Departamento de Medicina Experimental, Facultad de Medicina, Universidad Nacional Autónoma de México, Hospital General de México OD, México Distrito Federal, Mexico.
PLoS Negl Trop Dis. 2010 Nov 2;4(11):e871. doi: 10.1371/journal.pntd.0000871.
Leishmania mexicana (Lm) causes localized (LCL) and diffuse (DCL) cutaneous leishmaniasis. DCL patients have a poor cellular immune response leading to chronicity. It has been proposed that CD8 T lymphocytes (CD8) play a crucial role in infection clearance, although the role of CD8 cytotoxicity in disease control has not been elucidated. Lesions of DCL patients have been shown to harbor low numbers of CD8, as compared to patients with LCL, and leishmanicidal treatment restores CD8 numbers. The marked response of CD8 towards Leishmania parasites led us to analyze possible functional differences between CD8 from patients with LCL and DCL. We compared IFNγ production, antigen-specific proliferation, and cytotoxicity of CD8 purified from PBMC against autologous macrophages (MO) infected with Leishmania mexicana (MOi). Additionally, we analyzed tissue biopsies from both groups of patients for evidence of cytotoxicity associated with apoptotic cells in the lesions. We found that CD8 cell of DCL patients exhibited low cytotoxicity, low antigen-specific proliferation and low IFNγ production when stimulated with MOi, as compared to LCL patients. Additionally, DCL patients had significantly less TUNEL+ cells in their lesions. These characteristics are similar to cellular "exhaustion" described in chronic infections. We intended to restore the functional capacity of CD8 cells of DCL patients by preincubating them with TLR2 agonists: Lm lipophosphoglycan (LPG) or Pam3Cys. Cytotoxicity against MOi, antigen-specific proliferation and IFNγ production were restored with both stimuli, whereas PD-1 (a molecule associated with cellular exhaustion) expression, was reduced. Our work suggests that CD8 response is associated with control of Lm infection in LCL patients and that chronic infection in DCL patients leads to a state of CD8 functional exhaustion, which could facilitate disease spread. This is the first report that shows the presence of functionally exhausted CD8 T lymphocytes in DCL patients and, additionally, that pre-stimulation with TLR2 ligands can restore the effector mechanisms of CD8 T lymphocytes from DCL patients against Leishmania mexicana-infected macrophages.
墨西哥利什曼原虫（Lm）可引起局限性（LCL）和弥漫性（DCL）皮肤利什曼病。DCL 患者的细胞免疫反应较差，导致疾病慢性化。有人提出，CD8 T 淋巴细胞（CD8）在清除感染中发挥关键作用，尽管 CD8 细胞毒性在疾病控制中的作用尚未阐明。与 LCL 患者相比，DCL 患者的病变中 CD8 数量较低，而利什曼病治疗可恢复 CD8 数量。CD8 对利什曼原虫的显著反应促使我们分析 LCL 和 DCL 患者的 CD8 之间可能存在的功能差异。我们比较了从 PBMC 中纯化的 CD8 针对感染了墨西哥利什曼原虫的自体巨噬细胞（MOi）产生 IFNγ、抗原特异性增殖和细胞毒性的能力。此外，我们还分析了两组患者的组织活检，以寻找病变中与凋亡细胞相关的细胞毒性的证据。我们发现，与 LCL 患者相比，DCL 患者的 CD8 细胞在受到 MOi 刺激时表现出低细胞毒性、低抗原特异性增殖和低 IFNγ产生。此外，DCL 患者病变中的 TUNEL+细胞明显较少。这些特征类似于慢性感染中描述的细胞“衰竭”。我们试图通过用 TLR2 激动剂预先孵育 DCL 患者的 CD8 细胞来恢复其功能能力：Lm 脂磷壁酸（LPG）或 Pam3Cys。两种刺激物均可恢复对 MOi 的细胞毒性、抗原特异性增殖和 IFNγ 产生，而 PD-1（与细胞衰竭相关的分子）表达降低。我们的工作表明，CD8 反应与 LCL 患者中 Lm 感染的控制有关，而 DCL 患者中的慢性感染导致 CD8 功能衰竭状态，这可能促进疾病传播。这是首次报道表明 DCL 患者存在功能衰竭的 CD8 T 淋巴细胞，并且，预先刺激 TLR2 配体可以恢复 DCL 患者针对感染了墨西哥利什曼原虫的巨噬细胞的 CD8 T 淋巴细胞的效应机制。
