T cell receptor (TCR) Vβ gene usage in bronchoalveolar lavage and peripheral blood T cells from asthmatic and normal subjects

Abstract

T cells are thought to play an important regulatory role in asthma, but little is known about the T cell repertoire of the human lung or whether asthma is associated with any specific repertoire changes. Flow cytometry and MoAbs to TCR VB (TCRBV) families were used to quantify bronchoalveolar lavage (BAL) and blood T cells from normal and atopic individuals. Clonality was then assessed by polymerase chain reaction (PCR) amplification of cDNA and gene scanning using consensus and family-specific TCRBV primers and confirmed by sequence analysis. In addition, blood and BAL T cell populations were studied pre- and post-allergen challenge in four patients with allergic asthma. The majority of TCRBV families detected in blood by MoAb staining were also represented in BAL. While differences between BAL and blood populations were evident in each individual studied, these differences were not consistent between individuals or between CD4+ and CD8+ T cell subpopulations. These results are in broad agreement with other published studies, but in contrast to previous work we found a consistent difference between TCRBV7 family usage in blood and BAL in all individuals studied, and a consistently increased proportion of CD4+ BAL T cells bearing BV5S2/3 in asthmatics only. After allergen challenge, the pattern of TCRBV gene usage was largely unchanged as judged by flow cytometry. Gene scanning of PCR products generated from consensus VB primers revealed polyclonal lymphocyte populations in blood and BAL from all seven atopic individuals: in one normal tested polyclonal populations were found in blood and oligoclonal populations in BAL. Selected families amplified with family-specific primers BV5S2/3, BV6 and BV7 (chosen because of their predominance in BAL compared with blood) were more variable and revealed predominant polyclonal populations in blood and polyclonal or oligoclonal populations in BAL. In one asthmatic patient a clonal BV5S2 family was found in BAL. Following allergen challenge there were no significant changes in polyclonality/oligoclonality/clonality in three cases, but in one case a clonal BV5S2 population was found after challenge, that had not been evident beforehand. The lung T cell repertoire is thus broadly representative of blood T cells, but shows population differences that may result from response to persistent exposure to airborne antigens common to normal and atopic individuals. Oligoclonal TCRBV family expansion appears to be primarily lung-specific but independent of atopic asthma, although our challenge data in one case support the concept that clonal populations may follow local allergen challenge. These data are consistent with selection and amplification of specific T cell families in the lung in response to local antigenic exposure.

Molecular and flow cytometric analysis of the Vβ repertoire for clonality assessment in mature TCRαβ T-cell proliferations

“…18,21,22,26,29,30 However, a much faster and more quantitative analysis of the TCR␤ repertoire is now possible through the use of mAbs directed against the V␤ domains of TCR␣␤ molecules, which cover 65% to 70% of V␤ domains in blood T lymphocytes of children and adults. 10,[31][32][33][34] We performed parallel molecular and flow cytometric V␤ analyses in a series of SB-defined TCR␣␤ T-ALL samples and T-cell lines to validate both approaches for the detection of clonality. Indeed, clonal V␤-C␤ transcripts could easily be detected in all T-ALL samples and T-cell lines.…”

TCR usage and cytokine expression in peripheral blood and BAL T cells

“…Although we did not assess clonality in PB or BAL in this study, we have previously shown that the PB repertoire is polyclonal in both normal and asthmatic subjects, with a few clonal expansions in BAL before and after allergen challenge [20]. In the present study, we were particularly careful to study normal subjects and mild asthmatics, who had not had any recent respiratory infection and were stable as regards their disease activity status.…”

“…In each individual three TCR-Vb families, which had demonstrated elevated expression in peripheral blood by flow cytometry, were selected and examined in BAL T cells with regard to their cytokine profile. We felt justified in using this selection process as previous studies have demonstrated that the pattern of Vb expression is broadly similar in BAL and peripheral blood of asthmatics and normal individuals [20,21]. Thus, it can be assumed that those families found to be elevated in the peripheral blood of the patients studied will be elevated in BAL.…”

Does IL-4 Play a Role in the Expansion of Vβ8a T Cell Receptor-Bearing Cells?

“…To determine the relevance of different T cell receptors, polymerase chain reactions (PCR) can be helpful [6, 7, 12]. Using PCR, the Vβ8.2 gene segment was characterized as an important gene for IgE and IL-4 production in mice [21].…”

“…Changes in the T cell receptor repertoire in allergy or asthma have also been described in humans [10, 11, 12, 13]. Different results were found for blood cells or cells recovered by bronchoalveolar lavage.…”