Astrid G.S. van Halteren

Somatic activating ARAF mutations in Langerhans cell histiocytosis

The extracellular signal-regulated kinase (ERK) signaling pathway is activated in Langerhans cell histiocytosis (LCH) histiocytes, but only 60% of cases carry somatic activating mutations of BRAF. To identify other genetic causes of ERK pathway activation, we performed whole exome sequencing on purified LCH cells in 3 cases. One patient with wild-type BRAF alleles in his histiocytes had compound mutations in the kinase domain of ARAF. Unlike wild-type ARAF, this mutant was a highly active mitogen-activated protein kinase kinase in vitro and was capable of transforming mouse embryo fibroblasts. Mutant ARAF activity was inhibited by vemurafenib, a BRAF inhibitor, indicating the importance of fully evaluating ERK pathway abnormalities in selecting LCH patients for targeted inhibitor therapy.

MAP2K1 and MAP3K1 mutations in langerhans cell histiocytosis

Langerhans cell histiocytosis (LCH) is now understood to be a neoplastic disease in which over 50% of cases have somatic activating mutations of BRAF. However, the extracellular signal‐related (ERK) pathway is activated in all cases including those with wild type BRAF alleles. Here, we applied a targeted massively parallel sequencing panel to 30 LCH samples to test for the presence of additional genetic alterations that might cause ERK pathway activation. In 20 BRAF wild type samples, we found 3 somatic mutations in MAP2K1 (MEK1) including C121S and C121S/G128D in the kinase domain, and 56_61QKQKVG>R, an in‐frame deletion in the N‐terminal regulatory domain. All three variant proteins constitutively phosphorylated ERK in in vitro kinase assays. The C121S/G128D and 56_61QKQKVG>R variants were resistant to the mitogen‐activated protein kinase kinase (MEK) inhibitor trametinib in vitro. Within the entire sample set, we found 3 specimens with mutations in MAP3K1 (MEKK1), including two truncation mutants, T779fs and T1481fs; T1481fs encoded an unstable and nonfunctional protein when expressed in vitro. T779fs was present in a specimen carrying BRAF V600E. The third variant was a single nucleotide substitution, E1286V, which was fully functional and is likely a germline polymorphism. These results indicate that LCH cells can harbor additional genetic alterations in the RAS‐RAF‐MEK pathway which, in the case of MAP2K1, may be responsible for ERK activation in a wild type BRAF setting. The resistance of some of these variants to trametinib may also have clinical implications for the combined use of RAF and MEK inhibitors in LCH.

Langerhans cell histiocytosis: fascinating dynamics of the dendritic cell–macrophage lineage

Summary:u2002 In its rare occurrence, Langerhans cell histiocytosis (LCH) is a dangerous but intriguing deviation of mononuclear phagocytes, especially dendritic cells (DCs). Clinically, the disease ranges from self‐resolving or well manageable to severe and even fatal. LCH lesions in skin, bone, and other sites contain high numbers of cells with phenotypic features resembling LCs admixed with macrophages, T cells, eosinophils, and multinucleated giant cells. Here we review current progress in the LCH field based on two central questions: (i) are LCH cells intrinsically aberrant, and (ii) how does the lesion drive pathogenesis? We argue that LCH cells may originate from different sources, including epidermal LCs, tissue Langerin+ DCs, or mononuclear phagocyte precursors. Current and prospective in vitro and in vivo models are discussed. Finally, we discuss recent insights into plasticity of T‐helper cell subsets in light of the lesion microenvironment. LCH continues to provide urgent clinical questions thereby inspiring innovative DC lineage research.

