Double-negative T-cell Research Articles

Blocking of CD1d Decreases Trypanosoma cruzi-Induced Activation of CD4-CD8- T Cells and Modulates the Inflammatory Response in Patients With Chagas Heart Disease.

The control of inflammatory responses to prevent the deadly cardiac pathology in human Chagas disease is a desirable and currently unattained goal. Double-negative (DN) T cells are important sources of inflammatory and antiinflammatory cytokines in patients with Chagas heart disease and those with the indeterminate clinical form of Chagas disease, respectively. Given the importance of DN T cells in immunoregulatory processes and their potential as targets for controlling inflammation-induced pathology, we studied the involvement of CD1 molecules in the activation and functional profile of Trypanosoma cruzi-specific DN T cells. We observed that parasite stimulation significantly increased the expression of CD1a, CD1b, CD1c, and CD1d by CD14(+) cells from patients with Chagas disease. Importantly, among the analyzed molecules, only CD1d expression showed an association with the activation of DN T cells, as well as with worse ventricular function in patients with Chagas disease. Blocking of CD1d-mediated antigen presentation led to a clear reduction of DN T-cell activation and a decrease in the expression of interferon γ (IFN-γ) by DN T cells. Thus, our results showed that antigen presentation via CD1d is associated with activation of DN T cells in Chagas disease and that CD1d blocking leads to downregulation of IFN-γ by DN T cells from patients with Chagas heart disease, which may be a potential target for preventing progression of inflammation-mediated dilated cardiomyopathy.

Pro-inflammatory self-reactive Tcells are found within murine TCR-αβ(+) CD4(-) CD8(-) PD-1(+) cells.

TCR-αβ(+) double negative (DN) Tcells (CD3(+) TCR-αβ(+) CD4(-) CD8(-) NK1.1(-) CD49b(-) ) represent a minor heterogeneous population in healthy humans and mice. These cells have been ascribed pro-inflammatory and regulatory capacities and are known to expand during the course of several autoimmune diseases. Importantly, previous studies have shown that self-reactive CD8(+) Tcells become DN after activation by self-antigens, suggesting that self-reactive Tcells may exist within the DN T-cell population. Here, we demonstrate that programmed cell death 1 (PD-1) expression in unmanipulated mice identifies a subset of DN Tcells with expression of activation-associated markers and a phenotype that strongly suggests they are derived from self-reactive CD8(+) cells. We also found that, within DN Tcells, the PD-1(+) subset generates the majority of pro-inflammatory cytokines. Finally, using a TCR-activation reporter mouse (Nur77-GFP), we confirmed that in the steady-state PD-1(+) DN Tcells engage endogenous antigens in healthy mice. In conclusion, we provide evidence that indicates that the PD-1(+) fraction of DN Tcells represents self-reactive cells.

Abstract A176: Efficacy and safety of allogeneic double negative T cell as anti-AML therapy and its underlying mechanism

Abstract Acute myeloid leukemia (AML) is the most common form of adult acute leukemia that is associated with a low long-term survival rate. While chemotherapy achieves remission in the majority of AML patients, many relapse due to residual chemotherapy-resistant AML populations. Allogeneic hematopoetic stem cell transplantation is a potential curative treatment for AML that demonstrates the efficacy of a cell-mediated treatment for chemotherapy-resistant disease. However, its wide application is limited by suitable donor availability and associated toxicity such as graft-versus-host disease (GvHD). Hence, there is a need for a new treatment approach that targets chemotherapy-resistant AML blasts with minimal side effects. The goals of this study were to characterize allogeneic CD3+CD4-CD8- double negative T (DNT) cells as a potential new target of treatment for AML patients, including ones that are resistant to chemotherapy, and to dissect its underlying mechanisms. Using two-hour flow cytometry-based in vitro killing assay, we demonstrated that the allogeneic DNT cells expanded from healthy volunteers were cytotoxic against 23/29 primary AML patient blasts in a dose-dependent manner. Of those, 13 blasts were obtained from chemotherapy refractory or relapsing AML patients and nine of them were susceptible to DNT cells. The anti-leukemia activity of DNT cells was further validated in a AML-NSG xenograft model: a single infusion of DNT cells into mice pre-engrafted with primary AML blasts from chemoresponsive, chemorefractory, and relapsed patients significantly reduced the leukemia burden. Although residual blasts were observed from DNT cell-treated group, they did not develop resistance to DNT cells, as they remained susceptible to DNT cells in ex vivo killing assay. Further, we demonstrated the safety of allogeneic DNT cells, as DNT cells did not target allogeneic peripheral blood mononuclear cells (PBMC) and hematopoietic stem/progenitor cells (HSPC) in vitro. In addition, administration of allogeneic DNT cells into NSG mice, engrafted with human HSPC, had no effect on the engraftment level of human hematopoetic cells and their differentiation into different lineages. Further, infusion of human DNT cells did not cause xenogeneic GvHD in mice, collectively demonstrating the safety of allogeneic DNT cells. Using blocking assays, we showed the roles of HLA-class I, NKG2D, and DNAM-1 in DNT cell-mediated cytotoxicity against AML, whereas HLA-class II and T cell receptor were not involved. While IFNγ release correlated with cytotoxicity of DNT cells, IFNγ treatment alone did not induce AML cell death. Neutralizing IFNγ reduced susceptibility to DNT cell-mediated cytotoxicity while pretreating AML with recombinant IFNγ increased their susceptibility by inducing higher expression of NKG2D and DNAM-1 ligands, where NKG2D- and DNAM-1-blocking antibodies abrogated the effect of IFNγ pretreatment. Cytolytic activity of DNT cells was mediated in perforin-granzyme-dependent fashion, which subsequently activated caspase-8 and caspase-9 pathways in AML cells to induce AML cell death. Collectively, these studies demonstrated the safety and efficacy of allogeneic DNT cell therapy as a potential treatment for AML patients, including those with chemotherapy-resistant leukemia, and revealed important molecules for the anti-leukemia activity of DNT cells. Citation Format: Jong Bok Lee, Claire Weihsu Chen, Mark D. Minden, John E. Dick, Li Zhang. Efficacy and safety of allogeneic double negative T cell as anti-AML therapy and its underlying mechanism. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A176.

