Development of Tbet- and CD11c-expressing B cells in a viral infection requires T follicular helper cells outside of germinal centers.

Tbet+CD11c+ B cells arise during type 1 pathogen challenge, aging, and autoimmunity in mice and humans. Here, we examined the developmental requirements of this B cell subset. In acute infection, T follicular helper (Tfh) cells, but not Th1 cells, drove Tbet+CD11c+ B cell generation through proximal delivery of help. Tbet+CD11c+ B cells developed prior to germinal center (GC) formation, exhibiting phenotypic and transcriptional profiles distinct from GC B cells. Fate tracking revealed that most Tbet+CD11c+ B cells developed independently of GC entry and cell-intrinsic Bcl6 expression. Tbet+CD11c+ and GC B cells exhibited minimal repertoire overlap, indicating distinct developmental pathways. As the infection resolved, Tbet+CD11c+ B cells localized to the marginal zone where splenic retention depended on integrins LFA-1 and VLA-4, forming a competitive memory subset that contributed to antibody production and secondary GC seeding upon rechallenge. Therefore, Tbet+CD11c+ B cells comprise a GC-independent memory subset capable of rapid and robust recall responses.

Distinct Effector B Cells Induced by Unregulated Toll-like Receptor 7 Contribute to Pathogenic Responses in Systemic Lupus Erythematosus.

Systemic Lupus Erythematosus (SLE) is characterized by B cells lacking IgD and CD27 (double negative; DN). We show that DN cell expansions reflected a subset of CXCR5- CD11c+ cells (DN2) representing pre-plasma cells (PC). DN2 cells predominated in African-American patients with active disease and nephritis, anti-Smith and anti-RNA autoantibodies. They expressed a T-bet transcriptional network; increased Toll-like receptor-7 (TLR7); lacked the negative TLR regulator TRAF5; and were hyper-responsive to TLR7. DN2 cells shared with activated naive cells (aNAV), phenotypic and functional features, and similar transcriptomes. Their PC differentiation and autoantibody production was driven by TLR7 in an interleukin-21 (IL-21)-mediated fashion. An in vivo developmental link between aNAV, DN2 cells, and PC was demonstrated by clonal sharing. This study defines a distinct differentiation fate of autoreactive naive B cells into PC precursors with hyper-responsiveness to innate stimuli, as well as establishes prominence of extra-follicular B cell activation in SLE, and identifies therapeutic targets.

Molecular mechanisms controlling age-associated B cells in autoimmunity.

Age-associated B cells (ABCs) have emerged as critical components of immune responses. Their inappropriate expansion and differentiation have increasingly been linked to the pathogenesis of autoimmune disorders, aging-associated diseases, and infections. ABCs exhibit a distinctive phenotype and, in addition to classical B cell markers, often express the transcription factor T-bet and myeloid markers like CD11c; hence, these cells are also commonly known as CD11c+ T-bet+ B cells. Formation of ABCs is promoted by distinctive combinations of innate and adaptive signals. In addition to producing antibodies, these cells display antigen-presenting and proinflammatory capabilities. It is becoming increasingly appreciated that the ABC compartment exhibits a high degree of heterogeneity, plasticity, and sex-specific regulation and that ABCs can differentiate into effector progeny via several routes particularly in autoimmune settings. In this review, we will discuss the initial insights that have been obtained on the molecular machinery that controls ABCs and we will highlight some of the unique aspects of this control system that may enable ABCs to fulfill their distinctive role in immune responses. Given the expanding array of autoimmune disorders and pathophysiological settings in which ABCs are being implicated, a deeper understanding of this machinery could have important and broad therapeutic implications for the successful, albeit daunting, task of targeting these cells.

Regulation of B-cell function and expression of CD11c, T-bet, and FcRL5 in response to different activation signals.

