Langerhans Cell Histiocytosis Cells Research Articles

The plasma-soluble CSF1R level is a promising prognostic indicator for pediatric Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is a rare hematologic neoplasm characterized by the clonal proliferation of Langerhans-like cells. Colony-stimulating factor 1 receptor (CSF1R) is a membrane-bound receptor that is highly expressed in LCH cells and tumor-associated macrophages. In this study, a soluble form of CSF1R protein (sCSF1R) was identified by plasma proteome profiling, and its role in evaluating LCH prognosis was explored. We prospectively measured plasma sCSF1R levels in 104 LCH patients and 10 healthy children using ELISA. Plasma sCSF1R levels were greater in LCH patients than in healthy controls (p<.001) and significantly differed among the three disease extents, with the highest level in MS RO+ LCH patients (p<.001). Accordingly, immunofluorescence showed the highest level of membrane-bound CSF1R in MS RO+ patients. Furthermore, the plasma sCSF1R concentration at diagnosis could efficiently predict the prognosis of LCH patients treated with standard first-line treatment (AUC=0.782, p<.001). Notably, dynamic monitoring of sCSF1R levels could predict relapse early in patients receiving BRAF inhibitor treatment. In vitro drug sensitivity data showed that sCSF1R increased resistance to Ara-C in THP-1 cells expressing ectopic BRAF-V600E. Overall, the plasma sCSF1R level at diagnosis and during follow-up is of great clinical importance in pediatric LCH patients.

HDAC3 Is Required for Pathognomonic Features of Langerhans Cell Histiocytes

Langerhans cell histiocytosis (LCH) is a pediatric inflammatory myeloid neoplasm that develops due to dysregulated myeloid cell development. BRAFV600E is the most common disease-causing mutation and constitutively activates the mitogen-activated protein kinase (MAPK) pathway in myeloid lineage precursors leading to the key pathognomonic features of LCH cells. Enhanced myelopoiesis and reduced CCR7 expression promote accumulation of LCH cells in tissues by simultaneously increasing the production of pathological DCs and preventing tissue egress. Furthermore, LCH cells acquire an oncogene induced senescence-associated secretory phenotype (SASP) that depends on mammalian target of rapamycin (mTOR), which is hallmarked by increased expression of anti-apoptotic proteins, inflammatory cytokines, and matrix metalloproteinases enhancing survival of LCH cells and promoting recruitment of inflammatory immune cells forming characteristic granulomatous lesions. These pathognomonic features result in the accumulation of LCH cells in any organ causing a wide range of clinical symptoms. Frontline therapy for LCH involves combination chemotherapy and steroidal anti-inflammatories, or MAPK inhibitors, which have significant toxicity and fail to eliminate disease causing precursors. New therapeutic approaches are urgently needed. Here, using multiple genetic mouse models, we show that normal epidermal Langerhans cells (LCs) depend on HDAC3 for their development, differentiation, and survival. Integrative RNA and chromatin-immunoprecipitation-sequencing show loss of HDAC3 abrogates the expression of master regulators of myeloid development and function including Csf1r, Spi1, Id2 and Runx3. LCH cells are also known to rely on Csf1r and Pu.1 for their development and homeostasis, thus we hypothesized that LCH cells similarly rely on HDAC3 for their development and survival. CD11c Cre LSL-BRAFV600E ( BRAFV600E CD11c) mice develop severe multifocal LCH with pronounced lesion development in their livers and lungs, hepatosplenomegaly, and a reduced lifespan due to the accumulation of pathological dendritic cells (DCs). We generated BRAFV600E CD11c HDAC3 fl/fl ( BRAFV600E HD3KO) mice, which produce pathological DCs that simultaneously express BRAFV600E and harbor a conditional deletion in the deacetylase domain of HDAC3. Compared to BRAFV600E CD11c mice, BRAFV600E HD3KO mice exhibited significantly less hepatosplenomegaly, reduced lesional burden (Panel A), attenuated disease progression, and improved survival indicating reduced LCH disease burden. Compared to BRAFV600E CD11c flow cytometry showed BRAFV600E HD3KO had reduced numbers of LCH cells in lungs and livers linking improved disease outcomes to abrogation of pathological DCs. Flow cytometric analysis of circulating myeloid cells further found reduced frequency of circulating DCs and DC progenitors, indicating that a lack of HDAC3 activity prevents the development of pathological DCs (Panel B). LCH-like cells can be generated in vitro by culturing BRAFV600E CD11c bone marrow with granulocyte-monocyte colony-stimulating factor (GM-CSF), providing a valuable drug screening tool. Treating LCH-like cells with RGFP966, an HDAC3-specific inhibitor, increased apoptosis indicated by annexin-V and DAPI staining, reduced expression of Bcl-2, increased CCR7 expression, and decreased S6 phosphorylation (an indication of decreased mTOR activity), showing that pharmacological inhibition of HDAC3 may prove therapeutically efficacious by abrogating pathognomonic features of LCH cells. Together, our findings identify HDAC3 as a critical epigenetic regulator for both healthy and pathological LCs. We further show, HDAC3 is required for multiple pathognomonic features of LCH cells and could be a promising drug target. Furthermore, if HDAC3 is required for the development of pathological DC and DC progenitors, HDAC3 blockade would address a great need in treatment of patients with LCH.

