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

Phospholipase Cγ1 is essential for T cell development, activation, and tolerance

Phospholipase Cγ1 (PLCγ1) is an important signaling effector of T cell receptor (TCR). To investigate the role of PLCγ1 in T cell biology, we generated and examined mice with T cell–specific deletion of PLCγ1. We demonstrate that PLCγ1 deficiency affects positive and negative selection, significantly reduces single-positive thymocytes and peripheral T cells, and impairs TCR-induced proliferation and cytokine production, and the activation of ERK, JNK, AP-1, NFAT, and NF-κB. Importantly, PLCγ1 deficiency impairs the development and function of FoxP3+ regulatory T cells, causing inflammatory/autoimmune symptoms. Therefore, PLCγ1 is essential for T cell development, activation, and tolerance

Functional Dichotomy between NKG2D and CD28-Mediated Co-Stimulation in Human CD8+ T Cells

Both CD28 and NKG2D can function as co-stimulatory receptors in human CD8+ T cells. However, their independent functional contributions in distinct CD8+ T cell subsets are not well understood. In this study, CD8+ T cells in human peripheral blood- and lung-derived lymphocytes were analyzed for CD28 and NKG2D expression and function. We found a higher level of CD28 expression in PBMC-derived naïve (CD45RA+CD27+) and memory (CD45RA−CD27+) CD8+ T cells (CD28Hi), while its expression was significantly lower in effector (CD45RA+CD27−) CD8+ T cells (CD28Lo). Irrespective of the differences in the CD28 levels, NKG2D expression was comparable in all three CD8+ T cell subsets. CD28 and NKG2D expressions followed similar patterns in human lung-resident GILGFVFTL/HLA-A2-pentamer positive CD8+ T cells. Co-stimulation of CD28Lo effector T cells via NKG2D significantly increased IFN-γ and TNF-α levels. On the contrary, irrespective of its comparable levels, NKG2D-mediated co-stimulation failed to augment IFN-γ and TNF-α production in CD28Hi naïve/memory T cells. Additionally, CD28-mediated co-stimulation was obligatory for IL-2 generation and thereby its production was limited only to the CD28Hi naïve/memory subsets. MICA, a ligand for NKG2D was abundantly expressed in the tracheal epithelial cells, validating the use of NKG2D as the major co-stimulatory receptor by tissue-resident CD8+ effector T cells. Based on these findings, we conclude that NKG2D may provide an expanded level of co-stimulation to tissue-residing effector CD8+ T cells. Thus, incorporation of co-stimulation via NKG2D in addition to CD28 is essential to activate tumor or tissue-infiltrating effector CD8+ T cells. However, boosting a recall immune response via memory CD8+ T cells or vaccination to stimulate naïve CD8+ T cells would require CD28-mediated co-stimulation

HLA-DM Mediates Epitope Selection by a “Compare-Exchange” Mechanism when a Potential Peptide Pool Is Available

BACKGROUND: HLA-DM (DM) mediates exchange of peptides bound to MHC class II (MHCII) during the epitope selection process. Although DM has been shown to have two activities, peptide release and MHC class II refolding, a clear characterization of the mechanism by which DM facilitates peptide exchange has remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: We have previously demonstrated that peptide binding to and dissociation from MHCII in the absence of DM are cooperative processes, likely related to conformational changes in the peptide-MHCII complex. Here we show that DM promotes peptide release by a non-cooperative process, whereas it enhances cooperative folding of the exchange peptide. Through electron paramagnetic resonance (EPR) and fluorescence polarization (FP) we show that DM releases prebound peptide very poorly in the absence of a candidate peptide for the exchange process. The affinity and concentration of the candidate peptide are also important for the release of the prebound peptide. Increased fluorescence energy transfer between the prebound and exchange peptides in the presence of DM is evidence for a tetramolecular complex which resolves in favor of the peptide that has superior folding properties. CONCLUSION/SIGNIFICANCE: This study shows that both the peptide releasing activity on loaded MHCII and the facilitating of MHCII binding by a candidate exchange peptide are integral to DM mediated epitope selection. The exchange process is initiated only in the presence of candidate peptides, avoiding possible release of a prebound peptide and loss of a potential epitope. In a tetramolecular transitional complex, the candidate peptides are checked for their ability to replace the pre-bound peptide with a geometry that allows the rebinding of the original peptide. Thus, DM promotes a "compare-exchange" sorting algorithm on an available peptide pool. Such a "third party"-mediated mechanism may be generally applicable for diverse ligand recognition in other biological systems