An Ex Vivo Study of T Lymphocytes Recovered from the Lungs of I/St Mice Infected with and Susceptible to Mycobacterium tuberculosis

I/St mice, previously characterized as susceptible to Mycobacterium tuberculosis H37Rv, were given 10(3) or 10(5) CFU intravenously. At two time points postinoculation, the cell suspensions that resulted from enzymatic digestion of lungs were enumerated and further characterized phenotypically and functionally. Regarding the T-cell populations recovered at 2 and 5 weeks postinfection, two main results were obtained: (i) the population of CD44(−) CD45RB(+) cells disappeared within 2 weeks postinfection, while the number of CD44(+) CD45RB(−/low) cells slowly increased between weeks 2 and 5; (ii) when cocultured with irradiated syngeneic splenocytes, these lung T cells proliferated in the presence of H37Rv sonicate. Using H37Rv sonicate and irradiated syngeneic splenocytes to reactivate lung T cells, we selected five CD3(+) CD4(+) CD8(−) T-cell clones. In addition to the H37Rv sonicate, the five clones react to both a short-term culture filtrate and an affinity-purified 15- to 18-kDa mycobacterial molecule as assessed by the proliferative assay. However, there was a clear difference between T-cell clones with respect to cytokine (gamma interferon [IFN-γ] and interleukin-4 [IL-4] and IL-10) profiles: besides one Th1-like (IFN-γ(+) IL-4(−)) clone and one Th0-like (IFN-γ(+) IL-4(+) IL-10(+)) clone, three clones produced predominantly IL-10, with only marginal or no IL-4 and IFN-γ responses. Inhibition of mycobacterial growth by macrophages in the presence of T cells was studied in a coculture in vitro system. It was found that the capacity to enhance antimycobacterial activity of macrophages fully correlated with INF-γ production by individual T-cell clones following genetically restricted recognition of infected macrophages. The possible functional significance of cytokine diversity among T-cell clones is discussed.

先前已被证实对结核分枝杆菌H37Rv（Mycobacterium tuberculosis H37Rv）易感的I/St小鼠，经静脉注射给予10³或10⁵集落形成单位（Colony-Forming Unit，CFU）的菌液。在接种后的两个时间点，对经酶消化肺组织所得的细胞悬液进行计数，并进一步开展表型与功能特征鉴定。针对感染后2周和5周回收的T细胞群，得到两项核心结果：(i) CD44⁻CD45RB⁺细胞群在感染后2周内完全消失，而CD44⁺CD45RB⁻/low细胞群的数量在第2周至第5周间缓慢上升；(ii) 当与辐照同基因脾细胞共培养时，这些肺T细胞在结核分枝杆菌H37Rv超声裂解物存在的条件下发生增殖。利用结核分枝杆菌H37Rv超声裂解物与辐照同基因脾细胞对肺T细胞进行再激活，我们成功筛选得到5株CD3⁺CD4⁺CD8⁻ T细胞克隆。经增殖实验验证，除结核分枝杆菌H37Rv超声裂解物外，这5株克隆还可识别短期培养滤液以及经亲和纯化的15~18 kDa分枝杆菌分子。不过，各T细胞克隆在细胞因子（γ干扰素[IFN-γ]、白细胞介素4[IL-4]及白细胞介素10[IL-10]）表达谱上存在显著差异：除1株Th1样（Th1-like）克隆与1株Th0样（Th0-like）克隆外，其余3株克隆主要分泌IL-10，仅伴随极微量或无IL-4与IFN-γ应答。我们通过体外共培养体系，探究了T细胞存在时巨噬细胞对分枝杆菌生长的抑制作用。研究发现，单株T细胞克隆经遗传限制性识别受感染巨噬细胞后，其分泌IFN-γ的能力与其增强巨噬细胞抗分枝杆菌活性的能力完全正相关。本研究最后讨论了T细胞克隆间细胞因子表达多样性的潜在功能意义。