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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 孔祥智 | |
dc.contributor.author | Yi-Ting Chen | en |
dc.contributor.author | 陳宜婷 | zh_TW |
dc.date.accessioned | 2021-06-13T06:40:40Z | - |
dc.date.available | 2010-08-04 | |
dc.date.copyright | 2005-08-04 | |
dc.date.issued | 2005 | |
dc.date.submitted | 2005-08-01 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35090 | - |
dc.description.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。 | zh_TW |
dc.description.abstract | 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. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T06:40:40Z (GMT). No. of bitstreams: 1 ntu-94-D88449004-1.pdf: 1043567 bytes, checksum: ef1281a6bf37453d32373edb8487e504 (MD5) Previous issue date: 2005 | en |
dc.description.tableofcontents | List of Figures……………………………………………………... vii
List of Tables………………………………………………………. xii Introduction……………………………………………………….. 1 I. IL-4…………………………………………………………………….. 2 II. IgE antibody response…………………………………………………. 3 III. Nature killer T (NKT) cells……………………………………………. 4 IV. NK1-CD44lowCD4+CD8- thymocytes with TCR-stimulated IL-4 inducibility…………………………………………………………….. 6 V. TCR and hypervariable complementarity-determining region 3 (CDR3) ……………………………………………………………….. 8 VI. TCR-gd T cells………………………………………………………… 9 Materials and Methods……………………………………...……… 11 I. Mice…………………………………………………………………… 12 II. T cell isolation by panning…………..………………………...……… 13 III. Electronic cell sorting and flow cytometric analysis………………….. 14 IV. T cell stimulation…………………………………...…………………. 17 V. IL-4 and IL-2 bioassay……………………………………...………… 18 VI. IFN-g, IL-5, IL-10, and IL-13 detection by ELISA…………………… 19 VII. RNA extraction and reverse transcription…………………………… 20 VIII. IL-2 and IL-4 gene expression by competitive RT-PCR………… 20 IX. IL-12Rb1, IL-12Rb2, IFN-gR1, IFN-gR2, IL-4, GATA-3, cMaf, and JunB gene expression by real-time RT-PCR…………………….…... 21 X. Single-cell RT-PCR…………………..……………………………… 23 XI. TCR gene sequencing………………..………………………………. 26 XII. TCR transgenic mice………………………………………………….. 26 1. Generation of the TCR transgenic mice……………………… 26 2. Breeding and screening of the TCR transgenic mice……… 28 3. Genotyping of TCR transgenic mice on TCR-Ca-/-, TCR-Cb-/-, TCR-Ca-/-Cb-/-, and fyn-/- backgrounds………… 29 XIII. Serum IgE, IgG1 and IgG2a detection by ELISA…………………….. 30 XIV. Cell preparation from nonlymphoid tissues……………..…………... 31 XV. Bone marrow reconstitution………………………………………...… 32 XVI. Statistical analysis…………………………………….……………... 33 Results……………………………………………………………… 34 I. NK1−CD44lowCD4+CD8- thymocytes capable of TCR-stimulated IL-4 inducibility are distinct from NKT cells……………………………..… 35 1. Extremely low IL-4 production and mRNA expression by Va11+Vb3+NK1-CD4+CD8- thymocytes from I-Ek-restricted AND TCR transgenic mice…………………… 35 2. Biased TCR usage by IL-4 producing NK1-CD44lowCD4+CD8- thymocytes………………………… 37 3. Biased TCR usage by IL-4 producing NK1-CD44lowCD4+CD8- thymocytes in several mouse strains……………………………………………………………… 39 4. Post thymic down regulation of IL-4 gene inducibility in NK1-CD44lowCD4+CD8- T cells with biased TCR usage……………………………………………………………… 40 5. NK1−CD44lowCD4+CD8− thymocytes lack reactivity to aGalCer/CD1d complex………………………………………… 41 6. Failure by NK1-CD44lowCD4+CD8- thymus T cells to produce IFN-g in response to IL-12 + IL-18………………… 42 7. In contrast to NKT cells, NK1-CD44lowCD4+CD8- thymus T cells express extremely lower levels of IL-12Rb1, IL-12Rb2, and IFN-gR1……………………………………………………… 43 8. IL-4 inducibility in NK1-CD44lowCD4+CD8- thymocytes is not affected by removal of a-Galacotosylceramide-CD1d complex binding cells…………………………………………… 44 9. Minimal Va14-Ja18 TCR usage by IL-4 