Michael Gombert

Plasmacytoid predendritic cells initiate psoriasis through interferon-α production

Psoriasis is one of the most common T cell–mediated autoimmune diseases in humans. Although a role for the innate immune system in driving the autoimmune T cell cascade has been proposed, its nature remains elusive. We show that plasmacytoid predendritic cells (PDCs), the natural interferon (IFN)-α–producing cells, infiltrate the skin of psoriatic patients and become activated to produce IFN-α early during disease formation. In a xenograft model of human psoriasis, we demonstrate that blocking IFN-α signaling or inhibiting the ability of PDCs to produce IFN-α prevented the T cell–dependent development of psoriasis. Furthermore, IFN-α reconstitution experiments demonstrated that PDC-derived IFN-α is essential to drive the development of psoriasis in vivo. These findings uncover a novel innate immune pathway for triggering a common human autoimmune disease and suggest that PDCs and PDC-derived IFN-α represent potential early targets for the treatment of psoriasis.

CCL1-CCR8 Interactions: An Axis Mediating the Recruitment of T Cells and Langerhans-Type Dendritic Cells to Sites of Atopic Skin Inflammation

Atopic dermatitis represents a chronically relapsing skin disease with a steadily increasing prevalence of 10–20% in children. Skin-infiltrating T cells, dendritic cells (DC), and mast cells are thought to play a crucial role in its pathogenesis. We report that the expression of the CC chemokine CCL1 (I-309) is significantly and selectively up-regulated in atopic dermatitis in comparison to psoriasis, cutaneous lupus erythematosus, or normal skin. CCL1 serum levels of atopic dermatitis patients are significantly higher than levels in healthy individuals. DC, mast cells, and dermal endothelial cells are abundant sources of CCL1 during atopic skin inflammation and allergen challenge, and Staphylococcus aureus-derived products induce its production. In vitro, binding and cross-linking of IgE on mast cells resulted in a significant up-regulation of this inflammatory chemokine. Its specific receptor, CCR8, is expressed on a small subset of circulating T cells and is abundantly expressed on interstitial DC, Langerhans cells generated in vitro, and their monocytic precursors. Although DC maintain their CCR8+ status during maturation, brief activation of circulating T cells recruits CCR8 from intracytoplamic stores to the cell surface. Moreover, the inflammatory and atopy-associated chemokine CCL1 synergizes with the homeostatic chemokine CXCL12 (SDF-1α) resulting in the recruitment of T cell and Langerhans cell-like DC. Taken together, these findings suggest that the axis CCL1-CCR8 links adaptive and innate immune functions that play a role in the initiation and amplification of atopic skin inflammation.

Combined immunodeficiency with life-threatening EBV-associated lymphoproliferative disorder in patients lacking functional CD27

CD27, a tumor necrosis factor receptor family member, interacts with CD70 and influences T-, B- and NK-cell functions. Disturbance of this axis impairs immunity and memory generation against viruses including Epstein Barr virus (EBV), influenza, and others. CD27 is commonly used as marker of memory B cells for the classification of B-cell deficiencies including common variable immune deficiency. Flow cytometric immunophenotyping including expression analysis of CD27 on lymphoid cells was followed by capillary sequencing of CD27 in index patients, their parents, and non-affected siblings. More comprehensive genetic analysis employed single nucleotide polymorphism-based homozygosity mapping and whole exome sequencing. Analysis of exome sequencing data was performed at two centers using slightly different data analysis pipelines, each based on the Genome Analysis ToolKit Best Practice version 3 recommendations. A comprehensive clinical characterization was correlated to genotype. We report the simultaneous confirmation of human CD27 deficiency in 3 independent families (8 patients) due to a homozygous mutation (p. Cys53Tyr) revealed by whole exome sequencing, leading to disruption of an evolutionarily conserved cystein knot motif of the transmembrane receptor. Phenotypes varied from asymptomatic memory B-cell deficiency (n=3) to EBV-associated hemophagocytosis and lymphoproliferative disorder (LPD; n=3) and malignant lymphoma (n=2; +1 after LPD). Following EBV infection, hypogammaglobulinemia developed in at least 3 of the affected individuals, while specific anti-viral and anti-polysaccharide antibodies and EBV-specific T-cell responses were detectable. In severely affected patients, numbers of iNKT cells and NK-cell function were reduced. Two of 8 patients died, 2 others underwent allogeneic hematopoietic stem cell transplantation successfully, and one received anti-CD20 (rituximab) therapy repeatedly. Since homozygosity mapping and exome sequencing did not reveal additional modifying factors, our findings suggest that lack of functional CD27 predisposes towards a combined immunodeficiency associated with potentially fatal EBV-driven hemo-phagocytosis, lymphoproliferation, and lymphoma development.

