Characterization of peripheral and mucosal immune responses in rhesus macaques on long-term tenofovir and emtricitabine combination antiretroviral therapy

Abstract

Background: The goal of antiretroviral therapy (ART) is to suppress virus replication to limit immune system damage. Some have proposed combining ART with immune therapies to boost antiviral immunity. For this to be successful, ART must not impair physiological immune function.

Methods: We studied the impact of ART (tenofovir and emtricitabine) on systemic and mucosal immunity in uninfected and simian immunodeficiency (SIV)-infected Chinese rhesus macaques. Subcutaneous ART was initiated 2 weeks after tonsillar inoculation with SIVmac239.

Results: There was no evidence of immune dysregulation as a result of ART in either infected or uninfected animals. Early virus-induced alterations in circulating immune cell populations (decreased central memory T cells and myeloid dendritic cells) were detected, but normalized shortly after ART initiation. ART-treated animals showed marginal SIV-specific T-cell responses during treatment, which increased after ART discontinuation. Elevated expression of CXCL10 in oral, rectal, and blood samples and APOBEC3G mRNA in oral and rectal tissues was observed during acute infection and was down regulated after starting ART. ART did not impact the ability of the animals to respond to tonsillar application of polyICLC with increased CXCL10 expression in oral fluids and CD80 expression on blood myeloid dendritic cells.

Conclusion: Early initiation of ART prevented virus-induced damage and did not impede mucosal or systemic immune functions.

Figure 1. Plasma viral loads and changes in CD4+ T cells counts in untreated and ART-treated animals

(A) Plasma SIV RNA levels were determined by quantitative real time RT-PCR. The ART treatment period is indicated by the shaded grey box. Each symbol indicates an individual animal. Arrows indicate time-points of polyICLC application. Average geometric mean viral loads (±SEM) are shown for (B) all ART-treated animals and (C) ART-responding animals (excluding the two transient ART responders). Asterisks indicate significant differences between ART-naïve and ART-treated animals; * p< 0.05, **p< 0.01. (D) Absolute CD4+ T cell counts are shown for individual animals over time. AA47 in the untreated group had lower CD4 counts at baseline, showed rapid, progressive loss of CD4+ cells after infection and succumbed to AIDS-related pneumonia 33 w.p.i.

Figure 2. T cell immune responses to SIV- and Candida albicans in PBMCs during and after ART cessation

SIV- and Candida albicans-specific immune responses were detected in PBMCs by ICS at 26, 34, 51 and 61 w.p.i. PBMCs were gated based on forward/side scatter characteristics and subsequently on CD3+CD4+ or CD3+CD4- cells with doublet discrimination. Average percentages (±SEM) of TNF-α-, IFN-γ-, IL-2- and IL-17 single positive (A) SIV-specific CD3+CD4+ (upper panel) and CD3+CD4- (lower panel) T cells, (B) Candida-specific CD3+CD4+ (upper panel) and CD3+CD4- (lower panel) T cells are shown. Results are from 8 animals in the uninfected group (white bars), 4 in the SIV+ART- group (except for week 26, n=5; grey bars) and 5 in the SIV+ART+ group (black bars). (C) The percentages of single positive, double positive (IFN-γ-/IL-2, IFN-γ-/IL-17, IFN-γ-/TNF-α, IL-2/IL-17, and IL-2/TNF-α), triple positive (IFN-γ-/IL-2/IL-17, IFN-γ-/IL-2/TNF-α) and quadruple positive (IFN-γ-/IL-2/IL-17/TNF-α) CD4+ and CD4- cells specific for SIV or Candida are shown for the SIV-infected animals (± ART) at the 34 week time point.

Figure 3. Longitudinal assessment of the dynamics of CD4+ and CD8+ T cell subset frequencies in the blood during and after ART

Polychromatic flow cytometric analysis was performed to identify naïve, central and effector memory and regulatory T cells in PBMCs. PBMCs were gated on small lymphocytes and subsequently on CD3+ cells with doublet exclusion. CD4+ or CD4- cells were then forward gated based on the expression of CD28 and CD95. Naïve T cells were defined as CD28+CD95-, central memory T cells (TCM) as CD28+CD95+ and effector memory T cells (TEM) as CD28-CD95+ cells. FoxP3+CD25+ regulatory T cells (Treg) were identified in the CD4+ gate. Average fold changes (±SEM) of the frequency of the indicated T cell subset normalized on the baseline (three baseline time points were collected from each animal and the average was calculated and set as 1) are shown for uninfected animals (black, n=8), SIV-infected ART naïve animals (red, n=5 from baseline until week 32 and n=4 from week 36 until week 54), SIV-infected ART-treated animals (green, n=5). Asterisks indicate significant differences compared to uninfected animals; * p< 0.05, **p< 0.01.

Figure 4. Longitudinal assessment of the dynamics of DC subset frequencies in the blood during and after ART

DCs were detected in PBMCs using polychromatic flow cytometry by gating on forward/side scatter characteristics and subsequently on lineage- (CD3, CD14, CD20 negative) HLA-DR+ cells with doublet discrimination. In the lineage-HLA-DR+ DC gate mDCs were identified as CD11c+ and pDCs as CD123+. Numbers designate the average fold changes (±SEM) of the frequency of the indicated DC subset normalized on the baselines (two baseline time points were collected from each animal and the average was calculated and set as 1) for uninfected animals (black, n=8), SIV-infected ART-naïve animals (red, n=5 from baseline until week 32 and n=4 from week 36 until week 54) and SIV-infected ART-treated animals (green, n=5). Asterisks indicate significant differences compared to uninfected animals; * p< 0.05, ** p< 0.01.

Figure 5. Changes in the mRNA expression of innate and effector genes

Shown are normalized average fold changes (±SEM) of mRNA expression of innate and effector genes assessed in the oral and rectal mucosa as well as in PBMCs of infected macaques compared to uninfected controls at two different time points after infection. (A) 2 weeks post inoculation – uninfected animals (white bars, n=8), SIV-infected animals (black bars, n=10, except for PBMCs with n=6). (B) 35-36 weeks post inoculation – uninfected animals (white bars, n=8), SIV-infected ART-naïve group (grey bars, n=4), SIV-infected ART-treated group (black bars, n=5). Asterisks indicate significant differences between the groups; * p< 0.05, ** p< 0.01, *** p< 0.001.