Whole-Exome Analysis of Autoimmune Lymphoproliferative Syndrome-like Diseases

ALPS is characterized by chronic lymphoproliferation in combination with autoimmunity; mutations of molecules involved in FAS-dependent pathways play causative roles in this syndrome. The hallmarks of ALPS are an elevated CD4/CD8 double-negative T (DNT) cell count and attenuated induction of apoptosis by FAS stimulation. Autoimmune thrombocytopenia and/or hemolytic anemia are common in cases of ALPS; the combination of autoimmune thrombocytopenia and hemolytic anemia is referred to as Evans syndrome. ALPS is diagnosed in 47% of patients who present with Evans syndrome. Some patients with ALPS-like syndromes harbor mutations in RAS or PRKCD.In this study, we performed whole-exome analysis of undiagnosed patients exhibiting autoimmunity in combination with lymphoproliferation. Fifteen pediatric patients presenting with autoimmunity were enrolled in this study. Although not all of them fulfilled the diagnostic criteria of ALPS, most of them exhibited an ALPS-like phenotype, an autoimmune hematological disorder such as Evans syndrome, or immune thrombocytopenia with hepatosplenomegaly. Elevated counts of DNT cells among TCRαβ-positive cells were observed in some cases. All of the subjects screened negative for mutations in FAS, FASL, KRAS, and NRAS. These patients were subjected to whole-exome analysis, which revealed several mutations with known disease associations. One patient carried a mutation in CASP10, a causative gene for ALPS-CSAP (ALPS 2). This patient exhibited a typical ALPS phenotype with an elevated DNT cell count. In addition, we identified CTLA4 mutations in two patients, one of whom was described as ALPS type V (ALPS 5) and the other as CTLA4 haploinsufficiency with autoimmune infiltration (CHAI). A mutation in STAT3 was present in one patient. Dominant-negative mutations in the STAT3 gene result in hyper-IgE syndrome. Recently, an activating mutation of STAT3 was reported in infantile-onset multisystem autoimmune disease (ADMIO). It should be noted that an activating mutation of STAT3 results in a phenotype very similar to that of ALPS. Intriguingly, two autoinflammatory associated genes, PSTPIP1 and RNASEH2B were identified. Mutations in RNASEH2B causes Aicardi-Goutière syndrome (AGS), which phenotypically overlaps with SLE. Typical AGS develops as a results of biallelic mutations in causative genes. In this case, a heterozygous frame shift mutation was identified. Further evaluation is required to confirm whether this heterozygous frame shift mutation RNASEH2B really causes SLE or ALPS like symptoms. PSTPIP1 mutation causes pyogenic Arthritis, pyoderma gangrenosum and acne (PAPA) syndrome. The identified E250K mutation was one of the typical mutation in this disease. This patient exhibited SLE like symptom with hepatosplenomegaly without typical PAPA syndrome like symptoms, such as acne or pyoderma of the skin. Sequencing of the remaining patients yielded inconclusive results.The classical ALPS diagnostic procedure is well designed and suitable for identification of FAS-dependent apoptotic dysregulation. However, the measurement of FAS-dependent apoptosis requires technical skill, and the data quality is therefore dependent on the investigator. Moreover, RAS-associated ALPS-like disease (RALD, ALPS 3) and ALPS 5 (CHAI) cannot be identified by conventional ALPS diagnostic procedures. Therefore, the methods for diagnostic classification of these diseases need to be updated. It is interesting that mutations of autoinflammatory associated genes are involved in ALPS or SLE like patients. Our results reveal that a comprehensive genomic approach is a powerful tool for the characterization of ALPS or ALPS-like diseases. Together with recent progress in genome analysis in the PID field, our analyses provide an updated list of genes for use in differential diagnosis of ALPS-like diseases. This update will facilitate convenient genomic approaches such as comprehensive targeted sequencing focusing on genes involved in ALPS or ALPS-like diseases. This approach can be directly applied in the clinic to yield diagnostic benefits. DisclosuresNo relevant conflicts of interest to declare.

