Szandor Simmons

The sphingosine-1-phosphate transporter Spns2 expressed on endothelial cells regulates lymphocyte trafficking in mice

The bioactive lysophospholipid mediator sphingosine-1-phosphate (S1P) promotes the egress of newly formed T cells from the thymus and the release of immature B cells from the bone marrow. It has remained unclear, however, where and how S1P is released. Here, we show that in mice, the S1P transporter spinster homolog 2 (Spns2) is responsible for the egress of mature T cells and immature B cells from the thymus and bone marrow, respectively. Global Spns2-KO mice exhibited marked accumulation of mature T cells in thymi and decreased numbers of peripheral T cells in blood and secondary lymphoid organs. Mature recirculating B cells were reduced in frequency in the bone marrow as well as in blood and secondary lymphoid organs. Bone marrow reconstitution studies revealed that Spns2 was not involved in S1P release from blood cells and suggested a role for Spns2 in other cells. Consistent with these data, endothelia-specific deletion of Spns2 resulted in defects of lymphocyte egress similar to those observed in the global Spns2-KO mice. These data suggest that Spns2 functions in ECs to establish the S1P gradient required for T and B cells to egress from their respective primary lymphoid organs. Furthermore, Spns2 could be a therapeutic target for a broad array of inflammatory and autoimmune diseases.

Characterization of the IL-15 niche in primary and secondary lymphoid organs in vivo

Significance IL-15 is a cytokine critical for development and maintenance of T lymphoid cells. However, the identity and distribution of IL-15–expressing cells in lymphoid organs are not well understood. The present study reveals, by using IL-15–CFP knock-in mice that IL-15 was expressed in subsets of thymic epithelial cells, bone marrow stromal cells, lymph node stromal cells, and blood endothelial cells, a unique perspective of IL-15 niche in immune microenvironment. Taken together with our previous observation on IL-7–producing cells, this study suggests that some stromal cells express IL-7 and IL-15 differentially. Thus, the immune microenvironment appears to be consisted of functionally distinct subsets of stromal cells, expressing different cytokines. IL-15 is a cytokine critical for development, maintenance, and response of T cells, natural killer (NK) cells, NK T cells, and dendritic cells. However, the identity and distribution of IL-15–expressing cells in lymphoid organs are not well understood. To address these questions, we established and analyzed IL-15–CFP knock-in mice. We found that IL-15 was highly expressed in thymic medulla, and medullary thymic epithelial cells with high MHC class II expression were the major source of IL-15. In bone marrow, IL-15 was detected primarily in VCAM-1+PDGFRβ+CD31−Sca-1− stromal cells, which corresponded to previously described CXCL12-abundant reticular cells. In lymph nodes, IL-15–expressing cells were mainly distributed in the T-cell zone and medulla. IL-15 was expressed in some fibroblastic reticular cells and gp38−CD31− double-negative stromal cells in the T-cell zone. Blood endothelial cells, including all high endothelial venules, also expressed high IL-15 levels in lymph nodes, whereas lymphatic endothelial cells (LECs) lacked IL-15 expression. In spleen, IL-15 was expressed in VCAM-1+ stromal cells, where its expression increased as mice aged. Finally, IL-15 expression in blood and LECs of peripheral lymphoid organs significantly increased in LPS-induced inflammation. Overall, we have identified and characterized several IL-15–expressing cells in primary and secondary lymphoid organs, providing a unique perspective of IL-15 niche in immune microenvironment. This study also suggests that some stromal cells express IL-7 and IL-15 differentially and suggests a way to functionally classify different stromal cell subsets.

Biphenotypic B-lymphoid/myeloid cells expressing low levels of Pax5: potential targets of BAL development

The expression of Pax5 commits common lymphoid progenitor cells to B-lymphoid lineage differentiation. Little is known of possible variations in the levels of Pax5 expression and their influences on hematopoietic development. We have developed a retroviral transduction system that allows for the study of possible intermediate stages of this commitment by controlling the levels of Pax5 expressed in Pax5-deficient progenitors in vitro and in vivo. Retroviral transduction of Pax5-deficient pro-/pre-B cell lines with a doxycycline-inducible (TetON) form of the human Pax5 (huPax5) gene yielded cell clones that could be induced to different levels of huPax5 expression. Clones inducible to high levels developed B220(+)/CD19(+)/IgM(+) B cells, while clones with low levels differentiated to B220(+)/CD19(-)/CD11b(+)/Gr-1(-) B-lymphoid/myeloid biphenotypic cells in vitro and in vivo. Microarray analyses of genes expressed at these lower levels of huPax5 identified C/ebpα, C/ebpδ, Pu.1, Csf1r, Csf2r, and Gata-3 as myeloid-related genes selectively expressed in the pro-/pre-B cells that can develop under myeloid/lymphoid conditions to biphenotypic cells. Therefore, reduced expression of huPax5 during the induction of early lymphoid progenitors to B-lineage-committed cells can fix this cellular development at a stage that has previously been seen during embryonic development and in acute lymphoblastic lymphoma-like biphenotypic acute leukemias.

