Figure 1.
Fractionation of blood from HIV-1-infected patients for obtaining plasma and RBCnative in the presence or absence of EDTA.
Whole blood collected from a patient in the presence (A,B) or absence (C,D) of EDTA, and separated into four plasma and erythrocyte fractions, A, B, C, and D, as shown. Fraction D was treated with 5 mM EDTA, resulting in Fraction E. All of the samples were used immediately for co-culture with PBMC to detect capability for causing HIV-1 infection of the PBMC. Aliquots were stored at −20°C for viral RNA measurements. See Materials and Methods for further details.
Figure 2.
PLT-RBC is a subpopulation of cells in whole blood and in preparations of RBCnative from HIV-1-infected patients.
A–B. Flow cytometry was performed with EDTA-anticoagulated whole blood obtained from an HIV-positive individual. A. Representative dot plot shows scatter properties of the RBC, PLT-RBC, and PLT. CD41a+/CD235a+ cells (red) represent PLT-RBC; CD41a+/CD235a- cells (green) represent free PLT; CD41a-/CD235a+ cells (blue) represent RBC; and CD41a-/CD235a- particles (gray) represent small vesicles or debris. B. Quantification of cells shown in frame A that express CD41a and/or CD235a. C–D. Flow cytometry was performed with an EDTA-free RBCnative preparation (see fraction D in Figure 1) obtained from an HIV-positive individual. RBCnative were analyzed as in frames A–B.
Figure 3.
Enrichment of PLT-RBC as a subpopulation of normal (uninfected) cells by adding platelets to a preparation of RBCnative from a normal volunteer.
A. Normal RBCnative obtained from citrated blood from a normal (uninfected) volunteer were incubated with platelet-rich plasma, washed, and analyzed by flow cytometry. B. RBC, PLT-RBC, and PLT were separated by sorting the populations shown in frame A. C–E. Light microscopy of Giemsa-stained mixtures of normal RBCnative enriched with PLT-RBC. Magnification: 400X and 1000X, respectively. Arrows in frame C indicate platelets bound to RBC. F. Fluorescent microscopy of PLT-RBC visualized at 630X magnification. Left panel, bright field; 2nd panel, visualization of both FITC and PE signals; 3rd panel, visualization of FITC; right panel, visualization of PE signal. Arrows point to platelets.
Figure 4.
HIV-1 cell-cell infection of PBMC is dependent on PLT.
A. RBC, PLT-RBC, and PLT were separated by sorting with FACS as shown in Fig. 3B. After incubation with HIV-1Bal, followed by washing 3 times to remove unbound HIV-1, the cells were co-incubated with PBMC to determine HIV-1 infection. **Cell-cell infection with PLT-bound HIV was higher than with RBC-bound HIV (p = 0.0332) (one-way Anova with Tukey's multiple comparisons test); ***cell-cell infection with PLT-RBC-bound HIV was higher than with RBC-bound HIV (p = 0.0124) (one-way ANOVA with Tukey's multiple comparisons test). B. Mixtures of normal RBCnative enriched with PLT-RBC were prepared by pre-incubation of 0.3 ml containing 109 normal RBCnative with the indicated numbers of PLT in 0.3 ml RPMI dilutions of platelet-rich plasma, and then washed 3 times. The cells were then pre-incubated with HIV-1Bal, washed 3 times, and examined for HIV-1 infection of PBMC as described in Materials and Methods. Normal RBCnative not pre-incubated with PRP served as a control. *Cell-cell infection was increased as a function of the number of platelets added to RBCnative (p<0.0001, one way Anova).
Figure 5.
HIV-1 cell-cell infection of PBMC is eliminated by EDTA.
PLT-RBC enriched RBCnative were prepared by pre-incubation of normal RBCnative (109 cells) with the indicated numbers of PLT, washed 3 times, and pre-incubated with HIV-1Bal, washed 3 times, and then examined for HIV-1 infection of PBMC in the presence or absence of 5 mM EDTA. Cell-cell infection was eliminated by EDTA (p<0.0001, two-way Anova).
Figure 6.
Cell-cell infection of PBMC by RBCnative from HIV-positive patients.
A. EDTA-free RBCnative from 11 chronically-infected patients were obtained as in Fig. 1D and co-incubated with PBMC to examine infection of the PBMC. 25 µl of EDTA-free RBCnative were diluted in 75 µl of IL-2 medium and added to 50 µl of 1.5×105 PHA-stimulated PBMC/well in IL-2 medium. B. 100 µl of EDTA-free plasmas from the patients, obtained as in Fig. 1C and supplemented with 20 U/ml of recombinant IL-2, did not infect co-incubated PBMC. C. The RBCnative shown in frame A were treated with 5 mM EDTA followed by washing 3 times in IL-2 medium before infecting PBMCs (see Fig. 1E) showed no HIV-1 infection of co-incubated PBMC. The infection exhibited by the group of EDTA-free RBCnative (A) was significantly higher using a paired t-test, than the infection exhibited both by the group of EDTA-free plasma (B) and by the group of EDTA-stripped RBCnative (C) at 8 days post-infection (p = 0.0404) and 10 days post-infection (p = 0.038).
Table 1.
HIV-1 RNA in plasma and RBCnative from chronically infected HIV-1 patients.
Figure 7.
Binding of HIV-1 to RBCnative as well as cell-cell infection of PBMC is inhibited by anti-DC-SIGN monoclonal antibody.
A. RBCnative was pre-incubated with HIV-1Bal in the absence or presence of anti-DC-SIGN mAb in a final 50 µg/ml concentration, washed 3 times, and bound p24 was measured by ELISA. The binding was inhibited by pre-incubation of the cells with the monoclonal antibody (p<0.01, t-test), but not inhibited by pre-incubation of the virus with the monoclonal antibody. B. Cell-cell infection of PBMC was also inhibited in a dose-dependent manner by anti-DC-SIGN mAb (p<0.02 and p<0.01 respectively, paired t-test).