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.

摘要 i
Abstract ii
Table of Contents iv
List of Figures vi
Chapter I Introduction 1
1. Type I IFNs against HSV-1 infection 2
2. NK cells against HSV-1 infection 3
3. T cell immunity against HSV-1 infection 3
4. CD8+ T cell activation 5
5. Memory CD8+ T cell differentiation and maintenance 7
6. Rationale 9
Chapter II Materials and Methods 11
1. Establishment of HSV-1 zosteriform mouse model 11
2. Histological analysis 13
3. Quantification of tissue cytokine mRNAs 16
4. Quantification of tissue cytokines 19
5. Flow cytometry 20
6. Antibodies 26
7. Enzymes 29
8. Solution recipes 30
9. Chemicals, kits and reagents 33
Chapter III Results 38
1. P191 mice develop more severe lesional skin than B6 mice after HSV-1 infection 38
2. Severe skin inflammation in P191 accompanied by dramatic up-regulation of IL-1β and IL-6 transcripts 39
3. P191 mice exhibit reduced NK and CD44hiCD122hi CD8+ T cells and T cell activation and gB-specific CD8+ T cell generation are delayed after HSV-1 infection 40
4. The kinetics of early generation of gB-specific CD8+ T cells after HSV-1 infection in P191 mice is altered 43
5. CD8+ T cells from P191 spleen are less proliferative in response to in vitro restimulation with gB498-505 peptide, IL-2 and IL-15 45
6. IFN-γ production is reduced in P191 spleen 47
Chapter IV Discussion 49
1. Altered inflammatory responses and lesion resolution in P191 mice 49
2. Skin γδ T cells in inflammation and tissue repairing 51
3. Reduced surface marker up-regulation and generation of gB-specific CD8+ T cells in P191 mice 52
4. Lymph node retention of gB-specific CD8+ T cells 53
5. Antigen recall and IL-15 responsiveness of CD8+ T cells 55
6. Antigen-presenting and IL-15 trans-presenting cells in P191 mice 56
Chapter V Figures 59
Chapter VI References 76
Appendixes 87
Appendix 1. Establishment of HSV-1 infection mouse model via flank skin 87
Appendix 2. Sequences and Tm values of real-time PCR primers 88
Appendix 3. Histological analysis of TCRδc knockout mouse skin on day 7 after HSV-1 infection 89
Appendix 4. CD44hiCD62Lhi central memory CD8+ T cells are selectively reduced in P191 on day 29 after HSV-1 infection. 90

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