Figure 1.
Heterotypic fusion between infected T cells and macrophages is the most productive mode of HIV-1 cell-to-cell transfer toward macrophages. (A) Experimental design. Human monocytes isolated from blood were differentiated into macrophages for 7 d (MDMs), co-cultured during 24 h with CD4+ T cells (Jurkat cell line or autologous primary CD4+ T cells) infected with HIV-1 (NLAD8-VSVG) (for 2 or 7 d), CD4+ T cells were eliminated and MDM infection was analyzed. (B) Representative confocal image of a MDM pre-loaded with CellTracker Violet (red) infected by fusion with 2 d-infected Jurkat cells pre-loaded with CellTracker (green), HIV-p24 and nuclei (DRAQ5) are shown in gray. Merge: z projection along the dotted line is shown in lower panel. Scale bars, 20 and 10 µm. See also Video 1. (C) Quantification of the three mechanisms (fusion, phagocytosis, and synapse-like) of HIV-1 transfer from infected CD4+ T cells to MDMs as characterized in Fig. S1, C and D, among infected MDMs after a 24 h-co-culture with Jurkat cells infected for 2 d (HIV-2d) or 7 d (HIV-7d; n = 4 or 5 donors, mean ± SD). (D and E) Analysis of MDM infection after a 24 h-co-culture with uninfected Jurkat cells (NI) or Jurkat cells infected for 2 d (HIV-2d) or 7 d (HIV-7d) by flow cytometry (D, % of HIV-1-p24 + MDMs, n = 8 donors, mean ± SD) and fluorescence microscopy (E, MDM infection index, n = 11 donors, median ± interquartile range). (F) Analysis of MDM infection after a 24 h co-culture with uninfected autologous T cells (NI) or autologous primary CD4+ T cells infected for 2 d (HIV-2d) or 9 d (HIV-9d) by flow cytometry (n = 4 donors, median ± interquartile range). (G and H) Analysis of long-term MDM infection. After co-culture as in A, Jurkat cells were washed, and MDMs were kept in culture for additional 5 d. (G) Quantification of HIV-p24 concentration in the supernatant of MDMs (n = 6 donors, median ± interquartile range). (H) Representative microscopy images of MDM infection. F-actin (phalloidin, blue), HIV-p24 (yellow), nuclei (DAPI, gray). Inserts show magnifications of the white squares. Scale bars, 200 and 30 µm. Statistical analyses: Multiple comparison test (C) two-way Anova and Tukey, (D) one-way Anova and Tukey, (E–G) Kruskal-Wallis and Dunn’s, and (F) Friedman and Dunn’s. * P ≤ 0.05; ** P ≤ 0.01; **** P ≤ 0.0001.

Heterotypic fusion between infected T cells and macrophages is the most productive mode of HIV-1 cell-to-cell transfer toward macrophages. (A) Experimental design. Human monocytes isolated from blood were differentiated into macrophages for 7 d (MDMs), co-cultured during 24 h with CD4+ T cells (Jurkat cell line or autologous primary CD4+ T cells) infected with HIV-1 (NLAD8-VSVG) (for 2 or 7 d), CD4+ T cells were eliminated and MDM infection was analyzed. (B) Representative confocal image of a MDM pre-loaded with CellTracker Violet (red) infected by fusion with 2 d-infected Jurkat cells pre-loaded with CellTracker (green), HIV-p24 and nuclei (DRAQ5) are shown in gray. Merge: z projection along the dotted line is shown in lower panel. Scale bars, 20 and 10 µm. See also Video 1. (C) Quantification of the three mechanisms (fusion, phagocytosis, and synapse-like) of HIV-1 transfer from infected CD4+ T cells to MDMs as characterized in Fig. S1, C and D, among infected MDMs after a 24 h-co-culture with Jurkat cells infected for 2 d (HIV-2d) or 7 d (HIV-7d; n = 4 or 5 donors, mean ± SD). (D and E) Analysis of MDM infection after a 24 h-co-culture with uninfected Jurkat cells (NI) or Jurkat cells infected for 2 d (HIV-2d) or 7 d (HIV-7d) by flow cytometry (D, % of HIV-1-p24 + MDMs, n = 8 donors, mean ± SD) and fluorescence microscopy (E, MDM infection index, n = 11 donors, median ± interquartile range). (F) Analysis of MDM infection after a 24 h co-culture with uninfected autologous T cells (NI) or autologous primary CD4+ T cells infected for 2 d (HIV-2d) or 9 d (HIV-9d) by flow cytometry (n = 4 donors, median ± interquartile range). (G and H) Analysis of long-term MDM infection. After co-culture as in A, Jurkat cells were washed, and MDMs were kept in culture for additional 5 d. (G) Quantification of HIV-p24 concentration in the supernatant of MDMs (n = 6 donors, median ± interquartile range). (H) Representative microscopy images of MDM infection. F-actin (phalloidin, blue), HIV-p24 (yellow), nuclei (DAPI, gray). Inserts show magnifications of the white squares. Scale bars, 200 and 30 µm. Statistical analyses: Multiple comparison test (C) two-way Anova and Tukey, (D) one-way Anova and Tukey, (E–G) Kruskal-Wallis and Dunn’s, and (F) Friedman and Dunn’s. * P ≤ 0.05; ** P ≤ 0.01; **** P ≤ 0.0001.

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