To investigate whether different CD4⁺ T cell subsets differ in their susceptibility to DC-mediated HIV-1 transmission, we isolated by live sorting highly purified populations of CD45RA⁺ CD45RO^- naïve T cells (T_N) and CD45RA^- CD45RO⁺ memory T cells from pure CD4⁺ T cells. Based on the expression of CCR7, a homing receptor for secondary lymphoid tissue, the memory pool was further divided in CCR7⁺ central memory T cells (T_CM) and CCR7^- effector memory T cells (T_EM) 23. We subsequently incubated DC with the R5 virus JR-CSF isolate or the X4 virus LAI isolate for 2 hr, followed by washing steps to remove unbound virus. After addition of the respective T cell subsets, we determined the transmission efficiency by measuring the accumulation of HIV-1 capsid protein p24 (CA-p24) in T cells by FACS. To prevent subsequent rounds of HIV-1 replication after transmission in this single-cycle transmission assay, we added an inhibitor of the viral protease (saquinavir, 2526).

In a control experiment without HIV-1, no CA-p24 positive CD3⁺ T cells were scored (Fig. 1A). Addition of R5 HIV-1 resulted in high percentages of CA-p24⁺ T_EM cells, and hardly any CA-p24⁺ T_N cells (2.9 and 0.1 %, respectively). The transmission to T_CM cells was of intermediate efficiency (1.9%). With X4 HIV-1, the pattern was reversed: X4 HIV-1 was preferentially transmitted to T_N cells (4%), then to T_CM cells (2.2%), and the transmission to T_EM cells was least efficient (1.4%) (Fig. 1A). Overall, X4 transmission was more efficient than R5 transmission, and could take place to all subsets. For both viruses, the percentage CA-p24⁺ T cells reached a maximum value 2 days post transmission, and these data are quantified in Fig. 1B. This experiment demonstrates that there is not one exclusive T cell subset that is the preferred target of DC-mediated HIV-1 transmission, but that the efficiency depends on the tropism of the transmitted virus.

The different transmission patterns for R5 and X4 HIV-1 prompted us to investigate the co-receptor expression on each T cell subset (Fig. 2A). We found that the level of co-receptor expression for both CCR5 and CXCR4 correlates with the transmission efficiencies depicted in Fig. 1B: CCR5 expression is most pronounced on T_EM cells, and is undetectable on T_N cells; CXCR4 is detectable on all subsets, but its expression declines from T_N cells via T_CM to T_EM cells.

To investigate the role of co-receptor expression in DC-mediated HIV-1 transmission, we added the well-described inhibitors RANTES and AMD3100 in the single-cycle transmission assay. These compounds inhibit HIV-1 infection of T cells by blocking the co-receptors CCR5 and CXCR4, respectively 2728. Transmission of HIV-1 was completely blocked through the addition of these compounds (Fig. 2B, grey bars). We furthermore could block transmission completely with inhibitor T1249 (Fig. 2B, black bars). This peptide prevents fusion of viral and cellular membranes 29. Our results thus demonstrate that DC-mediated HIV-1 transmission requires 'regular' infection through CD4 and a co-receptor.

In addition to quantification of the transmission efficiency in a single-cycle transmission assay (Fig. 1 and 2), we followed viral replication after transmission (Fig. 3). In this spreading infection assay, we did not add saquinavir to allow cell-cell spread of newly produced virus. Replication of R5 and X4 HIV-1 in T_N, T_CM and T_EM cells following DC-mediated transmission reflects the results of the single-cycle transmission assay: R5 HIV-1 preferentially replicates in memory T cells, whereas X4 HIV-1 prefers T_N cells over the memory subsets (Fig. 3A and 3B).