Responses to Combination Chemotherapy with MAPK Pathway Inhibition in Relapsed and Refractory LCH

MAPK pathway inhibition (MAPKi) is associated with high response rates in patients with Langerhans cell histiocytosis (LCH), but monotherapy may not be curative and efficacy in LCH-associated neurodegeneration (LCH-ND) is not known. Based on upregulation of Bcl-xL by MAPK activation, we hypothesize that MAPKi may render LCH cells more sensitive to chemotherapy-induced cytotoxicity. We assessed the safety and efficacy of combination therapy in 10 patients with refractory systemic disease and LCH-ND. Records of patients treated with combination therapy at Texas Children's Hospital from October 2018 to July 2023 were reviewed for clinical responses as well as peripheral blood/tissue BRAFV600E mutation status, ataxia rating scores, and toxicity. The median age at the start of combination therapy was 6.82 years (range, 0.48-20.45 years) with a median of 4 treatments prior to this therapy regimen (range, 1-14). Almost all Clofarabine was administered outpatient (typical starting dose 25 mg/m2/day x 5 days). MAPK inhibitors included BRAFV600E inhibitors (50%) and MEK inhibitors (50%). Overall response was 8/10 (80%), with 5/5 (100%) objective response rate (ORR) for systemic disease. Further, 5/6 (83%) demonstrated reduced or extinguished peripheral BRAFV600E+ fraction by the end of combination therapy. Ataxia rating scores were improved in 2/3 (67%) individuals with clinical neurodegeneration. Common toxicities during treatment included fever, skin rash, arthralgia, and cytopenia. Combining therapy was well-tolerated and associated with clinical and molecular improvement in most patients with highly refractory systemic LCH and LCH-ND. Further prospective multi-center trials are required to optimize this approach, determine potential for combination therapy to achieve cure, and compare risks and benefits to chemotherapy or MAPK inhibitor monotherapy.

Opsin 3 expression in human Langerhans cell histiocytosis and its mediation of ELD-1 cellular function.

Langerhans cell histiocytosis (LCH) is a type of -histiocytic disorder characterized by aberrant function, differentiation or proliferation of mononuclear phagocyte system cells, however, the pathogenesis is not fully understood. Opsin 3 (OPN3) plays an important role in regulating cell function. We aimed to investigate OPN3 expression in LCH and Langerhans cells and evaluate its possible regulation of cellular function in a Langerhans cell-like cell line (ELD-1). Expression of OPN3 in LCH and paired adjacent healthy skin tissue was determined using microscopic tools (immunohistochemical and immunofluorescence staining) and RNA scope. OPN3 protein and mRNA levels in primary dendritic cells and ELD-1 were measured by real-time quantitative PCR and western blotting, respectively. The effects of reduced or over-expressed OPN3 mRNA level, via a lentiviral vector, were examined on ELD-1 proliferation, migration, cell cycle and apoptosis using the Cell Counting Kit 8, EdU-594 kit, Transwell assays and Cell Cycle Analysis Kit and Annexin V-PE apoptosis kit, respectively. Lastly, the signalling pathway mediating these functions was investigated via RNA sequencing and western blotting. OPN3 was highly expressed in human LCH tissue compared to healthy tissue, and was expressed in primary dendritic cells and ELD-1. Knockdown of OPN3 in ELD-1 inhibited cell proliferation, the cell cycle, and cell migration, while over-expression reversed these processes. These functions correlated with induction of the MAPK (p38/JNK/ERK) signalling pathway. Our results provide insight into the role of OPN3 in LCH which may become a molecular target for the clinical treatment of LCH.

