TCR Repertoire Clonality Analysis and Transcriptome Analyses of Immune Infiltrates in Patients with Langerhans Cell Histiocytosis Can Define Prognostic Biomarkers for Future Therapeutic Development

Langerhans Cell Histiocytosis (LCH) is a myeloproliferative disorder, most common in children, with an incidence of approximately 5 cases per million children and 1/10,000 live births per year - similar to the frequency of Hodgkin's lymphoma and AML in children. LCH lesions are comprised of clonal pathologic CD207+ dendritic cells (DCs) harboring mutually exclusive genomic alterations of MAPK pathway members along with a large inflammatory infiltrate comprised of macrophages, multinucleated giant cells, lymphocytes, and eosinophils that do not carry MAPK mutations. Mass cytometry revealed that in systemic LCH lesions, T lymphocytes are the most abundant cellular subsets among immune infiltrates and exhibit an exhausted phenotype characterized by expression of immune inhibitory receptors. Since inflammatory infiltrate composed of cells without MAPK pathway mutation is characteristic of LCH, it is likely that the pathogenic DCs in LCH mediate some of the pathologic manifestations through DC-T cell interactions. However, properties of tumor infiltrating T lymphocytes (TILs) in LCH are poorly understood, In this study we performed Bulk TCR alpha/ beta repertoire profiling using the SMARTer TCR a/b Profiling Kit (Takara Bio ) to identify the CDR3 and full length clonotypes in sorted CD8, CD4 and CD4 CD25 compartments isolated from lesions that were stimulated as well the repertoire from the matching white blood cells. We found that in the sorted CD8 cells and CD4 cells from all patient lesions, there was a characteristic clonality shift where-in the repertoire in the lesions were clonally expanded, and interestingly some of the V, J genes were shared among the patient lesions,

In addition to changes in the TCR repertoire, we performed bulk Transcriptome analyses to characterize the functional nature of the T-cell infiltrates, and we find that genes responsible for calcium mobilization and mitochondrial metabolism are significantly downregulated in LCH TILs, even after TCR stimulation. RANKL (encoding receptor activator of nuclear factor kappa-B ligand or tumor necrosis factor ligand superfamily member 11) expression, which is solely dependent on TCR activation‐induced calcium mobilization, is also downregulated in the LCH TILs. RANKL is widely known as a modulator of immune cell function by virtue of influencing the survival and T cell-stimulatory capacity of DCs. Impaired calcium mobilization and mitochondrial metabolism might explain the exhausted phenotype we observe in the LCH TILs. We are currently investigating whether the clonal signatures identified by TCR sequencing in the LCH TILs can be used for prognostic purposes, and the T cell functional states can also be exploited to identify biomarkers that can be used to stratify LCH patients for therapy.