Defect In Compartment Research Articles

Deletion of Micro RNA-15/16 Clusters Promotes Leukemic Initiation in AML Via the Deregulation of Hematopoietic Progenitors

The microRNA 15a/16-1 cluster on chromosome 13 is commonly lost in chronic lymphocytic leukemia (CLL). Even though this deletion is considered to be a CLL-associated genomic aberration, loss of miR-15/16 occurs in 79% of primary acute myeloid leukemia (AML) and in patients with myelodysplastic syndrome (MDS) that transformed into AML. A second highly homologous locus, miR-15b/16-2, is present in both human and mice, and deletion of either miR-15/16 cluster in mice leads to CLL. Unexpectedly, when both loci are deleted, only 23% of miR-15/16 double knock out (DKO) mice develop various non-Hodgkin's lymphomas, whereas 77% develop AML. Consistent with this, DKO mice have an expanded Gr-1+CD11b+ granulocytic population in both bone marrow (BM) and spleen. These observations led us to hypothesize that the miR-15/16 clusters can act coordinately to regulate lineage determination and proliferation in early hematopoiesis. Their loss might influence leukemogenesis in CLL and AML by altering the number and function of hematopoietic stem cells (HSCs) and/or their downstream lineages and potentially increasing a population of preleukemic stem cells. To test this, using flow cytometry, we analyzed the numbers and frequency of HSCs and progenitors in DKO and C57BL/6 (B6) mice. We discovered major changes in HSCs and the common myeloid progenitors (CMP) but similar numbers of common lymphoid progenitors. Therefore, we focused on the myeloid lineage, finding that CD16/32-CD34- megakaryocyte/erythroid progenitors (MEP) were significantly increased in the Lin- cKit+ compartment of DKO mice (44.9% vs 29.8% P = 0.01), but CD16/32+CD34+ granulocyte-macrophage progenitors (GMP) were decreased (19.1% vs 40.1% P = 0.01). This was surprising since we also found that a GMP progeny population, CD11b+Gr-1+ cells, was enriched in DKO compared to B6 BM (60-70% vs 40%). To determine if this was a result of a differentiation defect in an earlier progenitor, we assessed the frequency of Lin-Sca1+cKit+ (LSK) populations, finding that DKO mice had significantly more CD150+CD48-CD34- LT-HSCs among LSK cells (14.3% vs 7.8% P = 0.03), and fewer CD150-CD48+ multipotent progenitors (MPP) (58.5% vs 68.6% P = 0.04). Of note, there was an increased absolute count for all progenitors analyzed, possibly due to an increase in BM cells and/or disproportional increases of certain cell types. Since the observed myeloproliferative phenotype could be due to 1) an absolute increase of progenitor cells; 2) malfunctioning progenitor cells that aberrantly differentiate and proliferate; 3) or both, we tested whether DKO LSK cells had distinct functional characteristics in vitro. Methylcellulose replating of LT-HSC and MPP in the presence of SCF, IL-3, and IL-6 indicated that DKO LT-HSC gave rise to consistently fewer colonies as compared to B6 LT-HSC (21.5 vs 37.2 colonies/100 cells plated P = 0.02). Unexpectedly, we found the opposite for MPP, as DKO MPP consistently exhibited higher colony-forming activity than B6 MPP (16.6 vs 5.1 colonies/100 cells plated P = 0.04) over the course of serial replating experiments. This was surprising in light of the significant decrease in MPP frequency we had observed by immunophenotyping. Furthermore, B6 MPP cells ceased forming colonies after 3 rounds of replating, whereas DKO MPP continued for 5 rounds, suggesting that the absence of miR-15/16 increased the self-renewal potential of the CD150-CD48+ MPP cells. This was further supported by immunophenotyping analysis showing increased LSK cells derived from DKO MPP colonies (25.8% vs 4.4% P = 0.03). In concordance with the initial observation, an increase in DKO MPP colony-derived CD11b+Gr-1+ cells was at least partly responsible for the myeloproliferative defect in the mature cell compartment (76.1% vs 56.9%P = 0.01). Together, these results suggest that in miR-15/16 DKO mice, the phenotypically defined MPP compartment contains leukemia-initiating cells. Overall, our findings reveal a novel role for the miR-15/16 clusters at the top of the hematopoietic hierarchy. Future experiments focusing on these DKO progenitors that appears to be endowed with leukemic stem cell potential may provide clues to understand how the targets of miR-15a, miR-15b and miR-16 orchestrate their effects on lineage commitment and how their absence may lead to initiation of AML and CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Ipsilateral chondral lesions worsen the long-term prognosis following arthroscopic partial medial meniscectomy.