CD11c, FcRL5, or T-bet are commonly expressed by B cells expanding during inflammation, where they can make up >30% of mature B cells. However, the association between the proteins and differentiation and function in the host response remains largely unclear. We have assessed the co-expression of CD11c, T-bet, and FcRL5 in an in vitro B-cell culture system to determine how stimulation via the BCR, toll-like receptor 9 (TLR9), and different cytokines influence CD11c, T-bet, and FcRL5 expression. We observed different expression dynamics for all markers, but a largely overlapping regulation of CD11c and FcRL5 in response to BCR and TLR9 activation, while T-bet was strongly dependent on IFN-γ signaling. Investigating plasma cell differentiation and APC functions, there was no association between marker expression and antibody secretion or T-cell help. Rather the functions were associated with TLR9-signalling and B-cell-derived IL-6 production, respectively. These results suggest that the expression of CD11c, FcRL5, and T-bet and plasma cell differentiation and improved APC functions occur in parallel and are regulated by similar activation signals, but they are not interdependent.

Definition of a mouse microglial subset that regulates neuronal development and proinflammatory responses in the brain.

Expression of Itgax (encoding the CD11c surface protein) and Spp1 (encoding osteopontin; OPN) has been associated with activated microglia that can develop in healthy brains and some neuroinflammatory disorders. However, whether CD11c and OPN expression is a consequence of microglial activation or represents a portion of the genetic program expressed by a stable microglial subset is unknown. Here, we show that OPN production in the brain is confined to a small CD11c+ microglial subset that differentiates from CD11c- precursors in perinatal life after uptake of apoptotic neurons. Our analysis suggests that coexpression of OPN and CD11c marks a microglial subset that is expressed at birth and persists into late adult life, independent of environmental activation stimuli. Analysis of the contribution of OPN to the intrinsic functions of this CD11c+ microglial subset indicates that OPN is required for subset stability and the execution of phagocytic and proinflammatory responses, in part through OPN-dependent engagement of the αVß3-integrin receptor. Definition of OPN-producing CD11c+ microglia as a functional microglial subset provides insight into microglial differentiation in health and disease.

Emerging insights into atypical B cells in pediatric chronic infectious diseases and immune system disorders: T(o)-bet on control of B-cell immune activation.

Repetitive or persistent cellular stimulation in vivo has been associated with the development of a heterogeneous B-cell population that exhibits a distinctive phenotype and, in addition to classical B-cell markers, often expresses the transcription factor T-bet and myeloid marker CD11c. Research suggests that this atypical population consists of B cells with distinct B-cell receptor specificities capable of binding the antigens responsible for their development. The expansion of this population occurs in the presence of chronic inflammatory conditions and autoimmune diseases where different nomenclatures have been used to describe them. However, as a result of the diverse contexts in which they have been investigated, these cells have remained largely enigmatic, with much ambiguity remaining regarding their phenotype and function in humoral immune response as well as their role in autoimmunity. Atypical B cells have garnered considerable interest because of their ability to produce specific antibodies and/or autoantibodies and because of their association with key disease manifestations. Although they have been widely described in the context of adults, little information is present for children. Therefore, the aim of this narrative review is to describe the characteristics of this population, suggest their function in pediatric immune-related diseases and chronic infections, and explore their potential therapeutic avenues.

Hericenone C attenuates the second phase of formalin-induced nociceptive behavior by suppressing the accumulation of CD11c-positive cells in the paw epidermis via phosphorylated P65.

Hericenone C is one of the most abundant secondary metabolites derived from Hericium erinaceus, under investigation for medicinal properties. Here, we report that Hericenone C inhibits the second phase of formalin-induced nociceptive behavior in mice. As the second phase is involved in inflammation, in a mechanistic analysis on cultured cells targeting NF-κB response element (NRE): luciferase (Luc)-expressing cells, lipopolysaccharide (LPS)-induced NRE::Luc luciferase activity was found to be significantly inhibited by Hericenone C. Phosphorylation of p65, which is involved in the inflammatory responses of the NF-κB signaling pathway, was also induced by LPS and significantly reduced by Hericenone C. Additionally, in mice, the number of CD11c-positive cells increased in the paw during the peak of the second phase of the formalin test, which decreased upon Hericenone C intake. Our findings confirm the possibility of Hericenone C as a novel therapeutic target for pain-associated inflammation.

