Scheinberg Phillip, Price David A, Ambrozak David R, Barrett A John, Douek Daniel C
Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, MD 20892-1202, USA.
Exp Hematol. 2006 Jun;34(6):788-95. doi: 10.1016/j.exphem.2006.03.001.
The selective elimination of alloreactive T cells from donor stem cell grafts prior to hematopoietic stem cell transplantation (HSCT) is an important goal in the prevention of graft-vs-host disease (GVHD). However, in HLA-identical donor-recipient pairs, it has proven difficult to identify alloreactive T cells using in vitro systems pretransplant due, in part, to their low frequency and a lack of methodological standardization. To better understand the alloresponse between HLA-identical related pairs, we characterized the alloreactive T cells generated in a mixed lymphocyte reaction (MLR) assay system.
HSCT donor peripheral blood mononuclear cells (responder) were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFSE) dye and cocultured with irradiated HSCT recipient cells (stimulator) in a one-way MLR. Alloreactive T cells were sorted by upregulation of activation markers (CD25 in most cases) and the responding clonotypes were defined by sequencing the complementarity region 3 (CDR3) of the T cell receptor beta-chain.
We show that the recruitment of alloreactive CD4(+) T cells is highly variable. Oligoclonal CD4(+) T-cell expansions in repeated MLRs performed in the same donor-recipient pair showed inconsistent recruitment of clonotypes. The recruitment of alloreactive CD8(+) T cells was more consistent in repeated assays, with the same clonotypes identified in the same donor-recipient pair performed under different conditions.
Taken together, our data show that even in culture conditions constrained to eliminate background proliferation, stochastic events and low precursor frequencies preclude reproducible elicitation of immunodominant T cell clonotypes with the potential to cause GVHD.
在造血干细胞移植（HSCT）前从供体干细胞移植物中选择性清除同种异体反应性T细胞是预防移植物抗宿主病（GVHD）的一个重要目标。然而，在人类白细胞抗原（HLA）匹配的供受者对中，由于同种异体反应性T细胞频率低且缺乏方法标准化，移植前利用体外系统鉴定这些细胞已被证明很困难。为了更好地理解HLA匹配的相关供受者对之间的同种异体反应，我们对混合淋巴细胞反应（MLR）分析系统中产生的同种异体反应性T细胞进行了特征分析。
HSCT供体外周血单个核细胞（反应细胞）用羧基荧光素二乙酸琥珀酰亚胺酯（CFSE）染料标记，并在单向MLR中与经辐照的HSCT受者细胞（刺激细胞）共培养。通过激活标志物上调（大多数情况下为CD25）对同种异体反应性T细胞进行分选，并通过对T细胞受体β链互补决定区3（CDR3）进行测序来确定反应性克隆型。
我们发现同种异体反应性CD4(+) T细胞的募集具有高度变异性。在同一供受者对中重复进行的MLR中，寡克隆CD4(+) T细胞扩增显示克隆型的募集不一致。在重复试验中，同种异体反应性CD8(+) T细胞的募集更一致，在不同条件下对同一供受者对进行检测时可鉴定出相同的克隆型。
综上所述，我们的数据表明，即使在限制背景增殖的培养条件下，随机事件和低前体频率也会妨碍可重复诱导出具有引发GVHD潜力的免疫显性T细胞克隆型。