Myeloid cells from Langerhans cell histiocytosis patients exhibit increased vesicle trafficking and an altered secretome capable of activating NK cells.

Langerhans cell histiocytosis (LCH) is a potentially life-threatening inflammatory myeloid neoplasia linked to pediatric neurodegeneration, whereby transformed LCH cells form agglomerated lesions in various organs. Although MAP-kinase pathway mutations have been identified in LCH cells, the functional consequences of these mutations and the mechanisms that cause the pathogenic behavior of LCH cells are not well understood. In our study, we used an in vitro differentiation system and RNA-sequencing to compare monocyte-derived dendritic cells from LCH patients to those derived from healthy controls or patients with Crohn's disease, a non-histiocytic inflammatory disease. We observed that interferon-γ treatment exacerbated intrinsic differences between LCH patient and control cells, including strikingly increased endo- and exocytosis gene activity in LCH patients. We validated these transcriptional patterns in lesions and functionally confirmed that LCH cells exhibited increased endo- and exocytosis. Furthermore, RNA-sequencing of extracellular vesicles revealed the enrichment of pathological transcripts involved in cell adhesion, MAP-kinase pathway, vesicle trafficking and T-cell activation in LCH patients. Thus, we tested the effect of the LCH secretome on lymphocyte activity and found significant activation of NK cells. These findings implicate extracellular vesicles in the pathology of LCH for the first time, in line with their established roles in the formation of various other tumor niches. Thus, we describe novel traits of LCH patient cells and suggest a pathogenic mechanism of potential therapeutic and diagnostic importance.

Targeting of SIRPα as a potential therapy for Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is a rare neoplastic disorder characterized by inflammatory lesions arising from the anomalous accumulation of pathogenic CD1a+ CD207+ dendritic cells (DCs). SIRPα is a transmembrane protein highly expressed in myeloid cells such as DCs and macrophages. Here we show that SIRPα is a potential therapeutic target for LCH. We found that SIRPα is expressed in CD1a+ cells of human LCH lesions as well as in CD11c+ DCs in the spleen, liver, and lung of a mouse model of LCH (BRAFV600ECD11c mouse), in which an LCH-associated active form of human BRAF is expressed in a manner dependent on the mouse Cd11c promoter. BRAFV600ECD11c mice manifested markedly increased numbers of CD4+ T cells, regulatory T cells, and macrophages as well as of CD11c+ MHCII+ DCs in the spleen. Monotherapy with a mAb to SIRPα greatly reduced the percentage of CD11c+ MHCII+ DCs in peripheral blood, LCH-like lesion size in the liver, and the number of CD11c+ MHCII+ DCs in the spleen of the mutant mice. Moreover, this mAb promoted macrophage-mediated phagocytosis of CD11c+ DCs from BRAFV600ECD11c mice, whereas it had no effects on the viability or CCL19-dependent migration of such CD11c+ DCs or on their expression of the chemokine genes Ccl5, Ccl20, Cxcl11, and Cxcl12. Our results thus suggest that anti-SIRPα monotherapy is a promising approach to the treatment of LCH that is dependent in part on the promotion of the macrophage-mediated killing of LCH cells.

Clinicogenomic associations in childhood Langerhans cell histiocytosis: an international cohort study.

