Dang L H, Michalek M T, Takei F, Benaceraff B, Rock K L
Division of Lymphocyte Biology, Dana Farber Cancer Institute, Boston, MA 02115.
J Immunol. 1990 Jun 1;144(11):4082-91.
We report a methodology for selecting APC with mutations that have impaired their ability to present Ag to T cells. A20 B lymphoblastoid cells were mutagenized and then repeatedly cocultured with murine T-T hybridomas in the presence of specific Ag. During these cocultures, the T-T hybridomas kill the competent APC, allowing the outgrowth of inactive variants. Two variants, A20.M1 and A20.M2, were isolated and studied in detail. These variants are impaired in their ability to present multiple Ag to T cells. This defect is also observed for the presentation of processing independent peptides by fixed APC indicating that a lesion exists in a post-Ag processing step. The level of expression of MHC molecules is unaffected and the functional defect in the APC is not localized to a particular MHC molecule. In contrast, these mutants were found to have a selective decrease in the expression of the murine homolog of ICAM-1, and the residual ability of these cells to present Ag was not blocked by anti-ICAM-1 mAb. Conversely, Ag presentation by the wild-type A20 is inhibited by anti-ICAM-1 mAb. Similarly, anti-LFA-1 mAb inhibited the response of T cells to Ag presented by the wild-type A20 to a much greater degree than by the mutant cells, indicating that LFA-1 is involved in interaction of T cells with the former, but not latter, APC. In the apparent absence of a contribution of LFA-1 to the T cell-APC interaction, either as a result of mAb blocking or the disruption of the APC membrane, the mutant and wild-type APC have a similar level of Ag-presenting activity. Reconstitution of ICAM-1 expression in these mutants by transfection with murine ICAM-1 cDNA fully restores their ability to present Ag. Together these results demonstrate that a murine ICAM-1 homolog is expressed on A20 B cells, where it functions as a major cell interaction molecule. The degree of functional impairment in these mutant APC gives insight into the contribution of cell interaction molecules to efficient Ag presentation and T cell-B cell interaction. Finally, these results also demonstrate the feasibility of selecting APC with mutations affecting Ag presentation.
我们报告了一种选择具有向T细胞呈递抗原能力受损突变的抗原呈递细胞（APC）的方法。对A20 B淋巴母细胞进行诱变，然后在特异性抗原存在的情况下，将其与鼠T-T杂交瘤反复共培养。在这些共培养过程中，T-T杂交瘤杀死有功能的APC，使无活性变体得以生长。分离出两个变体A20.M1和A20.M2并进行了详细研究。这些变体向T细胞呈递多种抗原的能力受损。对于固定APC呈递不依赖加工的肽也观察到这种缺陷，这表明在抗原加工后步骤存在损伤。MHC分子的表达水平未受影响，APC中的功能缺陷并不局限于特定的MHC分子。相反，发现这些突变体中ICAM-1的鼠同源物表达有选择性降低，并且这些细胞呈递抗原的剩余能力未被抗ICAM-1单克隆抗体阻断。相反，野生型A20的抗原呈递被抗ICAM-1单克隆抗体抑制。同样，抗LFA-1单克隆抗体对T细胞对野生型A20呈递抗原的反应的抑制程度比对突变细胞的抑制程度大得多，这表明LFA-1参与T细胞与前者而非后者APC的相互作用。在明显不存在LFA-1对T细胞-APC相互作用的贡献的情况下，无论是由于单克隆抗体阻断还是APC膜的破坏，突变体和野生型APC具有相似水平的抗原呈递活性。通过用鼠ICAM-1 cDNA转染在这些突变体中重建ICAM-1表达，可完全恢复其呈递抗原的能力。这些结果共同表明，鼠ICAM-1同源物在A20 B细胞上表达，在那里它作为主要的细胞相互作用分子发挥作用。这些突变APC中的功能受损程度有助于深入了解细胞相互作用分子对有效抗原呈递和T细胞-B细胞相互作用的贡献。最后，这些结果也证明了选择具有影响抗原呈递突变的APC的可行性。