CC Chemokine Ligand 18, An Atopic Dermatitis-Associated and Dendritic Cell-Derived Chemokine, Is Regulated by Staphylococcal Products and Allergen Exposure

Atopic dermatitis is a chronic inflammatory skin disease with a steadily increasing prevalence. Exposure to allergens or bacterial superantigens triggers T and dendritic cell (DC) recruitment and induces atopic skin inflammation. In this study, we report that among all known chemokines CCL18/DC-CK1/PARC represents the most highly expressed ligand in atopic dermatitis. Moreover, CCL18 expression is associated with an atopic dermatitis phenotype when compared with other chronic inflammatory skin diseases. DCs either dispersed within the dermis or clustering at sites showing perivascular infiltrates are abundant sources of CCL18. In vitro, microbial products including LPS, peptidoglycan, and mannan, as well as the T cell-derived activation signal CD40L, induced CCL18 in monocytes. In contrast to monocytes, monocyte-derived, interstitial-type, and Langerhans-type DCs showed a constitutive and abundant expression of CCL18. In comparison to Langerhans cells, interstitial-type DCs produced higher constitutive levels of CCL18. In vivo, topical exposure to the relevant allergen or the superantigen staphylococcal enterotoxin B, resulted in a significant induction of CCL18 in atopic dermatitis patients. Furthermore, in nonatopic NiSO4-sensitized individuals, only relevant allergen but not irritant exposure resulted in the induction of CCL18. Taken together, findings of the present study demonstrate that CCL18 is associated with an atopy/allergy skin phenotype, and is expressed at the interface between the environment and the host by cells constantly screening foreign Ags. Its regulation by allergen exposure and microbial products suggests an important role for CCL18 in the initiation and amplification of atopic skin inflammation.

Topical Superantigen Exposure Induces Epidermal Accumulation of CD8+ T Cells, a Mixed Th1/Th2-Type Dermatitis and Vigorous Production of IgE Antibodies in the Murine Model of Atopic Dermatitis

Patients with atopic dermatitis (AD) have repeated cutaneous exposure to both environmental allergens and superantigen-producing strains of Staphylococcus aureus. We used a murine model of AD to investigate the role of staphylococcal enterotoxin B (SEB) in the modulation of allergen-induced skin inflammation. Mice were topically exposed to SEB, OVA, a combination of OVA and SEB (OVA/SEB), or PBS. Topical SEB and OVA/SEB exposure induced epidermal accumulation of CD8+ T cells and TCRVβ8+ cells in contrast to OVA application, which induced a mainly dermal infiltration of CD4+ cells. SEB and OVA/SEB exposure elicited a mixed Th1/Th2-associated cytokine and chemokine expression profile within the skin. Restimulation of lymph node cells from OVA- and OVA/SEB-exposed mice with OVA elicited strong production of IL-13 protein, whereas substantial amounts of IFN-γ protein were detected after SEB stimulation of cells derived from SEB- or OVA/SEB-exposed mice. Topical SEB treatment elicited vigorous production of SEB-specific IgE and IgG2a Abs and significantly increased the production of OVA-specific IgE and IgG2a Abs. The present study shows that topical exposure to SEB provokes epidermal accumulation of CD8+ T cells, a mixed Th2/Th1 type dermatitis and vigorous production of specific IgE and IgG2a Abs, which can be related to the chronic phase of atopic skin inflammation.