producing NK1−CD44lowCD4+CD8− thymocytes with biased TCR usage……………………………………………………………… 45 10. IL-4 gene inducibility and its biased TCR usage by IL-4-producing NK1−CD44lowCD4+CD8− thymus T cells does not require IL-4R-mediated signaling………………… 46 11. Neither IL-4 gene inducibility in NK1−CD44lowCD4+CD8− thymocytes nor its biased TCR usage is b2M or CD1d-dependent………………………………………………… 47 12. IL-4 producing MHC-II-/- NK1-CD44lowCD4+CD8- thymocytes do not show biased Vb2/Vb7/Vb8/Va3.2 usage……………………………………………………………… 49 13. IL-4 gene inducibility and its biased TCR usage by NK1−CD44lowCD4+CD8− thymocytes is Fyn-independent………………………………………………… 50 14. Vb(2/7/8)+NK1-CD44lowCD4+CD8- thymocytes are also capable of TCR-stimulated IL-5, IL-10, and IL-13 production………………………………………………………… 51 15. GATA-3, cMaf, and JunB mRNA expression by Vb(2/7/8)- and Vb(2/7/8)+ subsets of NK1-CD44lowCD4+CD8- thymocytes………………………………………………………… 52 16. IL-4 production by DO11.10 TCR (Vb8/Va3.1) transgenic CD4+CD8- thymocytes………………………………………… 53 17. Variable CDR3 sequences expression by IL-4-inducible NK1−CD44lowCD4+CD8− thymocytes………………………… 54 II. Generation of TCR-a, -b, and -ab transgenic mice using cDNAs cloned from single IL-4-producing and non-producing Va3.2+Vb8+NK1-CD4+CD8- thymocytes……………………………. 56 1. Cloning TCR-a and -b CDR3 from single IL-4-producing and non-producing Va3.2+Vb8+NK1-CD4+CD8- thymocytes by nested PCR and TCR transgene constructs………………… 56 2. Establishment of TCR-a, -b, and -ab transgenic mice……… 57 III. Analysis of IL-4 inducibility in NK1−CD44lowCD4+CD8− thymocytes from the “P”- and “N”-series TCR transgenic mice………………..… 58 1. Increased IL-4 inducibility in NK1−CD44lowCD4+CD8− T cells from “P-series”but not “N-series” TCR-ab transgenic mice………………………………………………………………… 58 2. IL-4 inducibility in spleen NK1-CD4+CD8- T cells from “P-series” TCR-ab transgenic mice………………………… 59 3. Markers expressed on CD4+CD8- T cells from P1 and P3 TCR-ab transgenic mice are similar to MHC-II-restricted conventional CD4+ T cells…………………………………… 61 4. Lack of a-GalCer-CD1d reactivity by NK1-CD44lowCD4+CD8- T cells from P3 TCR-ab transgenic mice………………………………………………………………… 61 IV. Analysis of serum IgE, IgG1, and IgG2a in “P” and “N” series TCR transgenic mice……………………………………………………….. 62 5. Elevated serum IgE in unimmunized P1, P2, P3 and P4 TCRab transgenic mice………………………………………… 62 6. Increased IgG1 and decreased IgG2a in unimmunized P1, P2 and P3 TCR-ab transgenic mice. ……………………………… 63 V. Genes that regulate the development and IL-4 inducibility of P3 TCR-ab transgenic T cells……………………………………………. 64 7. P3 TCR is MHC II- and not MHC-I- or CD1d-restricted…… 64 8. Lack of fyn requirement for P3 TCR-ab transgenic T cell development and IL-4 gene inducibility………………………… 65 9. Development of P3 TCR-ab transgenic CD4+CD8- thymocytes without IL-4 inducibility in RAG-1-/- background 66 10. P3 TCR-ab transgenic CD4+CD8- thymocytes and IL-4 inducibility can develop in Ca-/-, Cb-/-, Ca-/-Cb-/-,and IgH | |
dc.language.iso | en | |
dc.title | 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 | zh_TW |
dc.title | CDR3- and TCR-gd T cell-dependent developmental acquisition of IL-4 inducibility in a subset of MHC-II-restricted CD4+ T cells regulates serum IgE levels | en |
dc.type | Thesis | |
dc.date.schoolyear | 93-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 胡承波,林榮華,伍安怡,果伽蘭 | |
dc.subject.keyword | 輔助型 T 淋巴球,第四介白素,胸腺, | zh_TW |
dc.subject.keyword | CD4 T cells,IL-4,thymus,repertoire development, | en |
dc.relation.page | 169 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2005-08-01 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 免疫學研究所 | zh_TW |
顯示於系所單位: | 免疫學研究所 |
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