CDR3和TCR-gd T細胞促使受MHC-II限制的CD4+ T細胞亞羣經由發育過程得到IL-4分泌潛能進而調控IgE的產生

Abstract

受CD1d 限制發育的NKT細胞在給予TCR刺激時大量釋出IL-4，被認為在誘發IgE反應扮演重要角色。在各種常用的小鼠品系中，我發現NK1-CD44lowCD4+CD8- 胸線T細胞，具有在TCR刺激後快速且大量分泌IL-4的能力。擁有這種分泌IL-4潛能的NK1-CD44lowCD4+CD8- 胸線T細胞亞群主要表現的TCR為Va3.2與Vb2/7/8。這種具有分泌IL-4潛能的NK1-CD44lowCD4+CD8- 胸線T細胞亞群與NKT細胞不同的是，其發育的過程並不需要CD1d、b2m與fyn的幫助。這群NK1-CD44lowCD4+CD8- 胸線T細胞在受TCR刺激時也同時分泌IL-5、IL-10與IL-13，但只表現極少量的IFN-g。分析這群胸線細胞的TCR，發現不論是Va或Vb的CDR3序列都具有高度的變異性。 進一步分別從能分泌IL-4與不能分泌IL-4 的Va3.2+Vb8+NK1-CD44lowCD4+CD8- 胸線T細胞，得到其TCR a及b序列。接著以此序列建構可以大量表現此TCRa及b的質體，並利用此一質體生產基因轉殖小鼠，使其T細胞大量表現此Va3.2+Vb8+ TCR組合。由可以分泌IL-4（「P」系列）之胸線T細胞得到的四組TCR Va3.2及Vb8序列分別基因轉殖產生八種單一基因轉殖小鼠品系，之後再交配成「P」系列的四個TCR-ab雙重基因轉殖小鼠品系。此外，我也由不能分泌IL-4（「N」系列）之胸線T細胞得到三組TCR Va3.2及Vb8序列，分別基因轉殖產生六種單一基因轉置小鼠，之後再交配成「N」系列的三種TCR-ab雙重基因轉殖小鼠品系。所有的「P」系列TCR-ab雙重基因轉殖小鼠在其Va3.2+Vb8+CD4+CD8- T細胞，受TCR刺激時會有大量分泌IL-4的能力，而這些「P」系列基因轉殖小鼠血清中的IgE，也都較一般正常小鼠有明顯上升的現象。反之，「N」系列基因轉殖小鼠中的Va3.2+Vb8+胸線T細胞，受TCR刺激後並不具有大量分泌IL-4的能力，同時其血清中的IgE也沒有明顯的變化。「P3」TCR-ab基因轉殖之胸線T細胞於發育時的positive selection，是需要MHC II而不需要MHC I與CD1d。而發育時的positive selection，對這些「P3」TCR-ab基因轉殖T細胞具備分泌IL-4潛能而言，是必須但不夠充分的。這些「P3」TCR-ab基因轉殖T細胞，在發育時獲得具有分泌IL-4潛能的過程中，也需要TCR-gd T細胞的幫助。歸納以上結果發現：(1) 一群受MHC II限制的CD4+ T細胞亞群，具有大量分泌IL-4的潛能；(2) 特定TCR-a與-b的CDR3序列以及TCR-gd T細胞，對於這群CD4+CD8- T細胞獲得分泌IL-4的潛能，進而影響血清中的IgE含量，有重要性；(3) 由於這群具備分泌IL-4潛能的NK1-CD44lowCD4+CD8- T細胞，表現了高度變異性的TCR序列，推測這群T細胞可認得的抗原也非常多元，進而參與了免疫反應。

CD1d-restricted NKT cells respond to TCR-stimulation by prompt IL-4 production and were originally thought to play a major role in the initiation of IgE response. NK1-CD44lowCD4+CD8- thymocytes were also capable of TCR-stimulated prompt IL-4 inducibility in all common mouse strains examined. The property of IL-4 inducibility by these NK1-CD44lowCD4+CD8- thymocytes was found primarily in Va3.2+ and Vb2/7/8+ cells. Unlike NKT cells, the development of IL-4 inducibility+ NK1-CD44lowCD4+CD8- thymocytes was b2m-, CD1d-, and p59fyn-independent. NK1-CD44lowCD4+CD8- thymocytes also produced IL-5, IL-10, and IL-13, but very low levels of IFN-g in response to TCR stimulation. The IL-4 inducibility+ NK1-CD44lowCD4+CD8- thymocytes expressed highly diverse CDR3 in both TCR a and b chains. Va3.2 and Vb8 CDR3 from single IL-4 mRNA+ and IL-4 mRNA- cells were subcloned into full-length Va3.2 and Vb8 transgene constructs, and injected into fertilized eggs to produce TCR transgenic mice. A “P”-series of 8 independent TCR transgenic lines (4 each of TCR-a and -b) were produced from Va3.2 and Vb8 cloned from 4 randomly selected IL-4-producing single NK1-CD44lowCD4+CD8- thymocytes. Another “N”-series of 6 independent TCR transgenic lines (3 each of TCR-a and -b) were produced from Va3.2 and Vb8 cloned from 3 randomly selected IL-4-non-producing single NK1-CD44lowCD4+CD8- thymocytes. Single TCR-a and -b transgenic mice were crossed to generate double TCR-ab transgenic mice. All P-series TCR-ab transgenic mice displayed elevated IL-4 inducibility in Va3.2+Vb8+ thymocytes and increased serum IgE levels. All N-series TCR-ab transgenic mice, on the other hand, expressed low or negligible IL-4 inducibility in Va3.2+Vb8+ thymocytes and had normal serum IgE levels. Positive selection of P3 TCR-ab transgenic T cells was MHC-II-, but not MHC-I- or CD1d-dependent. Positive selection was necessary but not sufficient in conferring IL-4 inducibility which was dependent on the presence of TCR-gd T cells. These results demonstrate 1) the existence of a MHC-II-restricted CD4+ T cell subset capable of prompt IL-4 inducibility; 2) the critical roles played by CDR3 of both TCR-a and -b chains and by TCR-gd T cells in the acquisition of IL-4 inducibility and elevated serum IgE levels; and 3) the likelihood for the IL-4 inducibility+ NK1-CD44lowCD4+CD8- T cells to participate in immune response to a relatively large number of antigens due to their highly diverse TCR repertoire.