The effect of knee cartilage defects that are detected during partial meniscectomy remains controversial in terms of the long-term prognosis on knee function. This study aimed to investigate the effect of concurrent medial compartment focal cartilage lesions on the long-term prognosis of knee function in patients who underwent arthroscopic partial medial meniscectomy for traumatic medial meniscal tears. This retrospective study analyzed 46 patients who underwent arthroscopic partial medial meniscectomy between 1991 and 2008 by a single surgeon. Twenty-one patients who underwent arthroscopic partial medial meniscectomy due to traumatic meniscal tear had focal chondral lesions only at the medial compartment, and these patients were assigned to group A. Twenty-five patients who had no cartilage lesions in any compartments were assigned to group B. The age, sex, body mass index (BMI), follow-up time, age at the time of surgery, clinical and radiological scores, and perioperative arthroscopy findings were analyzed. The mean follow-up time was 20 ± 3.7years. No significant difference was found in the demographic data, and the average age of the patients at the time of operation was 35 ± 9.5years. Both groups had improved Lysholm score at the last follow-up. Although no difference was found between the groups during the preoperative period, group B had a higher Lysholm score at the last follow-up than group A. The mean International Knee Documentation Committee (IKDC) and Knee injury and Osteoarthritis Outcome Score (KOOS) scores at the last follow-up were significantly higher in group B. The mean Kellgren-Lawrence grades in the operated knees of group A were higher than those of group B. In group A, a negative correlation was found between the BMI and postoperative Lysholm (r = - 0.461, p = 0.03) IKDC (r = - 0.433, p = 0.05) and KOOS (r = - 0.565, p = 0.008) scores. In group B, no correlation was found between BMI and any score. Among patients who underwent arthroscopic partial medial meniscectomy with an average follow-up of 20years, those with concurrent focal cartilage defect in the medial compartment had clinically and radiologically worse outcomes than patients without any cartilage defect. Therefore, orthopedic surgeons should be meticulous before performing any arthroscopic partial medial meniscectomy in case of concurrent cartilage lesion. Level III.

Attenuated Interferon-α Targeted to CD38 Expressing Multiple Myeloma Tumor Cells Induces Robust and Durable Anti-Tumor Responses through Direct Anti-Proliferative Activity in Addition to Indirect Recruitment and Activation of M1 Macrophages

Despite recent advances in treatment, multiple myeloma (MM) remains an incurable disease. We previously reported that targeting an attenuated form of interferon-alpha (IFNα) directly to MM tumor cells via an anti-CD38 antibody induces robust and durable responses in murine xenograft MM models, eliminating large established human tumors in mice. We demonstrated that this attenuated immunocytokine, or Attenukine™, exhibits 10,000-fold increased specificity for CD38-positive cells in vitro compared to native IFNα and, importantly, is less toxic than native IFNα to normal bone marrow cells in vitro. Application of this therapy is predicted to significantly expand the therapeutic index of IFNα therapy beyond that of an immunocytokine, providing a rationale for clinical testing in MM.In this study, we explored potential mechanisms mediating the robust anti-MM tumor activity of the CD38-Attenukine™. In MM xenograft models, immunohistochemistry analysis of ex vivo tumors taken from treated and untreated mice revealed rapid and robust infiltration of M1 (iNOS positive) macrophages in treated MM tumors (shown in figure). Targeted depletion of macrophages in tumor bearing mice prior to treatment demonstrated the importance of macrophages for the robust and durable activity. Follow-on experiments compared the anti-tumor activity of the CD38-Attenukine™ in different mouse strains with varied degrees of immune competence. While treatment of SCID mice bearing NCI-H929 tumors with 5 mg/kg CD38-Attenukine™ eliminated tumors in all mice, tumors in only 2 of 10 mice were eliminated in the NOD-SCID background, and no tumors were eliminated in the NSG mouse strain. The NOD-SCID and NSG strains, which have defective macrophage populations, showed significantly less macrophage infiltration than was observed in the SCID mouse strain. Both the macrophage infiltration and antitumor activity of the CD38-Attenukine™ could be fully restored in NOD-SCID mice when co-treated with an anti-human CD47 antibody. Blocking CD47 signaling is thought to correct the defect in the NOD-SCID macrophage compartment. Interestingly, tumor sections from CD38-Attenukine™ treated mice were caspase-3 negative, suggesting that apoptosis may not be the major mechanism of tumor cell death in these models. In fact, based on the requirement for a glycosylated IgG Fc for robust and durable response, we predict that antibody-dependent cellular phagocytosis (ADCP) is a major mechanism for the observed tumor regression. Additional findings suggest that IFNα-induced chemokine production from tumor cells may signal M1 macrophage infiltration and activation following treatment. In conclusion, the potent anti-tumor response observed in CD38-Attenukine™ treated mice is the result of at least two complementary mechanisms, direct anti-proliferative activity of IFNα on tumor cells and indirect recruitment and activation of tumoricidal macrophages potentially through a chemokine gradient. These findings provide a solid framework supporting clinical evaluation of a CD38-Attenukine™ immunotherapy in MM patients. [Display omitted] DisclosuresPogue:Teva Pharmaceuticals: Employment. Bi:Teva Pharmaceuticals: Employment. Armanini:Teva Pharmaceuticals: Employment. Fatholahi:Teva Pharmaceuticals: Employment. Taura:Teva Pharmaceuticals: Employment. Valencia:Teva Pharmaceuticals: Employment. Yun:Teva Pharmaceuticals: Employment. Sho:Teva Pharmaceuticals: Employment. Jamin:Teva Pharmaceuticals: Employment. Nock:Teva Pharmaceuticals: Employment. Wilson:Teva Pharmaceuticals: Employment.