Lmo2's Oncogenic Function in T-Cell Leukemia Requires Ldb1

LIM domain Only-2 (LMO2) is one of the most frequently deregulated oncogenes in T-cell acute lymphoblastic leukemia (T-ALL). LMO2 encodes a small protein with 2 LIM domains that is part of a large multiprotein complex in hematopoietic stem and progenitor cells, where it is required for HSC specification and maintenance. Many of LMO2's protein partners in HSPCs are expressed in T-ALL implying that protein complexes similar to those nucleated by LMO2 in HSPCs also play a role in leukemia. In this study, we analyzed a critical component of the LMO2 associated complex, LIM domain binding1 (LDB1). LDB1 appears to be an obligate partner of LMO2 in HSPCs but it is not required for T-cell development from committed progenitors. LDB1 is concordantly expressed with LMO2 in human T-ALL although its expression is more widespread than LMO2. To further define Ldb1's role in leukemia, we induced its conditional knockout in CD2-Lmo2 transgenic mice. CD2-Lmo2 transgenic mice develop T-ALL with high penetrance and closely model the human disease. We discovered that Lmo2-induced T-ALL was markedly attenuated in penetrance and latency by Ldb1 deletion. Since Lmo2 induces a distinct differentiation arrest in T-cell progenitors prior to leukemic transformation, we analyzed the differentiation of T-cell progenitors in CD2-Lmo2 transgenic/floxed-Ldb1/Lck-Cre mice and in non-Lmo2 transgenics: floxed-Ldb1/Lck-Cre mice. Ldb1 deletion by Lck-Cre was efficient in double negative and double positive T-cell progenitors. In striking contrast, Ldb1 deletion could not be induced in CD2-Lmo2 transgenic T-cell progenitors. Consistent with this finding, T-ALLs that developed in CD2-Lmo2/floxed-Ldb1/Lck-Cre mice had incomplete deletion of Ldb1. These results imply that Ldb1 is a required factor for Lmo2 to induce T-ALL. Lastly, gene expression analysis of Lmo2-induced T-ALLs and ChIP-exonuclease analysis of Ldb1 occupancy in T-ALL suggested that the Lmo2/Ldb1 complex enforced a gene signature similar to that seen in HSPCs and in Early T-cell Precursor ALL. In conclusion, Ldb1 is a required partner for Lmo2 to induce T-ALL. Additionally, the HSPC function of Lmo2/Ldb1 complexes may be recapitulated in T-cell progenitors prior to T-ALL. DisclosuresNo relevant conflicts of interest to declare.

Autoimmune lymphoproliferative syndrome due to somatic FAS mutation (ALPS-sFAS) combined with a germline caspase-10 (CASP10) variation

Autoimmune lymphoproliferative syndrome (ALPS) is a primary immunodeficiency caused by impaired Fas/FasL-mediated apoptosis of lymphocytes and is characterized by chronic nonmalignant or benign lymphoproliferation, autoimmune manifestations and expansion of double negative (DN) T-cells (TCRαβ+CD4-CD8-). Most cases of ALPS are associated with germline (ALPS-FAS) or somatic (ALPS-sFAS) heterozygous FAS mutations or a combination of both.Here we report three unrelated patients with ALPS-sFAS. Only one of them showed impaired Fas function in PHA-activated T-cells. In this patient, the genetic analysis of the caspase-10 gene (CASP10) identified a heterozygous germline change in exon 9 (c.1337A>G) causing Y446C substitution in the caspase-10 protein. In addition, this patient had a dysregulated T- and B-cell phenotype; circulating lymphocytes showed expansion of T effector memory CD45RA+ (TEMRA) CD4 T-cells, effector memory CD8 T-cells, CD21low B-cells and reduced memory switched B-cells. Additionally, this patient showed altered expression in T-cells of several molecules that change during differentiation from naïve to effector cells (CD27, CD95, CD57 and perforin). Molecular alterations in genes of the Fas pathway are necessary for the development of ALPS and this syndrome could be influenced by the concurrent effect of other mutations hitting different genes involved in Fas or related pathways.

