1. Volume 24, Issue 8, Pages 2088-2100 (August 2018)
HIV-1-Infected CD4+ T Cells Facilitate Latent Infection of Resting CD4+ T Cells through Cell-Cell Contact 
Luis M. Agosto, Melissa B. Herring, Walther Mothes, Andrew J. Henderson 
Cell Reports 
Volume 24, Issue 8, Pages (August 2018)
DOI: /j.celrep
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2. Cell Reports 2018 24, 2088-2100DOI: (10.1016/j.celrep.2018.07.079)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1 In Vitro Model of HIV-1 Cell-to-Cell Transmission
See text and Experimental Procedures for details. A flow cytometry profile for HIV Gag expression in activated CD4+ T cells in the presence or absence of EFV is shown. The average percent of HIV-1 Gag expression ± SD from 8 experiments is indicated. A flow cytometry profile of activation marker expression among purified resting CD4+ T cells and activated CD4+ T cells is shown. We estimated the percent of target resting CD4+ T cells and their viability at the end of the co-culture period.
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4. Figure 2
HIV-Producing T Cells Transmit HIV-1 Directly to Resting CD4+ T Cells
(A) After co-culture, target resting CD4+ T cells were sorted to assess the level of HIV-1 integration within this population. The flow cytometry plots show the sorting strategy.
(B) Simplified diagram of the Alu-PCR strategy to specifically detect HIV integration. Shown are the relative primer locations.
(C) HIV-1 integration was assessed in co-cultures infected with HIVNL4-3 (X4) and HIVREJO (R5) by Alu-PCR. Parallel control co-cultures were treated with EFV. Symbols represent independent blood donors. Integration was measured 5 times for each sample. ∗∗p < 0.01.
Error bars represent the SD of the mean.
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5. Figure 3
Co-culture with Productively Infected Activated CD4+ T Cells Does Not Induce Robust Activation of Resting CD4+ T Cells
(A) We assessed the level of T cell activation among target resting CD4+ T cells at various time points post-co-culture based on the expression of CD69, HLA-DR, and CD25. The levels of activation based on the expression of activation markers (mean fluorescence index) were compared to activated CD4+ T cells treated with PHA, IL-2, and IL-7.
(B) Combined data from 3 individual experiments from three different blood donors. Cells cultured alone or in co-culture (CC) with infected CD4+ T cells are shown. The horizontal line represents the average of each population. ∗p < 0.05.
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6. Figure 4
HIV-1 Integrates in Resting CD4+ T Cells in a Cell-Contact-Dependent Manner
(A) A Transwell insert system was used to compare the relative efficiency of resting CD4+ T cell infection after co-culture with HIV-1-infected activated cells or when the infected activated cells were separated.
(B) HIV-1 integration was measured by Alu-PCR in the target resting CD4+ T cells after co-culture or Transwell-based infection. Note that the co-culture dataset is the same as Figure 1B for HIVNL4-3.
(C) To confirm that viral particles produced by infected activated CD4+ T cells can diffuse through the membrane, TZMbl cells were plated in the bottom well. Infection was assessed by luciferase expression. Error bars represent the SD of 3 measurements of luciferase. A parallel control culture was treated with EFV.
(D) Diagram of the HIVNL4-3-Gag-GFP genome.
(E and F) Activated CD4+ T cells producing HIVNL4-3-Gag-GFP (green, P) were co-cultured with target resting CD4+ T cells pre-labeled with CMAC (blue, T) and stained for CD4 (red). An example of a resting CD4+ T cell that carried HIV-1 Gag-GFP signal and was also in contact with an HIV-1-producer T cell is shown in (E). An example of a resting CD4+ T cell that was not in contact with HIV-1-producer T cells but carried HIV-1 Gag-GFP signals is shown in (F). The scale bars in the images represent 5 μm.
(G) The relative proportion of each is shown.