Regulatory T cells in patients with early untreated rheumatoid arthritis: Phenotypic changes in the course of methotrexate treatment

Rheumatoid arthritis (RA) is frequent systemic autoimmune disease characterized by excessive activation of collagen-specific T helper cells, and elevated level of autoantibodies in the serum. Development of RA is associated with defect in compartment of regulatory CD4+Foxp3+ T cells (Treg), but data concerning suppressive potential of Treg population in RA patients are contradictory and depend on the stage of disease. In this study we aimed to characterize abundance and phenotypic markers of CD4+Foxp3+ Treg in peripheral blood of healthy donors compared to untreated early RA patients to find potential correlations with the disease activity, antibody level, and absolute numbers and proportion of different subpopulations of T cells. Moreover, we assessed the influence of methotrexate (MT) treatment on percentage and absolute numbers of CD4+Foxp3+ Treg from the peripheral blood of untreated early RA patients. We demonstrate that increase and phenotypic changes in Treg population correlate well with response to MT. Analysis of the cohorts of matched RA patients (n = 45) and healthy controls (n = 20) revealed that patients with untreated early RA demonstrate substantial decrease in blood Treg percentage and absolute number, as well as low level of activated Treg surface markers in comparison to healthy control. The defect in Treg compartment negatively correlates with both RA activity and antibody level. MT treatment of patients with early untreated RA increases both proportion and absolute number of Treg with high level of activation markers, suggesting an increase of their functional capacity. Here we speculate the role of Tregs as specific cellular marker of successful RA treatment.

A gain-of-function RAC2 mutation is associated with bone-marrow hypoplasia and an autosomal dominant form of severe combined immunodeficiency.

Svere combined immunodeficiencies (SCID) constitute a heterogeneous group of life-threatening genetic disorders that typically present in the first year of life. They are defined by the absence of autologous T cells and the presence of an intrinsic or extrinsic defect in the B-cell compartment. In three newborns presenting with frequent infections and profound leukopenia, we identified a private, heterozygous mutation in the RAC2 gene (p.G12R). This mutation was de novo in the index case, who had been cured by hematopoietic stem cell transplantation but had transmitted the mutation to her sick daughter. Biochemical assays showed that the mutation was associated with a gain of function. The results of in vitro differentiation assays showed that RAC2 is essential for the survival and differentiation of hematopoietic stem/progenitor cells. Therefore, screening for RAC2 gain-offunction mutations should be considered in patients with a SCID phenotype and who lack a molecular diagnosis.

An Autosomal Dominant SCID Form Due to a Gain of Function Mutation in the RAC2 Gene

Severe combined immunodeficiencies (SCIDs) form a heterogeneous group of life-threatening genetic disorders defined by the absence of autologous T cells and an intrinsic or extrinsic defect in the B-cell compartment. In our cohort, three newborn patients with bone-marrow hypoplasia associated with clinical and biological features of SCID received allogenic hematopoietic stem cell transplantation in the first weeks of life. To identify the molecular defect involved in this life threatening SCID form, we performed whole-genome sequencing on patient's fibroblasts. The same heterozygous, dominant missense mutation was found in the RAC2gene encoding for the RAC2 GTPase (p.G12R). By biochemical analyses we demonstrated that arginine substitution at position 12 leads to sustained activation of the RAC2 signalling pathway; the G12R mutation is therefore a gain of function mutation. In cord blood hematopoietic cells, we observed that the mutation disrupts mitochondrial activity and blocks hematopoietic stem/progenitor cells (HSPCs) differentiation toward the myeloid and lymphoid lineages. Altogether, our data emphasize the involvement of the RAC2 signalling pathway during hematopoeisis and might thus explain the severity of the patients' clinical and immunological phenotype. The present study is the first to report an autosomal dominant form of SCID and suggest that RAC2 gene sequencing should be considered for patients with SCID clinical presentation. Disclosures Cavazzana: Smartimmune: Other: Founder of Smartimmune.