B cell-intrinsic IFN-γ promotes excessive CD11c+ age-associated B cell differentiation and compromised germinal center selection in lupus mice.

CD11c+ age-associated B cells (ABCs) have emerged as a key component in protective and autoreactive B cell responses. Lupus is an autoimmune disorder linked to reduced efficacy of vaccines and increased susceptibility to infections. Previously, we reported that excessive CD11c+ ABCs not only significantly contribute to autoantibody production but also promote aberrant T cell activation and compromised affinity-based germinal center selection in response to immunization in lupus mice. Yet, the regulation of CD11c+ ABC differentiation is not fully understood. In this study, we show that B cell-intrinsic IFN-γ is required for excessive CD11c+ ABC differentiation in lupus mice. B cell-intrinsic IFN-γ is mainly produced by CD11c+ ABCs. IFN-γ-deficiency leads to decreased expression of ABC characteristic genes. We further show that ablating IFN-γ can normalize T cell overactivation and rescue antigen-specific GC responses in lupus mice. Our study offers insight into the crucial role of B cell-intrinsic IFN-γ in promoting excessive CD11c+ ABC differentiation, which compromises affinity-based germinal center selection and affinity maturation in lupus, providing a potential strategy to normalize vaccine responses in lupus.

The role of M1 (CD11c) and M2 (CD163) interplay in the pathogenesis of oral submucous fibrosis and its malignant transformation: An immunohistochemical analysis.

OBJECTIVES: The M1/M2 macrophage framework is crucial in organ fibrosis and its progression to malignancy. This study investigated the possible role of M1/M2 macrophage interplay in the pathogenesis of oral submucous fibrosis (OSF) and its malignant transformation by analysing immunohistochemical expression of CD11c (M1) and CD163 (M2) markers. METHODS: Immunohistochemistry was performed using primary antibodies against CD11c and CD163 on ten formalin-fixed paraffin-embedded tissue blocks for each group: (i) Stage 1 OSF, (ii) Stage 2 OSF, (iii) Stage 3 OSF, (iv) Stage 4 OSF, (v) well-differentiated squamous cell carcinoma (WDSCC) with OSF, and (vi) WDSCC without OSF. Ten cases of healthy buccal mucosa (NOM) served as controls. RESULTS: Epithelial quick scores of M1 (CD11c) in NOM, Stages 1-4 OSF, and WDSCC with and without OSF were 0, 1.8, 2.9, 0.4, 0, 0, and 0, while connective tissue scores were 0, 3.2, 4.3, 2.7, 0.5, 1.2, and 2.4, respectively. Epithelial scores for M2 (CD163) were 0, 0.8, 0.8, 2.1, 0.6, 0.8, and 0.2, and connective tissue scores were 0, 1.8, 2.6, 3.9, 2.2, 5, and 4.4, respectively. Stages 3 and 4 OSF, WDSCC with and without OSF exhibited higher M2/M1 ratios compared to NOM and Stages 1-2 OSF. CONCLUSION: The interaction between M1 (CD11c) and M2 (CD163) macrophages, leading to M2 polarisation, plays a crucial role in the pathogenesis of OSF and its potential malignant transformation.

Distinct dynamics of antigen-specific induction and differentiation of different CD11c+Tbet+ B-cell subsets.

