Alina Lelic

Blood CD33(+)HLA‐DR(−) myeloid‐derived suppressor cells are increased with age and a history of cancer

As we age, the composition of our peripheral leukocytes changes dramatically. Many of these alterations contribute to the general immune dysfunction that burdens the elderly, which in turn, contributes to increased susceptibility to disease. MDSCs represent a heterogeneous population of immunosuppressive leukocytes that are elevated in the peripheral blood of cancer patients. Given the relation between cancer incidence and age, this study examined the frequency of peripheral blood CD33(+)HLA‐DR(−) MDSCs across three cohorts: healthy adults (19–59 years old), community‐dwelling seniors (61–76 years old), and frail elderly (67–99 years old). This analysis is the first to demonstrate that MDSCs and specifically the CD11b(+)CD15(+) MDSC subset are increased with age. Proinflammatory cytokines that are required for the differentiation of MDSCs (e.g., TNF‐α, IL‐6, and IL‐1β) were similarly found to be increased in the serum of the frail elderly. Furthermore, the proportion of MDSCs and the CD11b(+)CD15(+) subset were found to be elevated significantly in elderly donors with a history of cancer. This age‐related elevation in the frequency of MDSCs may contribute to the increased cancer incidence that occurs with age. Further investigation into the functional consequences of elevated MDSCs will provide valuable insight into the progression of age‐related pathologies.

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

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.

Epitope discovery in West Nile virus infection: Identification and immune recognition of viral epitopes

Cytotoxic T lymphocytes (CTL) play an important role in the control and elimination of infection by West Nile virus (WNV), yet the class I human leukocyte antigen (HLA)-presented peptide epitopes that enable CTL recognition of WNV-infected cells remain uncharacterized. The goals of this work were first to discover the peptide epitopes that distinguish the class I HLA of WNV-infected cells and then to test the T cell reactivity of newly discovered WNV epitopes. To discover WNV-immune epitopes, class I HLA was harvested from WNV (NY99 strain)-infected and uninfected HeLa cells. Then peptide epitopes were eluted from affinity-purified HLA, and peptide epitopes from infected and uninfected cells were comparatively mapped by mass spectroscopy. Six virus-derived peptides from five different viral proteins (E, NS2b, NS3, NS4b, and NS5) were discovered as unique to HLA-A*0201 of infected cells, demonstrating that the peptides sampled by class I HLA are distributed widely throughout the WNV proteome. When tested with CTL from infected individuals, one dominant WNV target was apparent, two epitopes were subdominant, and three demonstrated little CTL reactivity. Finally, a sequence comparison of these epitopes with the hundreds of viral isolates shows that HLA-A*0201 presents epitopes derived from conserved regions of the virus. Detection and recovery from WNV infection are therefore functions of the ability of class I HLA molecules to reveal conserved WNV epitopes to an intact cellular immune system that subsequently recognizes infected cells.

Alterations to the Frequency and Function of Peripheral Blood Monocytes and Associations with Chronic Disease in the Advanced-Age, Frail Elderly

Background 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. Design 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. Results 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. Conclusions 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.

The Memory T Cell Response to West Nile Virus in Symptomatic Humans following Natural Infection Is Not Influenced by Age and Is Dominated by a Restricted Set of CD8+ T Cell Epitopes

We examined the West Nile virus (WNV)-specific T cell response in a cohort of 52 patients with symptomatic WNV infections, including neuroinvasive and non-invasive disease. Although all virus proteins were shown to contain T cell epitopes, certain proteins, such as E, were more commonly targeted by the T cell response. Most patients exhibited reactivity toward 3–4 individual WNV peptides; however, several patients exhibited reactivity toward >10 individual peptides. The relative hierarchy of T cell reactivities in all patients showed a fixed pattern that was sustained throughout the 12-mo period of the current study. Surprisingly, we did not observe any relationship between age and either the breadth or magnitude of the T cell response following infection. We also did not observe a relationship between disease severity and either the breadth or magnitude of the T cell response. The T cell epitopes were distributed in a non-random fashion across the viral polyprotein and a limited number of epitopes appeared to dominate the CD8+ T cell response within our cohort. These data provide important new insight into the T cell response against WNV in humans.