Original concepts in anatomy, abdominal-wall surgery, and component separation technique and strategy.

The abdominal wall can be considered comprised of two compartments: an anterior and a posterior compartment. The anterior compartment includes the anterior rectus sheath and the rectus muscle. The posterior compartment comprises the posterior rectus sheath, the transversalis fascia, and the peritoneum. When a large defect in the anterior compartment has to be corrected, for example, a rectus diastasis or large incisional hernia, an action on the anterior compartment is necessary; therefore, an anterior component separation has to be considered. If a loss of substance is present in the posterior compartment, a trasversus abdominis release should be accomplished. We propose an original anterior compartment mobilisation, by a posterior approach. Dissection of the posterior rectus sheet proceeds until the linea semilunaris is reached. Incision of the anterior rectus sheath permits a mobilisation of the anterior compartment by a posterior approach. A mesh is placed in a sublay position. If the abdominal wall presents a loss of substance of the posterior compartment, a transversus abdominis release (TAR) can be performed in the same time. No hernia recurrences, no wound infection, and no mesh infection have been reported. The anterior compartment mobilization permits mobilization towards the midline of rectus muscle and restoration of anterior compartment, with low morbidity rate; it can be easily associated to a large sublay mesh placement, it allows the preservation of the neurovascular bundles and rectus muscle trophism, and it can be associated with a concomitant TAR procedure for the restoration of the PC, if necessary.

Combined autologous chondrocyte implantation and meniscus reconstruction for large chondral defect in the lateral compartment due to discoid lateral meniscus tear: A case report.

Discoid lateral meniscus tear leads to large chondral defect in the lateral compartment of the knee joint. There are few effective treatments for large chondral defect in both the tibial and femoral sides with severe degenerative lateral meniscus. We have developed a combined autologous chondrocyte implantation and meniscus reconstruction technique using hamstring tendon. This technique allows biological reconstruction and avoids knee arthroplasty.

Congenital erythrocytosis.

Congenital erythrocytosis is by definition present from birth. Patients frequently present in childhood or as young adults and a family history may be present. The erythrocytosis can be primary where there is a defect in the erythroid compartment of secondary where increased erythropoietin production produced due to the defect leads to an erythrocytosis. Primary causes include erythropoietin receptor mutations. Congenital secondary causes include mutations in the genes involved in the oxygen-sensing pathway and haemoglobins with abnormal oxygen affinity. Investigations for the cause include an erythropoietin level, oxygen dissociation curve, haemoglobin electrophoresis and sequencing for known gene variants. The finding of a known or new molecular variant confirms a diagnosis of congenital erythrocytosis. A congenital erythrocytosis may be an incidental finding but nonspecific symptoms are described. Major thromboembolic events have been noted in some cases. Low-dose aspirin and venesection are therapeutic manoeuvres which should be considered in managing these patients. Rare individuals presenting often at a young age may have a congenital erythrocytosis. Molecular investigation may reveal a lesion. However, in the majority, currently no defect is identified.

Role of CD47 in CD4+ T cell Proliferation after TCR Stimulation

CD47 is involved in adaptive immune responses and leukocyte homeostasis. Han and colleagues (JEM 209:1325, 2012) have reported that CD47‐/‐ mice are protected from (MOG)35‐55 peptide induced experimental autoimmune encephalomyelitis (EAE), but not from passive EAE, suggesting a defect in the immune cell compartment. They concluded a defect in T cell proliferation due to failed T cell activation. We also observed CD47‐/‐ are not susceptible to MOG‐induced EAE, and have reduced CD4 T cell proliferation after MOG immunization in vivo and MOG‐dependent recall proliferation assay in vitro. However, in our hands CD47‐/‐ T cells from MOG‐immunized mice express identical levels of surface activation markers (CD69, CD44, IL2R, PD‐1) vs WT cells, and similar levels of intracellular cytokine production. CD47‐/‐ T cells also have defective Ag independent expansion after TCR stimulation, even though proximal signaling events (p‐Lck, p‐ZAP‐70, p‐ERK1/2) are identical to WT cells using FACS and CyTOF mass spectrometry. Intriguingly, kinetics of CD47‐/‐ T cell proliferation and cell cycle analyzed by CSFE dye dilution and by DNA staining respectively, show a comparable proliferative response to WT cells in the first 48h, but fail to proliferate beyond day 3. Accordingly, CD47‐/‐ CD4 T cells show a 2.3 fold increase in Annexin‐V staining and a 40% reduced [H3]thymidine incorporation vs WT. In summary, CD47‐/‐ CD4 T cells become activated by MOG or TCR stimulation, but fail to maintain clonal expansion. We suggest this is due to defects in cell cycle machinery and/or increased cell apoptosis. This work was supported by NIH P01HL03628 and AHA 11POST7730055.