LRIG1 inhibits STAT3-dependent inflammation to maintain corneal homeostasis.

Corneal integrity and transparency are indispensable for good vision. Cornea homeostasis is entirely dependent upon corneal stem cells, which are required for complex wound-healing processes that restore corneal integrity following epithelial damage. Here, we found that leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is highly expressed in the human holoclone-type corneal epithelial stem cell population and sporadically expressed in the basal cells of ocular-surface epithelium. In murine models, LRIG1 regulated corneal epithelial cell fate during wound repair. Deletion of Lrig1 resulted in impaired stem cell recruitment following injury and promoted a cell-fate switch from transparent epithelium to keratinized skin-like epidermis, which led to corneal blindness. In addition, we determined that LRIG1 is a negative regulator of the STAT3-dependent inflammatory pathway. Inhibition of STAT3 in corneas of Lrig1-/- mice rescued pathological phenotypes and prevented corneal opacity. Additionally, transgenic mice that expressed a constitutively active form of STAT3 in the corneal epithelium had abnormal features, including corneal plaques and neovascularization similar to that found in Lrig1-/- mice. Bone marrow chimera experiments indicated that LRIG1 also coordinates the function of bone marrow-derived inflammatory cells. Together, our data indicate that LRIG1 orchestrates corneal-tissue transparency and cell fate during repair, and identify LRIG1 as a key regulator of tissue homeostasis.

miR-221 redirects precursor B cells to the BM and regulates their residence

Pluripotent hematopoietic stem cells and multipotent myeloid/lymphoid progenitors express miR‐221 and miR‐222. When Pax5 expression commits these progenitors to monopotent pre‐B lymphocytes the two microRNAs (miRNAs) are downregulated. Upon transplantation, stem cells and progenitors can reside in the BM, while pre‐B cells, after their commitment, no longer do so. Retrovirally transduced, doxycycline‐induced overexpression of either miR‐221 or miR‐222 in pre‐B‐I cells does not revert their monopotency to multipotency. However, upon transplantation miR‐221, but not miR‐222, transduced pre‐B‐I cells regain the capacity to reside in the BM. Upon subsequent termination of miR‐221‐expression by removal of doxycycline, the transplanted cells leave the BM again. Microarray analyses identified 25 downregulated miR‐221‐target genes, which could function to localize phases of B‐lymphocyte development in BM before and after commitment.

Integrin α9 on lymphatic endothelial cells regulates lymphocyte egress

Significance The lymphatic system plays critical roles in immune surveillance by providing a route for circulating immune cells. α9 integrin is a signature molecule of lymphatic endothelial cells (LECs), and has been suggested to have a significant role in lymphatic functions. Here we found that in draining lymph nodes (LNs) of mice treated with complete Freund adjuvant, blockade of α9 integrin induced deficiency of lymphocytes in medullary and cortical sinuses, which is the characteristic feature of impaired lymphocyte egress from LNs. In addition, we found that stimulation of embryo-derived LECs with the α9 ligand tenascin-C induced secretion of sphingosine 1-phosphate, which is the critical factor for lymphocyte egress. Thus, we suggest a possible role of α9 integrin-dependent lymphocyte egress from inflamed LNs. Sphingosine 1-phosphate (S1P) plays a role in lymphocyte egress from lymphoid organs. However, it remains unclear how S1P production and secretion are regulated. We show that under inflammatory conditions, α9 integrin, which is closely associated with activated β1 integrin, and its ligand, tenascin-C, colocalize on medullary and cortical sinuses of draining lymph nodes (dLNs), which is a gate for lymphocyte exit, and that inhibition of lymphocyte egress is evident by blockade of α9 integrin-mediated signaling at dLNs. Based on in vitro analysis using lymphatic endothelial cells obtained from mice embryos, we suggested the possibility that stimulation of lymphatic endothelial cells by tenascin-C enhances S1P secretion in an α9 integrin-dependent manner without affecting S1P synthesis and/or degradation. Blockade of α9 integrin-mediated signaling reduced lymphocyte egress from dLNs in several models, including experimental autoimmune encephalomyelitis, where it improved clinical scores and pathology. Therefore, manipulating α9 integrin function may offer a therapeutic strategy for treating various inflammatory disorders.