Langerhans cell histiocytosis (LCH) is a rare neoplastic disorder caused by somatic genetic alterations in hematopoietic precursor cells differentiating into CD1a+/CD207+ histiocytes. LCH clinical manifestation is highly heterogeneous. BRAF and MAP2K1 mutations account for ∼80% of genetic driver alterations in neoplastic LCH cells. However, their clinical associations remain incompletely understood. Here, we present an international clinicogenomic study of childhood LCH, investigating 377 patients genotyped for at least BRAFV600E. MAPK pathway gene alterations were detected in 300 (79.6%) patients, including 191 (50.7%) with BRAFV600E, 54 with MAP2K1 mutations, 39 with BRAF exon 12 mutations, 13 with rare BRAF alterations, and 3 with ARAF or KRAS mutations. Our results confirm that BRAFV600E associates with lower age at diagnosis and higher prevalence of multisystem LCH, high-risk disease, and skin involvement. Furthermore, BRAFV600E appeared to correlate with a higher prevalence of central nervous system (CNS)-risk bone lesions. In contrast, MAP2K1 mutations associated with a higher prevalence of single-system (SS)-bone LCH, and BRAF exon 12 deletions seemed to correlate with more lung involvement. Although BRAFV600E correlated with reduced event-free survival in the overall cohort, neither BRAF nor MAP2K1 mutations associated with event-free survival when patients were stratified by disease extent. Thus, the correlation of BRAFV600E with inferior clinical outcome is (primarily) driven by its association with disease extents known for high rates of progression or relapse, including multisystem LCH. These findings advance our understanding of factors underlying the remarkable clinical heterogeneity of LCH but also question the independent prognostic value of lesional BRAFV600E status.

Notch-dependent cooperativity between myeloid lineages promotes Langerhans cell histiocytosis pathology.

Langerhans cell histiocytosis (LCH) is a potentially fatal neoplasm characterized by the aberrant differentiation of mononuclear phagocytes, driven by mitogen-activated protein kinase (MAPK) pathway activation. LCH cells may trigger destructive pathology yet remain in a precarious state finely balanced between apoptosis and survival, supported by a unique inflammatory milieu. The interactions that maintain this state are not well known and may offer targets for intervention. Here, we used single-cell RNA-seq and protein analysis to dissect LCH lesions, assessing LCH cell heterogeneity and comparing LCH cells with normal mononuclear phagocytes within lesions. We found LCH discriminatory signatures pointing to senescence and escape from tumor immune surveillance. We also uncovered two major lineages of LCH with DC2- and DC3/monocyte-like phenotypes and validated them in multiple pathological tissue sites by high-content imaging. Receptor-ligand analyses and lineage tracing in vitro revealed Notch-dependent cooperativity between DC2 and DC3/monocyte lineages during expression of the pathognomonic LCH program. Our results present a convergent dual origin model of LCH with MAPK pathway activation occurring before fate commitment to DC2 and DC3/monocyte lineages and Notch-dependent cooperativity between lineages driving the development of LCH cells.

Optimal timing of liver transplantation for liver cirrhosis caused by sclerosing cholangitis in a patient with Langerhans cell histiocytosis: a case report.

Liver cirrhosis due to secondary sclerosing cholangitis caused by Langerhans cell histiocytosis (LCH) has a poor prognosis, and liver transplantation is the definitive treatment. However, the optimal timing has not been established. We report a 2-year-old girl with LCH-related liver cirrhosis who successfully underwent liver transplantation before progressing to severe liver dysfunction. Physical examination revealed a tumor on her palate. Biopsy was performed, and a diagnosis of LCH was established, together with hepatomegaly, splenomegaly, and rashes. Percutaneous liver biopsy before treatment revealed extreme fibrosis and absence of LCH cells. After beginning chemotherapy, she experienced several delays in treatment and dose reductions because of unacceptable bone marrow suppression, worsening liver dysfunction, and cholangitis. However, tumor shrinkage was observed in both magnetic resonance imaging and BRAF V600E mutant allele titers in her plasma. Given the good treatment response, liver transplantation was conducted. The postoperative course was uneventful, and chemotherapy was resumed 34days after liver transplantation. Subsequent maintenance treatment was completed with no severe adverse effects. To prevent perioperative complications due to exacerbation of liver dysfunction and possible discontinuation of chemotherapy, liver transplantation should be considered before development of end-stage liver failure, provided that the original disease is well controlled.

