Quantitative Measurement of Pathogen-Specific Human Memory T cell Repertoire Diversity Using a CDR3 beta-specific Microarray

BACKGROUND: Providing quantitative microarray data that is sensitive to very small differences in target sequence would be a useful tool in any number of venues where a sample can consist of a multiple related sequences present in various abundances. Examples of such applications would include measurement of pseudo species in viral infections and the measurement of species of antibodies or T cell receptors that constitute immune repertoires. Difficulties that must be overcome in such a method would be to account for cross-hybridization and for differences in hybridization efficiencies between the arrayed probes and their corresponding targets. We have used the memory T cell repertoire to an influenza-derived peptide as a test case for developing such a method. RESULTS: The arrayed probes were corresponded to a 17 nucleotide TCR-specific region that distinguished sequences differing by as little as a single nucleotide. Hybridization efficiency between highly related Cy5-labeled subject sequences was normalized by including an equimolar mixture of Cy3-labeled synthetic targets representing all 108 arrayed probes. The same synthetic targets were used to measure the degree of cross hybridization between probes. Reconstitution studies found the system sensitive to input ratios as low as 0.5% and accurate in measuring known input percentages (R2 = 0.81, R = 0.90, p \u3c 0.0001). A data handling protocol was developed to incorporate the differences in hybridization efficiency. To validate the array in T cell repertoire analysis, it was used to analyze human recall responses to influenza in three human subjects and compared to traditional cloning and sequencing. When evaluating the rank order of clonotype abundance determined by each method, the approaches were not found significantly different (Wilcoxon rank-sum test, p \u3e 0.05). CONCLUSION: This novel strategy appears to be robust and can be adapted to any situation where complex mixtures of highly similar sequences need to be quantitatively resolved

Age-Based Dynamics of a Stable Circulating Cd8 T Cell Repertoire Component

T-cell memory to pathogens can be envisioned as a receptor-based imprint of the pathogenic environment on the naive repertoire of clonotypes. Recurrent exposures to a pathogen inform and reinforce memory, leading to a mature state. The complexity and temporal stability of this system in man is only beginning to be adequately described. We have been using a rank-frequency approach for quantitative analysis of CD8 T cell repertoires. Rank acts as a proxy for previous expansion, and rank-frequency, the number of clonotypes at a particular rank, as a proxy for abundance, with the relation of the two estimating the diversity of the system. Previous analyses of circulating antigen-experienced cytotoxic CD8 T-cell repertoires from adults have shown a complex two-component clonotype distribution. Here we show this is also the case for circulating CD8 T cells expressing the BV19 receptor chain from five adult subjects. When the repertoire characteristic of clonotype stability is added to the analysis, an inverse correlation between clonotype rank frequency and stability is observed. Clonotypes making up the second distributional component are stable; indicating that the circulation can be a depot of selected clonotypes. Temporal repertoire dynamics was further examined for influenza-specific T cells from children, middle-aged, and older adults. Taken together, these analyses describe a dynamic process of system development and aging, with increasing distributional complexity, leading to a stable circulating component, followed by loss of both complexity and stability

Disproportional Effects in Populations of Concern for Pandemic Influenza: Insights from Seasonal Epidemics in Wisconsin, 1967-2004

Influenza infections pose a serious burden of illness in the United States. We explored age, influenza strains, and seasonal epidemic curves in relation to influenza associated mortality

Changes in Russian managerial values: a test of the convergence hypothesis?

This paper considers how Russian managerial values are developing in the context of the sweeping economic, political and social changes associated with the transition of Russia to a market economy. By replicating earlier research (Holt et al., 1994; Ralston et al., 1997), it was possible to overcome the weaknesses of previous cross-sectional studies by tracking changes in Russian managers' values over time. The paper concludes that some convergence between the values of Russian and US managers can be observed, but that the form of this convergence is not uniform. In addition, the way in which Russian managers act upon these values in the context of their own national context means that considerable divergence in managerial behaviour is still evident. Implications for international human resource management are discussed

Ecological and environmental transition across the forested-to-open bog ecotone in a west Siberian peatland

Climate change may cause increasing tree cover in boreal peatlands, and the impacts of this encroachment will be noted first at forested-to-open bog ecotones. We investigate key metrics of ecosystem function in five such ecotones at a peatland complex in Western Siberia. Stratigraphic analysis of three cores from one of these transects shows that the ecotone has been dynamic over time with evidence for recent expansion of forested peatland. We observed that the two alternative states for northern boreal peatlands (forested/open) clearly support distinct plant and microbial communities. These in turn drive and respond to a number of feedback mechanisms. This has led to steep ecological gradients across the ecotones. Tree cover was associated with lower water tables and pH, along with higher bulk density, aquatic carbon concentrations, and electrical conductivity. We propose that the conditions found in the forested peatland of Western Siberia make the carbon sink more vulnerable to warmer and drier conditions

A fractal clonotype distribution in the CD8+ memory T cell repertoire could optimize potential for immune responses

The nature of CD8(+) T cell memory is still incompletely understood. We have previously reported that the response to an HLA-A2-restricted influenza-derived peptide results in a complex T cell repertoire. In this study we extend this analysis and describe the repertoire with more rigor. In one individual we defined 141 distinct T cell clonotypes on the basis of the unique DNA sequence of the third complementarity-determining region of the TCR beta-chain. The frequency distribution of the clonotypes is not what is expected of a normal distribution but is characterized by a large low-frequency tail. The existence of a complex population indicates a mechanism for maintaining a large number of Ag-specific clonotypes at a low frequency in the memory pool. Ranking the clonotypes allowed us to describe the population in terms of a power law-like distribution with a parameter of decay of approximately 1.6. If the repertoire is divided into subsets, such as clonotypes that use BJ2.7 or those whose third complementarity-determining region encodes the amino acid sequence IRSS, the clonotype frequencies could also be described by a power law-like distribution. This indicates a self similarity to the repertoire in which smaller pieces are slightly altered copies of the larger piece. The power law-like description is stable with time and was observed in a second individual. The distribution of clonotypes in the repertoire could be mapped onto a polygonal spiral using a recursive algorithm. Self similarity, power laws, and recursive mapping algorithms are associated with fractal systems. Thus, Ag-specific memory CD8 T cell repertoires can be considered as fractal, which could indicate optimized flexibility and robustness