Quantitative longitudinal analysis of T cell receptor repertoire expression in HIV-infected patients on antiretroviral and interleukin-2 therapy.
ABSTRACT We have developed a single-step reverse transcriptase kinetic PCR assay (kRT-PCR) to accurately determine the expression of each of the 24 TCRbetaV gene families in CD8(+) cells. We analyzed the long-term effects of highly active antiretroviral therapy (HAART) on the stability of the CD8(+) T cell receptor (TCR) repertoire in a cohort of 15 treated and 10 untreated individuals diagnosed with human immunodeficiency virus (HIV) infection. The CD4(+) TCR repertoire was studied in a second cohort receiving interleukin-2 infusions in addition to HAART. Analysis was based on kinetic (quantitative) reverse-transcription PCR (kRT-PCR) of the TCR variable B gene (TCRbetaV). Expression of each of the 24 Vbeta families was assessed at baseline immediately after infection and following initiation of HAART at 2, 4, 12, 24, and up to 192 weeks in 24-week intervals. Statistically significant family-specific expression changes were observed between treated and untreated individuals for 10 TCRbetaV families. Overall, when compared to untreated patients, a more stable expression of TCR genes was observed for HAART-treated individuals. Interestingly, this difference did not correlate with either CD4 or CD8 counts, which follow the expected curves for treated and untreated patients. When we applied our quantitative analysis to IL-2-treated patients we observed a rapid polyclonal activation of the repertoire. These results suggest that homeostasis in the T cell receptor repertoire is more robust in those patients who stay on HAART for a long time and confirm the polyclonal stimulating capacity of IL-2.
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ABSTRACT: T cell receptor (TCR) diversity is critical for adaptive immunity. Existing methods for measuring such diversity are qualitative, expensive, and/or of uncertain accuracy. Here, we describe a method and associated reagents for estimating the absolute number of unique TCR Vβ rearrangements present in a given number of cells or volume of blood. Compared to next generation sequencing, this method is rapid, reproducible, and affordable. Diversity of a sample is calculated based on three independent measurements of one Vβ-Jβ family of TCR rearrangements at a time. The percentage of receptors using the given Vβ gene is determined by flow cytometric analysis of T cells stained with anti-Vβ family antibodies. The percentage of receptors using the Vβ gene in combination with the chosen Jβ gene is determined by quantitative PCR. Finally, the absolute clonal diversity of the Vβ-Jβ family is determined with the AmpliCot method of DNA hybridization kinetics, by interpolation relative to PCR standards of known sequence diversity. These three component measurements are reproducible and linear. Using titrations of known numbers of input cells, we show that the TCR diversity estimates obtained by this approach approximate expected values within a two-fold error, have a coefficient of variation of 20%, and yield similar results when different Vβ-Jβ pairs are chosen. The ability to obtain accurate measurements of the total number of different TCR gene rearrangements in a cell sample should be useful for basic studies of the adaptive immune system as well as in clinical studies of conditions such as HIV disease, transplantation, aging, and congenital immunodeficiencies.Journal of immunological methods 03/2011; 368(1-2):45-53. · 2.35 Impact Factor
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ABSTRACT: The diversity of T-cell populations is determined by the spectrum of antigen-specific T-cell receptors (TCRs) that are heterodimers of alpha and beta subunits encoded by rearranged combinations of variable (AV and BV), joining (AJ and BJ), and constant region genes (AC and BC). We have developed a novel approach for analysis of beta transcript diversity in mice with a real-time PCR-based method that uses a matrix of BV- and BJ-specific primers to amplify 240 distinct BV-BJ combinations. Defined endpoints (Ct values) and dissociation curves are generated for each BV-BJ combination and the Ct values are consolidated in a matrix that characterizes the beta transcript diversity of each RNA sample. Relative diversities of BV-BJ combinations in individual RNA samples are further described by estimates of scaled entropy. A skin allograft system was used to demonstrate that dissection of repertoires into 240 BV-BJ combinations increases efficiency of identifying and sequencing beta transcripts that are overrepresented at inflammatory sites. These BV-BJ matrices should generate greater investigation in laboratory and clinical settings due to increased throughput, resolution and identification of overrepresented TCR transcripts.Nucleic Acids Research 11/2008; 36(21):e140. · 8.28 Impact Factor
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ABSTRACT: Monodispersed zinc oxide (ZnO) particles with various morphologies were successfully prepared via a simple solution route at low temperature. SEM, XRD, TEM, HRTEM, SAED, FTIR and UV–Vis DRS were used to characterize the morphology and microstructure of the products. Photocatalysis of the as-prepared ZnO powders was evaluated by degradation of methyl orange in an aqueous solution exposed to ultraviolet light. Results suggested a close relationship between the photocatalytic activity and the particle morphology and size.Graphical AbstractMonodispersed ZnO particles with various morphologies were prepared via a simple solution route. Photocatalysis of the as-prepared ZnO powders was evaluated by degradation of methyl orange in an aqueous solution exposed to ultraviolet light. Results suggested a close relationship between the photocatalytic activity and the particle morphology and size.Research Highlights►ZnO particles with various morphologies were prepared via a simple solution route. ►The morphology of ZnO particles depends on the crystal nucleation and growth rates. ►Photocatalytic activity of ZnO particles is related to their morphology and size.Powder Technology. 01/2011; 207:140-144.