Sivan Cohen

Deficiency of caspase recruitment domain family, member 11 (CARD11), causes profound combined immunodeficiency in human subjects

BACKGROUND Profound combined immunodeficiency can present with normal numbers of T and B cells, and therefore the functional defect of the cellular and humoral immune response is often not recognized until the first severe clinical manifestation. Here we report a patient of consanguineous descent presenting at 13 months of age with hypogammaglobulinemia, Pneumocystis jirovecii pneumonia, and a suggestive family history. OBJECTIVE We sought to identify the genetic alteration in a patient with combined immunodeficiency and characterize human caspase recruitment domain family, member 11 (CARD11), deficiency. METHODS Molecular, immunologic, and functional assays were performed. RESULTS The immunologic characterization revealed only subtle changes in the T-cell and natural killer cell compartment, whereas B-cell differentiation, although normal in number, was distinctively blocked at the transitional stage. Genetic evaluation revealed a homozygous deletion of exon 21 in CARD11 as the underlying defect. This deletion abrogated protein expression and activation of the canonical nuclear factor κB (NF-κB) pathway in lymphocytes after antigen receptor or phorbol 12-myristate 13-acetate stimulation, whereas CD40 signaling in B cells was preserved. The abrogated activation of the canonical NF-κB pathway was associated with severely impaired upregulation of inducible T-cell costimulator, OX40, cytokine production, proliferation of T cells, and B cell-activating factor receptor expression on B cells. CONCLUSION Thus in patients with CARD11 deficiency, the combination of impaired activation and especially upregulation of inducible T-cell costimulator on T cells, together with severely disturbed peripheral B-cell differentiation, apparently leads to a defective T-cell/B-cell cooperation and probably germinal center formation and clinically results in severe immunodeficiency. This report discloses the crucial and nonredundant role of canonical NF-κB activation and specifically CARD11 in the antigen-specific immune response in human subjects.

c-Met and Its Ligand Hepatocyte Growth Factor/Scatter Factor Regulate Mature B Cell Survival in a Pathway Induced by CD74

The signals regulating the survival of mature splenic B cells have become a major focus in recent studies of B cell immunology. Durable B cell persistence in the periphery is dependent on survival signals that are transduced by cell surface receptors. In this study, we describe a novel biological mechanism involved in mature B cell homeostasis, the hepatocyte growth factor/scatter factor (HGF)/c-Met pathway. We demonstrate that c-Met activation by HGF leads to a survival cascade, whereas its blockade results in induction of mature B cell death. Our results emphasize a unique and critical function for c-Met signaling in the previously described macrophage migration inhibitory factor/CD74-induced survival pathway. Macrophage migration inhibitory factor recruits c-Met to the CD74/CD44 complex and thereby enables the induction of a signaling cascade within the cell. This signal results in HGF secretion, which stimulates the survival of the mature B cell population in an autocrine manner. Thus, the CD74–HGF/c-Met axis defines a novel physiologic survival pathway in mature B cells, resulting in the control of the humoral immune response.

The Cytokine Midkine and Its Receptor RPTPζ Regulate B Cell Survival in a Pathway Induced by CD74

Lasting B cell persistence depends on survival signals that are transduced by cell surface receptors. In this study, we describe a novel biological mechanism essential for survival and homeostasis of normal peripheral mature B cells and chronic lymphocytic leukemia cells, regulated by the heparin-binding cytokine, midkine (MK), and its proteoglycan receptor, the receptor-type tyrosine phosphatase ζ (RPTPζ). We demonstrate that MK initiates a signaling cascade leading to B cell survival by binding to RPTPζ. In mice lacking PTPRZ, the proportion and number of the mature B cell population are reduced. Our results emphasize a unique and critical function for MK signaling in the previously described MIF/CD74-induced survival pathway. Stimulation of CD74 with MIF leads to c-Met activation, resulting in elevation of MK expression in both normal mouse splenic B and chronic lymphocytic leukemia cells. Our results indicate that MK and RPTPζ are important regulators of the B cell repertoire. These findings could pave the way toward understanding the mechanisms shaping B cell survival and suggest novel therapeutic strategies based on the blockade of the MK/RPTPζ-dependent survival pathway.

