Gamma/Delta T cell mRNA levels decrease at mucosal sites and increase at lymphoid sites following an oral SIV infection of macaques

Abstract

The oral and esophageal mucosa have been identified as possible sites of HIV/SIV entry following oral infection. Here, gamma/delta (gammadelta) T cells, a multi-functional T cell subset, were assessed at oral/esophageal mucosa and lymphoid sites at the earliest times (1-14 days) post-oral SIV inoculation utilizing quantitative RT-PCR. During these earliest times post-infection, decreased gammadelta TCR mRNA levels were observed at the oral gingiva and esophageal mucosa, while increased levels were observed within regional lymph nodes (cervical and retropharyngeal). Higher lymph node gammadelta TCR levels were associated with increased mRNA expression of the lymphoid homing chemokine/receptor (CCL21/CCR7) pair in these lymph nodes. In contrast to gammadelta TCR levels, CD4 mRNA expression remained relatively stable through 4 days post-infection, and depletion of CD4 T cells was only evident after 7 or 14 days post-infection. The decrease of gammadelta T cell mRNA from mucosal sites and the corresponding increase at lymphoid sites suggest a rapid redistribution of these immune cells at these earliest times post-SIV infection.

Figure 1. Decreased Vδ1 and Vδ2 γδ TCR mRNA expression at the oral gingiva and esophageal mucosa during acute SIV infection

The fold change in Vδ1, Vδ2, and CD4 gene expression was assessed in oral (A and C) and esophageal (B and D) mucosal tissues of SIV+ rhesus macaques necropsied at the designated days following oral SIV inoculation. The mRNA levels shown are reported as fold change with regard to the average of mRNA levels in matched samples of four uninfected macaques. The grey shaded area represents a two standard deviation range of the average expression in uninfected macaques for Vδ1 (white bars) and Vδ2 (black bars) γδ T cells. Bars extending beyond the grey shaded area represent samples that are increased or decreased with regard to the uninfected controls.

Figure 2. Increased Vδ1 and Vδ2 γδ TCR mRNA expression detected in the cervical and retropharyngeal LNs during acute SIV infection

The fold change in Vδ1, Vδ2, and CD4 gene expression was assessed in the cervical (A and D), retropharyngeal (B and E), and axillary (C and F) lymph nodes of SIV+ rhesus macaques necropsied at the designated days following oral SIV inoculation. Changes in Vδ1 TCR expression are in white bars while changes in Vδ2 TCR expression are in black bars. The mRNA levels shown are reported as fold change with regard to the average of mRNA levels in matched samples of four uninfected macaques. The grey shaded area represents a two standard deviation range of the average expression in uninfected macaques. Bars extending beyond the grey shaded area represent samples that are increased or decreased with regard to the uninfected controls.

Figure 3. Decreased Vδ1 and Vδ2 γδ TCR mRNA expression at the oral gingiva and esophageal mucosa at 7 and 14 days post-oral SIV infection

The fold change in Vδ1, Vδ, and CD4 gene expression was assessed in oral gingiva and esophageal mucosa as well as the cervical and retropharyngeal lymph nodes at 7 (A and C) and 14 (B and D) days following SIV oral inoculation from samples obtained at necropsy. Changes in Vδ1 TCR expression are in white bars while changes in Vδ2 TCR expression are in black bars (A, B), mRNA expression of CD4 is also depicted (C, D). The mRNA levels shown are reported as fold change with regard to the average of mRNA levels in matched samples of four uninfected macaques. The asterisks (*) represent gene expression that was outside of the two standard deviation range determined from uninfected macaques.

Figure 4. Assessment of Vδ1 and Vδ2 mRNA levels within the peripheral blood during acute SIV infection

Quantitative real-time PCR was utilized to assess the mRNA levels of (A) Vδ1 and (B) Vδ2 in PBMC obtained from macaques analyzed throughout this study (RM1 – RM6) as well as from other macaques that had been orally inoculated with the same SIV isolate from which blood samples were available. Timepoints examined include 1 day post-infection (DPI), 2 DPI, 4 DPI, 7 DPI and 14–15 DPI. The mRNA levels shown are reported as fold change with regard to the average of mRNA levels in matched samples of seven uninfected macaques. The grey shaded area represents a two standard deviation range of the average expression from uninfected macaques. Bars extending beyond the grey shaded area represent samples that are increased or decreased with regard to the uninfected controls.

Figure 5. Increased CCL21 and CCR7 mRNA expression at the cervical and retropharyngeal LNs during acute SIV infection

Quantitative real-time PCR was utilized to assess the mRNA levels of (A) CCL21/6Ckine, (B) CCR7, (C) CCL5/RANTES and (D) CCR5 in the retropharyngeal (white bars) and cervical (black bars) LNs obtained at necropsied at the designated days following oral SIV inoculation of macaques. The mRNA levels shown are reported as fold change with regard to the average of mRNA levels in matched samples of four uninfected macaques. The grey shaded area represents a two standard deviation range of the average expression from uninfected macaques. Bars extending beyond the grey shaded area represent samples that are increased or decreased with regard to the uninfected controls.

Figure 6. Decreased percentage of γδ T cells from HIV+ patients expressing CCR7 and CD62L

Peripheral blood mononuclear cells from uninfected (n=13) and HIV+ (n=14) patients were assessed utilizing flow cytometry to identify the percentage of Vδ2+ and Vδ2^neg γδ T cells expressing either CCR7 or CD62L. The percentage of Vδ2+ (white bars) or Vδ2^neg (black bars) cells expressing either CCR7 or CD62L are shown. CCR7 expression (left panel) was significantly decreased on Vδ2^neg γδ T cells in HIV+ patients compared to uninfected controls. CD62L expression (right panel) was significantly decreased on both Vδ2+ and Vδ2^neg γδ T cells in HIV+ patients compared to uninfected controls. A Mann-Whitney t test at a 95% confidence interval was utilized to determine statistical significance (p < 0.05) between the uninfected and HIV+ groups.