokines abundantly expressed before HIV infection, CCL24 and CCL2 were preferentially produced in CD16+ Mo:T cocultures (Fig. 4, A and B). Quantification by ELISA confirmed significantly higher levels of CCL24 and CCL2 in CD16+ compared with CD16– Mo:T cocultures (Fig. 4E). Differential expression of CCL24 and CCL2 was also observed when these Mo subsets were cocultured with resting T cells (data not shown). Levels of CCL24 and CCL2 in Mo:T cocultures dramatically increased upon HIV exposure (Fig. 4, E and F). At the peak of HIV replication, higher levels of CCL24 expression were detected in CD16+ compared with CD16– Mo:T cocultures, whereas the highest CCL2 levels were observed in CD16– Mo:T cocultures (Fig. 4F). Two other chemokines, CCL22 and CCL17, known to be produced constitutively by DC (40, 41), were preferentially expressed in CD16+ Mo:T cocultures (Fig. 4, A and D). Levels of CCL22 were higher in CD16+ compared with CD16– Mo:T cocultures before HIV exposure, whereas CCL17 levels were close to the limit of detection (Fig. 4E). HIV dramatically up-regulated CCL22 and CCL17 expression, and levels were significantly higher in CD16+ compared with CD16– Mo:T cocultures (Fig. 4F). Levels of IL-2 in Mo:T cocultures were undetectable by ELISA before or after HIV exposure (data not shown). Taken together, these results demonstrate preferential expression of CCL24, CCL2, CCL22, CCL17, CCL4, CCL5, and CCL1 in CD16+ Mo:T cocultures before and/or after HIV infection, and identify CD16+ Mo/M{phi} as a major source of CCL24 and CCL2.

CCL24 and CCL2 are constitutively produced by CD16+ Mo/M{phi}

Intracellular staining with anti-chemokine Abs identified CCL24- and CCL2-producing cells in CD16+ Mo:T cocultures as Mo/M{phi} and Mo/M{phi}:T conjugates, but not T cells (Fig. 5, A and B). To investigate whether production of these chemokines requires cell-to-cell contact, we used Transwell filters to separate CD16+ Mo/M{phi} from T cells. Both CCL24 and CCL2 were produced in the Transwell system. CCL2 production increased significantly when CD16+ Mo/M{phi} were cultured in direct contact with T cells (143 ± 24 vs 466 ± 46 pg/ml; mean ± SD; n = 3; p < 0.05), whereas CCL24 production in CD16+ Mo:T cocultures was similar whether Mo/M{phi} and T cells were cultured in direct contact or physically separated (data not shown). To investigate whether CD16+ Mo/M{phi} constitutively produce these chemokines, Mo subsets were isolated from peripheral blood of seven uninfected individuals and three patients with AIDS (i.v. drug users with plasma viral loads <10,000 HIV RNA copies/ml and CD4 counts <200 CD4+ T cells/µl) and cultured alone for 3 days. CCL24 and CCL2 were detected almost exclusively in supernatants of CD16+ Mo/M{phi}, with higher chemokine levels produced by CD16+ Mo/M{phi} from HIV-infected compared with -uninfected individuals (Fig. 5, C and D). CCL24 and CCL2 were not detected in supernatants of CD4+ T cells isolated from HIV-infected and -uninfected individuals after 3 days of culture with PHA/IL-2 (data not shown). Thus, CD16+ Mo/M{phi} represent an important source of CCL24 and CCL2 in HIV-infected and -uninfected individuals.


Figure 5
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  		FIGURE 5. CCL2 and CCL24 are constitutively produced by CD16+ Mo/M{phi}. A and B, Mo:T cocultures were harvested before HIV infection, and Mo/M{phi} (CD33+CD3–), conjugates (CD33+CD3+), and T cells (CD33–CD3+) were analyzed by FACS for intracellular expression of chemokines. Results are representative of experiments performed with cells from two different donors. CD16+ and CD16– Mo were isolated from PBMC of (C) HIV-uninfected and (D) -infected individuals and cultured for 3 days. Chemokines levels in supernatants were quantified by ELISA. (Student’s t test; CD16+ vs CD16– Mo/M{phi}).