New role for the (pro)renin receptor in T-cell development

AbstractThe (pro)renin receptor (PRR) was originally thought to be important for regulating blood pressure via the renin-angiotensin system. However, it is now emerging that PRR has instead a generic role in cellular development. Here, we have specifically deleted PRR from T cells. T-cell–specific PRR-knockout mice had a significant decrease in thymic cellularity, corresponding with a 100-fold decrease in the number of CD4+ and CD8+ thymocytes, and a large increase in double-negative (DN) precursors. Gene expression analysis on sorted DN3 thymocytes indicated that PRR-deficient thymocytes have perturbations in key cellular pathways essential at the DN3 stage, including transcription and translation. Further characterization of DN T-cell progenitors leads us to propose that PRR deletion affects thymocyte survival and development at multiple stages; from DN3 through to DN4, double-positive, and single-positive CD4 and CD8. Our study thus identifies a new role for PRR in T-cell development.

The production of alpha/beta and gamma/delta double negative (DN) T-cells and their role in the maintenance of pregnancy.

The ability of the thymus gland to convert bone marrow-derived progenitor cells into single positive (SP) T-cells is well known. In this review we present evidence that the thymus, in addition to producing SP T-cells, also has a pathway for the production of double negative (DN) T-cells. The existence of this pathway was noted during our examination of relevant literature to determine the cause of sex steroid-induced thymocyte loss. In conducting this search our objective was to answer the question of whether thymocyte loss is the end product of a typical interaction between the reproductive and immune systems, or evidence that the two systems are incompatible. We can now report that “thymocyte loss” is a normal process that occurs during the production of DN T-cells. The DN T-cell pathway is unique in that it is mediated by thymic mast cells, and becomes functional following puberty. Sex steroids initiate the development of the pathway by binding to an estrogen receptor alpha located in the outer membrane of the mast cells, causing their activation. This results in their uptake of extracellular calcium, and the production and subsequent release of histamine and serotonin. Lymphatic vessels, located in the subcapsular region of the thymus, respond to the two vasodilators by undergoing a substantial and preferential uptake of gamma/delta and alpha/beta DN T- cells. These T- cells exit the thymus via efferent lymphatic vessels and enter the lymphatic system.The DN pathway is responsible for the production of three subsets of gamma/delta DN T-cells and one subset of alpha/beta DN T-cells. In postpubertal animals approximately 35 % of total thymocytes exit the thymus as DN T-cells, regardless of sex. In pregnant females, their levels undergo a dramatic increase. Gamma/delta DN T-cells produce cytokines that are essential for the maintenance of pregnancy.

Abstract W P87: Characteristics of T-cells Infiltrating Ruptured Intracranial Aneurysm

Introduction: Recently the role of adaptive immunity has been implied by microarray studies. But the results are contradictory. T-cell infiltration is a frequent histological finding in ruptured IA, T-cell phenotype, characteristic and true quantitation remains unknown. We preformed a prospective study to determine the subpopulation and expression of activation markers of T-cells infiltrating ruptured IA in relation to peripheral blood. Hypothesis: IA have different subsets and activation levels of T-cells than peripheral blood. Methods: We collected the tissue of ruptured IA of 8 patients operated on within 24 hours after subarachnoid hemorrhage symptoms onset. IA tissue was digested, stained with fluorescently labeled monoclonal antibodies and submitted to flow cytometry. In addition we collected and analyzed venous blood from 6 age, sex and risk factor-matched controls. Results: CD4+ cells are less prevalent in IA tissue than in peripheral blood (42.14±17.28 vs. 65.88±5.32%; p=0.011), while there was no difference in CD8+ T-cells infiltrating IA (30.28±9.07 vs. 27.78±5.45%; p=0.585), and double negative (CD4-CD8-CD3+) T-cells were more prevalent in wall of IA than in circulation, (15.68±11.94 vs. 2.81±1.32%; p=0.026). Importantly, CD4+ infiltrating IA wall showed higher expression of HLA-DR (25.9±6.42 vs. 9.19± 3.58%; p<0.001) higher expression of CD 69 (26.8±19.66 vs. 2.73±0.93%; p=0.014). Similarly, there significantly more CD8+ cells showed HLA-DR+ in the IA than in blood. (45.96±15.57 vs. 22.47±11.46%; p=0.018) and CD69 (30.32±22.73 vs. 5.03±1.55%; p=0.022). Double negative cells in IA also had higher expression of HLA-DR (46.56±21.40 vs. 22.58±5.1%; p=0.025), CD69 (31.05±16.79 vs. 7.83±2.05%; p=0.016). Conclusion: The tissue of ruptured IA is highly infiltrated by T-cells which show high expression of activation markers such as CD69 or HLA-DR. The importance of these cells to immunopathogenesis of intracranial aneurysm rupture should be further characterized.

