Correction: Minimally invasive monitoring of CD4 T cells at multiple mucosal tissues after intranasal vaccination in rhesus macaques.

[This corrects the article DOI: 10.1371/journal.pone.0188807.]

Minimally invasive monitoring of CD4 T cells at multiple mucosal tissues after intranasal vaccination in rhesus macaques.

Studies in nonhuman primates (NHP) for prospective immune cell monitoring subsequent to infection and/or vaccination usually rely on periodic sampling of the blood samples with only occasional collections of biopsies from mucosal tissues because of safety concerns and practical constraints. Here we present evidence in support of cytobrush sampling of oral, rectal, and genital mucosal tissues as a minimally invasive approach for the phenotypic analyses of different T cells subsets de novo as well as prospectively after intranasal immunization in rhesus macaques. Significant percentages of viable lymphocytes were obtained consistently from both naïve and chronically SIV-infected rhesus macaques. The percentages of CD3+ T cells in the blood were significantly higher compared to those in the mucosal tissues analyzed in the naïve animals, while in the SIV+ animals the CD3+ T cells were significantly elevated in the rectal tissues, relative to all other sites analyzed. In the naïve, but not SIV+ macaques, the rectal and vaginal mucosal tissues, compared to oral mucosa and blood, showed higher diversity and percentages of CD4+ T cells expressing the HIV entry co-receptor CCR5 and mucosal specific adhesion (CD103) as well as activation (HLA-DR) and proliferation (Ki67) markers. Sequential daily cytobrush sampling from the oral, rectal, and genital mucosal tissues was performed in SIV+ animals from an ongoing study where they were administered intranasal immunization with adenoviral vectored vaccines incorporating the green fluorescent protein (GFP) reporter gene. We detected a transient increase in GFP+ CD4 T cells in only oral mucosa suggesting limited mucosal trafficking. In general, CD4+ and CD8+ T cells expressing Ki67 transiently increased in all mucosal tissues, but those expressing the CCR5, HLA-DR, and CD103 markers exhibited minor changes. We propose the minimally invasive cytobrush sampling as a practical approach for effective and prospective immune monitoring of the oral-genital mucosal tissues in NHP

Gating strategy for the identification of T cells subsets from blood and mucosal cytobrush samples.

<p>Representative gating strategy to analyze for CD3+ T cells and the CD4+ T cells subsets expressing surface (CCR5, CD103, HLA-DR) and intracellular markers (Ki67) from (A) blood and (B) oral cytobrush. (A, bottom panel) A representative plot with the gating of FMO controls for CD103, Ki67, HLA-DR and CCR5 performed on PBMCs is shown.</p

Monitoring changes in T cells subsets after vaccination in the rectal mucosa.

<p>Percentages of different T cell subsets in the oral mucosa were quantified at different time points after vaccination. (A) Percentages of CD3+, CD4+, CD8+ (CD3+, CD4-) T cells and, the percentages of CCR5+, Ki67+, HLA-DR+ and CD103+ among (B) CD4+ T cells and (C) CD8+ T cells are shown. Data represented as means ± SD (n = 8). <i>P</i> values: *<i>p</i><0.05, ** <i>p</i><0.005. One-way ANOVA was performed to detect statistical difference between groups.</p

Monitoring changes in T cells subsets after vaccination in the oral mucosa.

<p>Percentages of different T cell subsets in the oral mucosa were quantified at different time points after vaccination. (A) Percentages of CD3+, CD4+, CD8+ (CD3+, CD4-) T cells and, the percentages of CCR5+, Ki67+, HLA-DR+ and CD103+ among (B) CD4+ T cells and (C) CD8+ T cells are shown. Data represented as means ± SD (n = 8). <i>P</i> values: *<i>p</i><0.05, ** <i>p</i><0.005. One-way ANOVA was performed to detect statistical difference between groups.</p

Monitoring of adenovirus-infected cells collected at different time points in the blood and mucosal tissues.

<p>Using the GFP reporter gene expression, the percentages of GFP+ cells among (A) CD4+ and (B) CD8+ (CD3+CD4-) T cells were quantified at different time points after vaccination in the blood and mucosal tissues as shown. (A—B) Data represented as means ± SD (n = 8). One-way ANOVA was performed to detect statistical difference between groups.</p

Timeline of SIV challenge, SIV viral loads, and Ad vaccine immunization.

<p>Plasma viral loads for individual macaques at different time points after SIVmac251 challenge are shown as RNA copy equivalents/ml. At week 25 the animals were administered intranasal Ad vaccine as described in the methods section and the viral loads were monitored for the next four weeks as shown. These assays were performed at the NIH Core Facility by Dr. Jeff Lifson's group. The threshold sensitivity of the assay is 30 viral RNA copy-equivalents/ml of plasma, and the inter-assay variation is <25% (coefficient of variation).</p

Gating strategy for the identification of T cells subsets from blood and mucosal cytobrush samples.

<p>Representative gating strategy to analyze for CD3+ T cells and the CD4+ T cells subsets expressing surface (CCR5, CD103, HLA-DR) and intracellular markers (Ki67) from (A) rectal, (B) vaginal and (C) urethral cytobrushes.</p

Monitoring changes in T cells subsets after vaccination in the genital mucosa.

<p>Percentages of different T cell subsets in the urethral (●) and vaginal (■) mucosa were quantified at different time points after vaccination. (A) Percentages of CD3+, CD4+, CD8+ (CD3+, CD4-) T cells and, the percentages of CCR5+, Ki67+, HLA-DR+ and CD103+ among (B) CD4+ T cells and (C) CD8+ T cells are shown. Data represented as means ± SD (n = 8).</p

Monitoring changes in T cells subsets after vaccination in the blood.

<p>Percentages of different T cell subsets in the blood were quantified at different time points after vaccination. (A) Percentages of CD3+, CD4+, CD8+ (CD3+, CD4-) T cells and, the percentages of CCR5+, Ki67+, HLA-DR+ and CD103+ among (B) CD4+ T cells and (C) CD8+ T cells are shown. Data represented as means ± SD (n = 8). <i>P</i> values: *<i>p</i><0.05, ** <i>p</i><0.005, ****<i>p</i><0.00005. One-way ANOVA was performed to detect statistical difference between groups.</p