Association of HY-restricting HLA class II alleles with pregnancy outcome in patients with recurrent miscarriage subsequent to a firstborn boy

Healthy females, pregnant with a boy, generate immune responses against male-specific minor histocompatibility (HY) antigens. The clinical importance of these responses is evident in stem cell transplantation. Birth of a boy prior to a series of miscarriages reduces the chance of a subsequent live birth. This study explores the putative impact of known HY-presenting HLA alleles on future pregnancy outcome in women with at least three consecutive miscarriages following a birth [secondary recurrent miscarriage (SRM)]. HLA-A, -B, -DRB1, DRB3-5 and DQB1 genotyping was performed in 358 SRM patients and in 203 of their children born prior to the miscarriages. The subsequent live birth in women with boys prior to the miscarriages compared with girls is lower in women with HY-restricting HLA class II alleles [odds ratio (OR): 0.17 (0.1-0.4), P = 0.0001]. One HY-restricting HLA class II allele in women with firstborn boys significantly reduces the chances of a live birth [OR: 0.46 (0.2-0.9), P = 0.02]. Two HY-restricting HLA class II alleles further reduced this chance [OR: 0.21 (0.1-0.7), P = 0.02]. HY-restricting HLA class II did not reduce the chances of a live birth in SRM women with firstborn girls. HY-restricting HLA class II alleles are associated with a decreased chance of a live birth in SRM women with firstborn boys. These findings strongly indicate an aberrant maternal immune reaction against fetal HY-antigens in SRM. The results may shed light on the as-yet unknown immunological causes of SRM and may help understand the successful maternal acceptance of the fetal semi-allograft.

Decreased Levels of Circulating IL17-Producing CD161+CCR6+ T Cells Are Associated with Graft-versus-Host Disease after Allogeneic Stem Cell Transplantation

The C-type lectin-like receptor CD161 is a well-established marker for human IL17-producing T cells, which have been implicated to contribute to the development of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (allo-SCT). In this study, we analyzed CD161+ T cell recovery, their functional properties and association with GVHD occurrence in allo-SCT recipients. While CD161+CD4+ T cells steadily recovered, CD161hiCD8+ T cell numbers declined during tapering of Cyclosporine A (CsA), which can be explained by their initial growth advantage over CD161neg/lowCD8+ T cells due to ABCB1-mediated CsA efflux. Interestingly, occurrence of acute and chronic GVHD was significantly correlated with decreased levels of circulating CD161+CD4+ as well as CD161hiCD8+ T cells. In addition, these subsets from transplanted patients secreted high levels of IFNγ and IL17. Moreover, we found that CCR6 co-expression by CD161+ T cells mediated specific migration towards CCL20, which was expressed in GVHD biopsies. Finally, we demonstrated that CCR6+ T cells indeed were present in these CCL20+ GVHD-affected tissues. In conclusion, we showed that functional CD161+CCR6+ co-expressing T cells disappear from the circulation and home to GVHD-affected tissue sites. These findings support the hypothesis that CCR6+CD161-expressing T cells may be involved in the immune pathology of GVHD following their CCL20-dependent recruitment into affected tissues.

HY Immune Tolerance Is Common in Women without Male Offspring

Background Sex difference is an established risk factor for hematopoietic stem cell transplantation (HSCT)-related complications like graft versus host disease (GVHD). CD8pos cytotoxic T cells specific for Y chromosome-encoded minor Histocompatibility antigens (HY) play an important role therein. Prior to HSC donation, female donors may encounter HY antigens through fetomaternal or transmaternal cell flow, potentially leading to the induction of HY-specific cytotoxic or regulatory immune responses. Whether HY priming occurs independent of parity, and whether HY priming is dependent on the presence of male microchimerism, is as yet unknown. Methods We investigated the presence of HY-specific regulatory T cells (Treg) and male microchimerism in 45 healthy women with a fully documented pregnancy and family history. HY peptide-induced linked suppression, a commonly reported functional feature of CD4pos and CD8pos Treg, was measured by trans vivo Delayed Type Hypersensitivity testing. As source of HY antigens, male microchimerism was analyzed by real-time PCR and defined by the presence of male DNA in at least one purified leukocyte cell type. Results HLA class I or class II restricted HY-specific Treg were detected in 26/42 (62%) women eligible for analysis. The prevalence of HY-specific Treg was significantly higher in women who had never given birth to sons than in women with male offspring (pu200a=u200a0.004). Male microchimerism could be detected in 24 out of 45 (53%) women but did not correlate with the presence of HY specific Treg. Conclusions HY-specific Treg in women with male offspring have been described previously. Here we show for the first time that, in fact, HY specific Treg are more common in nulliparous women and in parous women with female offspring. Their presence is independent of the presence of male microchimerism. Whether HY-specific Treg presence in female stem cell grafts might decrease the GVHD incidence in male HSCT recipients needs to be investigated.

