Hemophagocytic lymphohistiocytosis (HLH) is a syndrome characterized by pathologic immune activation in which prompt recognition and initiation of immune suppression is essential for survival. Children with HLH have many overlapping clinical features with critically ill children with sepsis and systemic inflammatory response syndrome (SIRS) in whom alternative therapies are indicated. To determine if plasma biomarkers could differentiate HLH from other inflammatory conditions and to better define a 'core inflammatory signature' of HLH, concentrations of inflammatory plasma proteins were compared in 40 patients with HLH to 47 pediatric patients with severe sepsis or SIRS. Fifteen of 135 analytes were significantly different in HLH plasma compared to SIRS/sepsis, including increased interferon-γ (IFN-γ)-regulated chemokines CXCL9, CXCL10 and CXCL11. Further, a two-analyte plasma protein classifier including CXCL9 and IL-6 was able to differentiate HLH from SIRS/sepsis. Gene expression in CD8+ T cells and activated monocytes from blood were also enriched for IFN-γ pathway signatures in peripheral blood cells from patients with HLH compared to SIRS/sepsis. This study identifies differential expression of inflammatory proteins as a diagnostic strategy to identify critically ill children with HLH, and comprehensive unbiased analysis of inflammatory plasma proteins and global gene expression demonstrates that IFN-γ signaling is uniquely elevated in HLH. In addition to demonstrating the ability of diagnostic criteria for HLH and sepsis or SIRS to identify groups with distinct inflammatory patterns, results from this study support the potential for prospective evaluation of inflammatory biomarkers to aid in diagnosis of and optimizing therapeutic strategies for children with distinctive hyperinflammatory syndromes.

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. Disclosures No relevant conflicts of interest to declare.

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by granulomatous lesions containing pathological CD207+ dendritic cells (DCs) with persistent mitogen-activated protein kinase (MAPK) pathway activation. Standard of care chemotherapy strategies are inadequate or overly toxic for the majority of patients with multisystem disease. The mechanisms underlying development of inflammation in LCH lesions are poorly understood, and potential for immunotherapy has not been determined. Analysis of the LCH lesion identified the most prominent immune cells as T lymphocytes, second only to pathologic CD207+/CD1a+ DCs (LCH DC). Both CD8+ and CD4+ T cells exhibited 'exhausted' phenotypes with high expression of the immune checkpoint inhibitor receptors. LCH DC cells also showed robust expression of ligands to checkpoint inhibitor receptors. Lesion CD8+ T cells exhibited blunted expression of Th1 cytokines with impaired effector function. In contrast, regulatory T cells (Tregs) isolated from LCH lesions demonstrated intact suppressive activity. Treatment of BRAFV600ECD11c LCH mice with anti-PD-1, anti-TIM-3 and MEKi decreased the lesion size: MEKi treatment resulted in reduction of the myeloid compartment; anti-PD-1 and anti-TIM-3 were associated with reduction in the lymphoid compartment. However, combined treatment with MEKi and either anti-PD-1 or anti-TIM-3 significantly decreased both CD8+ T cell and myeloid LCH cells in a synergistic fashion. These results thus indicate MAPK hyperactivation in myeloid LCH cells drives recruitment and activation of functionally exhausted T cells within the LCH microenvironment. While the results from this study did not demonstrate a significant impact of checkpoint inhibition alone on tumor burden in experimental mice, combined MAPK and checkpoint inhibition may be a potentially effective therapeutic strategy for patients with LCH. Disclosures Bollard: self: Patents & Royalties: patent on HIV T cells; NexImmune: Equity Ownership; Torque: Equity Ownership; Cellectis: Membership on an entity's Board of Directors or advisory committees; Caballetta: Equity Ownership; Mana Therapeutics: Equity Ownership.