BACKGROUND: CD11c+Tbet+ B cells are enriched in autoimmunity and chronic infections and also expand on immune challenge in healthy individuals. CD11c+Tbet+ B cells remain an enigmatic B-cell population because of their intrinsic heterogeneity. OBJECTIVES: We investigated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen-specific development and differentiation properties of 3 separate CD11c+ B-cell subsets-age-associated B cells (ABCs), double-negative 2 (DN2) B cells, and activated naive B cells-and compared them to their canonical CD11c- counterparts. METHODS: Dynamics of the response of the 3 CD11c+ B-cell subsets were assessed at SARS-CoV-2 vaccination in healthy donors by spectral flow cytometry. Distinct CD11c+ B-cell subsets were functionally characterized by optimized in vitro cultures. RESULTS: In contrast to a durable expansion of antigen-specific CD11c- memory B cells over time, both ABCs and DN2 cells were strongly expanded shortly after second vaccination and subsequently contracted. Functional characterization of antibody-secreting cell differentiation dynamics revealed that CD11c+Tbet+ B cells were primed for antibody-secreting cell differentiation compared to relevant canonical CD11c- counterparts. CONCLUSION: Overall, CD11c+Tbet+ B cells encompass heterogeneous subpopulations, of which primarily ABCs as well as DN2 B cells respond early to immune challenge and display a pre-antibody-secreting cell phenotype.

Associations of cortical SPP1 and ITGAX with cognition and common neuropathologies in older adults.

INTRODUCTION: The secreted phosphoprotein 1 (SPP1) gene expressed by CD11c+ cells is known to be associated with microglia activation and neuroinflammatory diseases. As most studies rely on mouse models, we investigated these genes and proteins in the cortical brain tissue of older adults and their role in Alzheimer's disease (AD) and related disorders. METHODS: We leveraged protein measurements, single-nuclei, and RNASeq data from the Religious Orders Study and Rush Memory and Aging Project (ROSMAP) of over 1200 samples for association analysis. RESULTS: Expression of SPP1 and its encoded protein osteopontin were associated with faster cognitive decline and greater odds of common neuropathologies. At single-cell resolution,  integrin subunit alpha X (ITGAX) was highly expressed in microglia, where specific subpopulations were associated with AD and cerebral amyloid angiopathy. DISCUSSION: The study provides evidence of SPP1 and ITGAX association with cognitive decline and common neuropathologies identifying a microglial subset associated with disease.

Absence of Aquaporin-4 (AQP4) Prolongs the Presence of a CD11c+ Microglial Population during Postnatal Corpus Callosum Development.

Aquaporin-4 (AQP4) expression is associated with the development of congenital hydrocephalus due to its structural role in the ependymal membrane. Gene expression analysis of periaqueductal tissue in AQP4-knockout (KO) mice at 11 days of age (P11) showed a modification in ependymal cell adhesion and ciliary protein expression that could alter cerebrospinal fluid homeostasis. A microglial subpopulation of CD11c+ cells was overexpressed in the periaqueductal tissue of mice that did not develop hydrocephalus, suggesting a possible protective effect. Here, we verified the location of this CD11c+ expression in the corpus callosum (CC) and cerebellum of AQP4-KO mice and analysed its time course. Immunofluorescence labelling of the CD11c protein in the CC and cerebellum of WT and KO animals at P3, P5, P7 and P11 confirmed an expanded presence of these cells in both tissues of the KO animal; CD11c+ cells appeared at P3 and reached a peak at P11, whereas in the WT animal, they appeared at P5, reached their peak at P7 and were undetectable by P11. The gene expression analysis in the CC samples at P11 confirmed the presence of CD11c+ microglial cells in this tissue. Among the more than 4000 overexpressed genes, Spp1 stood out with the highest differential gene expression (≅600), with other genes, such as Gpnmb, Itgax, Cd68 and Atp6v0d2, also identified as overexpressed. Therefore, CD11c+ cells appear to be necessary for normal corpus callosum development during postnatal life, and the absence of AQP4 prolonged its expression in this tissue.

High activation levels maintained in receptor-binding domain-specific memory B cells in people with severe coronavirus disease 2019.