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

Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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.

An Introduction to Automated Flow Cytometry Gating Tools and Their Implementation.

Current flow cytometry (FCM) reagents and instrumentation allow for the measurement of an unprecedented number of parameters for any given cell within a homogenous or heterogeneous population. While this provides a great deal of power for hypothesis testing, it also generates a vast amount of data, which is typically analyzed manually through a processing called “gating.” For large experiments, such as high-content screens, in which many parameters are measured, the time required for manual analysis as well as the technical variability inherent to manual gating can increase dramatically, even becoming prohibitive depending on the clinical or research goal. In the following article, we aim to provide the reader an overview of automated FCM analysis as well as an example of the implementation of FLOw Clustering without K, a tool that we consider accessible to researchers of all levels of computational expertise. In most cases, computational assistance methods are more reproducible and much faster than manual gating, and for some, also allow for the discovery of cellular populations that might not be expected or evident to the researcher. We urge any researcher who is planning or has previously performed large FCM experiments to consider implementing computational assistance into their analysis pipeline.

Deciphering epitope specificities within polyserum using affinity selection of random peptides and a novel algorithm based on pattern recognition theory.

While numerous strategies have been developed to map epitope specificities for monoclonal antibodies, few have been designed for elucidating epitope specificity within complex polysera. We have developed a novel algorithm based on pattern recognition theory that can be used to characterize the breadth of epitope specificities within a polyserum based on affinity selection of random peptides. To attribute these random peptides to a specific epitope, the sequences of the affinity-selected peptides were matched against a database of random peptides selected using well-described monoclonal antibodies. To test this novel algorithm, we employed polyserum from patients infected with West Nile virus and isolated 109 unique sequences which were recognized selectively by serum from West Nile virus-infected patients but not uninfected patients. Through application of our algorithm, it was possible to match 20% of the polyserum-selected peptides to the database of peptides isolated by affinity selection using monoclonal antibodies against the virus envelope protein. Statistical analysis demonstrated that the peptides selected with the polyserum could not be attributed to the peptide database by chance. This novel algorithm provides the basis for further development of methods to characterize the breadth of epitope recognition within a complex pool of antibodies.

Serum C-Reactive Protein and Congestive Heart Failure as Significant Predictors of Herpes Zoster Vaccine Response in Elderly Nursing Home Residents

Background Elderly long-term care residents often exhibit a myriad of risk factors for immune dysfunction, including chronic inflammation and multiple comorbid conditions, which undoubtedly contribute to their enhanced susceptibility to infection. Hence, understanding the factors required for optimal vaccine responsiveness is critical. Methods We examined 187 elderly nursing home residents (aged 80-102 years) and 50 community-dwelling seniors (aged 60-75 years) immunized with the live-attenuated varicella-zoster virus (VZV) vaccine. Specifically, we examined whether vaccine responsiveness was associated with serum C-reactive protein (CRP), tumor necrosis factor, interleukin 1β, 6, and 10, leukocyte telomere length, chronic disease status, and frailty. Results Elderly participants had significantly higher levels of CRP, tumor necrosis factor, and interleukin 6 and shorter leukocyte telomere length. Vaccine responsiveness was inversely related to the CRP level in elderly participants, but not seniors, and those with congestive heart failure were less likely to achieve a 2-fold response (odds ratio, 0.08). The latter relationship is probably due to immunosenescence, because heart failure was associated with increased senescent CD4+ T cells, and reduced naive and effector and central memory CD8+ T cells. Conclusions In summary, these data improve our understanding of vaccine responsiveness for those in long-term care, suggesting that certain risk factors are associated with a greater likelihood of vaccine failure.