Bronke Corine, Palmer Nanette M, Westerlaken Geertje H A, Toebes Mireille, van Schijndel Gijs M W, Purwaha Veenu, van Meijgaarden Krista E, Schumacher Ton N M, van Baarle Debbie, Tesselaar Kiki, Geluk Annemieke
Department of Clinical Viro-Immunology, Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam.
Hum Immunol. 2005 Sep;66(9):950-61. doi: 10.1016/j.humimm.2005.06.011. Epub 2005 Aug 16.
In order to detect epitope-specific CD4+ T cells in mycobacterial or viral infections in the context of human class II major histocompatibility complex protein human leukocyte antigen (HLA)-DR3, two HLA-DR3 tetrameric molecules were successfully produced. One contained an immunodominant HLA-DR3-restricted T-cell epitope derived from the 65-kDa heat-shock protein of Mycobacterium tuberculosis, peptide 1-13. For the other tetramer, we used an HLA-DR3-restricted T-cell epitope derived from cytomegalovirus (CMV) pp65 lower matrix protein, peptide 510-522, which induced high levels of interferon (IFN)-gamma-producing CD4+ T cells in three of four HLA-DR3-positive CMV-seropositive individuals up to 0.84% of CD4+ T cells by intracellular cytokine staining. In peripheral blood mononuclear cells from M. tuberculosis-exposed, Mycobacterium bovis bacille Calmette-Guérin (BCG)-vaccinated, or CMV-seropositive individuals, we were able to directly detect with both tetramers epitope-specific T cells up to 0.62% and 0.45% of the CD4+ T-cell population reactive to M. tuberculosis and CMV, respectively. After a 6-day culture with peptide p510-522, the frequency of CMV-specific tetramer-binding T cells was expanded up to 9.90% tetramer+ CFSElow (5,6-carboxyfluorescein diacetate succinimidyl ester) cells within the CD4+ T-cell population, further confirming the specificity of the tetrameric molecules. Thus, HLA-DR3/peptide tetrameric molecules can be used to investigate HLA-DR3-restricted antigen-specific CD4+ T cells in clinical disease or after vaccination.
为了在人类II类主要组织相容性复合体蛋白人类白细胞抗原（HLA）-DR3背景下检测分枝杆菌或病毒感染中的表位特异性CD4⁺ T细胞，成功制备了两种HLA-DR3四聚体分子。一种包含源自结核分枝杆菌65 kDa热休克蛋白的免疫显性HLA-DR3限制性T细胞表位，肽段1-13。对于另一种四聚体，我们使用了源自巨细胞病毒（CMV）pp65低基质蛋白的HLA-DR3限制性T细胞表位，肽段510-522，通过细胞内细胞因子染色，在4名HLA-DR3阳性CMV血清阳性个体中的3名中，该表位诱导产生高水平干扰素（IFN）-γ的CD4⁺ T细胞高达CD4⁺ T细胞的0.84%。在接触过结核分枝杆菌、接种过卡介苗（BCG）的牛分枝杆菌或CMV血清阳性个体的外周血单核细胞中，我们能够用这两种四聚体直接检测到表位特异性T细胞，分别占对结核分枝杆菌和CMV反应的CD4⁺ T细胞群体的0.62%和0.45%。用肽段p510-522培养6天后，CMV特异性四聚体结合T细胞的频率在CD4⁺ T细胞群体中扩大到高达9.90%的四聚体⁺ CFSE低（5,6-羧基荧光素二乙酸琥珀酰亚胺酯）细胞，进一步证实了四聚体分子的特异性。因此，HLA-DR3/肽四聚体分子可用于研究临床疾病或疫苗接种后HLA-DR3限制性抗原特异性CD4⁺ T细胞。
