改善人類周邊血液單核細胞建立的人類免疫系統小鼠模式

Abstract

人類免疫系統(HIS)小鼠模式是研究人類免疫治療的重要臨床前(preclinical)工具。使用人類周邊血液單核細胞(PBMC)在小鼠體內所重建的人類免疫系統，較能模擬病人的當前免疫狀態。然而，當前植入人類PBMC的HIS (Hu-PBL HIS)小鼠模式，有兩大限制：(1) 嚴重的異種移植物對抗宿主疾病(xeno-GVHD)，使小鼠提早死亡，而無法進行長期的免疫分析。(2) T細胞以外的人類免疫細胞族群無法在小鼠體內被完整重建。本篇研究的目的，是改善Hu-PBL HIS小鼠模式的兩大限制，期許在小鼠體內重建抗原呈現細胞，並使Hu-PBL HIS小鼠模式在免除xeno-GVHD時，能有效的發生抗原專一性免疫反應。為了避免xeno-GVHD的發生，我們使用了主要組織相容性複合物第一型與第二型缺失(MHC class I- and class II-deficient)的免疫缺陷NSG (NSG-dKO)小鼠，作為移植人類PBMC的宿主，NSG-dKO小鼠的種群擴增與分型正在進行。同時，我們用NSG小鼠當作宿主，探討提供人類細胞激素是否能促進在Hu-PBL HIS小鼠模式中移植率很低的免疫細胞族群的重建，例如：B細胞以及樹突細胞。腺相關病毒(AAV)載體可持續且全身性在體內表現基因，我們利用AAV載體在Hu-PBL HIS小鼠模式中表現人類細胞激素，證明人類細胞激素可以調控人類以及小鼠的免疫細胞。其中，介白素-4 (IL-4)可促進CD4 T細胞的增生以及抑制xeno-GVHD。植入人類脾臟細胞的HIS (Hu-SPL HIS)小鼠模式，具備人類細胞免疫以及體液免疫反應，為了仿造Hu-SPL HIS小鼠模式，本篇研究專注於使用人類IL-4, CD40配體 (CD40L)以及FMS樣酪氨酸激酶3配體 (Flt3L)來促進B細胞和樹突細胞的體內增生。但是，我們發現在NSG小鼠體內表現人類Flt3L會使小鼠的吞噬細胞大量增生，且此現象與人類PBMC的移植率呈現負相關。此策略還需要經過一些改善，才能看見人類細胞激素促進B細胞和樹突細胞的體內增生與存活的效果。本篇研究提出了新穎且技術上可行的方法，藉由表現人類IL-4, CD40L與Flt3L來改善抗原呈現細胞在Hu-PBL HIS小鼠模式中的低移植率。加上使用NSG-dKO小鼠作為宿主，此小鼠模式期望能在沒有xeno-GVHD的情況下，用來研究個人化的抗原專一性免疫反應。

Immunodeficient mice engrafted with human immune cells (HIS mice) serve as valuable tools for preclinical evaluation of immunotherapies. The use of human peripheral-blood mononuclear cells (PBMCs) to reconstitute human immune system in HIS mice enables the analysis of current immune status of individual patient. However, there are two major limitations of PBMC-engrafted HIS mouse model: (1) The rapid development of lethal xenogeneic graft-versus-host disease (GVHD) that hinders long-term and precise immune analysis, (2) incomplete reconstitution of immune subpopulations with few B cells and myeloid cells engrafted. We aimed to generate modified human PBMC-engrafted HIS mice with improved reconstitution of antigen presenting cells for the study of human antigen-specific immune response in the absence of xenogeneic GVHD. To avoid xenogeneic GVHD, we have obtained MHC class I- and class II-deficient NOD-scid IL-2rγnull (NSG-dKO) mice from collaborators as the host of human PBMC. Colony expansion and characterization of NSG-dKO mice is currently ongoing. In the meantime, we used NSG mice as the host to investigate whether supplementary of human cytokines can improve the poor engraftment rate of certain immune subpopulations in Hu-PBL HIS mice. Taking advantage of AAV vectors that allow sustained systemically expression of human cytokines in vivo, we demonstrated that AAV-delivered human cytokines regulated both murine and human leukocytes in HIS mice. The expression of human IL-4 significantly increased CD4 T cell expansion and suppressed GVHD. To mimic the features of human splenocyte-engrafted HIS mice, a model displays profound human humoral and cellular immune responses, we focused on expanding human B cells and dendritic cells using IL-4, CD40L and Flt3L. Interestingly, we found that the cell number of mouse phagocytes were greatly increased in human Flt3L expressing NSG mice. In addition, the phenomenon was negatively correlated with human PBMC engraftment rate. Modifications were needed to see the effect of human cytokines on supporting in vivo expansion and survival of B cell and dendritic cells. Collectively, we presented novel and technically feasible strategies to enhance the engraftment of antigen presenting cells in human PBMC-reconstituted HIS model by delivering human IL-4, CD40L and Flt3L. When combined with murine MHC deficiency, the HIS model will be able to recapitulate the personalized human antigen-specific immune responses without signs of xeno-GVHD.