Identification of rejection-associated indirect CD4 T cell epitopes provides a target for donor-specific transplant tolerance. Identification of rejection-associated indirect CD4 T cell epitopes provides a target for donor-specific transplant tolerance

Antibody-mediated rejection, caused by antibodies against the donor human leukocyte antigen (HLA) molecules plays a fundamental role in graft rejection in transplantation. Most significant is transplant patients who generate antibodies against the donor HLA-DQ have the highest risk of rejection. In this study, we investigated the role of indirect CD4 T cell epitopes in the formation of donor-specific antibodies (DSA). Using antigen mapping techniques in kidney and heart transplant recipients with HLA-DQ DSA, we identified two polymorphic hotspots in the HLA-DQ gene that generated alloreactive CD4 T cell responses. To study the functional significance of indirect CD4 T cells, we first mapped epitopes recognised by H2-Kb C57Bl6 mice against a skin graft from H2-Kd Balb/c mice. We identified a CD4 T cell epitope, specific for amino acids 287-301 derived from the H2-Kd protein, that generated tetramer-binding Kd287+ CD4 T cells during rejection. Importantly, the transfer of Kd287+ CD4 T cells into T cell-deficient mice was sufficient to drive an antibody response against the donor H2-Kd molecule. Lastly, we found that administration of systemic high-dose donor H2-Kd peptides combined with CTLA4-Ig reduced the formation of Kd287+ CD4 T cells and diminished DSA formation. Together, these findings demonstrate that the identification of donor antigens indicates the potential for inducing donor-specific immune tolerance in transplantation. Overall design: To investigate the TCR composition and diversity of alloreactive indirect CD4 T cells, we performed TCR sequencing on flow-sorted Kd287+ CD4 T cells isolated on day 14 after transplantation in mice that exhibited donor allograft rejection and underwent peptide treatment.

抗体介导的排斥反应由针对供体人类白细胞抗原（human leukocyte antigen, HLA）分子的抗体所引发，在移植移植物排斥过程中发挥核心作用。其中，产生针对供体HLA-DQ抗体的移植受者，其排斥风险最高，这一点最为关键。本研究探讨了间接途径CD4 T细胞表位在供体特异性抗体（donor-specific antibodies, DSA）形成过程中的作用。我们针对携带HLA-DQ型供体特异性抗体的肾与心脏移植受者，采用抗原定位技术，在HLA-DQ基因中鉴定出两个可诱导同种反应性CD4 T细胞应答的多态性热点区域。

为探究间接途径CD4 T细胞的功能意义，我们首先以H2-Kd型BALB/c小鼠的皮肤移植物为靶抗原，对H2-Kb型C57BL/6小鼠体内识别该抗原的表位进行了定位。我们成功鉴定出一个源自H2-Kd蛋白、对应氨基酸287-301位的CD4 T细胞表位，该表位可在排斥反应进程中诱导产生四聚体结合的Kd287+ CD4 T细胞。值得注意的是，将Kd287+ CD4 T细胞过继转移至T细胞缺陷小鼠体内，即可有效触发针对供体H2-Kd分子的抗体应答。

此外，我们发现联合给予全身性高剂量供体H2-Kd肽与细胞毒性T淋巴细胞相关抗原4-免疫球蛋白（CTLA4-Ig），可减少Kd287+ CD4 T细胞的生成，并抑制供体特异性抗体的形成。综上，本研究结果表明，鉴定供体抗原可为移植术中诱导供体特异性免疫耐受提供潜在方向。

整体实验设计：为探究同种反应性间接途径CD4 T细胞的T细胞受体（T cell receptor, TCR）组成与多样性，我们对移植后第14天、发生供体同种异体移植物排斥且接受肽治疗的小鼠体内，经流式分选得到的Kd287+ CD4 T细胞进行了TCR测序。