Error bars represent the SD of the mean. In (B), (C), and (G), ∗p < 0.05, ∗∗p < 0.01.
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7. Figure 5
Contacts between Productively Infected CD4+ T Cells and Uninfected Resting CD4+ T Cells Result in Polarization of HIV Gag to the Site of Cell-Cell Contact
(A) Live-cell imaging was conducted to observe conjugate formation between activated CD4+ T cells infected with HIVNL4-3-Gag-GFP (green) and uninfected resting CD4+ T cells in real time. Images were collected every 2.5 min. Shown are selected time points when the transfer of particles was detected (white arrows) and when polarization of HIV Gag-GFP to the site of cell-cell contact was detected (orange arrow). The full movie is provided as Video S1. The scale bars in the images represent 5 μm.
(B) Cell-cell contacts were identified and scored based on their duration. Contacts maintained for least 5 min were scored. The duration of cell-cell contacts that did not result in HIV Gag-GFP polarization were compared to the duration of contacts that resulted in polarization. Data shown represent all cell-cell contacts identified in 6 experiments from 2 blood donors. The horizontal line represents the average of each population. ∗∗p < 0.01.
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8. Figure 6
Visualization of Viral Cell-to-Cell Transmission to Resting CD4+ T Cells
(A) Example of polarized HIV-1 assembly as defined by the accumulation of HIV-1 Gag in HIV-1-producing activated cells (P) and CD4 in target cells (T) at the site of cell-cell contact (arrow).
(B) Example of resting CD4+ T cells carrying particles while in contact with productively infected T cells without polarization of viral assembly.
(C) Example of a resting CD4+ T cell carrying viral particles while in contact with uninfected activated T cells also carrying particles.
(D) The proportion of resting CD4+ T cells that acquired Gag-GFP from polarized viral assembly versus non-polarized viral assembly was quantified by counting cell-cell contacts.
The scale bars in the images represent 5 μm. Error bars represent the SD of the mean.
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9. Figure 7
HIV-1 Latent Infection Generated by Cell-to-Cell Transmission Is Less Inducible Than Latent Infection Generated by Cell-free HIV
(A) Co-cultures of productively infected activated CD4+ T cells with resting CD4+ T cells were stimulated with CD3/CD28 beads, IL-2, and IL-7 in the presence of raltegravir. At 24 hr post-stimulation, HIV-1 expression among target cells was assessed by intracellular HIV-1 Gag staining and flow cytometry analysis. A representative plot is shown comparing unstimulated with stimulated cells. A dot plot of the percent of target T cells expressing HIV Gag after stimulation or without stimulation is shown. Each symbol represents an independent experiment with different blood donors. The horizontal line represents the average of each population.
(B) Resting CD4+ T cells were spinoculated with HIVNL4-3 and incubated for 3 days. At this point, the cells were stimulated and analyzed for HIV-1 expression as in (A). A representative plot is shown comparing unstimulated with stimulated cells.
(C) Resting CD4+ T cells were infected with HIV-1 without spinoculation and incubated for 3 days. At this point, the cells were stimulated and analyzed for HIV-1 expression as in (A). A representative plot is shown comparing unstimulated with stimulated cells.
(D) HIV integration in target resting CD4+ T cells was measured under co-culture, spinoculation, and no-spinoculation infection conditions. Background PCR signal was determined based on samples treated with EFV.
(E) The proportion of inducible proviruses was calculated from the percent of HIV-positive cells based on flow cytometry after stimulation and after subtracting the background signal from unstimulated cells divided by the percent HIV-positive cells based on Alu-PCR. This fraction is plotted for 4 independent experiments done under co-culture conditions and 5 independent experiments for spinoculation and non-spinoculation infection conditions.
Error bar represent the SD of the mean. In (A), (B), (D), and (E), ∗p < 0.05, ∗∗p < 0.01.
Cell Reports  , DOI: ( /j.celrep )
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