 
CCL24 and CCL2 deliver costimulatory signals to T cells

To investigate whether the low levels of HIV replication in CD16– Mo:T cocultures are a consequence of low CCL24 and CCL2 expression by CD16– Mo/M{phi} (Figs. 4, A, B, and E, and 5), T cells were cocultured with CD16– Mo in the presence or absence of recombinant chemokines. Addition of these chemokines increased HIV replication to levels similar to those detected in CD16+ Mo:T cocultures (Fig. 6A). Increased HIV replication was observed with chemokine concentrations ranging from 10 to 100 ng/ml, regardless of whether CD16– Mo were cocultured with total or resting T cells, and was also observed when chemokines were added after HIV exposure (data not shown). Thus, CCL24 and CCL2 promote HIV replication in CD16– Mo:T cocultures and likely contribute to high levels of HIV replication in CD16+ Mo:T cocultures.


Figure 6
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  		FIGURE 6. CCL2 and CCL24 stimulate HIV replication in T cells cocultured with CD16– Mo or stimulated via CD3/CD28. A, CD16+ and CD16– Mo were cocultured with T cells for 3 days and then pulsed with HXBH10-89.6-GFP. CD16– Mo:T cocultures were maintained in the presence or absence of CCL24 and CCL2 (100 ng/ml). HIVp24 levels in supernatants were quantified by ELISA. Results (mean ± SD of duplicate wells) are representative of experiments performed with cells from three different donors. B, T cells were cocultured with CD16+ or CD16– Mo in direct contact or separated by a Transwell filter for 3 days and then pulsed with HXBH10-89.6-GFP. HIVp24 levels in supernatants were quantified by ELISA at day 9 postinfection (mean ± SD; n = 3). (Student’s t test; CD16+ Mo:T cocultures, cell-to-cell contact vs Transwell). C and D, T cells were stimulated with anti-CD3 (1 µg/ml) and/or -CD28 (1 µg/ml) Abs for 3 days in the presence or absence of CCL24 (20 ng/ml) and CCL2 (10 ng/ml). Cells were then pulsed with HXBH10-89.6-GFP, washed, and maintained in the presence of the same stimuli. C, HIVp24 levels in supernatants were quantified by ELISA (mean ± SD of duplicate wells). D, Shown are fluorescent and phase contrast pictures at day 6 postinfection. Results are representative of experiments performed with cells from two different donors.

 
To investigate whether HIV replication promoted by endogenously produced CCL24 and CCL2 also requires CD16+ Mo/M{phi}-T cell contact, T cells were cocultured with CD16+ Mo/M{phi} in direct contact or separated by a Transwell filter. Separation of T cells and CD16+ Mo/M{phi} resulted in a dramatic decrease in HIV replication (Fig. 6B). Similarly, productive HIV infection was not detectable in T cells maintained in the presence of CD16+ Mo:T coculture supernatant or stimulated with CCL24 or CCL2 in the absence of IL-2 (data not shown). These results demonstrate that chemokines alone are not sufficient to promote HIV replication in T cells and that cell-to-cell contact between Mo/M{phi} and T cells is required, possibly for TCR engagement (28, 29). To investigate this hypothesis, T cells were stimulated with CD3/CD28 Abs (42) in the presence or absence of CCL24 or CCL2 for 3 days, incubated with HXBH10-89.6-GFP, and maintained in the presence of the same stimuli. Low levels of GFP expression in cells and HIVp24 levels in supernatants were observed in T cells stimulated with CD3/CD28 Abs, but not in those stimulated with CD3 Ab alone (Fig. 6, C and D). Addition of CCL24 or CCL2 significantly up-regulated HIV replication in CD3/CD28-stimulated T cells starting at day 3 postinfection (Fig. 6, C and D). Thus, CCL24 and CCL2 provide costimulatory signals to T cells that, along with TCR engagement, dramatically increase productive HIV infection.