αβ-Double Negative CD4/CD8 (CD56) T cell (DNTs) in metastatic melanoma: basal frequency and behaviour during Ipilimumab treatment. Preliminary evaluations

Background The knowledge of the immune system role on melanoma has accelerated the translation of key advancements into medical breakthroughs like ipilimumab, an anti-CTLA4 immunomodulating antibody. Ipilimumab works amazingly well only in a limited number of patients and its effects on T-cell subpopulations as well as on immune response remains to be elucidated. Recently, it was described a new subset of immunomodulating T-cells, known as Double-negative T-cells (DNTs) expressing either ab or gδ T-cell receptors (TCR) but lacking CD4, CD8,CD56. The DNTs contribute specifically to antitumor immunity since involved in immune regulation and tolerance acting as regulatory T-cells (Treg) and/or cytotoxic T-cells and they contribute to in vivo anti-melanoma immunity as previously reported [1-5]. However no data are available on their frequency in melanoma, as well as the effects of ipilimimumab on DNTs functional attitude in immunomodulation and on modulating their expression during the therapy. We aimed to evaluate the modulation of DNT frequency in Metastatic Melanoma (MM) patients treated with ipilimumab during the therapy in order to explore their potential role on clinical outcome and therapy response.

Interleukin-7 Represses the Immunoregulatory Function of Human TCRαβ+ CD4- CD8- Double-Negative (DN) T Cells By Activating Akt/mTOR Signaling

Regulatory T (Treg) cells have been shown to be involved in downregulating immune responses in autoimmunity, transplant rejection, and graft-versus-host disease (GvHD). Recently, a novel subset of TCRαβ+ CD4- CD8- (double negative, DN) T cells has been described to possess a very potent suppressive activity on allogeneic T-cell responses in both mice and humans. Of interest, clinical studies in patients after allogeneic stem cell transplantation (SCT) revealed an inverse correlation between the frequency of circulating DN T cells and the severity of GvHD. Interleukin (IL)-7 is required for survival and homeostasis of mature T-cells and has been shown to modulate the functional activity of Treg cells. Here we asked whether IL-7 affects the suppressive activity of human DN T cells. We found that human DN T cells isolated from peripheral blood lymphocytes do express the IL-7 receptor and are able to transduce IL-7 downstream signaling. Intriguingly, IL-7 significantly diminished the suppressive activity of DN T cells towards allogeneic CD4+ and CD8+ T cells. Thereby, IL-7 did not render the responder T cells insensitive to suppression. Instead, blocking of key signaling molecules revealed that IL-7 has a direct impact on the suppressive activity of DN T cells. Further analyses demonstrated that IL-7 activates Akt/mTOR signaling in DN T cells. Importantly, selective inhibition of the Akt or mTOR protein kinase was able to reverse the IL-7 effect thereby restoring the suppressive function of DN T cells, whereas inhibition of other central T-cell signaling pathways did not. Thus, DN T-cell mediated suppression is impaired by IL-7 due to crucial activation of the Akt/mTOR signaling cascade. These findings indicate that IL-7 and Akt/mTOR signaling are critical factors for the suppressive capacity of DN T cells. Targeting of these pathways by pharmacological agents may restore and/or enhance DN T-cell functionality in GvHD. DisclosuresNo relevant conflicts of interest to declare.

Defective anti-polysaccharide response and splenic marginal zone disorganization in ALPS patients

AbstractAutoimmune lymphoproliferative syndrome (ALPS) caused by impaired FAS-mediated apoptosis of lymphocytes is characterized by lymphoproliferation, autoimmunity, but also an increased risk of invasive bacterial infection, notably following splenectomy. We surveyed a cohort of 100 ALPS patients (including 33 splenectomized) and found that 12 (10 splenectomized) had experienced 23 invasive bacterial infections mainly caused by Streptococcus pneumoniae. This vulnerability was associated with evidence of defective B-cell function characterized by low serum immunoglobulin (Ig) M, low IgM antibody production in response to S pneumoniae following nonconjugated immunization, and low blood memory B-cells counts (including marginal zone [MZ] B-cell counts). This immunodeficiency strongly correlated with intensity of lymphoproliferation. Spleen sections from 9 ALPS patients revealed double-negative T-cell (DN-T) infiltration of the MZ, which was depleted of B cells. MZ in ALPS patients contained an abnormally thick layer of MAdCAM-1(+) stromal cells and an excess of DN-Ts. DN-Ts were shown to express MAdCAM-1 ligand, the α4β7 integrin. These observations suggest that accumulating DN-Ts are trapped within stromal cell meshwork and interfere with correct localization of MZ B cells. Similar observations were made in spleens of fas-deficient mice. Our data revealed an unexpected mechanism by which ALPS results in anti-polysaccharide IgM antibody production–specific defect. Splenectomy should be avoided.