The long-term health consequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are still being understood. The molecular and phenotypic properties of SARS-CoV-2 antigen-specific T cells suggest a dysfunctional profile that persists in convalescence in those who were severely ill. By contrast, the antigen-specific memory B-cell (MBC) population has not yet been analyzed to the same degree, but phenotypic analysis suggests differences following recovery from mild or severe coronavirus disease 2019 (COVID-19). Here, we performed single-cell molecular analysis of the SARS-CoV-2 receptor-binding domain (RBD)-specific MBC population in three patients after severe COVID-19 and four patients after mild/moderate COVID-19. We analyzed the transcriptomic and B-cell receptor repertoire profiles at ~2 months and ~4 months after symptom onset. Transcriptomic analysis revealed a higher level of tumor necrosis factor-alpha (TNF-α) signaling via nuclear factor-kappa B in the severe group, involving CD80, FOS, CD83 and TNFAIP3 genes that was maintained over time. We demonstrated the presence of two distinct activated MBCs subsets based on expression of CD80hi TNFAIP3hi and CD11chi CD95hi at the transcriptome level. Both groups revealed an increase in somatic hypermutation over time, indicating progressive evolution of humoral memory. This study revealed distinct molecular signatures of long-term RBD-specific MBCs in convalescence, indicating that the longevity of these cells may differ depending on acute COVID-19 severity.

Detection of disease-associated microglia among various microglia phenotypes induced by West Nile virus infection in mice.

West Nile virus (WNV) has emerged as a significant cause of viral encephalitis in humans and horses. However, the pathogenesis of the West Nile encephalitis remains unclear. Microglia are activated by WNV infection, and the pathogenic involvement of their phenotypes is controversial. In this study, we examined the diversity of microglia phenotypes caused by WNV infection by assessing various microglia markers and identified disease-associated microglia in WNV-infected mouse brain tissue. Cells positive for general microglia markers such as Iba1, P2RY12, or TMEM119 were detected in the control and WNV-infected brain tissue. The morphology of the positive cells in brain tissue infected by WNV was different from that of control brain tissue, indicating that WNV infection induced activation of microglia. The activated microglia were classified into various phenotypes by investigation of specific marker expression. Among the activated microglia, disease-associated microglia that were positive for CD11c and weakly positive for TMEM119 were detected close to the WNV-infected cells. These results indicate that WNV infection induces activation of diverse microglia phenotypes and that disease-associated microglia may be associated with the pathogenicity of WNV infection in the mouse brain.

Leukocyte surface biomarkers implicate deficits of innate immunity in sporadic Alzheimer's disease.

INTRODUCTION: Blood-based diagnostics and prognostics in sporadic Alzheimer's disease (AD) are important for identifying at-risk individuals for therapeutic interventions. METHODS: In three stages, a total of 34 leukocyte antigens were examined by flow cytometry immunophenotyping. Data were analyzed by logistic regression and receiver operating characteristic (ROC) analyses. RESULTS: We identified leukocyte markers differentially expressed in the patients with AD. Pathway analysis revealed a complex network involving upregulation of complement inhibition and downregulation of cargo receptor activity and Aß clearance. A proposed panel including four leukocyte markers - CD11c, CD59, CD91, and CD163 - predicts patients' PET Aß status with an area under the curve (AUC) of 0.93 (0.88 to 0.97). CD163 was the top performer in preclinical models. These findings have been validated in two independent cohorts. CONCLUSION: Our finding of changes on peripheral leukocyte surface antigens in AD implicates the deficit in innate immunity. Leukocyte-based biomarkers prove to be both sensitive and practical for AD screening and diagnosis.

Expression and prognostic significance of chromatin modulators EHMT2/G9a and KDM2b in acute myeloid leukemia.

