Chan Kenneth S, Kaur Amitinder
Department of Immunology, New England Primate Research Center, Harvard Medical School, Southborough, MA 01772, USA.
J Immunol Methods. 2007 Aug 31;325(1-2):20-34. doi: 10.1016/j.jim.2007.05.011. Epub 2007 Jun 12.
Flow-cytometric conditions for detection of lysosomal-associated membrane proteins (LAMPs) on the surface of recently degranulated cells were optimized for rhesus macaques and used to investigate the functional properties of rhesus cytomegalovirus (rhCMV)-specific CD8+ T lymphocytes with regards to cytotoxicity and interferon (IFN)-gamma secretion in six asymptomatic CMV-seropositive rhesus macaques. Unlike humans, the rhesus macaque LAMP-1 protein CD107a underwent little or no endocytosis over a six to 18 h stimulation period. Following in vitro stimulation, rhCMV-specific CD8+ T lymphocytes were heterogeneous with regards to the composition of cells positive for CD107a and/or IFN-gamma, time to reach peak degranulation, and kinetics of IFN-gamma secretion relative to degranulation. Responder CD8+ T lymphocytes that underwent degranulation without IFN-gamma production (CD107a+IFN-gamma-) were predominantly composed of terminally differentiated effectors (CD28-CD45RA+). Moreover, they had significantly lower frequencies of effector memory (CD28-CD45RA-) cells compared to the IFN-gamma-secreting cells that did or did not undergo degranulation (CD107a+IFN-gamma+ or CD107a-IFN-gamma+). The perforin content of effector CD8+ T lymphocytes was significantly greater than that of effector memory CD8+ T lymphocytes in rhesus macaques, suggesting that they were more cytolytic. Our findings suggest that the composition of rhCMV-specific CD8+ T lymphocytes with regards to CD107a+IFN-gamma- responders may be an important determinant of their ability to control CMV replication.
针对恒河猴优化了用于检测近期脱颗粒细胞表面溶酶体相关膜蛋白（LAMPs）的流式细胞术条件，并用于研究6只无症状巨细胞病毒血清阳性恒河猴中恒河猴巨细胞病毒（rhCMV）特异性CD8⁺ T淋巴细胞在细胞毒性和干扰素（IFN）-γ分泌方面的功能特性。与人类不同，恒河猴LAMP-1蛋白CD107a在6至18小时的刺激期内几乎没有或没有发生内吞作用。体外刺激后，rhCMV特异性CD8⁺ T淋巴细胞在CD107a和/或IFN-γ阳性细胞的组成、达到脱颗粒峰值的时间以及相对于脱颗粒的IFN-γ分泌动力学方面存在异质性。发生脱颗粒但不产生IFN-γ（CD107a⁺IFN-γ⁻）的反应性CD8⁺ T淋巴细胞主要由终末分化效应细胞（CD28⁻CD45RA⁺）组成。此外，与发生或未发生脱颗粒的IFN-γ分泌细胞（CD107a⁺IFN-γ⁺或CD107a⁻IFN-γ⁺）相比，它们的效应记忆（CD28⁻CD45RA⁻）细胞频率显著更低。恒河猴中效应性CD8⁺ T淋巴细胞的穿孔素含量显著高于效应记忆性CD8⁺ T淋巴细胞，表明它们的细胞毒性更强。我们的研究结果表明，rhCMV特异性CD8⁺ T淋巴细胞中CD107a⁺IFN-γ⁻反应细胞的组成可能是其控制CMV复制能力的重要决定因素。
