Transplant Immunology Laboratory, Comprehensive Transplant Center, Cedars-Sinai Medical Center/UCLA School of Medicine, 8700 Beverly Blvd., SSB111, Los Angeles, CA 90048, USA.
Transpl Immunol. 2010 Feb;22(3-4):121-7. doi: 10.1016/j.trim.2009.11.004. Epub 2009 Nov 26.
Sensitization to HLA antigens (Ags) is a significant obstacle to kidney transplantation and risk factor for antibody-mediated rejection (AMR). Current screening methods to assess HLA Ag exposure include various antibody assays. However, tools to accurately measure cell-mediated immunity to allo-Ags in a clinical setting are lacking. Here we report on an intracellular cytokine flow cytometry (CFC) assay that detects intracellular gamma-interferon (IFNgamma) production in non-T cell populations (CD3-) that appears to assess sensitization from previous allo-Ag exposure.
Blood from 106 highly-HLA sensitized (HS) patients (pre-, post-IVIG-treatment [Rx] and/or post-transplant) and 14 3(rd) party normal controls (3(rd)N) were incubated with donor or 3(rd)N peripheral blood mononuclear cells (PBMCs), and IFNgamma+/CD3- cells were enumerated.
The percentage of IFNgamma+ cells in CD3- cells without stimulation in pre-IVIG-Rx HS patients was similar to normals, but significantly increased with incubation with donor and/or 3(rd)N PBMCs. Reactivity in normals was minimal. Reactivity was higher in HS females than HS males. Normal females with previous pregnancy (PG) showed significantly higher response than females without PG or non-sensitized normal males. Donor-specific reactivity in the CFC assay better correlated with donor-specific B cell crossmatch than total anti-HLA antibody levels or PRA. HS patients who developed AMR post-transplant showed significantly higher reactivity than those without AMR.
The CFC assay measures IFNgamma production in CD3- cells that may indicate a memory response to allo-Ags. This response is limited to HS patients and normal females with previous PG. Patients undergoing AMR show significantly higher reactivity. This assay may represent a novel approach to measurement of allo-sensitization with clinical utility in predicting those at risk for AMR.
对 HLA 抗原（Ags）的致敏是肾移植的一个重大障碍，也是抗体介导的排斥反应（AMR）的危险因素。目前评估 HLA Ag 暴露的筛选方法包括各种抗体检测。然而，在临床环境中准确测量针对同种异体抗原的细胞介导免疫的工具仍然缺乏。在这里，我们报告了一种细胞内细胞因子流式细胞术（CFC）检测，该检测可检测非 T 细胞群体（CD3-）中的细胞内γ干扰素（IFNgamma）产生，似乎可评估来自先前同种异体 Ag 暴露的致敏情况。
采集 106 名高度 HLA 致敏（HS）患者（预 IVIG 治疗[Rx]前、后和/或移植后）和 14 名第三方正常对照（3rdN）的血液，与供体或第三方外周血单个核细胞（PBMCs）孵育，并计数 IFNgamma+/CD3-细胞。
在预 IVIG-Rx HS 患者中，无刺激时 CD3-细胞中 IFNgamma+细胞的百分比与正常对照相似，但与供体和/或第三方 PBMC 孵育时显著增加。正常对照的反应性较小。HS 女性的反应性高于 HS 男性。有既往妊娠（PG）的正常女性的反应明显高于无 PG 或无致敏的正常男性。CFC 检测中的供体特异性反应与供体特异性 B 细胞交叉配型相比，与总抗 HLA 抗体水平或 PRA 相关性更好。移植后发生 AMR 的 HS 患者的反应性明显高于未发生 AMR 的患者。
CFC 检测可测量 CD3-细胞中 IFNgamma 的产生，这可能表明对同种异体抗原的记忆反应。这种反应仅限于 HS 患者和有既往 PG 的正常女性。发生 AMR 的患者表现出明显更高的反应性。该检测方法可能代表了一种测量同种异体致敏的新方法，在预测 AMR 风险方面具有临床应用价值。
