To investigate the importance of the ICAM-1— LFA-1 interaction in DC-mediated HIV-1 transmission, we performed transmission experiments with DC obtained from healthy donors and peripheral blood leukocytes (PBL) from LAD-1 patients or healthy controls. We isolated leukocytes from three different LAD-1 patients, whose characteristics are given in Table 1. To confirm the negative LFA-1 status of LAD-1 leukocytes, we performed FACS analysis on CD11a and CD18, of which one representative patient and control are depicted in Fig. 1, upper two panels. We further determined by FACS that the expression of CD4 and CXCR4 was comparable to healthy controls (results not shown). In order to test the transmission efficiency to LAD-1 T cells, we used DC stimulated by poly (I:C) since this subset expresses the highest level of ICAM-1 and is the most efficient HIV-1 transmitter 8.

We incubated the DC with HIV-1 for 2 hr, followed by washing steps to remove unbound virus. After addition of LAD-1 or control PBL, we determined the transmission efficiency by measuring the accumulation of HIV-1 capsid protein p24 (CA-p24) in T cells by FACS 1–3 days later. 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, 2324). To distinguish virus transmitted to T cells from HIV in DC, we co-stained with CD3 and DC-SIGN. DC and T cells tend to cluster, which would hamper an accurate estimation of transmission efficiency by FACS. We therefore added EDTA to our FACS buffer to reduce the amount of cell clustering. Indeed, the majority (95%) of the CD3 positive cells was negative for DC-SIGN, showing that only a few DC were attached to T cells during the FACS analysis (Fig. 2A). In uninfected controls of LAD-1 and healthy PBL only background percentages of CA-p24 positive T cells were scored (0.06%, Fig. 2B). Addition of HIV-1 resulted in an increase in CA-p24 positive T cells when using PBL from healthy controls (1.09%, Fig. 2C), whereas only a slight increase was observed in PBL from LAD-1 donors (0.14%, Fig. 2D). The intracellular CA-p24 levels reached a maximum two days after DC-mediated HIV-1 transmission for both control and LAD-1 PBL, which is depicted in Fig. 2E for one representative control and LAD-1 patient. On average, DC-mediated HIV-1 transmission to control T cells was nine times more efficient (Fig. 2F, n = 3).

In addition to quantification of the transmission efficiency in a single-cycle transmission assay (Fig. 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. The replication of HIV-1 after transmission to LAD-1 PBL is delayed with 1–2 days in comparison to healthy controls (Fig. 2), which reflects the lower transmission efficiency of figure 2. Since CA-p24 levels eventually reach a similar plateau, we conclude that LAD-1 cells are susceptible to HIV-1, but that the transmission is taking place at a lower efficiency.

In order to efficiently bind ICAM-1, LFA-1 needs to be activated. Cellular activation by chemokines from endothelial cells or by TCR/CD3 cross-linking results in a conformational change of LFA-1 from a low to a high ligand-binding state 122125. We questioned whether LFA-1 expression by T cells is sufficient for efficient HIV-1 transmission by DC, or that additional activation of LFA-1 is necessary. To investigate this, we used cells from a unique patient with mild LAD-1 symptoms (LAD-1/variant). The leukocytes from this patient express LFA-1 (Fig. 1 and Table 1), but the integrin cannot be induced into an active conformation 20. In comparison to the experiments with LFA-1 negative cells, we obtained similar results: HIV-1 transmission to LAD-1/variant T cells was impaired in the single-cycle assay (Fig. 4A) and replication after transmission was delayed with 2 days (Fig. 4B). This demonstrates that LFA-1 has to be activated to a high ligand-binding state in order to mediate HIV-1 transmission. As an additional control, we infected LAD-1/variant PBL with HIV-1 in the absence of DC (Fig 4C). HIV-1 replication in CD3/CD28 stimulated PBL from the LAD-1/variant patient was comparable to healthy controls, showing that not replication but efficient transmission by DC depends on LFA-1 activation.