Interferon-α acts on the S/G2/M phases to induce apoptosis in the G1 phase of an IFNAR2-expressing hepatocellular carcinoma cell line.

Background: The mode of action of interferon-α has been unknown. Results: Its point of action in the cell cycle was analyzed by single cell tracking using time lapse confocal imaging. Conclusion: Interferon-α activates p63 in S/G2/M and induces apoptosis and cell cycle arrest in the subsequent G1. Significance: Tracking cell cycle progression is crucial for understanding the mechanisms of interferon-α. Interferon-α (IFN-α) is used clinically to treat hepatocellular carcinoma (HCC), although the detailed therapeutic mechanisms remain elusive. In particular, IFN-α has long been implicated in control of the cell cycle, but its actual point of action has not been clarified. Here, using time lapse imaging analyses of the human HCC cell line HuH7 carrying a fluorescence ubiquitination-based cell cycle indicator (Fucci), we found that IFN-α induced cell cycle arrest in the G0/G1 phases, leading to apoptosis through an IFN-α type-2 receptor (IFNAR2)-dependent signaling pathway. Detailed analyses by time lapse imaging and biochemical assays demonstrated that the IFN-α/IFNAR2 axis sensitizes cells to apoptosis in the S/G2/M phases in preparation for cell death in the G0/G1 phases. In summary, this study is the first to demonstrate the detailed mechanism of IFN-α as an anticancer drug, using Fucci-based time lapse imaging, which will be informative for treating HCC with IFN-α in clinical practice.

The Non-Ig Parts of the VpreB and lambda 5 Proteins of the Surrogate Light Chain Play Opposite Roles in the Surface Representation of the Precursor B Cell Receptor

The VpreB and λ5 proteins, together with Igμ-H chains, form precursor BCRs (preBCRs). We established λ5−/−/VpreB1−/−/VpreB2−/− Abelson virus-transformed cell lines and reconstituted these cells with λ5 and VpreB in wild-type form or with a deleted non-Ig part. Whenever preBCRs had the non-Ig part of λ5 deleted, surface deposition was increased, whereas deletion of VpreB non-Ig part decreased it. The levels of phosphorylation of Syk, SLP65, or PLC-γ2, and of Ca2+ mobilization from intracellular stores, stimulated by μH chain crosslinking Ab were dependent on the levels of surface-bound preBCRs. It appears that VpreB probes the fitness of newly generated VH domains of IgH chains for later pairing with IgL chains, and its non-Ig part fixes the preBCRs on the surface. By contrast, the non-Ig part of λ5 crosslinks preBCRs for downregulation and stimulation.

Sphingosine-1-Phosphate: a Master Regulator of Lymphocyte Egress and Immunity

Abstract Sphingosine-1-phosphate (S1P) is a central factor responsible for lymphocyte distribution in the body. S1P is able to control the integrity of various effector cell populations within many lymphoid tissues by directing lymphocyte egress. In this review, we give an overview of the generation and degradation of S1P in specific lymphoid microenvironments. Furthermore, we discuss, sometimes contradictory, the functions of the five S1P receptors on different cells in diverse tissues and give an idea of additional counteracting chemotactic signals for lymphocyte immigration and emigration. We focus special attention to recent discoveries of S1P-specific transporters, like spinster-homolog-2 and the active secretion of S1P by endothelial cells, erythrocytes and platelets. In addition, we describe the microanatomical structures as well as entry and egress routes into lymphoid organs which lymphocytes use for efficient trafficking. Finally, we give an overview of open questions regarding the regulation of lymphocyte homing from primary lymphoid organs to secondary lymphoid organs and back again.

Reprint of: Environments of B cell development

B lymphocyte development in the mouse begins with the generation of long-term reconstituting, pluripotent hematopoietic stem cells, over multipotent myeloid/lymphoid progenitors and common lymphoid progenitors to B-lineage committed pro/pre B and pre B cells, which first express pre B cell receptors and then immunoglobulins, B cell receptors, to generate the repertoires of peripheral B cells. This development is influenced and guided by cells of non-hematopoietic and hematopoietic origins. We review here some of the recent developments, and our contributions in this fascinating field of developmental immunology.