Since this spreading infection assay involves two different steps, i.e. transmission and subsequent replication, we also studied R5 and X4 HIV-1 replication in T_N, T_CM and T_EM cells in a DC-independent system. Therefore, cellular proliferation was induced by cross linking of CD3 and CD28 on the T cells with antibodies (Fig. 3C and 3D). As expected, the susceptibility of all T cell subsets to R5 HIV-1 replication was low after CD3/CD28 stimulation. This phenomenon was previously described for CD4⁺ T cells in general, and is the consequence of CCR5 down regulation and production of natural CCR5 ligands that compete for co-receptor binding 3031. But despite this low replication capacity, the pattern of R5 replication was comparable to the replication after DC-mediated transmission of R5 HIV-1: replication was lower in T_N cells. Surprisingly, X4 replication in T_N cells was significantly delayed in comparison to T_CM and T_EM cells, which does not reflect the enhanced transmission and replication in T_N cells in the transmission experiments (Fig. 1 and 3B).

This discrepancy prompted us to compare HIV-1 replication in T cells stimulated by either DC or α-CD3/CD28 antibodies, without any complicating factors like transmission steps. We therefore stimulated all T cell subsets with DC, or alternatively, with α-CD3/CD28 antibodies and harvested the T cells after 4 days of proliferation. The cells were subsequently infected with X4 HIV-1. DC-stimulated T_N cells were more susceptible to X4 HIV-1 replication than the memory subsets (Fig. 4A), which reflects the replication after transmission (Fig. 3B). The reverse was observed with α-CD3/CD28 stimulated T cells (Fig. 4A), which is in concordance with the results of Fig. 3D in which the cells were infected immediately after stimulation. This indicates that the enhanced replication of X4 HIV-1 in T_N cells following DC-mediated transmission, is due to a higher HIV-1 susceptibility. It further demonstrates that crosslinking of CD3 and CD28 by antibodies is not comparable to DC-T cell stimulation, although this crosslinking is considered to mimic DC encounter. The difference between both stimulation methods is further manifested by the proliferative capacity of the T cells, as determined by ³H-thymidine incorporation (Fig 4B). The proliferation pattern of the different T cell subsets after DC or α-CD3/CD28 stimulation is clearly not the same.

The T_EM cell subset can be further divided into effector Th1 and Th2 cells 4. We generated in vitro polarized populations of pure Th1 and Th2 cells, or an unbiased population containing both types (Th0 cells), by culturing purified T_N cells with or without IL-12 or IL-4, as previously described 32. We next investigated whether HIV-1 is differently transmitted to these subsets of effector Th1, Th2 or Th0 cells. In addition, we tested different mature DC subsets. Depending on the type of pathogen and tissue factors, immature DC develop into mature effector DC that are specialized to stimulate naïve T cells to develop into IFNγ-producing Th1 cells or IL-4-producing Th2 cells, designated DC1 and DC2 respectively 33. DC0 induce an unpolarized response (Th0). DC0, DC1 and DC2 were generated by culturing immature DC with maturation factors (MF, IL-1β and TNFα) only (DC0), or MF with either IFNγ (DC1) or prostaglandin E₂ (DC2) 34.

The intracellular cytokine profiles of the effector Th cell populations were analyzed by FACS (Fig 5A). The Th1 population consists primarily of IFNγ producers, whereas the Th2 population contains mostly IL-4 producers. The unpolarized Th0 population is composed of both cell types. All T cell subsets expressed similar levels of CCR5 and CXCR4, and proliferated to a comparable extent, as determined by ³H incorporation (results not shown).

DC0, DC1 and DC2 were subsequently incubated with R5 and X4 HIV-1, followed by washing and addition of Th0, Th1 and Th2 cells. Two days later, the transmission efficiency was determined in the single-cycle transmission assay (Fig. 5B). Consistent with Fig. 1B, R5 virus was a bit more efficiently transmitted to these polarized T_EM cells than X4 HIV-1. More importantly, we found no significant differences in HIV-1 transmission efficiency to Th0, Th1 or Th2 cells within one DC subset, i.e. a particular DC subset transmits HIV-1 with equal efficiency to Th0, Th1 or Th2 cells. We also did not find a preference of HIV-1 transmission by a DC subset and its corresponding Th type: DC1 was the most efficient HIV-1 transmitter in all cases. The latter was previously demonstrated by us, using unpolarized peripheral blood leukocytes (PBL) and T cell lines 35. We now show that this also applies to polarized Th subsets.