CCR3 and CCR4 are preferentially expressed on HIV-infected T cells

We then assessed the expression of receptors for CCL24 (CCR3) (43) and CCL2 (CCR2 and CCR4) (44) on T cells cocultured with CD16+ and CD16– Mo before and after HIV infection. T cells cocultured with CD16+ or CD16– Mo for 3 days expressed moderate levels of CCR3, undetectable levels of CCR2, and high levels of CCR4 (Fig. 7A). However, at the peak of HIV replication expression of CCR3 and CCR4, but not CCR2, was higher on GFP+ T cells productively infected with HIV compared with GFP– T cells (Fig. 7B). We also compared HIV replication in FACS-sorted CCR4+ and CCR4– T cells cocultured with total Mo. HIV replication was 1.8-, 1.8-, and 1.6-fold greater at days 5, 8, and 11 postinfection, respectively, when cocultures were performed with CCR4+ compared with CCR4– T cells (data not shown). Taken together, these results identify CCR3+ and CCR4+ T cells as preferential sites for HIV replication and suggest that CCR3 and CCR4 ligands expressed in CD16+ Mo:T cocultures (i.e., CCL24, CCL2, CCL22, CCL17, or CCL5) (43, 44, 45) may act directly on T cells and promote their activation for productive HIV infection.


Figure 7
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  		FIGURE 7. CCR3 and CCR4 are preferentially expressed on HIV-infected T cells. A, CD16+ and CD16– Mo:T cocultures were harvested before HIV replication and stained with anti-CD3 and -CD14, and -CCR2, -CCR3, or -CCR4 Abs. CD3+CD14– T cells were analyzed by FACS for expression of CCR2–4. Results are representative of experiments performed with cells from three different donors. B, CD16+ Mo:T cocultures were harvested at the peak of HIV replication (day 9), and expression of CCR2–4 was analyzed on CD3+ T cells expressing or not GFP. Results are representative of experiments performed with cells from two different donors.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we demonstrate that CD16+ Mo differentiate into macrophages that activate resting CD4+ T cells for productive HIV infection by producing CCR3 and CCR4 ligands. Furthermore, we provide evidence that engagement of CCR3 and CCR4 on T cells by their chemokine ligands provides costimulatory signals that, along with TCR stimulation, enhance T cell susceptibility to HIV replication. These findings provide evidence that expansion of CD16+ Mo during HIV infection (14, 15, 16, 17) and their recruitment into tissues such as lymph nodes, brain, lung, and intestine (3, 9, 10, 11, 12, 13) contribute to HIV pathogenesis by promoting chronic immune activation and establishment of viral reservoirs in resting T cells.

Resting CD4+ T cells are resistant to productive HIV infection in vitro due to a postentry restriction (24, 25). In this study, we demonstrate that coculture with CD16+ Mo in the absence of exogenous Ag renders resting T cells highly permissive for productive HIV infection, while coculture with CD16– Mo results in low levels of HIV replication. Before HIV infection, CD16+ and CD16– Mo differentiated into M{phi} expressing CD14, CD40, CD80, and DC-SIGN, but not the DC markers CD1a and CD1c, and formed conjugates with T cells at a similar frequency. However, CD16+ Mo/M{phi} had a superior capacity to induce T cell proliferation and expression of activation markers such as HLA-DR, CXCR3, and CD25 (38, 46) on T cells and Mo/M{phi}-T cell conjugates. Thus, similar to DC, CD16+ Mo/M{phi} form conjugates with (30, 47) and activate T cells in an Ag-independent manner (41). Interactions between T cells and CD16+ vs CD16– Mo/M{phi} may involve distinct as yet unidentified molecules that influence T cell activation and/or cell-to-cell virus transmission. Neither CD16+ nor CD16– Mo/M{phi} showed evidence of productive infection. However, cell-to-cell contact with T cells was required for efficient HIV replication in CD16+ Mo:T cocultures. Furthermore, HIV replication was detected in CD16+ Mo/M{phi}:T conjugates. Similar conjugates harboring HIV DNA have been detected in peripheral blood from HIV-infected patients (35). Previous studies of HIV-pulsed Mo cocultured with activated T cells suggest that a low level of productive infection is initiated via a CD4-dependent pathway in Mo/M{phi}, with progeny virions transferred from Mo/M{phi} to T cells within conjugates (35). Thus, we cannot exclude the possibility that more efficient cell-to-cell HIV transmission may be an additional mechanism contributing to the superior capacity of CD16+ Mo/M{phi} to promote virus replication in T cells. However, uninfected CD16+ Mo/M{phi} promoted HIV replication in HIV-pulsed T cells, and induced expression of activation markers on T cells and T cell proliferation before HIV infection. Thus, the superior capacity of CD16+ Mo/M{phi} to promote productive HIV infection in resting T cells is likely to be explained by a greater ability to deliver activator signals to T cells.

