Prasad S, Humphreys I, Kireta S, Gilchrist R B, Bardy P, Russ G R, Coates P T H
Transplantation Immunology Laboratory, The Queen Elizabeth Hospital, Woodville, SA, Australia.
Tissue Antigens. 2007 Apr;69 Suppl 1:72-5. doi: 10.1111/j.1399-0039.2006.760_7.x.
The difficulties with using nonhuman primate species such as rhesus macaques and baboons have led us to investigate the common marmoset (Callithrix jacchus) as an alternative preclinical model for transplantation research. This requires reliable methods of detecting alloreactivity between donor and recipient pairs, particularly if colonies are inbred and share just a few common alleles for leucocyte antigens. We firstly identified marmoset major histocompatibility complex (MHC) Class II DRB genes (Caja-DRBW1201, Caja-DRB103, Caja-DRBW16) using sequence-based typing techniques. Genomic DNA (n= 49) was extracted from whole blood or spleen tissue. Exon 2 of target genes was amplified by PCR using primers specific for known marmoset alleles, and then sequenced using ABI PRISM((R)) Big Dye Terminator technology and Assign sequence analysis software. DRBW1201 was universally present. Eight DRBW16 alleles and five DRB103 alleles were identified in this colony. We also identified two previously unreported DRB*W16 alleles, and confirmed inheritance of these alleles within several sibling groups. Subsequently, we investigated whether matching at MHC Class II DRB loci alone could predict alloreactivity, as assessed in vitro by two-way mixed lymphocyte reactions (MLRs). Fully DRB-matched, partially mismatched and fully mismatched animal pairs were prospectively chosen. MLR was performed using mononuclear cells (MNC) isolated from whole blood by density gradient separation. T-cell proliferation after 5-day culture was measured by (3)H-thymidine incorporation. Combined MNC from fully mismatched and partially mismatched animal pairs exhibited significant in vitro T-cell proliferation above single cell controls (P < 0.01). MNC from fully DRB-matched (but unrelated) animal pairs exhibited no proliferation compared with controls (P= 0.3). Using DRB genotyping, suitably alloreactive donor-recipient pairs may therefore be rapidly and accurately identified for use in further studies of cellular and solid organ transplantation.
使用恒河猴和狒狒等非人灵长类动物存在困难，这促使我们研究普通狨猴（Callithrix jacchus）作为移植研究的替代临床前模型。这需要可靠的方法来检测供体和受体对之间的同种异体反应性，特别是如果群体是近交的并且白细胞抗原仅共享少数常见等位基因。我们首先使用基于序列的分型技术鉴定了狨猴主要组织相容性复合体（MHC）II类DRB基因（Caja-DRBW1201、Caja-DRB103、Caja-DRBW16）。从全血或脾脏组织中提取基因组DNA（n = 49）。使用针对已知狨猴等位基因的特异性引物通过PCR扩增靶基因的外显子2，然后使用ABI PRISM((R)) Big Dye Terminator技术和Assign序列分析软件进行测序。DRBW1201普遍存在。在这个群体中鉴定出八个DRBW16等位基因和五个DRB103等位基因。我们还鉴定出两个以前未报道的DRB*W16等位基因，并在几个同胞组中证实了这些等位基因的遗传。随后，我们研究了仅在MHC II类DRB位点进行匹配是否可以预测同种异体反应性，这通过双向混合淋巴细胞反应（MLR）在体外进行评估。前瞻性地选择了完全DRB匹配、部分不匹配和完全不匹配的动物对。使用通过密度梯度分离从全血中分离的单核细胞（MNC）进行MLR。通过(3)H-胸腺嘧啶核苷掺入测量5天培养后的T细胞增殖。完全不匹配和部分不匹配动物对的联合MNC在体外表现出明显高于单细胞对照的T细胞增殖（P < 0.01）。与对照相比，完全DRB匹配（但不相关）动物对的MNC没有增殖（P = 0.3）。因此，使用DRB基因分型，可以快速准确地鉴定出具有适当同种异体反应性的供体-受体对，用于细胞和实体器官移植的进一步研究。
