Antibody-Mediated CD4 Depletion Induces Homeostatic CD4+ T Cell Proliferation without Detectable Virus Reactivation in Antiretroviral Therapy-Treated Simian Immunodeficiency Virus-Infected Macaques

Abstract

A major barrier to human immunodeficiency virus (HIV) eradication is the long-term persistence of latently infected CD4⁺ T cells harboring integrated replication-competent virus. It has been proposed that the homeostatic proliferation of these cells drives long-term reservoir persistence in the absence of virus reactivation, thus avoiding cell death due to either virus-mediated cytopathicity or immune effector mechanisms. Here, we conducted an experimental depletion of CD4⁺ T cells in eight antiretroviral therapy (ART)-treated, simian immunodeficiency virus (SIV)-infected rhesus macaques (RMs) to determine whether the homeostatically driven CD4⁺ T-cell proliferation that follows CD4⁺ T-cell depletion results in reactivation of latent virus and/or expansion of the virus reservoir. After administration of the CD4R1 antibody, we observed a CD4⁺ T cell depletion of 65 to 89% in peripheral blood and 20 to 50% in lymph nodes, followed by a significant increase in CD4⁺ T cell proliferation during CD4⁺ T cell reconstitution. However, this CD4⁺ T cell proliferation was not associated with detectable increases in viremia, indicating that the homeostatic activation of CD4⁺ T cells is not sufficient to induce virus reactivation from latently infected cells. Interestingly, the homeostatic reconstitution of the CD4⁺ T cell pool was not associated with significant changes in the number of circulating cells harboring SIV DNA compared to results for the first postdepletion time point. This study indicates that, in ART-treated SIV-infected RMs, the homeostasis-driven CD4⁺ T-cell proliferation that follows experimental CD4⁺ T-cell depletion occurs in the absence of detectable reactivation of latent virus and does not increase the size of the virus reservoir as measured in circulating cells.IMPORTANCE Despite successful suppression of HIV replication with antiretroviral therapy, current treatments are unable to eradicate the latent virus reservoir, and treatment interruption almost invariably results in the reactivation of HIV even after decades of virus suppression. Homeostatic proliferation of latently infected cells is one mechanism that could maintain the latent reservoir. To understand the impact of homeostatic mechanisms on virus reactivation and reservoir size, we experimentally depleted CD4⁺ T cells in ART-treated SIV-infected rhesus macaques and monitored their homeostatic rebound. We find that depletion-induced proliferation of CD4⁺ T cells is insufficient to reactivate the viral reservoir in vivo Furthermore, the proportion of SIV DNA⁺ CD4⁺ T cells remains unchanged during reconstitution, suggesting that the reservoir is resistant to this mechanism of expansion at least in this experimental system. Understanding how T cell homeostasis impacts latent reservoir longevity could lead to the development of new treatment paradigms aimed at curing HIV infection.

Keywords: SIV; SIV reservoir; homeostatic proliferation.

FIG 1

Experimental design and ART treatment. (A) Study design. All animals were infected with SIV_mac239, 4 animals were treated with antiretroviral therapy (ART) at 2 months postinfection (early chronic treated animals), and another 4 animals were treated with ART at 10 months postinfection (late chronic treated animals). After ∼10 months of ART, when virus was fully suppressed, the CD4R1 anti-CD4-depleting antibody was administered in all animals (black arrow). Both early chronic and late chronic treated animals were monitored for 4 and 7 months, respectively. Small blood draws were collected every 2 weeks from all animals to monitor CD4 counts. Large blood draws (red arrows), rectal biopsy specimens (green arrows), and fine needle aspirates or lymph node biopsy specimens (blue arrows) were collected pre-CD4 depletion and post-CD4 depletion, at 4 or 5 months after CD4 depletion. (B) Plasma viral loads for individual animals using the standard assay (limit, 60 copies/ml plasma). (C) CD4 counts (cells/μl whole blood). (D) Percentage of CD4⁺ T cells. Open symbols represent early chronic treated animals, and closed symbols represent late chronic treated animals. Each symbol represents a different animal, and the line represents the median.

FIG 2

Dynamics of CD4⁺ T cell depletion and reconstitution. (A to C) CD4⁺ T cells expressed as counts (cells/μl whole blood) (A), frequency in PBMC (B), and a percentage of baseline CD4 counts (C). (D to G) CD4⁺ T cell memory subsets were measured during CD4 depletion and reconstitution, including naive (D), central memory (E), transitional memory (F), and effector memory T cells (G). (H to K) Tissue measures of CD4⁺ T cells. The frequency of CD4⁺ T cells in lymph nodes (H) and rectal biopsy specimens (I). The percentage of CD4⁺ T cells as a percentage of baseline in lymph nodes (J) and rectal biopsy specimens (K). Open symbols represent early chronic treated animals, and closed symbols represent late chronic treated animals. Each symbol represents a different animal, and the line represents the median. Statistical differences were determined using Wilcoxon rank sum test (*, P < 0.05).

FIG 3

Proliferation of CD4⁺ T cells after CD4 depletion. Expression of Ki67 on CD4⁺ T cells was measured in PBMC (A), LN (B), and RB (C) during CD4 depletion and reconstitution. (D to G) Ki67 was measured within each CD4⁺ T cell memory subset: naive (D), central memory (E), transitional memory (F), and effector memory (G). Expression of PD-1 on CD4⁺ T cells was measured in PBMC (H), LN (I), and RB (J) during CD4 depletion and reconstitution. Open symbols represent early chronic treated animals, and closed symbols represent late chronic treated animals. Each symbol represents a different animal, and the line represents the median.

FIG 4

CD4 depletion does not induce increases in SIV_mac239 viral loads. Viral load was measured using both the standard sensitivity (60 copies/ml of plasma) (A) and the ultrasensitive (3 copies/ml of plasma) viral load assay (B). Open symbols represent early chronic treated animals, and closed symbols represent late chronic treated animals. Each symbol represents a different animal, the solid line represents the median, and the horizontal dotted line represents the limit of detection (60 or 3 copies/ml of plasma).

FIG 5

SIV reservoir dynamics after CD4 depletion. Total cell-associated SIV DNA was measured in PBMC (A) and calculated in CD4⁺ T cells (B). (C) Quantitative SIV outgrowth assay. Open symbols represent early chronic treated animals, and closed symbols represent late chronic treated animals. Each symbol represents a different animal. Gray outlined animals were undetectable, the solid line represents the median, and horizontal dotted lines represent the limit of detection (60 or 3 copies/ml of plasma). Statistical differences were determined using the Friedman test (*, P < 0.05; **, P < 0.01).