Bone Marrow-Derived BRAFV600E-Mutated Cells Drive Neurodegeneration in a Mouse Model of Langerhans Cell Histiocytosis

Progressive neurodegeneration is a devastating and ultimately fatal complication in some survivors of multi-system high-risk Langerhans Cell Histiocytosis (LCH). Patients often develop LCH-ND years after the diagnosis and successful treatment of their systemic disease, and mechanisms that underlie LCH-ND remain largely undefined. BRAFV600E+ myeloid cells have been identified in areas of active neurodegeneration. However, it is unclear if the cell of origin of this disease is mutated microglia or circulating peripheral LCH cells invading the brain tissue. Embryonic origin (e.g. microglia) versus hematopoietic origin (or both) is a critical question to be able to identify, prevent and cure LCH-ND. We therefore tested the ability of bone marrow (BM)-derived BRAFV600E cells to migrate to the brain in a mouse model. We used BM chimera to study the transition of BRAFV600E-mutated cells to the brain as well as mechanisms of neurologic damage therein. Therefore, WT mice were conditioned with head-shielded Total Body Irradiation (TBI) and transplanted with BM from SCLCre-ER-TxBRAFV600ExRosa26YFP [LCH chimera] or SCLCre-ER-TxBRAFWTxRosa26YFP [control chimera]. Cre-recombination was induced after an engraftment period of 4 weeks. Mice were analyzed 4 months after Tamoxifen induction. LCH chimera exhibit a parenchymal infiltration with BM-derived BRAFV600E+ macrophages 4 months after Cre-recombination while control chimera do not have substantial numbers of reporter-tagged cells in the brain. The localization pattern of BRAFV600E+ macrophages in the brain recapitulate the pattern observed in humans, i.e. in the Brain Stem and Cerebellum. BM-derived BRAFV600E+ macrophages accumulate at the interface between the bloodstream and the central nervous system (CNS) parenchyma with 10-fold higher frequency than BM-derived BRAFWT macrophages and are associated with signs of local inflammation, astrocyte activation and decline in Purkinje cell density. Finally, LCH chimera exhibit signs of neurologic impairment (e.g. ataxia, decreased strength) that was partially reversed by targeted treatment. We conclude that BRAFV600E+ BM-derived myeloid progenitors reproducibly invade brain parenchyma where they differentiate into macrophages and induce inflammation and CNS injury. These novel insights inform previously unrecognized pathogenic mechanisms for LCH, with clinical implications in predicting LCH-ND risk, surveillance strategies, and novel therapeutic opportunities. Further, these data demonstrate potential for MAPK activation in hematopoietic myeloid progenitors to drive CNS inflammation and neurodegeneration, which may impact a broad range of neuropathological conditions beyond LCH.

Plasma Signaling Factors in Patients With Langerhans Cell Histiocytosis (LCH) Correlate With Relative Frequencies of LCH Cells and T Cells Within Lesions.

Langerhans cell histiocytosis (LCH) lesions contain an inflammatory infiltrate of immune cells including myeloid-derived LCH cells. Cell-signaling proteins within the lesion environment suggest that LCH cells and T cells contribute majorly to the inflammation. Foxp3+ regulatory T cells (Tregs) are enriched in lesions and blood from patients with LCH and are likely involved in LCH pathogenesis. In contrast, mucosal associated invariant T (MAIT) cells are reduced in blood from these patients and the consequence of this is unknown. Serum/plasma levels of cytokines have been associated with LCH disease extent and may play a role in the recruitment of cells to lesions. We investigated whether plasma signaling factors differed between patients with active and non-active LCH. Cell-signaling factors (38 analytes total) were measured in patient plasma and cell populations from matched lesions and/or peripheral blood were enumerated. This study aimed at understanding whether plasma factors corresponded with LCH cells and/or LCH-associated T cell subsets in patients with LCH. We identified several associations between plasma factors and lesional/circulating immune cell populations, thus highlighting new factors as potentially important in LCH pathogenesis. This study highlights plasma cell-signaling factors that are associated with LCH cells, MAIT cells or Tregs in patients, thus they are potentially important in LCH pathogenesis. Further study into these associations is needed to determine whether these factors may become suitable prognostic indicators or therapeutic targets to benefit patients.

Progress in the Treatment of Adult Langerhans Cell Histiocytosis --Review

Langerhans cell histiocytosis (LCH) is a rare proliferative disease dominated by the proliferation of Langerhans cells, which is inflammatory myeloid neoplasms. Its clinical manifestations are variable, occurring at any age and at any site, and it is rarer in adults than in children. The gold standard for diagnosis is histopathological biopsy. Due to the rarity of adult LCH and the heterogeneity of this disease, treatment of adult LCH should be developed according to the extent of the disease and risk stratification. With the discovery of MAPK, PI3K and c-KIT signaling pathway activation, especially BRAF V600E and MAP2K1 mutations, targeted therapy has become a hot spot for therapeutic research. Meanwhile, the discovery of high expression of M2-polarized macrophages and Foxp3+ regulatory T cells (Treg) in LCH has provided an important basis for the immunotherapy. In this article, we will focus on reviewing the latest research progress in the treatment of adult LCH in recent years, and provide a reference for clinical research on the treatment of adult LCH patients.

