Purpose: The majority of circulating human gdT lymphocytes are of the Vg9Vd2 lineage, and have T-cell receptor (TCR) specificity for nonpeptide phosphoantigens. Previous attempts to stimulate and expand these cells have therefore focused on stimulation using ligands of the Vg9Vd2 receptor, whereas relatively little is known about variant blood gdT subsets and their potential role in cancer immunotherapy.Experimental Design: To expand the full repertoire of gdT without bias toward specific TCRs, we made use of artificial antigen-presenting cells loaded with an anti gdTCR antibody that promoted unbiased expansion of the gdT repertoire. Expanded cells from adult blood donors were sorted into 3 populations expressing respectively Vd2 TCR chains (Vd2
Catalase is an endogenous antioxidant enzyme that neutralizes hydrogen peroxide and is induced by oxidative challenge. A À262C ! T polymorphism in the promoter region of the gene (CAT) is associated with risk of several conditions related to oxidative stress. We sought to determine the functional effects of the CAT polymorphism on enzyme activity in erythrocytes and the potential modifying effects of demographic and lifestyle factors on genotype/ phenotype relationships, using specimens and data from controls from breast and prostate cancer studies in Arkansas (n = 420). There was a dose-response reduction in catalase activity by genotype, with geometric means of 115.4 units/mg hemoglobin for those with CC genotypes, 82.1 units/mg for those with CT genotypes, and 73.5 units/mg for those with TT genotypes. Associations were only observed among Caucasians (P < 0.0001), with no effects among African Americans (P = 0.91), and were stronger among women than men, although numbers in stratified analyses were small. Differences in catalase activity by genotype were most pronounced among those in the highest tertiles of consumption of fruits and vegetables (À35%, P = 0.003), with weaker relationships among those who were lower consumers (À21.8%, P = 0.16). Among those with CC genotypes, there was no change in activity by consumption, but there were notable decreases in activity by tertiles of consumption for those with at least one T allele. These data indicate that the CAT À262C ! T polymorphism predicts a portion of catalase phenotype, which may be limited to Caucasians. Associations between genotype and phenotype were modified by dietary factors, illustrating the biochemical complexity of studies of genetic polymorphisms and disease risk. (Cancer Epidemiol Biomarkers Prev 2006;15(6):1217 -22)
BackgroundDeep-sequencing methods are rapidly developing in the field of B-cell receptor (BCR) and T-cell receptor (TCR) diversity. These promise to revolutionise our understanding of adaptive immune dynamics, identify novel antibodies, and allow monitoring of minimal residual disease. However, different methods for BCR and TCR enrichment and amplification have been proposed. Here we perform the first systematic comparison between different methods of enrichment, amplification and sequencing for generating BCR and TCR repertoires using large sample numbers.ResultsResampling from the same RNA or cDNA pool results in highly correlated and reproducible repertoires, but resampling low frequency clones leads to stochastic variance. Repertoires generated by different sequencing methods (454 Roche and Illumina MiSeq) and amplification methods (multiplex PCR, 5’ Rapid amplification of cDNA ends (5’RACE), and RNA-capture) are highly correlated, and resulting IgHV gene frequencies between the different methods were not significantly different. Read length has an impact on captured repertoire structure, and ultimately full-length BCR sequences are most informative for repertoire analysis as diversity outside of the CDR is very useful for phylogenetic analysis. Additionally, we show RNA-based BCR repertoires are more informative than using DNA.ConclusionsRepertoires generated by different sequencing and amplification methods are consistent, but we show that read lengths, depths and error profiles should be considered in experimental design, and multiple sampling approaches could be employed to minimise stochastic sampling variation. This detailed investigation of immune repertoire sequencing methods is essential for informing basic and clinical research.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-014-0029-0) contains supplementary material, which is available to authorized users.
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