Surface Phenotype and Functionality of WNV Specific T Cells Differ with Age and Disease Severity

West Nile virus (WNV) infection can result in severe neuroinvasive disease, particularly in persons with advanced age. As rodent models demonstrate that T cells play an important role in limiting WNV infection, and strong T cell responses to WNV have been observed in humans, we postulated that inadequate antiviral T cell immunity was involved in neurologic sequelae and the more severe outcomes associated with age. We previously reported the discovery of six HLA-A*0201 restricted WNV peptide epitopes, with the dominant T cell targets in naturally infected individuals being SVG9 (Env) and SLF9 (NS4b). Here, memory phenotype and polyfunctional CD8+ T cell responses to these dominant epitopes were assessed in 40 WNV seropositive patients displaying diverse clinical symptoms. The patients' PBMC were stained with HLA-I multimers loaded with the SVG9 and SLF9 epitopes and analyzed by multicolor flow cytometry. WNV-specific CD8+ T cells were found in peripheral blood several months post infection. The number of WNV-specific T cells in older individuals was the same, if not greater, than in younger members of the cohort. WNV-specific T cells were predominantly monofunctional for CD107a, MIP-1β, TNFα, IL-2, or IFNγ. When CD8+ T cell responses were stratified by disease severity, an increased number of terminally differentiated, memory phenotype (CD45RA+ CD27− CCR7− CD57+) T cells were detected in patients suffering from viral neuroinvasion. In conclusion, T cells of a terminally differentiated/cytolytic profile are associated with neuroinvasion and, regardless of age, monofunctional T cells persist following infection. These data provide the first indication that particular CD8+ T cell phenotypes are associated with disease outcome following WNV infection

Identification of CD8+ T Cell Epitopes in the West Nile Virus Polyprotein by Reverse-Immunology Using NetCTL

West Nile virus (WNV) is a growing threat to public health and a greater understanding of the immune response raised against WNV is important for the development of prophylactic and therapeutic strategies.In a reverse-immunology approach, we used bioinformatics methods to predict WNV-specific CD8(+) T cell epitopes and selected a set of peptides that constitutes maximum coverage of 20 fully-sequenced WNV strains. We then tested these putative epitopes for cellular reactivity in a cohort of WNV-infected patients. We identified 26 new CD8(+) T cell epitopes, which we propose are restricted by 11 different HLA class I alleles. Aiming for optimal coverage of human populations, we suggest that 11 of these new WNV epitopes would be sufficient to cover from 48% to 93% of ethnic populations in various areas of the World.The 26 identified CD8(+) T cell epitopes contribute to our knowledge of the immune response against WNV infection and greatly extend the list of known WNV CD8(+) T cell epitopes. A polytope incorporating these and other epitopes could possibly serve as the basis for a WNV vaccine

Alterations to the frequency and function of peripheral blood monocytes and associations with chronic disease in the advanced-age, frail elderly.

Circulating myeloid cells are important mediators of the inflammatory response, acting as a major source of resident tissue antigen presenting cells and serum cytokines. They represent a number of distinct subpopulations whose functional capacity and relative concentrations are known to change with age. Little is known of these changes in the very old and physically frail, a rapidly increasing proportion of the North American population.In the following study the frequency and receptor expression of blood monocytes and dendritic cells (DCs) were characterized in a sample of advanced-age, frail elderly (81-100 yrs), and compared against that of adults (19-59 yrs), and community-dwelling seniors (61-76 yrs). Cytokine responses following TLR stimulation were also investigated, as well as associations between immunophenotyping parameters and chronic diseases.The advanced-age, frail elderly had significantly fewer CD14(++) and CD14(+)CD16(+), but not CD14(++)CD16(+) monocytes, fewer plasmacytoid and myeloid DCs, and a lower frequency of monocytes expressing the chemokine receptors CCR2 and CX3CR1. At baseline and following stimulation with TLR-2 and -4 agonists, monocytes from the advanced-age, frail elderly produced more TNF than adults, although the overall induction was significantly lower. Finally, monocyte subset frequency and CX3CR1 expression was positively associated with dementia, while negatively associated with anemia and diabetes in the advanced-age, frail elderly.These data demonstrate that blood monocyte frequency and phenotype are altered in the advanced-age, frail elderly and that these changes correlate with certain chronic diseases. Whether these changes contribute to or are caused by these conditions warrants further investigation

Associations between blood monocyte and DC markers and disease in the advanced-age, frail elderly.