Autoimmune lymphoproliferative syndrome-like disease in patients with LRBA mutation.

Mutations in LPS-responsive and beige-like anchor (LRBA) gene were recently described in patients with combined immunodeficiency, enteropathy and autoimmune cytopenia. Here, we extend the clinical and immunological phenotypic spectrum of LRBA associated disorders by reporting on three patients from two unrelated families who presented with splenomegaly and lymphadenopathy, cytopenia, elevated double negative T cells and raised serum Fas ligand levels resembling autoimmune lymphoproliferative syndrome (ALPS) and one asymptomatic patient. Homozygous loss of function mutations in LRBA were identified by whole exome analysis. Similar to ALPS patients, Fas mediated apoptosis was impaired in LRBA deficient patients, while apoptosis in response to stimuli of the intrinsic mitochondria mediated apoptotic pathway was even enhanced. This manuscript illustrates the phenotypic overlap of other primary immunodeficiencies with ALPS-like disorders and strongly underlines the necessity of genetic diagnosis in order to provide early correct diagnosis and subsequent care.

Genomics and drug profiling of fatal TCF3-HLF-positive acute lymphoblastic leukemia identifies recurrent mutation patterns and therapeutic options

TCF3-HLF−positive acute lymphoblastic leukemia (ALL) is currently incurable. Using an integrated approach, we uncovered distinct mutation, gene expression and drug response profiles in TCF3-HLF−positive and treatment-responsive TCF3-PBX1−positive ALL. We identified recurrent intragenic deletions of PAX5 or VPREB1 in constellation with the fusion of TCF3 and HLF. Moreover somatic mutations in the non-translocated allele of TCF3 and a reduction of PAX5 gene dosage in TCF3-HLF ALL suggest cooperation within a restricted genetic context. The enrichment for stem cell and myeloid features in the TCF3-HLF signature may reflect reprogramming by TCF3-HLF of a lymphoid-committed cell of origin toward a hybrid, drug-resistant hematopoietic state. Drug response profiling of matched patient-derived xenografts revealed a distinct profile for TCF3-HLF ALL with resistance to conventional chemotherapeutics but sensitivity to glucocorticoids, anthracyclines and agents in clinical development. Striking on-target sensitivity was achieved with the BCL2-specific inhibitor venetoclax (ABT-199). This integrated approach thus provides alternative treatment options for this deadly disease.

Infection Exposure Is a Causal Factor in B-cell Precursor Acute Lymphoblastic Leukemia as a Result of Pax5-Inherited Susceptibility.

UNLABELLED Earlier in the past century, infections were regarded as the most likely cause of childhood B-cell precursor acute lymphoblastic leukemia (pB-ALL). However, there is a lack of relevant biologic evidence supporting this hypothesis. We present in vivo genetic evidence mechanistically connecting inherited susceptibility to pB-ALL and postnatal infections by showing that pB-ALL was initiated in Pax5 heterozygous mice only when they were exposed to common pathogens. Strikingly, these murine pB-ALLs closely resemble the human disease. Tumor exome sequencing revealed activating somatic, nonsynonymous mutations of Jak3 as a second hit. Transplantation experiments and deep sequencing suggest that inactivating mutations in Pax5 promote leukemogenesis by creating an aberrant progenitor compartment that is susceptible to malignant transformation through accumulation of secondary Jak3 mutations. Thus, treatment of Pax5(+/-) leukemic cells with specific JAK1/3 inhibitors resulted in increased apoptosis. These results uncover the causal role of infection in pB-ALL development. SIGNIFICANCE These results demonstrate that delayed infection exposure is a causal factor in pB-ALL. Therefore, these findings have critical implications for the understanding of the pathogenesis of leukemia and for the development of novel therapies for this disease.