HIV replication in CD16+ Mo:T cocultures was associated with expression of high levels of specific chemokines (i.e., CCL24, CCL5, CCL2, CCL8, CCL17, CL22, CCL1, CXCL1–3, CXCL8, CCL3, and CCL4) and cytokines (i.e., IL-10, IL-13, GM-CSF, IFN-{gamma}, and IL-6). Among these chemokines, CCL24 and CCL2 were selectively expressed by CD16+ Mo/M{phi} and Mo/M{phi}:T conjugates before exposure of Mo:T cocultures to HIV. In addition, CD16+ but not CD16– Mo/M{phi} isolated from HIV-infected and -uninfected individuals constitutively produced CCL24 and CCL2. Thus, CD16+ Mo/M{phi}, similar to DC and M{phi} (32, 34), produce soluble factors that may promote HIV replication in T cells. Mo-T cell contact up-regulated CCL2, but not CCL24, production by CD16+ Mo/M{phi}. Previous studies demonstrated that Mo/M{phi} produce CCL2 upon CD40 engagement (48, 49), while CCL24 production is triggered by ligation of TLR2/TLR4 (48, 49). However, the signaling pathways that lead to CCL24 and CCL2 production by CD16+ Mo/M{phi} remain to be determined. HIV exposure dramatically increased CCL24 and CCL2 levels in Mo:T cocultures. Furthermore, CD16+ Mo isolated from HIV-infected compared with -uninfected individuals produced higher levels of CCL2 and CCL24. These findings are consistent with previous reports demonstrating induction of CCL2 following HIV infection in M{phi} (50, 51) and indicate a higher state of Mo activation during HIV infection. At the peak of HIV replication, differences in HIV p24 levels in CD16+ and CD16– Mo:T cocultures correlated positively with CCL24 concentrations in supernatants. In contrast, high levels of CCL2 were detected in CD16– Mo:T cocultures. One possible explanation for the discrepancy between high levels of CCL2 production and low HIV replication in CD16– Mo:T cocultures is that chemokine production precedes T cell activation. In particular, T cell activation for susceptibility to HIV infection may occur earlier in CD16+ compared with CD16– Mo:T cocultures because CCL24 and CCL2 are constitutively produced by CD16+ Mo/M{phi}.

