臺灣博碩士論文加值系統

調節性 T 細胞 (regulatory T cells) 為 T 細胞中具有免疫抑制功能的一亞群，同時扮演維持免疫系統自體耐受性 (self-tolerance) 的角色。初步發現，存在於成人周邊血 (adult peripheral blood) 中的人類調節性 T 細胞，表現 CD4 分子與高量 IL-2 受體  次單元 (CD25; CD4+CD25high)。近年來的報告顯示人類臍帶血 (umbilical cord blood) 中這一群細胞比例較多，可作為良好的調節性 T 細胞的來源。前人的報導顯示，體外培養調節性 T 細胞在外加 IL-2 的條件之下，可以增加調節性T 細胞的數目。但是因為 IL-2 容易使得活化的 T 細胞凋亡 (activation induced cell death)，而 IL-15 則較不易。為了提高人類臍帶血調節性 T 細胞的應用性，本論文利用希望藉由 IL-15 的幫助在體外培養並增加人類臍帶血調節性 T 細胞的數量。本論文發現受 IL-15 刺激增生的臍帶血及成人周邊血中 CD4+CD25+ T 細胞，呈現與 IL-2 刺激的臍帶血及成人周邊血CD4+CD25+ T 細胞相似的外表型。相較於成人周邊血 CD4+CD25+ T 細胞，於體外刺激增生的臍帶血 CD4+CD25+ T 細胞分泌較高量的 TGF-。在體外受到刺激增生的 CD4+CD25+ T 細胞能夠抑制同種異體的成人CD4+ T 細胞的增生以及IFN- 的分泌。儘管在 1/1 抑制/反應細胞的比例之下，體外增生的臍帶血 CD4+CD25+ T 細胞呈現較成人周邊血 CD4+CD25+ T 細胞更高的抑制能力，但是在 1/8 抑制/反應細胞的比例下兩者都會失去抑制功能。本論文發現無論是臍帶血或成人周邊血，在體外受到 IL-15 刺激增生的 CD4+CD25+ T 細胞與前人報導受到 IL-2 刺激增生的 CD4+CD25+ T 細胞一致，都需要透過細胞與細胞之間的作用達成其抑制效果。本研究提出了在體外受到 IL-2 或 IL-15 刺激增生的臍帶血 CD4+CD25+ T 細胞，相較於成人血 CD4+CD25+ T 細胞具有更高的免疫抑制功能，因此可作為調節性 T 細胞的來源的證據。在體外刺激增的 CD4+CD25+ T 細胞或許可以進一步應用於自體免疫疾病的治療。

Regulatory T (Treg) cells are a subpopulation of T cells with immunosuppressive function and play a pivotal role in the maintenance of self-tolerance. Human Treg cells were primarily defined by the expression of CD4 molecules together with intensive expression of interleukin (IL)-2 receptor  chains (CD25; CD4+CD25high) from adult peripheral blood (APB). Umbilical cord blood (UCB) has recently been reported to be a good source for Treg cells. Although exogenous IL-2 was required for the expansion of Treg cells in vitro, the presence of IL-2 usually induces activation induced cell death (AICD) during T cell activation. IL-15, however, has similar potent to activate T cells with less effect on AICD. For potential usage of UCB Treg cells, we applied IL-15 as well as IL-2 to expand CD4+CD25+ cells in vitro. The phenotypes of IL-15 expanded CD4+CD25+ cells were similar to IL-2 expanded CD4+CD25+ cells in both UCB and APB. However, in vitro expanded UCB CD4+CD25+ cells secreted higher level of TGF- than APB. The in vitro expanded CD4+CD25+ cells suppressed the proliferation of allogeneic adult CD4+ cells and reduced the accumulation of IFN-. Despite, the UCB CD4+CD25+ cells exerted higher suppressive activity than APB CD4+CD25+ cells at 1/1 suppressor-responder ratio. Both cell populations lost their suppressive function at 1/8 suppressor-responder ratio. The suppressive function of both IL-2 and IL-15 expanded CD4+CD25+ cells were mediated through cell-cell contact manners. These evidences supported that UCB CD4+CD25+ cells with IL-2 or IL-15 treatment will be a feasible source of Treg cells for the therapy of autoimmune disorders.