Peripheral blood mononuclear cells (PBMC) were isolated by density centrifugation on Lymphoprep (Nycomed, Torshov, Norway). Subsequently, PBMC were layered on a Percoll gradient (Pharmacia, Uppsala, Sweden) with three density layers (1.076, 1.059, and 1.045 g/ml). The light fraction with predominantly monocytes was collected, washed, and seeded in 24-well culture plates (Costar, Cambridge, MA, USA) at a density of 5 × 10⁵ cells per well. After 60 min at 37°C, non-adherent cells were removed, and adherent cells were cultured to obtain immature DC in Iscove's modified Dulbecco's medium (IMDM; Life Technologies Ltd., Paisley, United Kingdom) with gentamicin (86 μg/ml; Duchefa, Haarlem, The Netherlands) and 10% fetal calf serum (HyClone, Logan, UT, USA), supplemented with GM-CSF (500 U/ml; Schering-Plough, Uden, The Netherlands) and IL-4 (250 U/ml; Strathmann Biotec AG, Hannover, Germany). At day 3, the culture medium with supplements was refreshed. At day 6, maturation was induced by culturing the DC with maturation factors only (MF; IL-1β (10 ng/ml) and TNFα(50 ng/ml); Strathmann Biotec AG), or MF with either IFNγ (1000 U/ml; Strathmann Biotec AG), or prostaglandin E₂ (10^-6 M; Sigma-Aldrich, St. Louis, MO), see results for more details 34. After two days, mature CD14^- CD1b⁺ CD83⁺ DC were obtained. All subsequent tests were performed after harvesting and extensive washing of the cells to remove all factors. Mature DC were analysed for the expression of cell surface molecules on a FACScan (BD Biosciences, San Jose, CA, USA). Mouse anti-human mAbs were used against the following molecules: CD14 (BD Biosciences), CD1b (Diaclone, Besançon, France), CD83 (Immunotech, Marseille, France) and ICAM-1 (CD54) (Pelicluster, Sanquin, Amsterdam, The Netherlands). All mAb incubations were followed by incubation with FITC-conjugated goat F(ab')₂ anti-mouse IgG and IgM (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).

Naïve and memory T cells were live sorted from pure CD4⁺ T cells on a FACS ARIA (BD Biosciences). The following mouse-anti-human antibodies were used: CD45RA-FITC (Coulter, Hialeah, FL, USA), CD45RO-APC (BD Biosciences), CD4-PE-Cy7 (BD Biosciences). Rat-anti-human CCR7 (BD Biosciences) incubation was followed by biotin-rabbit-anti-rat (Zymed Laboratories Inc., San Francisco, CA, USA) and streptavidin-PerCp-Cy5.5 (BD Biosciences) incubation. CD4⁺ CD45RA⁺ CD45R0^- cells were considered naïve T cells (T_N). CD4⁺ CD45RA^- CD45R0⁺ cells (the memory population) was separated into central memory (T_CM) (CCR7⁺) and effector memory (T_EM) (CCR7^-) cells, according to the classification described by Sallusto et al 2. Polarized Th1 and Th2 cells, and an unpolarized population containing both types (Th0 cells) were generated from purified T_N cells as previously described 32. In short, T_N cells (10⁵/200 μl) were stimulated with immobilized α-CD3 (CLB-T3/3; 1 μg/ml) and α-CD28 (CLB-CD28/1; 2 μg/ml) (both from Sanquin, Amsterdam, The Netherlands) and cultured for 10 days in the absence (Th0) or presence of IL-12 (100 U/ml; a gift from Dr. M. K. Gately, Hoffma-La Roche) or IL-4 (1000 U/ml) for Th1 and Th2 cells respectively. To generate fully polarized Th cells, the cells were restimulated with PHA (10 μg/ml; Difco, Detroit, MI, USA) and 3000 rad-irradiated feeder cells (PBMC of two unrelated donors and EBV-B cells (JY cells)) in the presence of IL-4 for Th0 cells; IL-4 neutralizing antibodies (CLB_IL-4/6, Sanquin) plus IL-12 for Th1 cells; and IL-12 neutralizing antibodies (U-CyTech, Utrecht, the Netherlands) plus IL-4 for Th2 cells. All T cells were cultured in IMDM with 10% FCS, gentamycin and IL-2 (Cetus, Emeryville, CA, USA). During co-culture with DC, Staphylococcus enterotoxin B (SEB; Sigma-Aldrich; final concentration, 10 pg/ml) was added. α-CD3/CD28 stimulation of T cells for viral replication experiments was done with mouse mAb to human CD28 (CLB-CD28/1) and human CD3 (CLB-T3/4E-1XE, Sanquin).