IL-10/Janus kinase/signal transducer and activator of transcription 3 signaling dysregulates Bim expression in autoimmune lymphoproliferative syndrome

Autoimmune lymphoproliferative syndrome (ALPS) is a human disorder of T cell homeostasis caused by mutations that impair FAS-mediated apoptosis. A defining characteristic of ALPS is the expansion of double negative T cells (DNTC). Relatively little is known about how defective FAS-driven cell death and the Bcl-2 apoptotic pathway intersect in ALPS patients. We studied changes in Bcl-2 family member expression in ALPS to determine whether the Bcl-2 pathway might provide a therapeutic target. We used flow cytometry to analyze the expression of pro- and anti-apoptotic Bcl-2 family members in T cells from 12 ALPS patients and determined the in vitro sensitivity of ALPS DNTC to the pro-apoptotic BH3 mimetic, ABT-737. The pro-apoptotic molecule, Bim, was significantly elevated in DNTC. Although no general pattern of individual anti-apoptotic Bcl-2 family members emerged, increased expression of Bim was always accompanied by increased expression of at least 1 anti-apoptotic Bcl-2 family member. Strikingly, Bim levels in DNTC correlated significantly with serum IL-10 in ALPS patients, and IL-10 was sufficient to mildly induce Bim in normal and ALPS T cells via a Janus kinase/signal transducer and activator of transcription 3-dependent mechanism. Finally, ABT-737 preferentially killed ALPS DNTC in vitro. Combined, these data show that an IL-10/Janus kinase/signal transducer and activator of transcription 3 pathway drives Bim expression in ALPS DNTC, which renders them sensitive to BH3 mimetics, uncovering a potentially novel therapeutic approach to ALPS.

Compound heterozygous CORO1A mutations in siblings with a mucocutaneous-immunodeficiency syndrome of epidermodysplasia verruciformis-HPV, molluscum contagiosum and granulomatous tuberculoid leprosy.

Coronin-1A deficiency is a recently recognized autosomal recessive primary immunodeficiency caused by mutations in CORO1A (OMIM 605000) that results in T-cell lymphopenia and is classified as T(-)B(+)NK(+)severe combined immunodeficiency (SCID). Only two other CORO1A-kindred are known to date, thus the defining characteristics are not well delineated. We identified a unique CORO1A-kindred. We captured a 10-year analysis of the immune-clinical phenotypes in two affected siblings from disease debut of age 7 years. Target-specific genetic studies were pursued but unrevealing. Telomere lengths were also assessed. Whole exome sequencing (WES) uncovered the molecular diagnosis and Western blot validated findings. We found the compound heterozygous CORO1A variants: c.248_249delCT (p.P83RfsX10) and a novel mutation c.1077delC (p.Q360RfsX44) (NM_007074.3) in two affected non-consanguineous siblings that manifested as absent CD4CD45RA(+) (naïve) T and memory B cells, low NK cells and abnormally increased double-negative (DN) ϒδ T-cells. Distinguishing characteristics were late clinical debut with an unusual mucocutaneous syndrome of epidermodysplasia verruciformis-human papilloma virus (EV-HPV), molluscum contagiosum and oral-cutaneous herpetic ulcers; the older female sibling also had a disfiguring granulomatous tuberculoid leprosy. Both had bilateral bronchiectasis and the female died of EBV+ lymphomas at age 16 years. The younger surviving male, without malignancy, had reproducibly very short telomere lengths, not before appreciated in CORO1A mutations. We reveal the third CORO1A-mutated kindred, with the immune phenotype of abnormal naïve CD4 and DN T-cells and newfound characteristics of a late/hypomorphic-like SCID of an EV-HPV mucocutaneous syndrome with also B and NK defects and shortened telomeres. Our findings contribute to the elucidation of the CORO1A-SCID-CID spectrum.

CD4+ T-Cell Converted Double Negative T-Cells Suppress B-Cells Proliferation and IGG Production in Mice.

We have reported that CD4+ T-cells could be converted to CD3+CD4-CD8-(DN) T-cells, the converted DN T-cells were very potent in suppressing antigen-specific T cells responses in vitro and prolonged islet allograft survival in vivo. In this study, we further tested the functional potential of CD4+ T-cells converted DN T-cells on B-cells. We found that the proliferation of B-cells were significantly suppressed by DN T-cells and IgG secretion of stimulated B cells was also inhibited by DN T-cells in vitro and in vivo. The suppression was cell dose dependent and was relied on cell-cell contact. Perforin, a cytotoxic lymphocyte related cytokine, was highly expressed by DN T-cells, played an important role in DN T-cell mediated suppression, the potency of DN T-cells derived from perforin KO mice to suppress the proliferation of B-cells was significantly lower than that from wild type mice. Our study suggested that ex vivo CD4+ T-cells converted DN T-cells have the potential to down-regulate immune responses by suppressing B cells proliferation and IgG production. It supports the concept and the feasibility of potentially utilizing this novel cell-based therapeutic approach clinically for the prevention and treatment of B-cell mediated rejection.