DC attract T cells and form large clusters in vivo and in vitro, a process that is dependent on cell-cell adhesion 2627. We studied the clusters of DC and leukocytes 24 hr after DC-mediated HIV-1 transmission. The cluster size of DC with T cells from healthy individuals was clearly larger than the clusters with T cells from LAD-1 and LAD-1/variant T cells, as is shown in the photographs (Fig 5A). Quantitative determination of the amount and diameter of clusters showed that although the number of clusters was only slightly reduced (30, 26 and 25 clusters on average for control, LAD-1 and LAD-1/variant respectively), the mean cluster diameter of control cells was significantly larger (9.1 versus 5.6 and 6.2 in arbitrary units for LAD-1 and the variant respectively; p < 0.001 and <0.002). We subsequently grouped the clusters according to diameter (Fig. 5B), and clearly demonstrate that their actual number is not reduced for the LAD-1 and LAD-1/variant T cells, but that they are significantly smaller in size.

Peripheral blood mononuclear cells (PBMC) were isolated from blood of healthy donors 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 or 6-well culture plates (Costar, Cambridge, MA, USA) at a density of 5 × 10⁵ cells or 2,5 × 10⁶ per well, respectively. After 60 min at 37°C, nonadherent 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 clone serum (HyClone, Logan, UT, USA) and 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 cells with poly (I:C) (20 μg/ml; Sigma-Aldrich, St. Louis, MO, USA). 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 by FACS. Mouse anti-human mAbs were used against the following molecules: CD14 (BD Biosciences, San Jose, CA, USA), 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). Samples were analysed on a FACScan (BD Biosciences).

Peripheral Blood Leukocytes (PBL) were isolated by layering PBMC from healthy donors and LAD-1 patients on a Percoll gradient. The heavy fraction with predominantly PBL was collected and stored at -150°C. PBL were cultured in IMDM with 10% FCS, gentamycin, 10 U/ml IL-2 (Cetus, Emeryville, CA, USA) and Staphylococcus enterotoxin B (SEB; Sigma-Aldrich; final concentration, 10 pg/ml). Mouse mAb to human CD28 (CLB-CD28/1) and human CD3 (CLB-T3/4E-1XE) were obtained from Sanquin (Amsterdam, The Netherlands).

C33A cervix carcinoma cells or PM1 T cells were transfected using calcium phosphate or electroporation respectively with 5 μg of the molecular clone of HIV-1 LAI. Since any of the patients could bear one or two mutant alleles for the CCR5 co-receptor, resulting is decreased susceptibility to CCR5-using HIV-1, we chose to use CXCR4-using HIV-1 LAI. The virus containing supernatant was harvested 3 to 5 days post transfection, filtered and stored at -80°C. The concentration of virus was determined by CA-p24 ELISA. C33A and PM1 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) or Roswell Park Memorial Institute (RPMI) medium 1640 (Life Technologies) respectively, both 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, Breda, The Netherlands).

Fully matured DC were incubated in a 96-well-plate (40–50 × 10³ DC/50 μl/well) with PM1 produced virus (10 ng CA-p24/well) or C33A produced virus (20 ng) for 2 hr at 37°C. The DC were washed with PBS after centrifugation at 400 × g to remove unbound virus. Washing was repeated, followed by addition of 50 × 10³ PBL. Prior to addition to DC the PBL were analyzed by FACS with the following mouse anti-human antibodies: FITC-labeled CD11a (Pelicluster, Sanquin), APC-labeled CD4 (BD Biosciences) and PE-labeled CXCR4 (BD Biosciences). CD18 (Pelicluster, Sanquin) incubation was followed by incubation with FITC-conjugated goat F(ab')₂ anti-mouse IgG and IgM (Jackson ImmunoResearch Laboratories). 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, United Kingdom at 0.2 μM) was added to prevent cell-to-cell spread of newly produced virions. After 48 hr, the cells were harvested and stained with FITC-labeled CD3 (BD Biosciences) and APC-labeled DC-SIGN (R7D Systems, MN, USA), 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-labelled CA-p24 (KC57-RD1, Coulter, Hialeah, FL, USA) followed by washing with successively perm/wash- and washing buffer. Cells were then analysed by FACS.

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.