Lateral Opening-wedge Distal Femoral Osteotomy: Pain Relief, Functional Improvement, and Survivorship at 5Years.

Distal femoral varus osteotomy may be used to treat valgus knee malalignment or to protect a knee compartment in which cartilage restoration surgery (such as osteochondral or meniscus allografting) has been performed. Medial closing-wedge osteotomy has demonstrated good success in treatment of osteoarthritis in published series, but few studies have evaluated distal femoral lateral opening-wedge osteotomy in terms of correction of deformity, pain and function, and survivorship. (1) Does lateral opening-wedge osteotomy lead to accurate correction? (2) What pain and function levels do patients experience after lateral opening-wedge osteotomy? (3) What are the nonunion, complication, and reoperation rates after lateral opening-wedge osteotomy? Between 2000 and 2010, we performed 40 distal femoral osteotomies. Two knees (two patients) underwent a medial closing-wedge osteotomy and were excluded from the present study. Thirty-eight knees (97%) in 36 patients were lateral opening-wedge varus-producing osteotomies; of those, 31 knees (82%) in 30 patients had followup at a minimum of 2years (mean, 5years; SD, 2; range, 2-12years) and comprised the study population. The indications for osteotomy included symptomatic lateral compartment arthritis with clinical valgus deformity or a cartilage or meniscal defect in the lateral compartment with clinical valgus alignment. The study population was stratified into two groups based on reason for osteotomy: patients with isolated symptomatic lateral compartment arthritis (arthritis group; 19 knees [61%]) and patients who underwent joint preservation procedures including osteochondral allograft transplantation or meniscal allograft transplantation (joint preservation group; 12 knees [39%]). Data collection from our institution's osteotomy database included patient demographics, lower extremity coronal alignment, and operative details. Pain and function were measured preoperatively and postoperatively using the International Knee Documentation Committee (IKDC) score. Time to radiographic union, complications, and reoperations were recorded. Twenty-one of 31 knees had postoperative radiographic data available for review. Of these, seven of 15 knees in the arthritis group and three of six knees in the joint preservation group were within the correction goal of±3° from neutral mechanical alignment. In the arthritis group, the mean IKDC total score improved from 47 (SD, 15) preoperatively to 67 (SD, 10) postoperatively. In the joint preservation group, the mean IKDC total score improved from 36 (SD, 12) preoperatively to 62 (SD, 18) postoperatively. One nonunion occurred in the arthritis group. No postoperative complications were experienced. Ten knees in the arthritis group and six knees in the joint preservation group had additional surgery after the osteotomy, consisting primarily of hardware removal, arthroscopy for cartilage-related conditions, or conversion to arthroplasty. Survivorship at 5years, with conversion to arthroplasty as the endpoint, was 74% in the arthritis group and 92% in the joint preservation group. Lateral opening-wedge distal femoral osteotomy was less accurate in correction of valgus deformity than expected, but the procedure was associated with improved knee pain and function scores. Our clinical and radiographic results are comparable to published series evaluating medial closing-wedge distal femoral osteotomy. Achieving our desired correction of±3° from neutral alignment was clinically difficult. An improved method of preoperative templating and refinement of the intraoperative technique may improve this. Future studies with more patients and longer followup will provide clarity on this topic. Level IV, therapeutic study.

Genetic control of susceptibility to Candida albicans in SM/J mice.

In the immunocompromised host, invasive infection with the fungal pathogen Candida albicans is associated with high morbidity and mortality. Sporadic cases in otherwise normal individuals are rare, and they are thought to be associated with genetic predisposition. Using a mouse model of systemic infection with C. albicans, we identified the SM/J mouse strain as unusually susceptible to infection. Genetic linkage studies in informative [C57BL/6JxSM/J]F2 mice identified a major locus on distal chromosome 15, given the appellation Carg5, that regulates C. albicans replication in SM/J mice. Cellular and molecular immunophenotyping experiments, as well as functional studies in purified cell populations from SM/J and C57BL/6J, and in [C57BL/6JxSM/J]F2 mice fixed for homozygous or heterozygous Carg5 alleles, indicate that Carg5-regulated susceptibility in SM/J is associated with a complex defect in the myeloid compartment of these mice. SM/J neutrophils express lower levels of Ly6G, and importantly, they show significantly reduced production of reactive oxygen species in response to stimulation with fMLF and PMA. Likewise, CD11b(+)Ly6G(-)Ly6C(hi) inflammatory monocytes were present at lower levels in the blood of infected SM/J, recruited less efficiently at the site of infection, and displayed blunted oxidative burst. Studies in F2 mice establish strong correlations between Carg5 alleles, Ly6G expression, production of serum CCL2 (MCP-1), and susceptibility to C. albicans. Genomic DNA sequencing of chromatin immunoprecipitated for myeloid proinflammatory transcription factors IRF1, IRF8, STAT1 and NF-κB, as well as RNA sequencing, were used to develop a "myeloid inflammatory score" and systematically analyze and prioritize potential candidate genes in the Carg5 interval.