Natural history of autoimmune lymphoproliferative syndrome associated with FAS gene mutations

AbstractAutoimmune lymphoproliferative syndrome (ALPS) presents in childhood with nonmalignant lymphadenopathy and splenomegaly associated with a characteristic expansion of mature CD4 and CD8 negative or double negative T-cell receptor αβ+ T lymphocytes. Patients often present with chronic multilineage cytopenias due to autoimmune peripheral destruction and/or splenic sequestration of blood cells and have an increased risk of B-cell lymphoma. Deleterious heterozygous mutations in the FAS gene are the most common cause of this condition, which is termed ALPS-FAS. We report the natural history and pathophysiology of 150 ALPS-FAS patients and 63 healthy mutation-positive relatives evaluated in our institution over the last 2 decades. Our principal findings are that FAS mutations have a clinical penetrance of <60%, elevated serum vitamin B12 is a reliable and accurate biomarker of ALPS-FAS, and the major causes of morbidity and mortality in these patients are the overwhelming postsplenectomy sepsis and development of lymphoma. With longer follow-up, we observed a significantly greater relative risk of lymphoma than previously reported. Avoiding splenectomy while controlling hypersplenism by using corticosteroid-sparing treatments improves the outcome in ALPS-FAS patients. This trial was registered at www.clinicaltrials.gov as #NCT00001350.

Recurrent Oral Inflammation in Autoimmune Lymphoproliferative Syndrome

Abstract Background and aim: Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of abnormal lymphocyte survival caused by dysregulation of the Fas apoptotic pathway. In ALPS defective lymphocyte apoptosis manifests as a chronic, nonmalignant lymphadenopathy and/or splenomegaly/hepatosplenomegaly, expansion of double negative T cell (DNTC) – CD4 - CD8 - TCRI±I²+ T cells, autoimmune cytopenias and other autoimmune diseases. Patients demonstrate oral lesions which have not yet been reported in the literature. The aim of the study was to present the oral status of children with ALPS, associated mainly with haematologic and immunologic disorders. Material and methods: Among almost 1500 patients with primary immune deficiency diagnosed between 1980 and 2012 at the Department of Immunology, Children's Memorial Hospital, Warsaw (Poland) 7 cases of ALPS were identified according to ALPS diagnostic criteria. Routine immunological parameters were evaluated (serum immunoglobulin levels, immunophenotyping of peripheral lymphocytes, DNT cells, proliferative response to mitogens), vitamin B12 level, haematological and biochemical laboratory tests. Clinical course was retrieved retrospectively from medical records and evaluated during control visits. All patients were offered dental evaluation and treatment. Clinical examinations included the status of marginal gingiva, oral mucosa (the presence and type of lesions) and presence of caries. Results and conclusions: Almost all patients presented with leukopenia and granulocytopenia during control visits. Unsatisfactory oral hygiene status, carious cavities and gingivitis were found in all patients. In six subjects oral ulcers were also noticed. In two of them gingivitis was assessed as severe (with redness, swelling and spontaneous gingival bleeding), in other - as mild. An association between hematological and immune disorders and the health status of the oral mucosa and gingivae was observed in all but one patient. Findings included pale oral mucosa, a smooth tongue, recurrent mucosal ulcers, and gingivitis with bleeding. Their onset was accompanied by deterioration in the patients' general health condition and the presence of local causative factors. R ecurrent oral inflammation/aphtae can be the first symptom of ALPS and should be taken into account in differential diagnosis by paediatrician or general practitioner. It indicates strong need for cooperation between the dentists and immunologists and haematologists.

Autoimmune lymphoproliferative syndrome presenting as urticaria

Autoimmune lymphoproliferative syndrome (ALPS) is an inherited disorder characterized by chronic splenomegaly, lymphadenopaphy hypergammaglobulinemia and autoimmune phenomena especially immune-mediated cytopenias. The disease is characterized by presence of double-negative (CD3 + CD4 − CD8 − ) T-cells in the peripheral blood and lymphoid tissue. Rarely it can also present as urticaria. We report a child of ALPS who presented to us with fever, generalized lymphadenopathy, chronic urticaria and hepatosplenomegaly.

