Lu L, Hsieh M, Oriss T B, Morel P A, Starzl T E, Rao A S, Thomson A W
Pittsburgh Transplantation Institute, University of Pittsburgh, Pennsylvania.
Immunology. 1995 Jan;84(1):127-34.
In all tissues that have been studied to date, dendritic leucocytes constitute only a small proportion of total cells and are difficult both to isolate and purify. This study reports on a method for the propagation of large numbers of dendritic cells (DC) from mouse spleen using granulocyte-macrophage colony-stimulating factor (GM-CSF) and their characteristics. Within a few days of liquid culture in GM-CSF, B10 BR (H-2k, I-E+) mouse splenocytes formed loosely adherent myeloid cell clusters. Mononuclear progeny released from these clusters at and beyond 4 days exhibited distinct dendritic morphology and strongly expressed leucocyte common antigen (CD45), CD11b, heat-stable antigen, Pgp-1 (CD44) and intercellular adhesion molecule-1 (ICAM-1; CD54). The intensity of expression of the DC-restricted markers NLDC 145 and 33D1, the macrophage marker F4/80, and Fc gamma RII (CDw32) was low to moderate, whereas the cells were negative for CD3, CD45RA and NK1.1. High and moderate levels, respectively, of cell surface staining for major histocompatibility complex (MHC) class II (I-Ek) and the B7 antigens (counter-receptors of CTLA4, a structural homologue of CD28) were associated with potent stimulation of unprimed, allogeneic T cells (B10; H-2b, I-E-). DC propagated in a similar fashion from DBA/2 mouse spleen proved to be strong antigen-presenting cells (APC) for MHC-restricted, syngeneic T-helper type 2 (Th2) cell clones specifically responsive to sperm whale myoglobin. Footpad or intravenous injection of GM-CSF-stimulated B10.BR spleen-derived DC into B10 (H-2b, I-E-) recipients resulted in homing of the allogeneic cells to T-cell-dependent areas of lymph nodes and spleen, where they strongly expressed donor MHC class II antigen 1-2 days later. These findings indicate that cells can be propagated from fresh splenocyte suspensions that exhibit distinctive features of DC, namely morphology, motility, cell-surface phenotype, potent allogeneic and syngeneic APC function and in vivo homing ability. Propagation of DC in this manner from progenitors present in lymphoid tissue provides an alternative and relatively convenient source of high numbers of these otherwise difficult to isolate but functionally important APC.
在迄今所研究的所有组织中，树突状白细胞仅占细胞总数的一小部分，并且难以分离和纯化。本研究报告了一种使用粒细胞 - 巨噬细胞集落刺激因子（GM - CSF）从小鼠脾脏中大量增殖树突状细胞（DC）的方法及其特性。在GM - CSF中进行液体培养的几天内，B10 BR（H - 2k，I - E +）小鼠脾细胞形成了松散附着的髓样细胞簇。在4天及以后从这些簇中释放的单核后代表现出明显的树突状形态，并强烈表达白细胞共同抗原（CD45）、CD11b、热稳定抗原、Pgp - 1（CD44）和细胞间粘附分子 - 1（ICAM - 1；CD54）。DC特异性标志物NLDC 145和33D1、巨噬细胞标志物F4/80以及FcγRII（CDw32）的表达强度低至中等，而细胞对CD3、CD45RA和NK1.1呈阴性。主要组织相容性复合体（MHC）II类（I - Ek）和B7抗原（CTLA4的反受体，CD28的结构同源物）的细胞表面染色分别为高水平和中等水平，这与对未致敏的同种异体T细胞（B10；H - 2b，I - E -）的有效刺激相关。以类似方式从DBA/2小鼠脾脏中增殖的DC被证明是针对MHC限制的、对抹香鲸肌红蛋白特异性反应的同基因T辅助2型（Th2）细胞克隆的强抗原呈递细胞（APC）。将GM - CSF刺激的B10.BR脾脏来源的DC足垫注射或静脉注射到B10（H - 2b，I - E -）受体中，导致同种异体细胞归巢到淋巴结和脾脏的T细胞依赖区域，1 - 2天后它们在那里强烈表达供体MHC II类抗原。这些发现表明，可以从新鲜脾细胞悬液中增殖出具有DC独特特征的细胞，即形态、运动性、细胞表面表型、强大的同种异体和同基因APC功能以及体内归巢能力。以这种方式从淋巴组织中的祖细胞增殖DC提供了一种替代且相对方便的来源，可获得大量原本难以分离但功能重要的APC。