Naturally acquired microchimerism: Implications for transplantation outcome and novel methodologies for detection

Microchimerism represents a condition where one individual harbors genetically distinct cell populations, and the chimeric population constitutes <1% of the total number of cells. The most common natural source of microchimerism is pregnancy. The reciprocal cell exchange between a mother and her child often leads to the stable engraftment of hematopoietic and non-hematopoietic stem cells in both parties. Interaction between cells from the mother and those from the child may result in maternal immune cells becoming sensitized to inherited paternal alloantigens of the child, which are not expressed by the mother herself. Vice versa, immune cells of the child may become sensitized toward the non-inherited maternal alloantigens of the mother. The extent of microchimerism, its anatomical location, and the sensitivity of the techniques used for detecting its presence collectively determine whether microchimerism can be detected in an individual. In this review, we focus on the clinical consequences of microchimerism in solid organ and hematopoietic stem cell transplantation, and propose concepts derived from data of epidemiologic studies. Next, we elaborate on the latest molecular methodology, including digital PCR, for determining in a reliable and sensitive way the extent of microchimerism. For the first time, tools have become available to isolate viable chimeric cells from a host background, so that the challenges of establishing the biologic mechanisms and function of these cells may finally be tackled.

In Situ Detection of HY-Specific T Cells in Acute Graft-versus-Host Disease–Affected Male Skin after Sex-Mismatched Stem Cell Transplantation

HY-specific T cells are presumed to play a role in acute graft-versus-host disease (aGVHD) after female-to-male stem cell transplantation (SCT). However, infiltrates of these T cells in aGVHD-affected tissues have not yet been reported. We evaluated the application of HLA-A2/HY dextramers for the in situ detection of HY-specific T cells in cryopreserved skin biopsy specimens. We applied the HLA-A2/HY dextramers on cryopreserved skin biopsy specimens from seven male HLA-A2(+) pediatric patients who underwent stem cell transplantation with confirmed aGVHD involving the skin. The dextramers demonstrated the presence of HY-specific T cells. In skin biopsy specimens of three male recipients of female grafts, 68% to 78% of all skin-infiltrating CD8(+) T cells were HY-specific, whereas these cells were absent in biopsy specimens collected from sex-matched patient-donor pairs. Although this study involved a small and heterogeneous patient group, our results strongly support the hypothesis that HY-specific T cells are actively involved in the pathophysiology of aGVHD after sex-mismatched stem cell transplantation.

Erratum to: Intact IFN-γR1 Expression and Function Distinguishes Langerhans Cell Histiocytosis From Mendelian Susceptibility to Mycobacterial Disease