Correction of hemophilia as a proof of concept for treatment of monogenic diseases by fetal spleen transplantation

Previous clinical attempts to correct genetic deficiencies such as hemophilia or Gaucher disease by transplantation of allogeneic spleen were associated with aggressive graft versus host disease, mediated by mature T cells derived from the donor spleen. We show that a fetal pig spleen harvested at the embryonic day 42 stage, before the appearance of T cells, exhibited optimal growth potential upon transplantation into SCID mice, and the growing tissue expressed factor VIII. Transplantation of embryonic day 42 spleen tissue into hemophilic SCID mice led to complete alleviation of hemophilia within 2–3 months after transplant, as demonstrated by tail bleeding and by assays for factor VIII blood levels. These results provide a proof of principle to the concept that transplantation of a fetal spleen, obtained from a developmental stage before the appearance of T cells, could provide a novel treatment modality for genetic deficiencies of an enzyme or a factor that can be replaced by the growing spleen tissue.

Pig Embryonic Pancreatic Tissue as a Source for Transplantation in Diabetes: Transient Treatment With Anti-LFA1, Anti-CD48, and FTY720 Enables Long-Term Graft Maintenance in Mice With Only Mild Ongoing Immunosuppression

OBJECTIVE Defining an optimal costimulatory blockade–based immune suppression protocol enabling engraftment and functional development of E42 pig embryonic pancreatic tissue in mice. RESEARCH DESIGN AND METHODS Considering that anti-CD40L was found to be thrombotic in humans, we sought to test alternative costimulatory blockade agents already in clinical use, including CTLA4-Ig, anti-LFA1, and anti-CD48. These agents were tested in conjunction with T-cell debulking by anti-CD4 and anti-CD8 antibodies or with conventional immunosuppressive drugs. Engraftment and functional development of E42 pig pancreatic tissue was monitored by immunohistology and by measuring pig insulin blood levels. RESULTS Fetal pig pancreatic tissue harvested at E42, or even as early as at E28, was fiercely rejected in C57BL/6 mice and in Lewis rats. A novel immune suppression comprising anti-LFA1, anti-CD48, and FTY720 afforded optimal growth and functional development. Cessation of treatment with anti-LFA1 and anti-CD48 at 3 months posttransplant did not lead to graft rejection, and graft maintenance could be achieved for >8 months with twice-weekly low-dose FTY720 treatment. These grafts exhibited normal morphology and were functional, as revealed by the high pig insulin blood levels in the transplanted mice and by the ability of the recipients to resist alloxan induced diabetes. CONCLUSIONS This novel protocol, comprising agents that simulate those approved for clinical use, offer an attractive approach for embryonic xenogeneic transplantation. Further studies in nonhuman primates are warranted.

Regulation of CLL survival by hypoxia‐inducible factor and its target genes

Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the Western world, is characterized by the progressive accumulation of small mature CD5+B lymphocytes in the peripheral blood, lymphoid organs, and bone marrow (BM). The main feature of the disease is decreased apoptosis, resulting in the pathologic accumulation of these malignant cells. Appropriate cellular responses to changes in oxygen tension during normal development or pathological processes, such as cardiovascular disease and cancer, are ultimately regulated by the transcription factor, hypoxia‐inducible factor (HIF). Unlike their normal counterparts, CLL cells express HIF‐1α even under normoxia. In addition, overexpression of HIF‐1α has been observed in leukemic cells in BM specimens from CLL patients. The HIF transcription factor has been implicated in controlling the expression of a wide variety of genes implicated in apoptosis, angiogenesis, invasion, and metastasis. This review describes pathways regulating CLL survival with a focus on HIF‐1α and its target genes, MIF and Midkine (MK), and the potential cross‐talk between these factors.

