Fusion-induced apoptosis contributes to thymocyte depletion by a pathogenic human immunodeficiency virus type 1 envelope in the human thymus

Abstract

The mechanisms of CD4(+) T-cell depletion during human immunodeficiency virus type 1 (HIV-1) infection remain incompletely characterized. Of particular importance is how CD4(+) T cells are depleted within the lymphoid organs, including the lymph nodes and thymus. Herein we characterize the pathogenic mechanisms of an envelope from a rapid progressor (R3A Env) in the NL4-3 backbone (NL4-R3A) which is able to efficiently replicate and deplete CD4(+) thymocytes in the human fetal-thymus organ culture (HF-TOC). We demonstrate that uninterrupted replication is required for continual thymocyte depletion. During depletion, NL4-R3A induces an increase in thymocytes which uptake 7AAD, a marker of cell death, and which express active caspase-3, a marker of apoptosis. While 7AAD uptake is observed predominantly in uninfected thymocytes (p24(-)), active caspase-3 is expressed in both infected (p24(+)) and uninfected thymocytes (p24(-)). When added to HF-TOC with ongoing infection, the protease inhibitor saquinavir efficiently suppresses NL4-R3A replication. In contrast, the fusion inhibitors T20 and C34 allow for sustained HIV-1 production. Interestingly, T20 and C34 effectively prevent thymocyte depletion in spite of this sustained replication. Apoptosis of both p24(-) and p24(+) thymocytes appears to be envelope fusion dependent, as T20, but not saquinavir, is capable of reducing thymocyte apoptosis. Together, our data support a model whereby pathogenic envelope-dependent fusion contributes to thymocyte depletion in HIV-1-infected thymus, correlated with induction of apoptosis in both p24(+) and p24(-) thymocytes.

FIG. 1.

CD4⁺ thymocyte depletion is dependent on sustained viral replication. (A and B) Thymocytes from mock-, NL4-R3A-, and NL4-R3B-infected HF-TOC were analyzed using flow cytometry for forward and side scatter (% gated live) (A) and expression of CD4 on cells which were gated live (B). Shown are data from at least seven independent experiments. (*, P < 0.05 for the NL4-R3A trend line relative to mock and NL4-R3B.) (C and D) Saquinavir was added to NL4-R3A-infected HF-TOC 6 (C) or 8 (D) days postinfection and each day thereafter, with the first day of drug addition indicated by the arrow. CD4⁺ thymocyte depletion was assessed at the indicated times. Error bars are from quadruplicate samples (*, P < 0.05 by the student's t test for NL4-R3A with drug relative to NL4-R3A without drug).

FIG. 2.

Increase in 7AAD⁺ thymocytes in NL4-R3A-infected HF-TOC is predominantly in the p24⁻ population. (A) Cells in the scatter-defined live cell gate were stained for 7AAD to measure dead cells and p24 to measure productively infected cells. Shown are representative plots for mock-, NL4-R3A-, and NL4-R3B-infected thymus at 9 and 12 dpi. (B) The percentage of 7AAD⁺ thymocytes in the live gate for mock-, NL4-R3A-, and NL4-R3B-infected thymus over time. (*, P < 0.05 for NL4-R3A trend line relative to mock and NL4-R3B.) (C) The increase in 7AAD⁺ cells occurs predominantly in the uninfected (p24⁻) population of the NL4-R3A-infected thymus. The proportion of uninfected thymocytes (p24⁻ thymocytes) and infected thymocytes (p24⁺ thymocytes) which are 7AAD⁺ is shown. (B and C) Data are from seven independent experiments. (*, P < 0.05 for p24⁻ trend line relative to p24⁺.)

FIG. 3.

Infection with NL4-R3A increases the frequency of thymocytes expressing active caspase-3. (A) 7AAD-negative live cells were stained for p24 and active caspase-3. Shown is a representative of seven independent experiments from 9 and 12 dpi for mock-, NL4-R3A-, and NL4-R3B-infected thymus. (B) NL4-R3A increases the frequency of total thymocytes with active caspase-3 expression (*, P < 0.05 for NL4-R3A trend line relative to mock and NL4-R3B.) (C) The increase in active caspase-3⁺ cells in NL4-R3A-infected HF-TOC occurs in both uninfected (p24⁻) and infected (p24⁺) thymocytes in proportion to the level of NL4-R3A replication. (*, P < 0.05 for the strength of significance for each trend line.) (B and C) Data are from seven independent experiments.

FIG. 4.

Most caspase-3⁺ thymocytes in NL4-R3A-infected HF-TOC do not express p24. Fragments from mock- and NL4-R3A-infected thymus at 12 dpi were stained for p24 (green; Fluor 546) and active caspase-3 (red; Fluor 488). Shown is a representative of two independent experiments. Low (×20) and high (×60) magnifications are shown. Isotype control antibodies showed no specific signals (data not shown).

FIG. 5.

Prevention of thymocyte depletion in T20- and C34-treated HF-TOC in spite of sustained replication. (A to C) NL4-R3A-infected HF-TOC was treated with C34, T20, or saquinavir from 7 dpi for 6 days. Arrows indicate the day of drug addition. (A) Viral load was quantitated by ELISA detection of Gag antigen in the HF-TOC supernatant on the indicated days. Error bars are derived from triplicate samples. (*, P < 0.05 by the student's t test for drug treatment relative to no drug.) (B and C) Similar percentages of CD4⁺ thymocyte protection after T20, C34, and saquinavir treatment were detected 6 days after drug treatment by CD4 and CD8 staining. Error bars are derived from triplicate samples. Shown is a representative of three independent experiments. (*, P < 0.05 by the student's t test relative to no drug.)

FIG. 6.

Apoptosis in NL4-R3A-infected HF-TOC is inhibited by T20. (A) NL4-R3A-infected HF-TOC was treated with saquinavir or T20 at 6 dpi for up to 4 days. Thymocytes were stained with 7AAD, p24, and active caspase-3. Shown is a representative of five experiments for 7AAD⁻ live cells 3 days after drug addition. (B) The frequency of bystander apoptosis (percent caspase-3⁺ p24⁻) was determined for each experimental treatment. The combined data for all time points from five independent experiments are shown with standard error bars. (mean percentage p24⁺ of 14% for no drug, 4.5% for saquinavir, and 5.7% for T20). (C) Saquinavir increased, but T20 decreased, apoptosis of infected thymocytes. To compare HF-TOC with similar levels of replication, only samples with less than 7% p24⁺ were considered for “no drug” treatment (mean percent p24⁺ of 4.4% for no drug, 4.5% for saquinavir, and 5.7% for T20). (*, P < 0.05 by the student's t test relative to no drug for panels B and C.)