Article Figures & Data
Figures
- FIG 1
HIV-2 infects human thymic tissue and isolated thymocytes. TOCs were infected with R5- or X4-tropic HIV-2 or HIV-1 primary isolates and cultured for 10 or 11 days on Millipore inserts. Single-cell suspensions of total thymocytes or CD4SP thymocytes, sorted as CD3high CD8neg cells, were also infected with the above-mentioned viruses and cultured for 10 days. Graphs show total HIV DNA (A), as assessed by quantitative real-time PCR, or viral gag mRNA (B), as assessed by real-time RT-PCR (the level of mRNA expression was normalized to the level of GAPDH expression). Each dot represents a single thymus. Lines indicate median values. *, P < 0.05; **, P < 0.01.
- FIG 2
Viral production in HIV-2-infected TOCs. TOCs infected with R5- or X4-tropic HIV-2 or HIV-1 primary isolates were analyzed for viral production by immunohistochemistry using anti-Gag antibodies (brown) and hematoxylin counterstain (blue). (A) Viral production at days 1 and 11 after infection with HIV-2 (top) or HIV-1 (bottom), as assessed using an anti-p27 MAb (ARP396/397 from MRC) or an anti-p24 MAb (Kal-1 from Dako), respectively. (B) Extracellular (arrow) and cytoplasmic (arrowhead) Gag expression in R5- or X4-tropic HIV-2-infected TOCs at day 11 (determined with the ARP396/397 anti-p27 antibody).
- FIG 3
Limited replication of HIV-2 in human thymocytes. Viral production at the single-cell level was determined by flow cytometry using the anti-Gag antibody (Ab) KC57 at 10 days postinfection of TOCs, total thymocytes, or CD4SP thymocytes with R5- or X4-tropic HIV-1 and HIV-2 primary isolates. Representative dot plots of intracellular Gag expression (numbers show the proportion of cells inside the gate) (A) and frequency of productively infected KC57-positive thymocytes (B) under each condition are shown. Flow cytometric analysis was performed after exclusion of dead cells and aggregates. Each dot represents a single thymus. Lines indicate median values. *, P < 0.05; **, P < 0.01. FSC, forward scatter.
- FIG 4
Distinct cytopathic impact of HIV-2 infection on TOCs and thymocytes. HIV-infected TOCs (A and B) or thymocyte suspensions (C and D) were cultured for 10 days. Representative dot plots of CD4 and CD8 expression in cells in TOCs (A) and in total thymocyte suspensions (C), with the graphs showing the mean frequency of thymocyte subsets in all thymuses analyzed (n = 4 for panel B and n = 8 for panel D). TOC analysis was performed in CD14neg CD16neg CD19neg CD123neg cells. Dead cells and aggregates were excluded from the flow cytometric analysis. Numbers in dot plots represent the proportion of cells inside quadrants. (E) Frequency of CD4neg cells within Gag-positive (KC57 antibody-positive) thymocytes in HIV-infected thymocyte suspensions. (F) Fold change in the frequency of live cells in TOCs relative to that in the uninfected controls, as assessed by flow cytometry. (G) Fold change in the number of live thymocytes in culture relative to that in the uninfected control. Lines indicate median values. *, P < 0.05.
- FIG 5
The X4-tropic HIV-2 envelope per se does not impact the thymocyte distribution in TOCs. TOCs were cultured for 7 days with medium only (control) or in the presence of the recombinant envelope protein HIV-2 gp105ROD, HIV-1 gp120Ba-L, or HIV-1 gp120IIIB (all at 1 μg/ml) or anti-CD4 MAb. The dot plots show a representative example (from one of three independent experiments with different thymuses) of the frequency of subsets in TOCs, as determined by CD4 and CD8 expression. Numbers correspond to the proportion of cells inside gates.
Tables
- TABLE 1
HIV isolates used in the study
Virus Major coreceptor(s) used Source Primary isolates HIV-192US660 R5 NIHa HIV-192HT599 X4 NIHa,b HIV-260415K R5 NIHa,c HIV-220.04d X4 Nuno Taveira Lab-adapted strains HIV-1NL4-3 X4 MRCe HIV-2ROD10 R5, X4 MRCe ↵a From the Multicenter AIDS Cohort Study, NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH.
↵b Provided by Neal Halsey.
↵c Provided by Feng Gao and Beatrice Hahn.
↵d Previously represented as PTHCC20/2004 (26) and 19/2004 (27).
↵e NIBSC, Centre for AIDS Reagents, Medical Research Council, United Kingdom.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
Table S1 (Primer and probe sequences.)
Fig. S1 (Comparison of anti-Gag antibodies for the detection of HIV-2 and HIV-1 infection by immunohistochemistry.)
Fig. S2 (Low to undetectable intracellular Gag protein expression at the single-cell level in HIV-2-infected thymocytes.)
Fig. S3 (Validation of KC57 (anti-Gag) antibody for HIV-2 detection by flow cytometry.)
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- Supplemental file 1 -