指導教授推薦書 …………………………………………………………口試委員會審定書 ………………………………………………………授權書 ………………………………………………………………... iii 中文摘要 ……………………………………………………………... iv
英文摘要 ……………………………………………………………… v 誌謝 …………………………………………………………………... vi

Introduction …………………………………………………………… 1
Application of umbilical cord blood ……………………………….. 1
Regulatory T cells ………………………………………………….. 2
Interleukin-15 …………………………….………………………… 5
Materials and Methods ………………………………………………... 8
Magnetic cell sorting (MACS) purification of CD25+ and CD25- from CD4+ T cells ………………………………………………………... 8
The culture of UCB and APB CD4+CD25+ cells …………………... 9
The proliferation of CD4+CD25+ and CD4+CD25- cells ………….. 10
Antibodies and flow cytometry …………………………………… 11
Cytokine analysis by ELISA ……………………………………… 13
Suppressive function assay ……………………………………….. 15
Statistical analysis ………………………………………………… 17

Results ………………………………………………………………. 18
Enriched UCB CD4+CD25+ T cells contained more CD25high and FoxP3+ cells than enriched APB CD4+CD25+ cells ……………… 18
UCB and APB CD4+CD25+ T cells were expanded by TCR and CD28 signaling in the presence of exogenous IL-2 and IL-15 in vitro …. 19
Exogenous IL-2 and IL-15 contributed to the living cells percentage of expanded UCB and APB CD4+CD25+ T cells …………………… 20
Exogenous IL-2 and IL-15 promoted the proliferation of UCB and APB CD4+CD25+ T cells …………………………………………. 21
IL-2 and IL-15 promoted the phenotypes of in vitro expanded CD4+CD25+ T cells to Treg cells like ……………………………… 22
In vitro expanded UCB CD4+CD25+ cells expressed higher TGF- but lower IL-10 than expanded APB CD4+CD25+ cells …………….... 23
In vitro expanded CD4+CD25+ cells suppressed the proliferation of allogeneic adult CD4+ T cells …………………………………….. 24
In vitro expanded CD4+CD25+ cells reduced the interferon (IFN)- production of allogeneic adult CD4+ T cells ……………………… 26
In vitro expanded UCB CD4+CD25+ T cells exerted higher suppressive activity than APB CD4+CD25+ T cells ………………………..….. 27 TRIL-15 cells inhibited the proliferation of responder cells through cell contact-dependent manner as well as TRIL-2 cells …………………. 28

Discussion ……………………………………………………………. 29

Table and Figures …………………………………………………….. 36
Table 1. The summary of Treg cells associated characteristics of in vitro expanded UCB and APB CD4+CD25+ T cells …………………….. 36
Fig. 1 UCB and APB CD4+CD25+ cells were enriched from peripheral blood mononuclear cells (PBMCs) ………………………………... 37
Fig. 2 Exogenous IL-2 and IL-15 contributed to the expansion of UCB and APB CD4+CD25+ cells in vitro ……………………………….. 39
Fig. 3 The exogenous IL-2 and IL-15 enhanced the survivals of expanded UCB and APB CD4+CD25+ T cells in vitro ……………. 40
Fig. 4 Exogenous IL-2 and IL-15 promoted the proliferation of CD4+CD25+ T cells in vitro ……………………………………….. 42
Fig. 5 Exogenous IL-2 and IL-15 increased the expression of Treg cells related markers on in vitro expanded UCB and APB CD4+CD25+ T cells ………………………………………………………………... 44
Fig. 6 Both exogenous IL-2 and IL-15 increased the production of IL-10 from either UCB or APB CD4+CD25+ T cells and expanded UCB CD4+CD25+ T cells secreted higher TGF- than APB ……… 46
Fig. 7 In vitro expanded UCB and APB CD4+CD25+ T cells suppressed the proliferation of allogeneic adult CD4+ T cells ………………… 47
Fig. 8 In vitro expanded UCB CD4+CD25+ T cells suppressed the proliferation of allogeneic adult CD4+ T cells substantially and also APB CD4+CD25+ T cells slightly ………………………………… 48
Fig. 9 The production of interferon- from allogeneic adult CD4+ cells was inhibited by in vitro expanded UCB and APB CD4+CD25+ but not CD4+CD25- cells …………………………………………………. 49
Fig. 10 In vitro expanded UCB CD4+CD25+ T cells exerted higher suppressive activities than APB ………………………………….. 50
Fig. 11 Both UCB (A) and APB (B) TRIL-15 cells as well as TRIL-2 cells suppressed the proliferation of responders through cell contact-dependent manners ………………………………………. 51

Reference ……………………………………………………………. 52

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