MRNA sequencing analysis and growth inhibitory effects of palbociclib on cell lines from canine histiocytic proliferative disorders.

Canine histiocytic proliferative disorders include aggressive and fatal diseases, such as histiocytic sarcoma (HS) and histiocytosis (SyH). The molecular mechanisms underlying cell proliferation need to be elucidated for the development of effective treatments. In the present study, mRNA expression levels were comprehensively analysed in cell lines derived from localized HS, disseminated HS, SyH and Langerhans cell histiocytosis (LCH) in dogs. Based on the results obtained, the growth inhibitory effects of palbociclib, a CDK4/6 inhibitor, were verified with the cell lines in vitro and in xenograft mouse model. Hierarchical clustering and principal component analysis plots of mRNA expression profiles divided the cell lines into three groups: a localized HS group, disseminated HS/SyH group, and LCH. The results of an ingenuity pathway analysis suggested that the MAPK signalling pathway was activated in the localized HS and LCH cell lines, and the PI3K signalling pathway in the disseminated and localized HS cell lines. In all cell lines, the expression of the tumour suppressor genes TP53, CDKN2A and CDKN1A was down-regulated, whereas that of Rb was preserved. In vitro assessments revealed the growth inhibitory effects of palbociclib in all cell lines examined. In a xenograft mouse model using a cell line from disseminated HS, palbociclib exerted significant growth inhibitory effects. These results suggest the potential of palbociclib as a therapeutic drug candidate for the treatment of malignant histiocytic proliferative disorders of the dog.

Langerhans cell histiocytosis of the colon: A case report

Langerhans cell histiocytosis (LCH) is a neoplastic proliferation of myeloid dendritic cells that can be confined to a single organ system (usually bone) or multisystemic. We present a rare case of LCH encountered as a solitary ulcer in the transverse colon of a 45-year-old woman. Histological examination revealed expansion of the submucosa by a mixed infiltrate, including sheets of histiocytoid ‘LCH’ cells and numerous eosinophils. The LCH cells had abundant pale cytoplasm, folded nuclear contours, open chromatin and inconspicuous nucleoli.

Elucidated pathogenesis and therapeutic prospects in Langerhans cell histiocytosis

Langerhans cell histiocytosis (LCH) is characterized by immature dendritic cell proliferation, which is currently classified as an inflammatory myeloid neoplasm. Clinical features and outcomes vary from spontaneously regressing isolated bone disease to fatal liver, spleen, or hematopoietic system (risk organ) involvement-positive multisystem disease. LCH cells have the only mutation in the mitogen-activated protein kinase (MAPK) signaling pathway gene, represented by the BRAF V600E mutation, which is the driver mutation. The type of disease depends on the stage of hematopoietic cell differentiation at which the mutation occurs. LCH cells acquire anti-apoptosis and senescence-associated secretory phenotype by oncogene-induced senescence, with migration failure to lymph nodes. These cause LCH cell accumulation and various inflammatory cell recruitment in the lesion, resulting in severe inflammation. Tissue damage in LCH is due to this inflammation, not the LCH cell proliferation. Patients with a risk of organ involvement without the initial treatment response may be rescued by allogeneic hematopoietic stem cell transplantation after reducing the disease activity with MAPK inhibitors. Intravenous zoledronic acid and intrathecal cytarabine injections have been introduced into the ongoing clinical trial in Japan to reduce bone recurrence and prevent neurodegeneration as sequelae.