PurposePoly-ostotic Langerhans Cell Histiocytosis (LCH) can be difficult to distinguish clinically and histologically from disseminated infection in manifesting specific subtypes of Mendelian Susceptibility to Mycobacterial Disease (MSMD). In MSMD-patients, dominant negative germline mutations in the IFN-γR1 gene, in particular in exon 6, lead to autosomal dominant IFN-γ receptor 1 deficiency (ADIFNGR1) and can mimic LCH. We hypothesized that similar defects might underlie the pathogenesis of LCH.MethodsIFN-γR1 expression was immunohistochemically determined at disease onset in biopsies from 11 LCH-patients and four ADIFNGR1-patients. IFN-γR1 function was analyzed in 18 LCH-patients and 13 healthy controls by assessing the IFN-γ-induced upregulation of Fc-gamma-receptor I (FcγRI) expression on monocytes. Pro-inflammatory cytokine production was measured after stimulation of whole blood with LPS and IFN-γ. Exon 6 of the IFN-γR1 gene was sequenced in 67 LCH-patients to determine whether mutations were present.ResultsIFN-γR1 expression was high in three LCH-affected biopsies, similar to ADIFNGR1-affected biopsies, but varied from negative to moderate in eight other LCH-affected biopsies. No functional differences in IFN-γ signaling were detected between LCH-patients with active or non-active disease and healthy controls. No germline mutations in exon 6 of the IFN-γR1 gene were detected in any of the 67 LCH-patients.ConclusionsIn contrast to ADIFNGR1-patients, IFN-γ signaling is fully functional in LCH-patients. Either performed before, during or after treatment, these non-invasive functional assays can distinguish LCH-patients from ADIFNGR1-patients and thereby facilitate correct therapy regimens for patients with recurrent osteolytic lesions.Poly-ostotic Langerhans Cell Histiocytosis (LCH) can be difficult to distinguish clinically and histologically from disseminated infection in manifesting specific subtypes of Mendelian Susceptibility to Mycobacterial Disease (MSMD). In MSMD-patients, dominant negative germline mutations in the IFN-γR1 gene, in particular in exon 6, lead to autosomal dominant IFN-γ receptor 1 deficiency (ADIFNGR1) and can mimic LCH. We hypothesized that similar defects might underlie the pathogenesis of LCH. IFN-γR1 expression was immunohistochemically determined at disease onset in biopsies from 11 LCH-patients and four ADIFNGR1-patients. IFN-γR1 function was analyzed in 18 LCH-patients and 13 healthy controls by assessing the IFN-γ-induced upregulation of Fc-gamma-receptor I (FcγRI) expression on monocytes. Pro-inflammatory cytokine production was measured after stimulation of whole blood with LPS and IFN-γ. Exon 6 of the IFN-γR1 gene was sequenced in 67 LCH-patients to determine whether mutations were present. IFN-γR1 expression was high in three LCH-affected biopsies, similar to ADIFNGR1-affected biopsies, but varied from negative to moderate in eight other LCH-affected biopsies. No functional differences in IFN-γ signaling were detected between LCH-patients with active or non-active disease and healthy controls. No germline mutations in exon 6 of the IFN-γR1 gene were detected in any of the 67 LCH-patients. In contrast to ADIFNGR1-patients, IFN-γ signaling is fully functional in LCH-patients. Either performed before, during or after treatment, these non-invasive functional assays can distinguish LCH-patients from ADIFNGR1-patients and thereby facilitate correct therapy regimens for patients with recurrent osteolytic lesions.

Gastrointestinal Acute Graft-versus-Host Disease in Children: Histology for Diagnosis, Mesenchymal Stromal Cells for Treatment, and Biomarkers for Prediction of Response

Steroid-nonresponsive acute graft-versus-host disease (aGVHD) after hematopoietic stem cell transplantation carries a poor prognosis. Various groups have reported beneficial effects of mesenchymal stromal cell (MSC) infusion as salvage treatment. Response to treatment is typically evaluated using the diagnostic clinical criteria for aGVHD. In this study, we evaluated the usefulness of additional gastrointestinal biopsy specimens paired with serum biomarkers. In a cohort of 22 pediatric patients, persistent or recurrent diarrhea was seen in 18 children treated with MSC infusion for steroid-refractory aGVHD. To exclude other causes of gastrointestinal pathology, patients were biopsied for histological analysis. Eight of 12 patients exhibited residual tissue damage and villous atrophy, but no active aGVHD. Serum biomarkers have been identified as an alternative tool for monitoring the response to aGVHD treatment. The value of biomarkers for inflammation, tissue, and endothelial cell damage was evaluated in our cohort. Although predictive of response to treatment and survival, these markers lack the necessary specificity and sensitivity to predict response to MSC therapy. Objective endpoints for clinical trials on the treatment of steroid-refractory aGVHD remain to be defined. Thus, we recommend including biopsies and biomarkers to discriminate between ongoing aGVHD and postinflammatory malabsorption.