利用ENU突變鼠發現IL-15異構體能影響HSV-1感染後皮膚的發炎反應與CD8+ T細胞的生成

Abstract

Interleukin-15 (IL-15)是一種多功能的細胞激素，對於宿主的先天性及後天性免疫反應都扮演重要角色。中研院基因突變鼠動物模式核心實驗室 (MMPCF)利用ENU的致突變機制產生出表現顯著多量的IL15選擇性剪接異構體mRNA，簡稱IL-15∆E7的P191品系，在感染第一型疱疹病毒(HSV-1)後，皮膚出現比B6小鼠更為嚴重的傷口，病毒蛋白的表現量較高，cDNA微陣列分析和即時定量PCR的結果也顯示P191皮膚中有顯著高量的IL-1β和IL-6。IL-15∆E7如何影響IL-15的功能，並造成宿主在感染HSV-1後產生更嚴重的發炎反應，還需要進一步的探討。 和MMPCF的結果相符，我們利用流式細胞儀分析P191小鼠淋巴結、脾臟和週邊血中CD8+ T細胞的組成，也發現P191 CD8+ T細胞中表現CD44hiCD122hi的百分比較低。利用Kb-HSV-gB498-505四聚體分子，我們發現具gB抗原特異性的CD8+ T細胞在P191小鼠體內生成的時間比野生型小鼠晚。然而，這組細胞在感染後第十天P191小鼠脾臟中的總細胞數明顯比在B6小鼠還高，顯示這些細胞在感染早期能夠很有效率地在P191的脾臟中增生。當在活體外以gB抗原及IL-2和IL-15刺激CD8+ T細胞時，我們發現P191小鼠的CD8+ T細胞無論在感染前後，接受抗原及IL-15刺激後分裂的能力都比B6小鼠的CD8+ T細胞還差，顯示P191的CD8+ T細胞可能較不具抗原記憶性。利用FlowCytomix檢測B6和P191小鼠脾臟中細胞激素表現的情形，發現在HSV-1感染第七天時，P191小鼠脾臟中IFN-γ表現量顯著低於B6小鼠。P191小鼠脾臟中IFN-γ表現量的下降和具抗原特異性CD8+ T細胞的增生能力是否有任何關聯，仍需要更進一步的研究。 我們的實驗結果顯示P191小鼠在感染HSV-1後，皮膚發炎反應的情形及所產生具抗原特異性CD8+ T細胞的性質都和B6小鼠很不一樣，究竟IL-15異構體如何造成這些變化，是很值得研究的課題。

Interleukin-15 (IL-15) is a pleiotropic cytokine that plays an important role in mediating innate and adaptive immunity in the host. The pedigree 191 (P191) of the ENU-mutagenized mice, generated by the Mouse Mutagenesis Program Core Facility (MMPCF) has been identified and predominantly express an alternatively spliced IL-15 mRNA called IL-15 ∆E7. Infection of P191 mutant mice via flank skin with herpes simplex virus-1 (HSV-1) showed a much more severely disrupted lesional skin than in B6 wildtype mice accompanied with enhanced HSV viral protein expression as well as elevated expressions of IL-1β and IL-6 in P191 lesional skin by cDNA microarray and real-time PCR analysis. How the function of IL-15 is affected and/or regulated by IL-15 ∆E7 and thus results in the altered inflammatory response against HSV-1 infection will be further investigated. Consistent with the depressed CD44 expression on CD8+ T cells in P191, we also found that the percentages of CD8+ T cells from lymph nodes, spleen and peripheral blood expressing CD44hiCD122hi were reduced in P191 mice by flow cytometric analysis. Using Kb-HSV-gB498-505 tetramer reagent, we found that gB-specific CD8+ T cells were generated in a delayed kinetics in P191 as compared to wildtype mice. However, these gB-specific CD8+ T cells significantly expanded in the spleen of P191 on day 10 after infection and the absolute numbers of gB-specific CD8+ T cells were higher than these in B6 mice, indicating that these cells efficiently proliferated in P191 spleen on early times of infection. In proliferation experiment, CFSE-labeled T cells were stimulated with gB498-505 peptide in rIL-2 and rIL-15. Whereas HSV-primed CD8+ T cells from B6 mice proliferated to gB antigen in vitro, proliferation of HSV-primed CD8+ T cells from P191 was significantly reduced given with sufficient IL-15. This suggested that gB-specific CD8+ T cells generated in P191 were poorly responsive to recall antigen. Using FlowCytomix to profile cytokine expression in B6 and P191 mice after HSV-1 infection, we have found that the level of IFN-γ in spleen was significantly reduced in P191 as compared to B6 spleen. How the reduced production of IFN-γ is associated with less proliferation of antigen-specific CD8+ T cells from P191 requires further investigation. Results from current experiments have shown that inflammatory response in skin and the properties of antigen-specific CD8+ T cells induced by HSV-1 infection are both altered in P191. How expression of IL-15 splice variant is involved in the control of these phenotypes remains to be clarified.