CD8 Cells of Patients with Diffuse Cutaneous Leishmaniasis Display Functional Exhaustion: The Latter Is Reversed, In Vitro, by TLR2 Agonists

Abstract

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.

Author Summary

Leishmania mexicana causes localized and diffuse cutaneous leishmaniasis. Whereas the former is a benign form the disease, diffuse cutaneous leishmaniasis is a chronic disfiguring disease, for which no cure is available, and the immune cells of these patients respond poorly to the parasite. It has been proposed that the elimination of Leishmania-infected cells by CD8 T cells is crucial for disease control. We compared the functional characteristics of CD8 T cells from patients with localized and diffuse cutaneous leishmaniasis. We found that CD8 T cells from patients with diffuse cutaneous leishmaniasis were functionally exhausted, as compared to patients with the benign form of the disease. We were able to restore functional capacity of these cells by culturing them with molecules that stimulate TLR2. This is the first report showing that stimulation of the TLR2 can restore effector mechanisms in functionally exhausted CD8 cells from patients with diffuse cutaneous leishmaniasis. This finding will help design novel treatment schemes for patients infected with the parasite Leishmania mexicana who have the progressive, incurable form of diffuse cutaneous leishmaniasis.