Hypomorphic mutation in the RAG2 gene affects dendritic cell distribution and migration

OS is a severe combined immunodeficiency characterized by erythrodermia and protracted diarrhea as a result of infiltration of oligoclonal-activated T cells, caused by hypomorphic mutations in RAGs. The RAG2(R229Q) mouse model fully recapitulates the clinical OS phenotype. We evaluated whether T and B cell defects, together with the abnormal lymphoid structure, could affect DC homeostasis and function. High density of LCs was observed in skin biopsies of Omenn patients and in the derma of RAG2(R229Q) mice, correlating with the presence of erythrodermia. In vivo models of cutaneous skin painting and CHS demonstrated a decreased migration of RAG2(R229Q) DCs-in particular, LCs-into draining LNs. Interestingly, at steady state, RAG2(R229Q) mice showed a reduction in DC number in all hematopoietic organs except LNs. Analysis of the MHCII marker revealed a diminished expression also upon the LPS-driven inflammatory condition. Despite the decreased number of peripheral DCs, BM pre-cDCs were present in normal number compared with RAG2(+/+) controls, whereas pDCs and monocytes were reduced significantly. Overall, these results point to a secondary defect in the DC compartment, which contributes to clinical manifestations and autoimmunity in OS.

Coactivator-Associated Arginine Methyltransferase 1 Regulates Fetal Hematopoiesis and Thymocyte Development

Coactivator-associated arginine methyltransferase 1 (CARM1) is a protein arginine methyltransferase that methylates histones and transcriptional regulators. We previously reported that the absence of CARM1 partially blocks thymocyte differentiation at embryonic day 18.5 (E18.5). In this study, we find that reduced thymopoiesis in Carm1(-/-) mice is due to a defect in the fetal hematopoietic compartment rather than in the thymic stroma. To determine the cellular basis for impaired thymopoiesis, we examined the number and function of fetal liver (FL) and bone marrow cells. Despite markedly reduced cellularity of hematopoietic progenitors in E18.5 bone marrow, the number of long-term hematopoietic stem cells and downstream subsets was not reduced in Carm1(-/-) E14.5 or E18.5 FL. Nevertheless, competitive reconstitution assays revealed a deficit in the ability of Carm1(-/-) FL cells to contribute to hematopoiesis. Furthermore, impaired differentiation of Carm1(-/-) FL cells in a CARM1-sufficient host showed that CARM1 is required cell autonomously in hematopoietic cells. Coculture of Carm1(-/-) FL cells on OP9-DL1 monolayers showed that CARM1 is required for survival of hematopoietic progenitors under conditions that promote differentiation. Taken together, this report demonstrates that CARM1 is a key epigenetic regulator of hematopoiesis that affects multiple lineages at various stages of differentiation.

IRF7-dependent IFNβ production in response to RANK facilitates medullary thymic epithelial cell development. (60.3)

Abstract The signal transducer and activator of transcription 1 (STAT1) is an essential part of signaling cascades induced by type I and II interferons. Deficiency of STAT1 results in heightened susceptibility to infections and autoimmunity in mouse and humans. While the role of IFNγ in T cell development is well known, the role of type I interferons in thymic development has largely remained unexplored. Here we show that STAT1 and IFNAR-deficient mice harbor a defect in medullary compartment characterized by a significant reduction in self-antigen presenting, AIRE expressing medullary thymic epithelial cells. Using a novel reporter mouse we demonstrate that constitutive IFNAR signaling is taking place in the thymic medulla in the absence of any infection or inflammatory signals. In addition, we found that RANK stimulation in thymic stromal cells results in the upregulation of IFNβ which in turn is inhibits RANK signaling thereby facilitating AIRE expression. Finally, we show that IRF7 is required for the induction of IFNβ by RANKL, and that IRF7-, but not IRF3-deficient mice display a defect in thymic architecture and medullary thymic epithelial cell differentiation similar to that observed in IFNAR- or STAT1-deficient animals. These results illustrate that a spatially and temporally coordinated crosstalk between the RANKL/RANK signaling pathway and the IRF7/IFNβ/IFNAR/STAT1 axis is essential for the differentiation of AIRE-expressing medullary thymic epithelial cells.