Epigenetics factors are critical for normal cell function and their regulation is sensitive to malignancy development. EHMT2/G9a and KDM2b are key epigenetics players in different cancer types. However, their expression profiles and related consequences in acute myeloid leukemia (AML) patients have not been known yet. In addition to routine lab work, expression levels of EHMT2/G9a and KDM2b were determined in 110 adult and pediatric patients with De Novo AML. Relations between their expression and patients' clinical data were tested by statistical methods. EHMT2/G9a and KDM2b were highly expressed in AML patients against control cases and associated with the presence of adverse genomic alterations. In response to induction chemotherapy, EHMT2/G9a and KDM2b showed to be significantly high in resistant and relapsed patients in comparison to the complete remission group. KDM2b overexpression was associated with CD11c (integrin alpha X) downregulation. Kaplan-Meier analysis indicated that EHMT2/G9a and KDM2b overexpression was correlated with poor survival status in AML patients. We conclude that EHMT2/G9a and KDM2b expression levels could be used as independent prognostic factors for AML disease.

NFAT5 contributes to the pathogenesis of experimental autoimmune encephalomyelitis (EAE) and decrease of T regulatory cells in female mice.

Multiple sclerosis disproportionally affects women. The present study was undertaken to determine whether NFAT5 contributed to the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, and if it did, whether the impact was sex associated. NFAT5 haplodeficiency reduced the disease severity only in female mice. This effect was associated with significant increases in frequency of T regulatory (Treg) cells in the CNS (from 1.45 ± 0.39% to 3.73 ± 0.94%) and spleen from (0.31 ± 0.06% to 0.94 ± 0.29%) without significantly affecting the CNS CD4+ subsets frequency. NFAT5 haploinsufficiency also significantly reduced the frequency of CD11c+CD8α+ dendritic cells in the female CNS. However, increase of their frequency in the CNS via intraperitoneal Flt3L injection at peak EAE had no significant effect on the disease courses. We conclude that NFAT5 contributes to pathogenesis of EAE in female mice, possibly through decreasing tissue specific frequency of Treg cells.

High levels of thyroid hormones promote recurrence of Graves' disease via overexpression of B-cell-activating factor.

BACKGROUND: Elevated thyroid hormone (TH) levels have been suggested to be associated with the pathological progression of Graves' disease (GD). However, direct evidence from clinical studies remains unclear. METHODS: Peripheral blood samples were collected from patients with or without the recurrence of Graves' hyperthyroidism (GH) and healthy donors. Thyroid tissue samples were obtained from patients with benign thyroid nodules. To assess the differentiation of autoreactive B cells, the expression of B-cell-activating factor (BAFF) and the proportion of CD11c+/-IgG+/- subsets of B cells stimulated by high levels of triiodothyronine (T3) in vivo and in vitro were examined by ELISA, flow cytometry, western blotting, and qRT-PCR. RESULTS: Serum BAFF levels in patients with GD were significantly and positively correlated with FT3, FT4, and TRAb levels. Furthermore, the ratio of abnormally differentiated CD11c+ autoreactive B cells positively correlated with BAFF and TRAb. High levels of triiodothyronine (T3) induced BAFF overexpression in thyroid follicular cells and mononuclear cells of the normal thyroid in vitro, thereby promoting the differentiation of CD11c+IgG+ autoreactive secretory B cells (ASCs). However, the precise knockdown of BAFF expression significantly inhibited the abnormal differentiation of ASCs. CONCLUSION: The pathological progression of GD was prolonged and exacerbated by autoimmune positive feedback modulation caused by high TH levels. BAFF could be considered a potential target for localized thyroid immunosuppressive treatment of Graves' hyperthyroidism recurrence.

Excessive CD11c+Tbet+ B cells promote aberrant TFH differentiation and affinity-based germinal center selection in lupus.

