Lesional senescent CD4+ T cells mediate bystander cytolysis and contribute to the skin pathology of human cutaneous leishmaniasis

Abstract

Cytotoxic activity is a hallmark of the immunopathogenesis in human cutaneous leishmaniasis (CL). In this study, we identified accumulation of CD4⁺ granzyme B producing T cells with increased cytotoxic capacity in CL lesions. These cells showed enhanced expression of activating NK receptors (NKG2D and NKG2C), diminished expression of inhibitory NKG2A, along with the upregulation of the senescence marker CD57. Notably, CD4⁺ T cells freshly isolated from CL lesions demonstrated remarkable capacity to mediate NL-like bystander cytolysis. Phenotypic analyses revealed that lesional CD4⁺ T cells are mainly composed of late-differentiated effector (CD27-CD45RA-) and terminally differentiated (senescent) TEMRA (CD27-CD45RA+) subsets. Interestingly, the TEMRA CD4⁺ T cells exhibited higher expression of granzyme B and CD107a. Collectively, our results provide the first evidence that senescent cytotoxic CD4⁺ T cells may support the skin pathology of human cutaneous leishmaniasis and, together with our previous findings, support the notion that multiple subsets of cytotoxic senescent cells may be involved in inducing the skin lesions in these patients.

Keywords: CD4-CTL; Leishmania braziliensis; bystander cytotoxicity; cutaneous leishmaniasis; senescent cells.

Figure 1

Cytotoxic signature is found in CL lesions and positively correlates with CD4 T cells. (A) Cytotoxic signature score expressed in skin biopsies from healthy controls (HC, light blue) and lesions from LCL patients (LCL, red). Gene signature scores statistical differences were calculated using Wilcoxon’s Rank Sum test. (B) The heatmap of cytotoxicity and activation genes differentially expressed in skin lesions represented as relative log2 normalized and variance stabilized (vst) values, where healthy controls (HC, light blue) and lesions from LCL patients (LCL, red) are compared. (C) Spearman’s correlation test between cytotoxic score and estimated cell (NK, CD4⁺ and CD8⁺) proportions obtained by cell deconvolution, where ρ represents Spearman’s Rho value. The p values were calculated using ANOVA test with Bonferroni correction **** p < 0.0001.

Figure 2

Lesional skin-infiltrating CD4+ T cells from CL-patients exhibit enhanced expression of cytotoxicity and natural killer (NK) markers. (A) Representative image of cellular infiltration, dispersion of granzyme B positive cells (red) and cytotoxic CD4⁺ T cells in CL lesions (green arrows indication). Staining was performed using immunofluorescence assay and analysed by Halo software. (B) Frequency of granzyme B and (C) cells expressing granzyme B in the blood and lesions of CL patients and (D) granzyme B differential expression (fold change) within circulating and lesional CD4⁺ and CD8⁺ T cells. The graphs show the mean ± SD. The p values were calculated using t test or ANOVA with Bonferroni correction (*p< 0,05, **p<0.01) ***p<0.001).

Figure 3

Lesional CD4 T cells mediate bystander cytotoxicity through NK receptors. Frequency of (A) NKG2D, (B) NKG2C, (C) CD57 and (D) NKG2A within lesional CD4+ T cell. (E) Lesional CD4⁺ T cell degranulation capacity accessed by CD107 mean fluorescence intensity (MFI); representative histogram of CD107a expression (blue line represents FMO; dashed line represents blood cells; solid line represents skin cells) and (F) bystander cytolysis capacity against K562 cell line. Purified CD4⁺ T cells (over 95% purity) from CL lesions were co-cultured with K562 target cells (effector: target ratio of 10:1) for 18 hr. The cytotoxic activity assessed by calcein-release lysis assay. The graphs show the mean ± SD. The p values were calculated using paired t test or ANOVA with Bonferroni correction (*p< 0,05, **p<0.01) ***p<0.001).

Figure 4

Skin lesion-derived CD4⁺ CTL T cells are predominantly found in the senescent-EMRA memory subset and demonstrate significant cytotoxic capacity. (A) Representative FACS plot and frequencies of memory T cell subsets defined by CD45RA/CD27 expression as: Naive (CD27 + 45RA+; Blue color), Central Memory (CD27+CD45RA-;Green color), Effector Memory (CD27-CD45RA; Red color), and Terminal Effector Memory EMRA (CD27-CD45RA+; Orange color) within CD4⁺ T cells and (B) cumulative data of memory subsets frequencies from blood and skin of CL patients. (C) Frequency (%) and representative histogram of granzyme B mean fluorescence intensity (MFI). (D) Cumulative data and representative histogram of Mean Fluorescence Intensity (MFI) of the expression of CD107a. Cumulative data of (E) NKG2C, (F) NKG2D and (G) CD57 expression within memory CD4⁺ T cells subsets. The p values were calculated using ANOVA test with Bonferroni correction (*p< 0,05, **p<0.01, ***p<0.001, **** p < 0.0001).

Figure 5

Leishmania infection leads to the infiltration of cytotoxic CD4 T cells (CD4-CTL) with senescent features, which aberrantly target and kill stromal and immune cells, contributing to the excessive tissue damage observed in cutaneous leishmaniasis lesions. These CD4-CTLs, expressing various NK receptors (NKRs), including NKG2D and NKG2C, interact with resident skin cells. This interaction results in bystander activation and skin cells killing, exacerbating off-target tissue pathology.