Defects within the Myeloid Derived Supressor Cells (MDSCS) Compartment may Facilitate Aberrant Immune Responses in Systemic Lupus Erythematosus (SLE)

Background and objectivesThere is a currently unmet need to define the mechanisms involved in the homeostatic control of the immune response in autoimmune diseases. MDSCs (characterised in mice as CD11b+Gr1+...

A Spontaneous Mutation of the Rat Themis Gene Leads to Impaired Function of Regulatory T Cells Linked to Inflammatory Bowel Disease

Spontaneous or chemically induced germline mutations, which lead to Mendelian phenotypes, are powerful tools to discover new genes and their functions. Here, we report an autosomal recessive mutation that occurred spontaneously in a Brown-Norway (BN) rat colony and was identified as causing marked T cell lymphopenia. This mutation was stabilized in a new rat strain, named BNm for “BN mutated.” In BNm rats, we found that the T cell lymphopenia originated in the thymus, was intrinsic to CD4 T lymphocytes, and was associated with the development of an inflammatory bowel disease. Furthermore, we demonstrate that the suppressive activity of both peripheral and thymic CD4+ CD25bright regulatory T cells (Treg) is defective in BNm rats. Complementation of mutant animals with BN Treg decreases disease incidence and severity, thus suggesting that the impaired Treg function is involved in the development of inflammatory bowel disease in BNm rats. Moreover, the cytokine profile of effector CD4 T cells is skewed toward Th2 and Th17 phenotypes in BNm rats. Linkage analysis and genetic dissection of the CD4 T cell lymphopenia in rats issued from BNm×DA crosses allowed the localization of the mutation on chromosome 1, within a 1.5 megabase interval. Gene expression and sequencing studies identified a frameshift mutation caused by a four-nucleotide insertion in the Themis gene, leading to its disruption. This result is the first to link Themis to the suppressive function of Treg and to suggest that, in Themis-deficient animals, defect of this function is involved in intestinal inflammation. Thus, this study highlights the importance of Themis as a new target gene that could participate in the pathogenesis of immune diseases characterized by chronic inflammation resulting from a defect in the Treg compartment.

Bim is a component of the endosomal-lysosomal interface in Interleukin-7 dependent T cells (57.10)

Abstract Bim is a key protein in T cell homeostasis. Bim deficiency prevents activated T cell death, and protects splenic T cells in IL-7R/Bim double knockout mice through an undetermined mechanism. We propose a novel function for Bim in T cells involving lysosomal development in both apoptotic and non-apoptotic cells. Using IL-7 dependent T cell lines, Bim-containing and Bim-deficient (Bim-/-), we determined that the transcription of bim increased in the absence of IL-7 and the loss of Bim protected cells from apoptosis induced by IL-7 withdrawal. Treatment of Bim-containing cells with Bim siRNA confirmed these results. Bim-/- cells had increased endosomal and decreased lysosomal proteins, suggesting that Bim plays a role in endosomal to lysosomal trafficking and maturation. Subcellular organelle fractionation revealed that the BimL isoform associated with both endosomal and lysosomal fractions in non-apoptotic cells. However, during IL-7 withdrawal, the BimEL isoform associated with lysosomal fractions and less so with endosomal fractions. In fact, reduced co-localization of endosomes and lysosomes in the absence of Bim was observed by confocal microscopy, suggesting a defect in an endocytic compartment. Overexpression of the individual Bim isoforms restored acidic organelle distribution with BimL contributing the most in non-apoptotic cells. These results demonstrate that Bim has both apoptotic and non-apoptotic activities in T cells that affects lysosomal equilibrium.

