IL-32 producing CD8+ memory T cells and Tregs define the IDO1 / PD-L1 niche in human cutaneous leishmaniasis skin lesions
Human cutaneous leishmaniasis (CL) is characterised by chronic skin pathology. Experimental and clinical data suggest that immune checkpoints (ICs) play a crucial role in disease outcome but the cellular and molecular niches that facilitate IC expression during leishmaniasis are ill-defined. We previously showed that in Sri Lankan patients with CL, indoleamine 2,3-dioxygenase 1 (IDO1) and programmed death-ligand 1 (PD-L1) are enriched in lesion skin and that reduced PD-L1 expression early after treatment onset predicted cure rate following antimonial therapy. Here, we use spatial cell interaction mapping to identify IL-32-expressing CD8+ memory cells and regulatory T cells as key components of the IDO1 / PD-L1 niche in Sri Lankan CL patients and in patients with distinct forms of dermal leishmaniasis in Brazil and India. Furthermore, the abundance of IL-32+ cells and IL-32+CD8+ T cells at treatment onset was prognostic for rate of cure in Sri Lankan patients. This study provides a unique spatial perspective on the mechanisms underpinning IC expression during CL and a novel route to identify additional biomarkers of treatment response. Overall design: Samples: SL2 cohort, BR cohort and IN cohort. Study design Sri Lanka The study design for the Sri Lankan cohort was initially published as part of the validation cohort details in Dey et al, 2021 32. Briefly, individuals who provided written informed consent and exhibited clinically highly suspected CL lesions were enrolled. On day 0, punch biopsies were collected and intralesional sodium stibogluconate (SSG) treatment was administered. Biopsies were processed into formalin-fixed and embedded in paraffin (FFPE) format and then shipped to the University of York (UoY) for subsequent immunological studies described here. These enrolled patients were then monitored for up to 6 months to assess complete clinical cure, and given weekly intralesional SSG injections. Of these, 24/25 patients had achieved complete clinical cure by 6 months, and 1/25 patients at 6.5 months. Out of the 25 patients in this cohort, 23/25 patient sections were included in the neighbourhood image analysis aimed at identifying neighbours of IDO1 and PD-L1 in stained sections; two were excluded due to insufficient material in the blocks. For including samples for 10x Visium analysis, these 23 patients were further screened for the presence of parasites by using an RNA probe against Amastin to detect parasites in 5u thick sections as described previously 32. 6/23 were Amastin+ and were included for Visium spatial analysis (P1-P6). Furthermore, out of the 6 samples included for Visium analysis, only four samples could be included in the CosMx analysis due to the availability of sufficient material in the blocks for follow up studies.  Brazil Patient cohort from Brazil involved 20 patients that agreed to participate by signing the informed consent Form. Patients were diagnosed on the basis of positive polymerase chain reaction and/or indirect immunofluorescence and/or ELISA directed to Leishmania. Depending on the size and location of the lesion and the clinical condition of the patients, treatment was done with systemic pentavalent antimonials (Glucantime) for 20 days; or intralesional pentavalent antimonials (Glucantime) with one application per week for 4 weeks; or liposomal amphotericin B for 10 days. Biopsy samples of the lesion were collected on day 0 when the diagnosis was established and patients were followed up after treatment for 6 months to assess clinical cure. To understand IDO1 and CD274 microenvironments in healing and non-healing chronic skin L.(V)b. lesions, 20 patient samples were screened for double positivity against diagnostic PCR and ELISA (12/20) and were then stratified on the basis of visual treatment score at 3 months; healed if visual score=0 at 3 months and not healed if visual score >0 at 3 months. Of 12 samples, only 2 patients had healing score>0 at 3 months and were included in the study. 2 patients with visual score=0 at 3 months with comparable age, lesion size and lesion duration were then selected from the bigger cohort (12) to include a total of 4 samples that were processed through 10x Visium.  India The initial diagnosis was based on clinical features suggestive of PKDL (presence of papules, nodules and/or hypopigmented macular lesions), a prior history of VL, rK-39 positivity, and/or if they resided in an area endemic for VL. In general, cases with hypopigmented patches were considered as macular PKDL, whereas cases with an assortment of papules, nodules, plaques, and/or macules were termed as polymorphic PKDL. Based on these criteria, individuals who were willing and provided written informed consent were enrolled for this study. The patients were examined by a clinician, clinical history recorded, and biological samples (blood and punch biopsy) collected. Subsequently, the diagnosis was confirmed by qPCR 91. 2 samples with typical histopathology of PKDL lesions and comparable RNA quality were chosen. These two patients were enrolled between November 2019 and August 2021. 5u thick sections were cut and processed through 10x Visium.