In addition to CCL24 and CCL2, two other chemokines, CCL22 and CCL17, were preferentially expressed in CD16+ Mo:T cocultures. CCL22 was produced before HIV infection, while CCL22 and CCL17 expression increased dramatically upon HIV infection. CCL22 and CCL17 are markers for Langerhans cells and Mo-derived DC (40, 41). GM-CSF, a cytokine that drives Mo differentiation into DC (8), is expressed in CD16+ Mo:T cocultures at the peak of HIV replication, raising the possibility that CD16+ Mo/M{phi} in Mo:T cocultures may acquire features typical of DC upon exposure to HIV (52, 53). However, we found that CD16+ Mo acquire M{phi} but not DC characteristics in culture. The effect of GM-CSF on Mo differentiation may be overcome by the presence of IL-6, a cytokine that switches the differentiation of Mo from DC to M{phi} (54). Chemokines not only orchestrate T cell chemotaxis (45) but also regulate DC immunogenic potential by acting at multiple levels (40, 41, 42, 55, 56). Accordingly, CCL24, CCL2, CCL22, and CCL17, and possibly other chemokines produced by M{phi} or DC derived from CD16+ Mo, may prime T cell responses by triggering chemotaxis, polarization, formation of an immunological synapse (IS), and/or activation of intracellular pathways that potentiate signals delivered via TCR and costimulatory molecules.

CD16+ Mo are a major source of CCR3 and CCR4 ligands, whereas CD16– Mo produce low to undetectable levels of these chemokines. Addition of CCL24 and CCL2 dramatically increased HIV replication in T cells cocultured with CD16– Mo, suggesting that these chemokines stimulate HIV replication in CD16+ Mo:T cocultures. CCL2 has been shown to enhance HIV replication in T cells (57, 58). In HIV-infected patients, plasma levels of CCL2 correlate with viral loads (59) and high levels of CCL2 in brain or cerebrospinal fluid are associated with HIV-associated dementia (60). Moreover, a CCR2 polymorphism and a mutant CCL2 promoter allele linked to increased CCL2 levels are associated with accelerated progression to AIDS and increased risk of HIV-associated dementia (60, 61). Whereas CCL2 plays a well-recognized role in HIV pathogenesis, our report is the first to demonstrate a direct link between HIV replication and CCL24, a chemokine known to trigger T cell chemotactic migration and to inhibit myelopoiesis (62). Our study also provides evidence for possible roles of CCL22 (63) and CCL17 in HIV pathogenesis.

Consistent with the absolute requirement for TCR engagement in T cell activation (28, 29), signals delivered by CCL24 and CCL2 were not sufficient to promote HIV infection in T cells cultured alone, but dramatically enhanced HIV replication in T cells cocultured with CD16– Mo or stimulated via CD3/CD28. Recent studies support the idea that chemokines, in addition to their role in regulating leukocyte trafficking (45), act as immunotransmitters within the IS between DC and T cells (64). CCL3, CCL4, CCL5, CCL2, and CXCL12 provide costimulatory signals to T cells for enhanced IL-2 production and T cell proliferation (55, 65), while CCL5 and CCL22 strengthen the IS (66). In addition, CCL5 and CXCL12 produced by Mo-derived DC trigger accumulation of their counterreceptors, CCR5 and CXCR4, respectively, on the T cell side of the IS and deliver costimulatory signals to T cells that lower the threshold for TCR-mediated T cell activation (41, 42). Consistent with these findings, we provide evidence suggesting that CCR3 and CCR4 ligands produced by CD16+ Mo/M{phi} deliver costimulatory signals to T cells that increase their susceptibility to productive HIV infection.