Excessive self-reactive and inadequate affinity-matured antigen-specific antibody responses have been reported to coexist in lupus, with elusive cellular and molecular mechanisms. Here, we report that the antigen-specific germinal center (GC) response-a process critical for antibody affinity maturation-is compromised in murine lupus models. Importantly, this defect can be triggered by excessive autoimmunity-relevant CD11c+Tbet+ age-associated B cells (ABCs). In B cell-intrinsic Ship-deficient (ShipΔB) lupus mice, excessive CD11c+Tbet+ ABCs induce deregulated follicular T-helper (TFH) cell differentiation through their potent antigen-presenting function and consequently compromise affinity-based GC selection. Excessive CD11c+Tbet+ ABCs and deregulated TFH cell are also present in other lupus models and patients. Further, over-activated Toll-like receptor signaling in Ship-deficient B cells is critical for CD11c+Tbet+ ABC differentiation, and blocking CD11c+Tbet+ ABC differentiation in ShipΔB mice by ablating MyD88 normalizes TFH cell differentiation and rescues antigen-specific GC responses, as well as prevents autoantibody production. Our study suggests that excessive CD11c+Tbet+ ABCs not only contribute significantly to autoantibody production but also compromise antigen-specific GC B-cell responses and antibody-affinity maturation, providing a cellular link between the coexisting autoantibodies and inadequate affinity-matured antigen-specific antibodies in lupus models and a potential target for treating lupus.

Juvenile and adult xanthogranuloma: A 30-year single-center experience and review of the disorder and its relationship to other histiocytoses.

BACKGROUND: Juvenile xanthogranuloma (JXG) is the most common type of non-Langerhans cell histiocytosis whose cell of origin, etiology and pathogenesis are not fully understood. We aimed to provide an update on histopathologic and immunophenotypic profile of this well-characterized entity whose relationship to the other histiocytoses has received renewed attention in light of recent molecular genetic studies. MATERIALS AND METHODS: A retrospective review of all the cases with the pathologic diagnosis of "xanthogranuloma" was performed on our archives from 1989 to 2019. RESULTS: A total of 525 patients with 547 lesions diagnosed as JXG were identified with the median age of 4.5 years, a male predominance (M:F ratio 1.3:1) and a predilection for the head and neck region (40.8%). Cutaneous lesions comprised 76.8% cases and another 15.7% presented within soft tissues. The most common non-soft tissue, extracutaneous lesions included the brain (2.6%), and lungs (1.8%). Three basic histopathologic patterns were identified: early classic (EJXG) (14.2%), classic (CJXG) (45.3%), and transitional JXG (TJXG) (40.5%). Multinucleated giant cells, either Touton or non-Touton, were most frequently present in CJXG followed by TJXG. Mitosis was rare (<1/10 high-power field) among different patterns. There was an association among the patterns and lymphocytic infiltrates (P = 0.036), and presence of Touton or non-Touton giant cells (P < 0.001 for both) but not for mitotic count (P = 0.105) or eosinophilic infiltrates (P = 0.465). Additionally, there was a correlation between age groups and presence of non-Touton giant cells (P = 0.012) but not for Touton cells (P = 0.127). We have demonstrated that immunophenotypic expression of the lesion was not associated with age at diagnosis nor morphologic pattern: factor XIIIa 192/204 (94.1%), CD11c 75/77 (97.4%), CD4 82/84 (97.6%), CD68 200/201 (99.5%), CD163 15/15 (100%), CD1a 1/110 (0.9%), S-100 48/152 (31.6%), CD31 15/21 (71.4%), and vimentin 104/105 (99.0%). CONCLUSION: We have documented in a substantial series of cases of JXG that there is a correlation between one of the three basic histopathologic patterns with age at diagnosis, but with a consistent immunophenotype among the three patterns. Considering sensitivity and specificity rates, we suggest that a combination of CD11c, CD4, CD1a and either CD163 (preferred) or CD68 stains provides more specific diagnostic yield in the differentiation of JXG from other histiocytic disorders. JXG is also discussed in terms of its relationship and distinction from other similar histiocytic disorders in the context of MAPK/ERK pathway mutations.