Apparent Lack of BRAF^V600E Derived HLA Class I Presented Neoantigens Hampers Neoplastic Cell Targeting by CD8⁺ T Cells in Langerhans Cell Histiocytosis

Langerhans Cell Histiocytosis (LCH) is a neoplastic disorder of hematopoietic origin characterized by inflammatory lesions containing clonal histiocytes (LCH-cells) intermixed with various immune cells, including T cells. In 50–60% of LCH-patients, the somatic BRAF^V600E driver mutation, which is common in many cancers, is detected in these LCH-cells in an otherwise quiet genomic landscape. Non-synonymous mutations like BRAF^V600E can be a source of neoantigens capable of eliciting effective antitumor CD8⁺ T cell responses. This requires neopeptides to be stably presented by Human Leukocyte Antigen (HLA) class I molecules and sufficient numbers of CD8⁺ T cells at tumor sites. Here, we demonstrate substantial heterogeneity in CD8⁺ T cell density in n = 101 LCH-lesions, with BRAF^V600E mutated lesions displaying significantly lower CD8⁺ T cell:CD1a⁺ LCH-cell ratios (p = 0.01) than BRAF wildtype lesions. Because LCH-lesional CD8⁺ T cell density had no significant impact on event-free survival, we investigated whether the intracellularly expressed BRAF^V600E protein is degraded into neopeptides that are naturally processed and presented by cell surface HLA class I molecules. Epitope prediction tools revealed a single HLA class I binding BRAF^V600E derived neopeptide (KIGDFGLATEK), which indeed displayed strong to intermediate binding capacity to HLA-A^*03:01 and HLA-A^*11:01 in an in vitro peptide-HLA binding assay. Mass spectrometry-based targeted peptidomics was used to investigate the presence of this neopeptide in HLA class I presented peptides isolated from several BRAF^V600E expressing cell lines with various HLA genotypes. While the HLA-A^*02:01 binding BRAF wildtype peptide KIGDFGLATV was traced in peptides isolated from all five cell lines expressing this HLA subtype, KIGDFGLATEK was not detected in the HLA class I peptidomes of two distinct BRAF^V600E transduced cell lines with confirmed expression of HLA-A^*03:01 or HLA-A^*11:01. These data indicate that the in silico predicted HLA class I binding and proteasome-generated neopeptides derived from the BRAF^V600E protein are not presented by HLA class I molecules. Given that the BRAF^V600E mutation is highly prevalent in chemotherapy refractory LCH-patients who may qualify for immunotherapy, this study therefore questions the efficacy of immune checkpoint inhibitor therapy in LCH.