Immune Checkpoint Inhibitors Restore Function of Lesion Infiltrating T Lymphocytes in Langerhans Cell Histiocytosis

Langerhans Cell Histiocytosis (LCH) is characterized by inflammatory lesions with pathologic CD207+ dendritic cells (DCs) and constitutively activated ERK. LCH lesions are associated with a local cytokine storm and exhibit a heterogeneous collection of inflammatory infiltrates (eosinophils, neutrophils and lymphocytes). The critical role of the immune system in regulating tumor growth and progression has been highlighted with the extraordinary impact of dis-inhibiting cytotoxic T cells in some adult cancers. However, the mechanisms behind tumor formation, both accumulation of pathologic CD207+ DCs as well as recruitment of inflammatory infiltrate in LCH, remain to be defined. Mutually exclusive somatic mutations in MAPK pathway genes have been identified in approximately 75% of LCH cases, including recurrent BRAF -V600Eand MAP2K1 mutations. However, the inflammatory infiltrate within LCH lesions is composed of cells without any BRAF mutation or ERK activation. We hypothesized that the pathologic inflammation in LCH is orchestrated by interactions between the pathologic MAPK-activated DCs and normal immune cells. Defining the nature and function of infiltrating T cells in LCH has the potential to not only provide insights into mechanisms of pathogenesis and potential therapeutic targets, but may also inform novel physiologic mechanisms of DC-mediated T cell function. Clonality, investigated by exploring T cell receptor beta (TCRB) rearrangement in LCH patients, identified clonal populations among LCH lesion-infiltrating CD3+ T cells. The complementarity-determining region 3 (CDR3) of the TCRBV07 gene demonstrated marked monoclonal expansion, suggesting a potential involvement of antigen selection in the pathogenesis of LCH. High dimensional mass cytometric analysis identified multiple tumor-infiltrating T lymphocyte subsets in LCH. Functional analysis indicated hypercytokinemia and defective alloreactive CD8 effector T cells. Spanning tree progression of density normalized events (SPADE) analysis of infiltrating CD3+ T cells revealed high expression of T cell immunoglobulin mucin (TIM-3), lymphocyte activated gene 3 (LAG3) and programmed cell death protein 1 (PD-1) compared to healthy controls, indicating that effector T cell exhaustion might be an important mechanism of immune dysregulation in LCH patients. In addition we found pathologic DCs in LCH lesions expressing ligands for the inhibitory receptors (PDL-1/L2, Galectin-9 and MHC Class II). We also found that patients harboring BRAF -V600E mutation express higher ligand expression compared to patients with wild type BRAF . Checkpoint inhibitor therapy by inhibiting PD-1 in an ex vivo setting to reinvigorate exhausted T cells only partially rescued CD8 effector cells alloreactivity in LCH patients. We are currently evaluating the efficacy of combination therapy in restoring effector T lymphocyte function. The results from this study thus support a model in which LCH lesion DCs with activated MAPK signaling select and recruit activated T cells with potential for clonal expansion to LCH lesions. While the CD207+ DCs in LCH lesions is histologically uniform across the disease spectrum, the composition of the immune microenvironment is variable. The variability of the inhibitory receptor ligand expression in BRAF -V600E vs BRAF wild-type lesions suggests that specific mechanisms of MAPK activation may have some distinct impact on T cell recruitment and activation. Finding more effective ways to reinvigorate T cells and have them regain functions could prove to be incredibly useful for efficacious therapeutic intervention in patients with LCH. DisclosuresNo relevant conflicts of interest to declare.

Hyper MAPK/ERK signaling in DCs causes increased LPS-induced TNFa at the post-transcriptional level

Abstract Background: Langerhans Cell Histiocytosis (LCH) is an inflammatory disease driven by abnormal dendritic cells (DCs) with hyper-MAPK/ERK signaling, usually due to Braf-V600E mutation. Since DCs are poised with Toll like Receptors (TLRs), which utilize MAPK/ERK signaling to incite an immune response, we hypothesize that LCH cells have a hyper-TLR response. Methods: We use an animal model of LCH (CD11c-Cre:BRAFV600E-flox; which expresses Braf-V600E in DCs) to measure serum TNFa and tnfa transcripts in CD11c+ splenocytes after LPS injection. Cultured BMDCs were pre-treated with/without a V600E-inhibitor before LPS stimulation. We measured intracellular TNFa, secretion and reuptake of TNFa, tnfa transcripts, NFkB signaling, TNFa degradation, and TACE activity. Results: LCH mice have increased LPS-induced circulating TNFa compared to WT. LCH-BMDCs have reversible LPS-induced increase in TNFa secretion and intracellular accumulation, but decreased tnfa and tlr4 transcripts and NFkB signaling compared to WT. There is no difference in TACE activity, TNFa reuptake, or protein degradation. Conclusions: Hyper-MAPK/ERK signaling in DCs causes a hyper LPS-induced TNFa response, despite diminished LPS signaling. Increased intracellular TNFa indicates a cell intrinsic, post-transcriptional mechanism. Current experiments are analyzing translation of TNFa, which is implicated by MAPK/ERK signaling. These data indicate novel effects of Braf-V600E on LPS-induced TNFa production, and current studies are investigating whether this is a more global phenomenon.

