Fig 1.
KLRK1 is expressed in lesions and is correlated with cytolytic genes.
RNAseq analysis from 7 healthy skin (HS) and 21 lesions from L. braziliensis-infected patients (CL). (A) Gene expression of KLRK1, which encodes NKG2D. (B) Correlation shows KLRK1 expression compared to GZMB, GNLY, and PRF1. (C) Correlation shows KLRK1 expression compared to MCP counter abundance scores for CD8 T cell and CTL score. Gene expression is represented as counts per million (CPM) in the log2 scale. (D and E) Cells isolated from lesions or PBMC obtained from HS and L. braziliensis patients were stained for flow cytometry directly ex vivo and depicted are representative flow cytometry plots (D) and scatter plots (E) of NKG2D expression in CD8 T cells. Data were obtained from 5 HS and 9 L. braziliensis patients. HS, healthy subjects; CL, cutaneous leishmaniasis; PBMC, peripheral blood mononuclear cells. *p<0.05. *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < .0001.
Fig 2.
NKG2D ligands are enriched in lesions from L. braziliensis-infected patients.
RNAseq analysis from 7 healthy skin (HS) and lesions from 21 L. braziliensis-infected patients (CL). (A) Gene expression of MICA, MICB, ULBP1, ULBP2, ULBP3, and RAET1E (ULBP4). (B and C) Immunohistochemistry for MICA/B in biopsies from L. braziliensis patients’ lesions. Data were obtained from 3 HS and 3 L. braziliensis lesions. (D and E) Cells isolated from lesions or PBMC obtained from L. braziliensis patients were stained for flow cytometry directly ex vivo and depicted are representative flow cytometry plots (D) and scatter plots (E) of MICA/B expression in CD11b+ cells. Data were obtained from 6 PBMC and 5 skin lesions. HS, healthy skin; CL, cutaneous leishmaniasis; PBMC, peripheral blood mononuclear cells. *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < .0001.
Fig 3.
IL-15 induces NKG2D expression on CD8 T cells from L. braziliensis-infected patients.
RNAseq analysis from 7 healthy skin (HS) and lesions from 21 L. braziliensis-infected patients (CL). (A) Gene expression of IL15, IL15RA, IL2RG, and IL2RG in the skin of HS and lesions from L. braziliensis-infected patients. (B) Correlation between KLRK1 expression and IL15, IL15RA, IL2RB, and IL2RG expression in the lesions. Gene expression is represented as counts per million (CPM) in the log2 scale. (C–F) PBMCs from L. braziliensis-infected patients and healthy subjects were cultured with IL-15 for 18h and stained for flow cytometry. Dot plots (C and E) and graph bars represent (D and F) NKG2D expression by CD8 T cells after IL-15 stimulation. Data were obtained from 5 healthy subjects and 7 L. braziliensis-infected patients. HS, healthy skin; PBMC, peripheral blood mononuclear cells. **p ≤ 0.01, ***p ≤ 0.001, ****p < .0001.
Fig 4.
IL-1β induces MICA/B expression in L. braziliensis patients.
RNAseq analysis from 7 healthy skin (HS) compared to lesions from 21 L. braziliensis-infected patients. (A) Gene expression of IL1B in HS and lesions from L. braziliensis-infected patients (CL). (B) Correlation between MICB expression and IL1B expression at the lesion. Gene expression is represented as counts per million (CPM) in the log2 scale. (C–F) PBMCs from L. braziliensis infected patients (Lb) (C and D) or healthy subjects (E and F) were cultured in the presence of IL-1β recombinant for 18h. LPS was used as a positive control of MICA/B induction. Data were obtained from 7 L. braziliensis infected patients and 5 healthy subjects. Representative histogram of MICA/B expression of L. braziliensis infected patients (C) and healthy subjects (E) gated in CD11b+ CD14+ cells. PBMC, peripheral blood mononuclear cells. **p ≤ 0.01, ***p ≤ 0.001, ****p < .0001.
Fig 5.
NKG2D promotes CD8 T cell degranulation in a subset of L. braziliensis-infected patients and is associated with treatment failure.
Cells isolated from the lesions of L. braziliensis patients were incubated with anti-CD107a and cultured with or without anti-NKG2D antibody. Flow cytometry plots (A and C) and scatter plots (B and D) of CD107a expression on the surface of CD8 T cells are depicted. Data were obtained from 10 L. braziliensis-infected lesions. (E) KLRK1 log2 counts per million (log2 CPM) expression in patients that cured (n = 14) or failed (n = 7) the first round of treatment with antimony. *p < 0.05, **p<0.01.