To the Editor

It is well established that canonical Wnt signalling plays essential roles during embryogenesis and the self-renewal of adult tissues such as the skin and the gut. In addition, in a review in this journal, Staal and Sen have highlighted an important role of this pathway for haematopoiesis in general 1. Although we agree with this conclusion, we believe that it can currently not be deduced that canonical Wnt signalling within haematopoietic cells, as opposed to supporting non-haematopoietic cells, is essential for haematopoiesis in the adult. When considering the existing literature (as shown in Fig. 2 of 1), it is evident that the role of canonical Wnt signalling in haematopoiesis has been deduced to a large extent from gain-of-function experiments, i.e. the enforced expression of components of this pathway or ex vivo treatment of cells by Wnt ligands or inhibitors. Although some of these manipulations have yielded striking phenotypes, the relevance of these findings to normal haematopoiesis is uncertain. Indeed, as a precedent, while gain-of-function experiments suggested that Notch signals play a role in expanding hematopoietic progenitors, Notch signalling in HSC plays no essential role in the maintenance of HSC 2. There are few reports on loss-of-function experiments of genes implicated in the Wnt pathway that show clear-cut effects in the haematopoietic system. However, for most of these “clear-cut” phenotypes, it was not determined whether Wnt signalling was actually involved. This is important as many of these Wnt-signalling genes play roles outside the Wnt pathway. In addition, even when specific Wnt signalling components such as Fzd or Wnts were deleted, it was often not analysed whether the effects found involved canonical, β-catenin-mediated signalling or non-canonical, β-catenin independent signalling. Indeed, a recent study, which looked at this aspect in more detail, reported that Wnt4 affects haematopiesis via non-canonical signalling 3. Finally, in most cases where a phenotype was detected, it was not determined whether the defect was intrinsic to haematopoietic cells or whether it was indirect, e.g. due to a defect in stromal cells. The deletion of two nuclear effectors of canonical Wnt signalling, LEF-1 and/or TCF-1, resulted in impaired lymphocyte development, which was based on a defect(s) in the haematopoietic compartment 4. However, these transcription factors can interact with several regulatory proteins and repress transcriptional activity in the absence of Wnt signalling. It was thus possible that the loss of this repressive function caused the phenotype. Indicative of a role of canonical Wnt signal transduction in haematopoeisis, we have shown that thymopoiesis is dependent on the N-terminal domain of TCF-1. This domain binds β- and γ-catenin 5, 6, the two known transducers of canonical Wnt signals in vertebrates. However, we and others have recently reported that lymphopoiesis and haematopoiesis were normal in the combined absence of β- and γ-catenin in the hematopoietic compartment 5, 6. Importantly, we further showed that haematopoietic cells lacking β- and γ-catenin retained significant TCF/LEF reporter gene activity when stimulated ex vivo by Wnt ligands 5. We concluded that canonical Wnt signals can be transduced in the absence of β- and γ-catenin. However, based on these findings, it can currently not be concluded that canonical Wnt signalling in haematopoietic cells is essential for normal haematopoiesis. Although full-size β-catenin was absent, we reported the presence of a shorter β-catenin species upon cre-mediated deletion of exons III-VI of the Ctnnb1 gene specifically in haematopoietic cells 5. It was thus possible that TCF/LEF reporter gene activity in our mice was mediated by this truncated β-catenin protein. However, this β-catenin species does not bind to TCF-1 (5 and Fig. 1) nor does it activate a TCF/LEF reporter 5. Obviously, while it cannot be excluded that the truncated β-catenin protein has some other effects, our study 5 rules out the possibility that it transduces canonical Wnt signals. Truncated β-catenin does not bind TCF-1. HEK 293T cells were transiently transfected with myc-tagged TCF-1 alone (lane 1) or together with truncated β-catenin (lane 2) or with full-length β-catenin (lane 3). Anti-myc immunoprecipitates (i.p.) and whole cell lysates were analysed by Western blot (WB) specific for β-catenin. Based on these findings, we proposed the existence of an additional, currently unidentified transducer of Wnt signals in haematopoietic cells. Consistent with this possibility, an additional β-catenin-like Wnt transducer was indeed identified in Caenorhabditis elegans 7. However, even if an additional transducer of canonical Wnt signals is found in vertebrates, this still does not prove that canonical Wnt signalling plays a role in haematopoietic cells. Future studies, using a complete block of canonical Wnt signalling selectively in hamatopoietic cells, will be needed to determine the precise role of this signalling pathway for normal haematopoiesis in the adult

Ectopic expression of C/EBP and ID1 is sufficient to restore defective neutrophil development in low-risk myelodysplasia

In patients with myelodysplasia, a general defect in the multipotent stem-cell compartment results in disturbed proliferation and differentiation of the erythroid, megakaryocytic and myeloid lineages. Although a number of genetic defects in myelodysplastic progenitor cells have been described, the intracellular signaling pathways underlying aberrant regulation of myelopoiesis remain relatively undefined. Here, an ex vivo differentiation system was used to selectively screen for molecules improving defective hematopoiesis in myelodysplastic CD34(+) progenitor cells. Bone marrow-derived CD34(+) cells isolated from patients with low-risk myelodysplastic syndrome showed impaired capacity to proliferate and differentiate as well as increased levels of apoptosis. In an attempt to improve the expansion and differentiation of the myelodysplastic CD34(+) progenitors, cells were treated with the p38MAPK pharmacological inhibitor SB203580, or retrovirally transduced to ectopically express active protein kinase B (PKB/c-akt), or the transcriptional regulators STAT5, C/EBPalpha or ID1. Whereas treatment of progenitors with SB203580, PKB or STAT5 did not enhance neutrophil development, ID1- and C/EBPalpha-transduced cells exhibited increased granulocyte/macrophage colony formation. Furthermore, ectopic expression of C/EBPalpha resulted in improved neutrophil maturation. These data suggest that targeting the ID1 and C/EBPalpha transcriptional regulators may be of benefit in the design of novel therapies for low-risk myelodysplasia.