Overcoming T-cell exhaustion in LCH: PD-1 blockade and targeted MAPK inhibition are synergistic in a mouse model of LCH

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by granulomatous lesions containing pathological CD207+ dendritic cells (DCs) with persistent MAPK pathway activation. Standard-of-care chemotherapies are 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 the 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. Intralesional CD8+ T cells showed blunted expression of Tc1/Tc2 cytokines and impaired effector function. In contrast, intralesional regulatory T cells 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 cells 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 they highlight combined MAPK and checkpoint inhibition as a potential therapeutic strategy.

Expression of CD1 molecules and colony-stimulating factor 1 receptor in indeterminate cell histiocytosis.

Indeterminate cell histiocytosis (ICH) and Langerhans cell histiocytosis (LCH) are rare histiocyte proliferating disorders with unknown etiologies. However, both tumor cells immunophenotypically share some features of Langerhans cells (LC), thereby expressing CD1a. Recent transcriptome analysis revealed that circulating CD1c+ myeloid dendritic cells are the potential precursor of LCH tumor cells. LC express CD1a as well as CD1c, but not CD1b. We discovered that both tumor cells express CD1c, but not CD1b, similar to LC. Moreover, like LC, both tumor cells express colony-stimulating factor 1 receptor (CSF-1R). Considering the crucial role of the interleukin (IL)-34/CSF-1R axis for the development and survival of LC, CSF-1R on both tumor cells might facilitate their survival and proliferation in situ. These data provide additional evidence to support the fact that ICH and LCH share immunophenotypical features with LC. In addition, we hypothesized that tumor cells in ICH and LCH survive and proliferate through IL-34-mediated CSF-1R signaling.

Differential In Situ Cytokine Profiles of Langerhans-Like Cells and T Cells in Langerhans Cell Histiocytosis: Abundant Expression of Cytokines Relevant to Disease and Treatment

AbstractThe pathogenesis of Langerhans cell histiocytosis (LCH) remains poorly understood. To further elucidate LCH pathogenesis, we analyzed the expression of 10 cytokines relevant to cellular recruitment and activation at the protein level in 14 patients and identified the lesional cells responsible for cytokine production in situ by immunohistochemistry. The cytokines investigated included the hematopoietic growth factors interleukin-3 (IL-3), IL-7, and granulocyte-macrophage colony-stimulating factor (GM-CSF); the lymphocyte regulatory cytokines IL-2, IL-4, and IL-10; the inflammatory regulators IL-1α and tumor necrosis factor-α (TNF-α); and the effector cell-activating cytokines IL-5 and interferon-γ (IFN-γ). In all specimens, CD1a+ histiocytes (LCH cells) and CD3+ T cells produced large amounts of cytokines, creating a true cytokine storm. IL-2, IL-4, IL-5, and TNF-α were produced exclusively by T cells, whereas only IL-1α was produced by LCH cells. Equal numbers of LCH cells, T cells, and macrophages produced GM-CSF and IFN-γ. Equal numbers of LCH cells and macrophages produced IL-10, whereas IL-3 was produced by T cells and macrophages. IL-7 was only produced by macrophages. Eosinophils, present in some specimens, were partially responsible for the production of IL-5, IFN-γ, GM-CSF, IL-10, IL-3, and IL-7. Expression of all cytokines, abundant in most biopsies, was irrespective of age, gender, or site of biopsy. These findings emphasize the role of T cells in LCH. The juxtaposition of T cells and LCH cells suggests that both cells interact in a cytokine amplification cascade, resulting from stimulation of autocrine and paracrine stimulatory loops. This cascade can be linked directly to the development of LCH through recruitment, maturation, and proliferation of LCH cells. The cytokines studied are known to be involved in the development of other characteristic features of LCH, such as fibrosis, necrosis, and osteolysis.