Embryonic Porcine Liver as a Source for Transplantation: Advantage of Intact Liver Implants over Isolated Hepatoblasts in Overcoming Homeostatic Inhibition by the Quiescent Host Liver

Cell therapy as an alternative to orthotopic liver transplantation represents a major challenge, since negligible proliferation of isolated hepatocytes occurs after transplantation because of the stringent homeostatic control displayed by the host liver. Thus, different modalities of liver injury as part of the pretransplant conditioning are a prerequisite for this approach. The major objective of the present study was to test whether xenotransplantation of pig fetal liver fragments, in which potential cell‐cell and cell‐stroma interactions are spared, might afford more robust growth and proliferation compared with isolated pig fetal hepatoblasts. After transplantation into SCID mice, fetal liver tissue fragments exhibited marked growth and proliferation, in the setting of a quiescent host liver, compared with isolated fetal hepatoblasts harvested at the same gestational age (embryonic day 28). The proliferative advantage of fetal pig liver fragments was clearly demonstrated by immunohistochemical and morphometric assays and was observed not only after implantation into the liver but also into extrahepatic sites, such as the spleen and the subrenal capsule. The presence of all types of nonparenchymal liver cells that is crucial for normal liver development and regeneration was demonstrated in the implants. Preservation of the three‐dimensional structure in pig fetal liver fragments enables autonomous proliferation of transplanted hepatic cells in the setting of a quiescent host liver, without any requirement for liver injury in the pretransplant conditioning. The marked proliferation and functional maturation exhibited by the pig fetal liver fragments suggests that it could afford a preferable source for transplantation.

Embryonic pig pancreatic tissue for the treatment of diabetes: potential role of immune suppression with "off-the-shelf" third-party regulatory T cells.

Background. Xenogeneic embryonic pancreatic tissue can provide an attractive alternative for organ replacement therapy. However, immunological rejection represents a major obstacle. This study examines the potential of regulatory T cells (Tregs) in the prevention of E42 pancreas rejection. Methods. To develop new approaches to combat rejection, we evaluated engraftment, growth, and development of E42 pig pancreatic tissue in mice treated with ex vivo expanded Tregs in combination with T-cell debulking and the conventional immunosuppressive drugs, rapamycin and FTY720. Results. Transplantation of E42 pig pancreas into C57BL/6 mice immunosuppressed by this protocol resulted in complete rejection within less than 6 weeks. In contrast, additional treatment with a single infusion of ex vivo expanded third-party Tregs markedly delayed the onset of graft rejection to 10 weeks. The infusion of Tregs was associated with a significant reduction in CD4+ and CD8+ expansion in the lymph nodes and other peripheral organs at the priming stages after implantation. Freezing and thawing of the Tregs did not affect their efficacy, indicating the potential of Tregs banking. Conclusion. Considering the technical difficulties encountered in the generation of Tregs from patients or from specific donors, our results demonstrate the feasibility of using “off-the-shelf” fresh or frozen third-party Tregs to control rejection in organ transplantation.

CD151 Regulates T-Cell Migration in Health and Inflammatory Bowel Disease.

Abstract:The continuous recirculation of mature lymphocytes and their entry into the peripheral lymph nodes are crucial for the development of an immune response to foreign antigens. Occasionally, the entry and the subsequent response of T lymphocytes in these sites lead to severe inflammation and pathological conditions. Here, we characterized the tetraspanin molecule, CD151, as a regulator of T cell motility in health and in models of inflammatory bowel disease. CD151 formed a cell surface complex with VLA-4 and LFA-1 integrins, and its activation led to enhanced migration of T cells. Picomolar levels of CCL2 that were previously shown to inhibit T-cell migration to lymph nodes suppressed CD151 expression and dissociated CD151-integrin complexes in T lymphocytes, resulting in attenuated migration toward T-cell attractant chemokines. To directly inhibit CD151 function, a truncated CD151 peptide fragment mimicking of the CD151 extracellular loop was designed. CD151 extracellular loop inhibited T-cell migration in vitro and in vivo and attenuated the development of dextrane sulfate sodium-induced colitis. Thus, CD151 is a key orchestrator of T cell motility; interference with its proper function results in attenuated progression of inflammatory bowel disease.

Midkine as a regulator of B cell survival in health and disease.

In healthy individuals, the pool of peripheral lymphocytes is constant in size. The control of lymphoid homeostasis is the result of a very fine balance between lymphocyte production, survival and proliferation. Survival factors have been shown to play a critical role in maintaining the correct size of lymphocyte populations. Midkine, a heparin‐binding cytokine was recently shown to be involved in cell proliferation, differentiation and apoptosis in various cell types including normal and malignant B cells. This review focuses on the role of midkine in the regulation of peripheral B cell survival in health and disease.