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.
"Moreover, while it is difficult to amplify the full TCR repertoire without either bias or compromised sensitivity, it is relatively straightforward to amplify specific VβCβ or VβJβ gene families with defined primer pairs with high sensitivity and minimal bias. Quantitative PCR methods could potentially be substituted to estimate Vβ usage (Sriram et al., 2007) or VβJβ usage (Wettstein et al., 2008) if live cells or flow cytometry equipment were not available. Finally, it can be very costly to prepare samples that contain equal, large numbers of wellpurified T cell subpopulations that are required for fully measuring absolute repertoire diversity. "
[Show abstract][Hide abstract] 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.
"Despite the capacity of spectratyping to both evaluate diversity and antigen-driven selection, technical complexities appear to have limited its wide-spread application, as evidenced by the drive to develop the alternative, hybridization-based approaches described above (7–9). Methods that utilize quantitative (real-time) RT-PCR have more recently been developed to quantitate the relative percentages of T cells that express individual BV genes (17,18), but these methods lack the resolution required to effectively evaluate repertoire diversity and efficiently identify overrepresented transcripts due to their BV gene-specific amplifications. The capacity of real-time PCR methodology to simultaneously monitor amplification in multiple reactions with high sensitivity offers an opportunity to develop TCR repertoire analysis with improved levels of resolution, speed and cost. "
[Show abstract][Hide abstract] 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. DOI:10.1093/nar/gkn634 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A subset of HIV-infected individuals who receive antiretroviral therapy (ART) develop a paradoxical pathological response that significantly increases morbidity and sometimes mortality. Following the induction of highly active ART, a rapid decline in the viral load results within weeks and coincides with a steep rise in the CD4(+) T-cell counts and immune hyperactivation. Although no mechanistic pathway has been elucidated for the development of immune reconstitution inflammatory syndrome (IRIS), it is thought that change in the nature of the immune response is a predominant factor in the development of reconstitution disease. In this article, we review the current state of knowledge in this field and provide a model for the development of IRIS.
International Journal of STD & AIDS 08/2009; 20(7):447-52. DOI:10.1258/ijsa.2009.008521 · 1.05 Impact Factor
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