<p>Odds ratios and 95% confidence intervals presented. Only comparison-wise (logistic regression) significance at p<0.25 shown;</p><p>*p<0.05,</p><p>**p<0.01. Bolded observations indicate experimental-wise (Benjamini-Hochberg FDR) significance at p<0.15. CD45%, percentage of cells relative to CD45+ PBMCs; MFI, mean fluorescent intensity.</p

The Polyfunctionality of Human Memory CD8+ T Cells Elicited by Acute and Chronic Virus Infections Is Not Influenced by Age

<div><p>As humans age, they experience a progressive loss of thymic function and a corresponding shift in the makeup of the circulating CD8+ T cell population from naïve to memory phenotype. These alterations are believed to result in impaired CD8+ T cell responses in older individuals; however, evidence that these global changes impact virus-specific CD8+ T cell immunity in the elderly is lacking. To gain further insight into the functionality of virus-specific CD8+ T cells in older individuals, we interrogated a cohort of individuals who were acutely infected with West Nile virus (WNV) and chronically infected with Epstein Barr virus (EBV) and Cytomegalovirus (CMV). The cohort was stratified into young (<40 yrs), middle-aged (41–59 yrs) and aged (>60 yrs) groups. In the aged cohort, the CD8+ T cell compartment displayed a marked reduction in the frequency of naïve CD8+ T cells and increased frequencies of CD8+ T cells that expressed CD57 and lacked CD28, as previously described. However, we did not observe an influence of age on either the frequency of virus-specific CD8+ T cells within the circulating pool nor their functionality (based on the production of IFNγ, TNFα, IL2, Granzyme B, Perforin and mobilization of CD107a). We did note that CD8+ T cells specific for WNV, CMV or EBV displayed distinct functional profiles, but these differences were unrelated to age. Collectively, these data fail to support the hypothesis that immunosenescence leads to defective CD8+ T cell immunity and suggest that it should be possible to develop CD8+ T cell vaccines to protect aged individuals from infections with novel emerging viruses.</p> </div

Cytokine production, but not induction, is elevated in monocytes from the advanced-age, frail elderly as compared to young adults.

<p>PBMCs were stimulated with mock (PBS), and TLR-2 (Pam3CSK4, Pam) and TLR-4 (LPS) agonists, and the production of A) IL-1β, B) IL-6, C) IL-8 and D) TNF in classical (CLS), intermediate (INT) and non-classical (NON) monocytes was measured by flow cytometry. Relative to mock, the induction of E) TNF was significantly lower in the advanced-age, frail elderly. n = 5–8 per group, per treatment. Comparison-wise p-value, ***p<0.001, **p<0.01, *p<0.05.</p

Representation of the differences in A) the expression of CX₃CR1 on the classical (CLS), intermediate (INT) and non-classical (NON) monocyte subsets and B) the classical to intermediate monocyte ratio, between cases (grey) and controls (white) for dementia, diabetes mellitus and anemia.

<p>Comparison-wise p-value, **p<0.01, *p<0.05.</p

Summary of the gating strategy to define blood monocyte (upper panel) and dendritic cell (lower panel) subsets.

<p>Monocytes were defined as CD45 and HLA-DR expressing and lineage (CD2, CD3, CD15, CD19, CD56, NKp46) negative. Dendritic cell subsets were defined as CD45 and HLA-DR expressing, lineage (CD3, CD15, CD19, CD56) negative, and CD123 bright plasmacytoid dendritic cells (pDCs) or CD123 low, and CD1c or CD141 expressing myeloid dendritic cells (mDCs).</p