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 is inadequate for most patients with multisystem disease, and optimal strategies for relapsed and refractory disease are not defined. The mechanisms underlying development of inflammation in LCH lesions, the role of inflammation in pathogenesis and potential for immunotherapy are unknown. Analysis of the immune infiltrate in LCH lesions identified the most prominent immune cells as T lymphocytes. Both CD8+ and CD4+ T cells exhibited 'exhausted' phenotypes with high expression of the immune checkpoint receptors. LCH DCs showed robust expression of ligands to checkpoint receptors. Intra-lesional CD8+ T cells showed blunted expression of Tc1/Tc2 cytokines and impaired effector function. In contrast, intra-lesional regulatory T cells (Tregs) demonstrated intact suppressive activity. Treatment of BRAFV600ECD11c LCH mice with anti-PD-1 or MAPK inhibitor reduced lesion size, but with distinct responses: whereas MAPK inhibitor treatment resulted in reduction of the myeloid compartment, anti-PD-1 treatment was associated with reduction in the lymphoid compartment. Notably, combined treatment with MAPK inhibitor and anti-PD-1 significantly decreased both CD8+ T cell and myeloid LCH cells in a synergistic fashion. These results are consistent with a model that MAPK hyperactivation in myeloid LCH cells drives recruitment of functionally exhausted T cells within the LCH microenvironment and highlight combined MAPK and checkpoint inhibition as a potential therapeutic strategy.

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Background Inhibition of IL-6 receptor with tocilizumab (TCZ) is an effective strategy for treating rheumatoid arthritis (RA) patients. However, the effect of TCZ on lymphocytes remains poorly studied. Regulatory T cells (Tregs) and NK cytotoxicity are known to be impaired in RA. Objectives The objective of the present study was to analyze the effect of TCZ on B, T, NK and NKT cells and to find predictive factors of response. Methods To be included, patients should meet ACR/EULAR criteria, require the introduction of TCZ, have corticosteroid doses below 10 mg per day, stable for more than a week and should not have received rituximab in the previous year. Extra-cellular staining with antibodies directed against CD19, CD24, CD27, CD38, IgD, CD3, CD4, CD8, CD25, CD127, CD56, CD16, g9d2 and intra-cellular staining of Granzyme B and Perforine A were performed after ficoll at baseline, 3 and 6 months. IL-10 was measured in sera by ELISA. Results Twenty-three patients requiring a treatment with TCZ as well as 31 healthy controls were included. Patients with RA had significantly lower percentage of Tregs as defined by CD4+CD25HiCD127lo (3.8 [2.7-4.9] vs 4.8 [3.5-6.0]% of CD4+ lymphocytes; respectively, p=0.007). After 3 months of TCZ, Tregs significantly increased (+1.27±1.00% of CD4+ cells; p<0.001), meeting normal values. Tregs and IL-10 were correlated at baseline (r=0.52; p=0.01) and variations of Tregs were correlated with variations of IL-10 after 3 months of TCZ (r=0.69; p<0.01). No change in percentage of B, NK and NKT cells subsets was observed. Patients in EULAR remission at 3 months had higher percentage of CD3-CD56+ NK cells at baseline (10.4±7.1 vs 3.6±3.1% of lymphocytes; p=0.01), especially CD56BriCD16- NK cells (0.88±0.68 vs 0.28±0.28% of CD3- cells; p=0.04) than patients with a DAS28 at 3 months above 2.6. NK cells were more frequently positive for perforine A and tended to be more frequently positive for granzyme B after TCZ treatment (+6.8 ±2.9% of CD3-CD56+ cells; p=0.02 and +7.1 ±3.8% of CD3-CD56+ cells; p=0.06, respectively). Conclusions TCZ increased the number of phenotypic Tregs. Significant correlation between variation of IL-10 and variations of Tregs suggest than these Tregs are probably functional, at least in terms of IL-10 production. TCZ also seem to restore NK cells cytotoxicity, known to be impaired in RA. Values of NK cells at baseline could help to predict remission after 3 months of treatment. Acknowledgements To the French Society of Rheumatology and Roche-Chugaï (France) for their financial support Disclosure of Interest: None Declared

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.