Immune biomarkers predictive of respiratory viral infection in elderly nursing home residents

Abstract

Objective: To determine if immune phenotypes associated with immunosenescence predict risk of respiratory viral infection in elderly nursing home residents.

Methods: Residents ≥ 65 years from 32 nursing homes in 4 Canadian cities were enrolled in Fall 2009, 2010 and 2011, and followed for one influenza season. Following influenza vaccination, peripheral blood mononuclear cells (PBMCs) were obtained and analysed by flow cytometry for T-regs, CD4+ and CD8+ T-cell subsets (CCR7+CD45RA+, CCR7-CD45RA+ and CD28-CD57+) and CMV-reactive CD4+ and CD8+ T-cells. Nasopharyngeal swabs were obtained and tested for viruses in symptomatic residents. A Cox proportional hazards model adjusted for age, sex and frailty, determined the relationship between immune phenotypes and time to viral infection.

Results: 1072 residents were enrolled; median age 86 years and 72% female. 269 swabs were obtained, 87 were positive for virus: influenza (24%), RSV (14%), coronavirus (32%), rhinovirus (17%), human metapneumovirus (9%) and parainfluenza (5%). In multivariable analysis, high T-reg% (HR 0.41, 95% CI 0.20-0.81) and high CMV-reactive CD4+ T-cell% (HR 1.69, 95% CI 1.03-2.78) were predictive of respiratory viral infection.

Conclusions: In elderly nursing home residents, high CMV-reactive CD4+ T-cells were associated with an increased risk and high T-regs were associated with a reduced risk of respiratory viral infection.

Figure 1. Gating strategy for T-cell phenotypes.

T cell phenotypes were defined using the flowDensity software package. Lymphocytes were first gated from non-margin events, and then singlets were gated. CD45RA thresholds were calculated based on singlet lymphocytes FMO. CD3+ cells were gated and then separated into CD4+CD8- and CD4-CD8+. Expression of CD57, CD28, CD45RA and CCR7 was analyzed on either CD4+CD8- or CD8-CD4+.

Figure 2. Gating strategy of T-reg.

Lymphocytes and singlets were selected. Gates were then set up for CD3+ cells and CD4+. To identify the T-regs, a gate was set up to select CD25+CD127 ^lo/− and T-regs were defined as CD25+CD127 ^lo/− FOXP3+.

Figure 3. Gating strategy for CMV-reactive T cells.

PBMC were stimulated with pp65 peptides to identify CMV-reactive T-cells. As a negative control, PBMC were stimulated with DMSO. Subsequently, the T-cells were stained for surface markers and intracellular cytokines. To define the CMV-reactive T-cells, the flow data was gated on singlet lymphocytes (as shown in Figures 1 and 2) and subsequently gated on CD3+CD8+ cells and CD3+CD4+. The plots show intracellular cytokine staining results for a single patient. CMV-reactive T-cells were defined as IFN-γ+ TNF-α+.

Figure 4. Time to respiratory viral infection stratified by a) T-reg%, adjusted for age, sex, frailty and CMV-reactive CD4+ T-cell%) and b) CMV-reactive CD4+ T-cell%, adjusted for age, sex, frailty and T-reg%.