Langerhans cell histiocytosis (LCH) is a rare disease involving inflammatory lesions that mostly affect children but can also occur in adults. Lesions comprise CD1a-expressing ‘LCH’ cells that often harbour mitogen-activated protein kinase cell-signalling pathway mutations, along with immune cells such as T cells and an inflammatory cytokine milieu. This composition suggests that both LCH cells and T cells contribute to the local inflammation. Our study aimed to understand which T cell and LCH cell associated proteins are upregulated in response to microenvironmental factors in LCH lesions. Multiplex digital spatial profiling is a novel proteomics approach that allows for high-throughput quantitative and spatial analysis of formalin-fixed, paraffin-embedded tissue samples. Using this technique, we conducted a deep characterization of LCH lesion tissue (n =4). We segmented regions of interest by expression of CD3 and CD1a and used a 71-protein immuno-oncology panel to examine expression of proteins adjacent to T cells (CD3+) in CD3 rich areas and in CD1a (LCH cell) rich areas, and expression of LCH cell (CD1a) adjacent proteins in CD3 rich areas and CD1a rich areas. We found 14 proteins significantly upregulated adjacent to T cells in CD1a rich regions of tissue and 15 proteins significantly upregulated adjacent to LCH cells in CD3 rich regions of tissue. Significance was based on a fold change >2 and p <0.05. This study highlights proteins that are potentially important in LCH pathogenesis. Further study into these associations is needed to determine whether the proteins or their associated pathways are suitable prognostic indicators or therapeutic targets to benefit patients.