12 days after the second stimulation round, resting T cells were restimulated with PMA (10 ng/ml) and ionomycin (1 μg/ml) for 6 hr, the last 4.5 hr in the presence of Brefeldin A (10 μg/ml) (all Sigma-Aldrich). Cells were fixed in 2% PFA, permeabilized with 0.5% saponin (Sigma-Aldrich), and stained with anti-IFNγ -FITC and anti-IL4-PE (both BD Biosciences). Cells were then analysed by FACS.

C33A cervix carcinoma cells were transfected using calcium phosphate with 5 μg of the molecular clone of CXCR4-using HIV-1 LAI or CCR5-using HIV-1 JR-CSF. The virus containing supernatant was harvested 3 days post transfection, filtered and stored at -80°C. The concentration of virus was determined by CA-p24 ELISA. C33A cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen, Breda, the Netherlands), supplemented with 10% FCS, 2 mM sodium pyruvate, 10 mM HEPES, 2 mM L-glutamine, penicillin (100 U/ml) (Sigma-Aldrich) and streptomycin (100 μg/ml; Invitrogen).

Fully matured DC (IFNγ/MF if indicated otherwise) were incubated in a 96-well-plate (45 × 10³ DC/100 μl/well) with HIV-1 (15 ng CA-p24/well) for 2 hr at 37°C. The DC were washed with PBS after centrifugation at 400 × g to remove unbound virus. Washing was repeated 2 times, followed by addition of 50 × 10³ T_N, T_CM or T_EM cells. In some experiments, T1249 (250 ng/ml; Trimeris, Durham, NC, USA), RANTES (500 ng/ml, R&D Systems, Abingdon, UK) or AM3100 (10 μg/ml, Sigma-Aldrich) was added. The latter two were pre-incubated with the T cells for 30 min at 37°C. Prior to addition to DC, the T cells were analyzed by FACS with the following mouse anti-human antibodies: CD4-PE, CCR5-PE and CXCR4-PE (all BD Biosciences). Viral replication after transmission was followed by measuring CA-p24 in the culture supernatant by ELISA. To determine intracellular CA-p24 in the single-cycle transmission assay, saquinavir (Roche, London, UK at 0.2 μM) was added to prevent cell-to-cell spread of newly produced virions. After 48 hr, the T cells were harvested and stained with FITC-labeled CD3 (BD Biosciences), followed by fixation with 4% PFA and washing with washing buffer (PBS with 2 mM EDTA and 0.5% BSA). Fixated cells were then washed with perm/wash buffer (BD Biosciences), and incubated with PE-labeled CA-p24 (KC57-RD1, Coulter) followed by washing with successively perm/wash- and washing buffer. Cells were then analysed by FACS.

Fully matured DC (45 × 10³ DC/well) were incubated in a 96-well-plate with T_N, T_CM, T_EM cells, or polarized Th cells (50 × 10³ T cells/well) in a final volume of 200 μl. After 2 days, cell proliferation was assessed by the incorporation of [³H]-TdR after a pulse with 13 KBq/well during the last 16 hr of the co-culture, as measured by scintillation spectroscopy. Alternatively, T_N, T_CM or T_EM cells were stimulated with α-CD3/CD28 antibodies, followed by the [³H]-TdR pulse 2 days later.

Data were analysed for statistical significance (GraphPad InStat, Inc, San Diego, CA, USA) using ANOVA. A p value < 0.05 was considered to be significant.