促進人類免疫系統小鼠模式之後天免疫

Abstract

帶有人類免疫系統（Human immune system ,HIS）的小鼠，主要透過移植人類造血幹細胞至免疫缺陷小鼠來建立，為研究人類免疫系統在體內功能的重要工具；然而，如何在HIS小鼠建立具有功能的人類後天性免疫系統，依然存在許多挑戰。細胞激素在免疫系統的發展與功能上扮演重要的角色，因此HIS小鼠後天性免疫的缺陷，可能是因為小鼠細胞激素不能有效的和人類細胞受器結合所致。除此之外，HIS小鼠T細胞功能的缺失，可能與人類T細胞在小鼠胸腺，是接受小鼠主要組織相容性複合物（major histocompatibility complex, MHC）教育而非人類白血球抗原（human leukocyte antigen, HLA）有關，這些由小鼠MHC教育出的T細胞，可能失去和人類抗原呈現細胞上之HLA結合的能力，導致無法有效的被活化。因此，我們認為在建立HIS小鼠時，額外提供促進T細胞和B細胞發育的人類細胞激素與HLA，或許能幫助建立具有功能的後天性免疫系統。 在本篇研究的第一部份，我們成功建立了從臍帶血分離人類CD34+造血幹細胞的方法，當這些造血幹細胞移植到放射線照射過的四週齡NSG免疫缺陷小鼠(NOD.Cg‑Prkdcscid Il2rgtm1Wjl)，或是沒有照射過放射線的NSG新生鼠，皆可於移植後約一個月，在小鼠周邊血中測得一定比例的CD45+人類白血球細胞，且所占比例隨時間而增多；分析這些HIS小鼠的免疫器官，也可在脾臟、肝臟、胸腺與骨髓中看到成熟的CD4＋及CD8＋T 細胞。然而，我們卻發現在造血幹細胞移植後約兩個月，有照射過放射線的小鼠會變得虛弱甚至死亡，分析其血液組成後，判斷死亡原因可能與放射線照射，造成紅血球及血小板數量大量減少有關；由於照射放射線會減短小鼠壽命，不利於進行需要長時間觀測的實驗，因此，我們決定之後的實驗都以沒有照射放射線的新生鼠作為建立HIS小鼠的移植宿主，與研究人類後天免疫系統的對象。 為了促進HIS小鼠的後天性免疫系統，我們利用腺相關病毒為載體，表現人類細胞激素（GM-CSF, IL-7, BLyS 和 IL-4）和人類第一型及第二型HLA蛋白。體內與體外的實驗結果顯示，這些AAV載體確實可表現所攜帶的人類細胞激素和HLA基因。由於T細胞必須在胸腺表現接受HLA的教育，因此我們將AAV/HLA直接以胸腺注射的方式送入，而表達細胞激素的AAV則以腹腔注射，使之在小鼠全身表達細胞激素。我們比較只有移植造血幹細胞的HIS小鼠，與有施打表現HLA和細胞激素的AAV之HIS-AAV小鼠，其血液中CD45+人類白血球細胞比例與免疫球蛋白的含量。實驗總共進行了三次，但結果並不一致。其中兩次實驗的結果，顯示HIS-AAV小鼠較早產生CD45+人類白血球，其所占比例也比沒有施打細胞激素和的HIS小鼠高，此外，在其中一次的實驗中HIS-AAV小鼠可測到高量的人類IgG免疫球蛋白，而HIS小鼠完全測不到人類免疫球蛋白。但是，另外一次的實驗卻沒能再現這些結果，我們正努力找出造成實驗結果不一致的原因，會在日後繼續尋找促進HIS小鼠產生具有功能的後天性免疫系統的辦法。

Human immune system (HIS) mice, generated by transplanting human hematopoietic stem cells to immunodeficient mice, are a powerful tool to study human immune functions in vivo. However, how to generate HIS mice containing functional adaptive immunity remains challenges. It is well known that cytokines play a critical role in directing immune cell development and functions. The defect in adaptive immunity in current HIS mice may be due to the limited cross-reactivity between mouse cytokines and human receptors. Another potential cause of T cell impairment in HIS mice is that human T cells are educated by mouse major histocompatibility complex (MHC) in the thymus of HIS mice, which may result in their T cell receptors not being able to recognize human antigen-presenting cells that express human leukocyte antigen (HLA). We hypothesized that by providing HIS mice with a combination of human cytokines that are important for T and B cell development and human HLA molecules, we may be able to generate HIS mice with improved adaptive immunity. In the first part of this study, we established protocols to successfully isolate CD34+ hematopoietic stem cells from the umbilical cord blood. Transplantation of these hematopoietic stem cells into irradiated young adult or neonatal immunodeficient NOD.Cg‑Prkdcscid Il2rgtm1Wjl （NSG）mice led to significant amounts of human CD45+ leukocyte cells in the blood and the percentage of these human cells increased with time. Mature human CD4+ T cells and CD8+ T cells were also found in the spleen, liver, bone marrow and thymus. However, these HIS mice became ill and died within two months after transplantation. The cause of death was likely due to radiation which resulted in extremely low levels of red blood cells and platelets in these mice. Because radiation pretreatment will shorten mice lifespan, which is unfavorable for long term experiment observation. Therefore, we adopted a protocol which used neonatal mice without radiation pretreatment for generating HIS mice. This HIS neonatal mice were successfully generated and we took it as our modal for investigate human adaptive immune system in the later experiment. To improve the adaptive immunity in HIS mice, we used adeno-associated viral (AAV) vectors to deliver human cytokines (GM-CSF, IL-7, BLyS, and IL-4) and both class I and class II HLA molecules. In vitro and in vivo assays demonstrated that these AAV vectors were able to express their corresponding cytokines and HLA proteins. Since expression of HLA within the thymus is essential for human T cells to be properly educated, the AAV/HLA vectors were directly injected into the thymus. AAV vectors that express cytokines were intraperitoneally injected which allows cytokine expression systemically. We compared the percentage of human CD45+ cells and the total human immunoglobulin levels in the blood of the HIS mice with and without transduction of AAV vectors expressing cytokines and HLAs. So far, three experiments have been performed with inconsistent results. In two experiment, AAV-cytokine and HLA transduced HIS mice showed much higher percentage of human CD45+ cells as compared with HIS mice without AAV transduction. Analysis of serum immunoglobulin levels also showed high titers of human IgG levels in one experiment, while no human immunoglobulins can be detected in HIS mice without AAV transduction. Unfortunately, these results can not be reproduced in another experiment. We are currently clarifying the possible causes of these inconsistent results and will continue to work on generating HIS mice with an improved adaptive immunity.