Inherited susceptibility to pre B-ALL caused by germline transmission of PAX5 c.547G>A

A single-nucleotide polymorphism (SNP) in PAX5 leading to an amino-acid change in the octapeptide domain at position c.547G>A (p.Gly183Ser) has recently been described to confer an inherited susceptibility for childhood pre B-ALL.1 This susceptibility was transmitted autosomal dominant in two independent families with variable penetrance and aberrations of chromosomal part 9p resulting in loss of the wild-type (wt) PAX5 allele occurruring simultaneously in the leukemic cells.

ALK fusion genes in children with atypical myeloproliferative leukemia

The receptor tyrosine kinase ALK has a pivotal function in various malignant diseases, and its exceptional behavior is documented by the fact that chromosomal ALK rearrangements have been described in completely different malignancies, namely inflammatory myofibroblastic tumors, anaplastic large-cell lymphomas and lung cancer of the NSCLC subtype. Recently, the expression of a particular ALK fusion protein, namely TPM4-ALK, in esophageal squamous cell carcinoma was suggested. ALK belongs to the insulin-receptor superfamily, and its expression is normally restricted to specific regions of the central nervous system. Chromosomal translocations fusing and activating the ALK locus foster tumor genesis by its constitutive tyrosine kinase activity. In cells with ALK fusion proteins, a number of overlapping and interconnected signaling pathways are subsequently activated, for example RAS-ERK, JAK-STAT and PI3K-AKT. Enhanced proliferation and survival through inhibition of apoptosis are key features of this ALK-mediated activation of signaling cascades. An involvement of ALK in myeloid malignancies, however, has not yet been reported. Here, we show that ALK is rarely but recurrently rearranged in children with myeloid diseases, indicating the great clinical potential that anti-ALK therapies may acquire in the near future. Among 1708 successfully analyzed karyotypes of children with myeloproliferative diseases (1278 AML, 430 MDS), provided by classical cytogenetics, six were found to have an abnormality involving the chromosomal band 2p23 (Table 1; Supplementary Table 1). Two children showed a t(2;4)(p23;q27) (pt. 1) and a del(2)(p23) (pt. 2) as a part of a complex karyotype. In three of the remaining four (pts. 3–5), an inv(2)(p23q13) was detected, whereas patient 6 had a t(2;2)(p23;q11B13). Interestingly, in three of the latter (pts. 3, 4, 6), a monosomy 7 ( 7) was found in addition. To confirm the breakpoint in 2p23, FISH analysis was performed using the LSI ALK Dual Color Break Apart Rearrangement Probe on bone marrow cells of all six patients. A splitting of the green and red signal was found in patients 3, 4 and 6 (Figure 1), whereas in the remaining three patients, two fusion signals appeared, indicating no involvement of the ALK gene. By the use of chromosome arm-specific painting probes, translocation t(2;2) in pt. 6 was also proved (Figure 1). Of special interest were the cytogenetic findings of pt. 4, who had two aberrant clones, one with the inv(2)(p23q13) only and another clone with the additional loss of chromosome 7. To prove that the 7 abnormality occurred secondary to the ALK splitting, we performed co-hybridization of the ALK probe together with a chromosome 7 centromere probe (Supplementary Figure 1). Whereas almost no cells with 7 as sole abnormality were found (far below the cutoff of 6%), we counted 25% of cells with an ALK splitting only but two normal centromere signals for chromosome 7. In all, 42% of the cells had both aberrations, an ALK rearrangement and a 7. The remaining cells revealed a wild-type configuration at both loci. Thus, we conclude that the ALK rearrangement preceded the monosomy 7 in this case. In contrast, at subsequent relapse, the same patient showed ALK splitting in conjunction with the 7 in all malignant cells. This finding was confirmed by G-banding showing the karyotype 45,XY,inv(2)(p23q13), 7[8]/46,XY[3] at this time point. On the basis of an earlier report in which a translocation t(2;2)(p23;q13) resulted in a RAN-binding protein 2 (RanBP2)ALK fusion mRNA, we performed an RT-PCR analysis for the presence of such a chimeric mRNA. This analysis showed a distinct band of 240 bp for patients 3, 4, and 6, whereas pts. 1 and 2 were negative (Supplementary Figure 2). Unfortunately, there was no material left from pt. 5 for this analysis. Subsequent