Mølvig J, Sønderstrup McDevitt G, Thomsen M, Bosmann B, Blangero C, de Preval C, Baek L, Nerup J
Steno Memorial Hospital, Gentofte, Denmark.
Scand J Immunol. 1989 Nov;30(5):615-29. doi: 10.1111/j.1365-3083.1989.tb02469.x.
The particular susceptibility to insulin-dependent diabetes mellitus (IDDM) conferred by HLA-DR3,4 heterozygosity has been suggested to be an effect of transcomplementation of HLA class II molecules. To test this hypothesis of special IDDM-specific hybrid determinants and to evaluate the T-cell repertoire towards a specific antigen in IDDM patients we generated a total of 352 PPD-specific T-cell lines by the soft-agar cloning technique and studied their restriction by HLA class II molecules. Of these lines, 227 were from nine IDDM patients, of whom six were DR3,4 heterozygotes, and 125 from 10 healthy controls. Forty-six T-cell lines elicited specific responses in at least two experiments and in addition to T-cell lines demonstrating class-II-restricted PPD specificity, lines with an alloreactivity occurred. HLA-DQ-restricted PPD-specific T-cell lines were not identified and a possible DP restriction (DPw2) was only observed with one line. These data indicate that PPD is preferentially presented to T cells in the context of HLA-DR/Dw. Presentation of PPD by hybrid molecules in IDDM patients or by IDDM-specific class II epitopes recognized by the T-cell lines was not demonstrated. By restriction fragment length polymorphism analysis using a probe for the joining region of the T-cell receptor gamma gene, T-cell lines generated by the soft-agar cloning technique were found to be oligoclonal. It is concluded that soft-agar cloning should be followed by subsequent limiting dilution in order to assure monoclonality. Different preparations of antigen-presenting cells (APC) were tested. In several cases the T-cell lines were not able to respond to PPD presented by Epstein-Barr-virus-transformed lymphoblastoid cell lines (LCL). It was demonstrated that lipopolysaccharides (LPS) of E. coli potently reduce the proliferative response of antigen-specific and alloreactive T cells when T-cell-depleted peripheral blood mononuclear cells (E- cells) were used as APC, whereas only limited inhibition was observed when LCL were used as APC in the presence of LPS. This effect of LPS is suggested to be mediated by increased prostaglandin secretion by monocytes among the E- cells since indomethacin abolished the effect of LPS. This observation may have implications for T-cell cloning procedures since we have found that most commercially available culture media are heavily contaminated with endotoxin.
HLA - DR3,4杂合性赋予的对胰岛素依赖型糖尿病（IDDM）的特殊易感性被认为是HLA II类分子反式互补的结果。为了验证这种特殊的IDDM特异性杂交决定簇的假说，并评估IDDM患者针对特定抗原的T细胞库，我们通过软琼脂克隆技术共产生了352个PPD特异性T细胞系，并研究了它们受HLA II类分子的限制情况。这些细胞系中，227个来自9名IDDM患者，其中6名是DR3,4杂合子，125个来自10名健康对照。46个T细胞系在至少两个实验中引发了特异性反应，除了显示II类限制的PPD特异性的T细胞系外，还出现了具有同种异体反应性的细胞系。未鉴定出HLA - DQ限制的PPD特异性T细胞系，仅在一个细胞系中观察到可能的DP限制（DPw2）。这些数据表明，PPD在HLA - DR/Dw的背景下优先呈递给T细胞。未证明IDDM患者中由杂交分子或T细胞系识别的IDDM特异性II类表位呈递PPD。通过使用T细胞受体γ基因连接区探针的限制性片段长度多态性分析，发现通过软琼脂克隆技术产生的T细胞系是寡克隆的。得出的结论是，为了确保单克隆性，软琼脂克隆之后应进行后续的有限稀释。测试了不同的抗原呈递细胞（APC）制剂。在几种情况下，T细胞系无法对由爱泼斯坦 - 巴尔病毒转化的淋巴母细胞系（LCL）呈递的PPD作出反应。已证明，当使用T细胞耗竭的外周血单核细胞（E细胞）作为APC时，大肠杆菌的脂多糖（LPS）会强烈降低抗原特异性和同种异体反应性T细胞的增殖反应，而当在LPS存在下使用LCL作为APC时，仅观察到有限的抑制作用。LPS的这种作用被认为是由E细胞中的单核细胞增加前列腺素分泌介导的，因为吲哚美辛消除了LPS的作用。这一观察结果可能对T细胞克隆程序有影响，因为我们发现大多数市售培养基都被内毒素严重污染。
