Mechanism of T cell activation in the airways of asthma

哮喘的特征是呼吸道炎症，伴有明显的嗜酸性粒细胞和CD4T细胞的渗透。然而，非特应性哮喘患者T细胞被激活的机制尚不清楚。为确定非特应性哮喘患者T细胞是否被不明抗原克隆性激活，我们采用RT-PCR和随后的单链构象多态性(SSCP)分析方法对非特应性哮喘患者支气管肺泡灌洗液(BALF)中T细胞的T细胞受体(TCR)Vbeta基因进行了分析。PCR-SSCP分析显示，在非特应性哮喘患者的BALF和PBL中，大多数Vbeta基因的涂片中都有几条克隆型条带。在7名受试者中，有4名受试者BALF中各Vbeta基因的编码条带数显著高于PBL(P&lt；0.05)。此外，BALF中表达TCR Vbeta6、Vbeta12和Vbeta17基因的T细胞克隆数显著高于PBL(p&lt；0.05)。此外，从BALF累积的T细胞克隆中对TCR Vbeta基因CDR3区的测序分析表明，在6个受试者中发现了几个增加的氨基酸基序，其频率约为测序克隆的8%-9%，这些氨基酸基序是在每个患者中特异发现的。有趣的是，在一名患者中检测到两个克隆，它们在CDR3区具有相同的Pf基序，但Jbeta基因的用途不同。此外，在两名具有共同的HLA-DR等位基因的患者中还检测到一个保守的氨基酸序列PTGTAG。这些结果提示，非特应性哮喘患者气道内的T细胞可能识别呼吸道内相对有限的抗原表位，并在抗性腺激素的刺激下扩增。

Asthma is characterized by airway inflammation with prominent infiltrates of eosinophils and CD4 T cells. However, the mechanism by which T cells of nonatopic asthmatics are activated is unknown. To determine whether T cells are clonally activated by an unidentified antigen in nonatopic asthma, we analyzed T cell receptor (TCR) Vbeta genes of T cells in bronchoalveolar lavage fluids (BALF) of nonatopic asthmatics by using RT-PCR and subsequent single-strand conformation polymorphism (SSCP) analysis. PCR-SSCP analysis showed that several clonotypic bands in smears were found in most of Vbeta genes from BALF and PBL of nonatopic asthma patients. The numbers of the bands encoding each Vbeta gene from BALF significantly increased as compared with those from PBL in four of seven subjects (P<0.05). In addition, the numbers of T cell clones expressing TCR Vbeta6, Vbeta12, and Vbeta17 genes in BALF significantly increased in comparison to those in PBL (p<0.05). Moreover, sequencing analysis of CDR3 region of TCR Vbeta genes from BALF-accumulated T cell clones showed that several increased amino acid motifs were found in six subjects analyzed at a frequency of approximately 8-9% of sequenced clones, which were specifically found in each individual patient. Interestingly, two clones sharing an identical motif PF in CDR3 region with different Jbeta gene usage were detected in one patient. Moreover, one conserved amino acid sequence PTGTAG was detected in two patients who shared a common HLA-DR allele. These results suggest that infiltrating T cells in the airways of nonatopic asthmatics might recognize relatively limited epitopes of antigens in the airways and expand by the antigendriven stimulation.