Generation and Characterization Of a CD30 Positive T-Cell Lymphoma Mouse Model Resembling Human Anaplastic Large Cell Lymphoma

Anaplastic large cell lymphomas (ALCL) are a subgroup of aggressive Non-Hodgkin-Lymphomas mainly affecting children and young adults. In 60 % of systemic ALCLs, a translocation t(2;5) (p23;q35) resulting in NPM-ALK fusion gene expression is found. The constitutively activation of ALK tyrosine kinase expressed from the NPM-promoter causes increased proliferation and inhibition of apoptosis thereby promoting cell survival and tumorigenesis. Immunphenotypic characterization of human ALCLs revealed highly CD30-positive cells of T- or Null-Cell-origin and resulted in promising clinical trials with CD30-coupled antibodies. However, the impact of CD30 on diseases development as well as NPM-ALK signal transduction in course of disease remains unclear and appropriate mouse models to answer these questions are missing.In this regard, we established a retroviral murine bone marrow (BM) transplantation model resembling a human ALCL-like T-cell neoplasia. Therefore we use an inducible Cre/loxP system where NPM-ALK expression is controlled and expressed in a special type of early T-cells. For generation of this vector, we inserted a floxed translational ‘stop-cassette' between the retroviral promoter MSCV-LTR and the NPM-ALK cDNA, which guaranties specific expression of NPM-ALK only in cells, where the enzyme Cre-recombinase is expressed. Recognition of the loxP-sites by Cre-recombinase leads in our system to deletion of the stop-cassette and consequently NPM-ALK expression. Using different Cre-expressing cell types allowed us to study pathogenesis of ALCL in more detail.In our recent study, we infected bone marrow of transgenic mice expressing Cre-recombinase under the control of the Lck-promotor with our MSCV-Stop-NPM-ALK-IRES-EGFP (MSNAIE) vector and transplanted it into lethally irradiated C57Bl6 recipient mice. With a latency of 4-5 months, these mice developed Thy1.2-positive lymphomas and died from neoplastic infiltration of bone marrow and lymphatic organs with T-cells. Immunphenotypic analyses confirmed T-Cell origin of the lymphomas and showed importantly highly CD30-expression. Staining of the different T-cell-subpopulations demonstrated highest NPM-ALK expression in immature CD4/CD8 double negative T-cells and not fully differentiated CD4/CD8 double positive T-cells. Interestingly, FACS-staining of the proliferation marker Ki-67 revealed highest expression in CD4/CD8 double negative T-cells, in contrast to the other subpopulations where Ki-67 is less detected. Therefore we hypothesized, that the lymphoma initiating cell (LIC) must be within this early T-cell population. Most interestingly we found highest CD30-expression just in the same CD4/CD8 negative T-cell population, pointing to a crucial role of CD30 in lymphoma initiation.To further substantiate our hypothesis we performed secondary and tertiary transplantations with different sorted T-Cell subpopulation and indeed, the immature CD4/CD8 double negative population was able to initiate lymphoma growth in recipient mice. Further transplantations by limited dilution will help to identify the leukemia initiating cell in this model.Taken together, our murine LckCre-NPM-ALK bone marrow transplantation model represents a precise and versatile tool to study disease initiation and development resembling human ALCL. Moreover, the impact of specific proteins (e.g. CD30) in the course of disease can be addressed by combining Knockout (e.g. CD30)/LckCre transgenic mice with our model. To this end we crossed CD30/Lck-Cre mice, and preliminary analysis indicate that CD30 expression seems not to be required for the initial onset of disease. Further characterization of the role of CD30 in ALCL is ongoing. Disclosures:No relevant conflicts of interest to declare.

Sequential decisions on FAS sequencing guided by biomarkers in patients with lymphoproliferation and autoimmune cytopenia

Clinical and genetic heterogeneity renders confirmation or exclusion of autoimmune lymphoproliferative syndrome difficult. To re-evaluate and improve the currently suggested diagnostic approach to patients with suspected FAS mutation, the most frequent cause of autoimmune lymphoproliferative syndrome, we prospectively determined 11 biomarkers in 163 patients with splenomegaly or lymphadenopathy and presumed or proven autoimmune cytopenia(s). Among 98 patients sequenced for FAS mutations in CD3(+)TCRα/β(+)CD4(-)CD8(-) "double negative" T cells, 32 had germline and six had somatic FAS mutations. The best a priori predictor of FAS mutations was the combination of vitamin B12 and soluble FAS ligand (cut-offs 1255 pg/mL and 559 pg/mL, respectively), which had a positive predictive value of 92% and a negative predictive value of 97%. We used these data to develop a web-based probability calculator for FAS mutations using the three most discriminatory biomarkers (vitamin B12, soluble FAS ligand, interleukin-10) of the 11 tested. Since more than 60% of patients with lymphoproliferation and autoimmune cytopenia(s) in our cohort did not harbor FAS mutations, 15% had somatic FAS mutations, and the predictive value of double-negative T-cell values was rather low (positive and negative predictive values of 61% and 77%, respectively), we argue that the previously suggested diagnostic algorithm based on determination of double-negative T cells and germline FAS sequencing, followed by biomarker analysis, is not efficient. We propose vitamin B12 and soluble FAS ligand assessment as the initial diagnostic step with subsequent decision on FAS sequencing supported by a probability-calculating tool.