The preferential expression of CCR3 and CCR4 on HIV-infected T cells at the peak of HIV replication suggests that ligands for CCR3 (62) (i.e., CCL24 and CCL5) and CCR4 (44, 67) (i.e., CCL2, CCL22, CCL17, and CCL5) act directly on T cells to enhance productive HIV infection. CCR3 and CCR4 are markers for T cells producing Th2 cytokines (43, 44, 45, 67). The expression of CCR3 and CCR4 ligands (44, 45), together with Th2 cytokines, such as IL-10, IL-13, and IL-6 (68), in CD16+ Mo:T cocultures, indicates that CD16+ Mo drive polarization of T cells toward a Th2 profile, which may be favorable for HIV replication (69, 70, 71). The preferential expression of CCR3 and CCR4 on HIV-infected T cells in CD16+ Mo:T cocultures suggests that T cells expressing these receptors may be preferential targets for HIV replication. CD16+ Mo/M{phi}-induced T cell activation may also be mediated by engagement of CCR1, CCR5, and/or CCR8 (44, 67) via CCL1, CCL4, CCL5, CCL17, and/or CCL22, as these chemokines were also detected in cell culture supernatants (Fig. 4, A–D, and data not shown). Previous reports demonstrated that CCL5 enhances T cell susceptibility to infection with X4 HIV strains, while inhibiting replication of R5 HIV strains (32, 57, 72). Thus, high levels of CCL5 in CD16+ Mo:T cocultures may inhibit infection by R5 HIV strains (73). Consistent with this possibility, levels of HIV replication were low when CD16+ Mo:T cocultures were exposed to an R5 HIV strain (BaL). Whether higher levels of replication of an R5X4 compared with an R5 strain were due to the presence of CCR5-binding chemokines or higher expression of CXCR4 compared with CCR5 on T cells in CD16+ Mo:T cocultures (Fig. 3C, top panel) remains an open question that merits further investigation. Given the well-documented role of nef in inducing T cell activation (33, 34), it will also be important to perform future studies using HIV strains expressing nef.

Our data support a model in which CD16+ Mo are recruited into peripheral tissues, differentiate into M{phi}, and produce CCL24 and CCL2, which trigger chemotactic migration of CCR3+ and CCR4+ T cells (Fig. 8). Cell-to-cell contact between CD16+ Mo/M{phi} and T cells results in T cell stimulation via TCR, costimulatory molecules, and/or adhesion molecules, and further production of CCR3 and CCR4 ligands. Abundant expression of these chemokines in the CD16+ Mo/M{phi}-T cell microenvironment contributes to a positive feedback loop of T cell activation and increased susceptibility to productive HIV infection. Our findings provide evidence for the contribution of CD16+ Mo to chronic immune activation and HIV replication during the natural course of infection and also suggest that T cells expressing CCR3 and CCR4 may be preferential sites for productive HIV infection in vivo. Thus, new therapies aimed at decreasing the frequency of CD16+ Mo in peripheral blood and/or blocking T cell activation via CCR3 and CCR4 ligands may be beneficial for HIV-infected patients.


Figure 8
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  		FIGURE 8. Model of CD16+ Mo/M{phi}-induced T cell activation for productive HIV replication. Left, CCL24 and CCL2 produced by CD16+ Mo/M{phi} trigger chemotactic migration of CCR3+ and CCR4+ T cells. Right, Contact between CD16+ Mo/M{phi} and T cells creates a microenvironment rich in CCR3 and CCR4 ligands including CCL2, CCL5, CCL17, CCL22, and CCL24. Engagement of CCR3 and CCR4, together with signals delivered by CD16+ Mo/M{phi} via TCR and other immune synapse molecules, activate resting T cells for susceptibility to HIV infection.

 

   Acknowledgments
 
We thank R. P. Sekaly, A. Mehle, E. Thomas, and H. Wilson for critical reading of the manuscript and valuable discussions, H. Gottlinger for the gift of HXBH10-89.6-GFP, M. Ocana and M. Chafel for technical assistance with confocal microscopy analysis, and Sandra Lee for assistance with statistical analysis.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health DA016549. Core facilities were supported by the Harvard Medical School Center for AIDS Research and Dana-Farber Cancer Institute/Harvard Center for Cancer Research grants and the Harvard Center for Neurodegeneration and Repair. Back

2 Address correspondence and reprint requests to Dr. Dana Gabuzda, Dana-Farber Cancer Institute, 44 Binney Street, JFB816, Boston, MA 02115. E-mail address: dana_gabuzda{at}dfci.harvard.edu Back

3 Abbreviations used in this paper: Mo, monocyte; DC, dendritic cell; M{phi}, macrophage; tMo, total Mo; RT, reverse transcriptase; FSC, forward scatter; SSC, side scatter; IS, immune synapse. Back

Received for publication November 16, 2005. Accepted for publication February 22, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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