Defining human skin immunity to cutaneous leishmaniasis via systems immunology approaches

National Institute Of Allergy And Infectious Diseases

Investigators

Abstract

(1) Construction of a single cell transcriptomic atlas of human cutaneous leishmaniasis. In FY25, we continued our collaborative project with Drs. Elise O’Connell and Janitzio Guzmán (LPD) through the NIH Leishmaniasis Clinic and expanded our cohort of patients to construct a single cell transcriptomic atlas of human CL. Over the past year we evaluated ten patients, of whom nine were confirmed by PCR to have Leishmania infection. Of these, eight patients consented to the collection of research biopsies. Skin punch biopsies from both a CL lesion and from healthy skin (as a negative control) were collected from each patient and cryopreserved for single cell RNA-seq (scRNA-seq) analysis. Of the eight patients who provided skin biopsy samples, two had infections with Eastern hemisphere Leishmania species, five had infections with Western hemisphere species, and one had an infection that could not be speciated, but was most likely acquired in the Western hemisphere. Five patients presented with a primary lesion, while three patients had treatment-refractory or relapsed CL. There is now a total of 23 patients in the study cohort, with skin samples from 11 patients currently being sequenced as of this writing (NCI Frederick Sequencing Facility) and samples from the final 3 patients scheduled to be sequenced in October 2025. Also in FY25, we continued our scRNA-seq analysis of CL lesions from a case series of three patients with L. braziliensis seen at the NIH Clinical Center. This analysis was led by a new member of our research group, Julius Herzog (postbaccalaureate IRTA) in collaboration with Dr. Justin Lack and Jian Sun (NIAID, RTB, IDSS). We performed scRNA-seq on skin punch biopsies from patient CL lesions and matched healthy skin using the 10X Genomics 5’ High Throughput (HT) NextGEM v2 assay. A total of 66,019 cells passed quality control and were included in the atlas. For cell clustering, we applied a novel algorithm called CHOIR which uses a random forest classifier with iterative pruning of a hierarchical clustering tree to rigorously and reproducibly identify statistically distinct cell subsets and cell states. CHOIR analysis revealed 160 distinct cell states in the atlas distributed across six major cell types. IFN-γ-induced gene expression programs are highly upregulated across both immune and stromal cell types. T cells compose the largest proportion of cells in the CL lesion, with the novel identification of γδ T cells, MAIT cells, and iNKT cells in human CL lesions. The T cell subsets in the lesion are notable for large populations of exhausted cytotoxic T cells, regulatory T cells, and T resident memory cells. T cells exhibited gene expression signatures consistent with the formation of tertiary lymphoid structures, correlating with the presence of IgG-producing plasma cells in the lesions. Dual host-pathogen scRNA-seq successfully identified parasite-derived transcripts in infected macrophages in the atlas. Remarkably, infected macrophages formed a distinct cluster in the dataset, typified by downregulation of the MHC class II pathway, IFN-γ-responsive genes, and M1 polarization. Finally, there is a global shift across multiple cell types towards glycolytic metabolism associated with induction of HIF-1α and upregulation of pro-fibrotic gene programs in lesion macrophages, potentially leading to excessive scarring during CL lesion healing. In summary, this scRNA-seq atlas offers insights into the novel immune cell subsets, formation of tertiary lymphoid structures, and hypoxic metabolic alterations involved in the pathogenesis of human CL caused by L. braziliensis. (2) Microbiopsies of human cutaneous leishmaniasis lesions. In FY25, we collected skin microbiopsies from 7 patients, including one patient who declined a standard skin punch research biopsy. All patients tolerated the procedure well without complications and with normal healing of all the microbiopsy puncture sites. In collaboration with Drs. O’Connell and Guzmán in the Nutman Laboratory (LPD), we sought to determine whether microbiopsies could be used as a novel, non-invasive diagnostic approach for the clinical diagnosis of CL by PCR, as an alternative to conventional skin punch biopsy, which requires local anesthesia and leaves a permanent scar. We determined that microbiopsies provide a sufficient amount of skin from the lesion site to detect Leishmania by qPCR with a sensitivity up to 88% and a specificity of 100% versus unrelated protozoa using a novel tandemly repeated sequence called CL3 that is conserved across all Leishmania species which infect humans. Interestingly, parasite DNA was detected in healthy appearing skin in one patient, suggesting that microbiopsies are sensitive enough to detect asymptomatic disseminated infection. Thus, microbiopsies represent a promising non-invasive tool to diagnose and speciate CL in resource-limited field settings.