Masahiro Kitabatake

Chemokine-mediated rapid turnover of myeloid-derived suppressor cells in tumor-bearing mice

Tumor growth is associated with aberrant myelopoiesis, including the accumulation of CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs) that have the potential to promote tumor growth. However, the identity, growth, and migration of tumor-associated MDSCs remain undefined. We demonstrate herein that MDSCs at tumor site were composed primarily of bone marrow-derived CD11b(+)Gr-1(hi)Ly-6C(int) neutrophils and CD11b(+)Gr-1(int/dull)Ly-6C(hi) macrophages. Unexpectedly, in vivo bromodeoxyuridine (BrdU) labeling and parabiosis experiments revealed that tumor-infiltrating macrophages were replenished more rapidly than neutrophils. CCR2 deficiency caused striking conversion of infiltrating cellular dominance from macrophages to neutrophils in the tumor with the excessive production of CXCR2 ligands and granulocyte-colony stimulating factor in the tumor without affecting tumor growth. Overall, our data established the identity and dynamics of MDSCs in a tumor-bearing host mediated by chemokines and elucidated unexpected effects of the paucity of macrophages on tumor development.

Defective B1 cell homing to the peritoneal cavity and preferential recruitment of B1 cells in the target organs in a murine model for systemic lupus erythematosus.

We previously reported that B lymphocyte chemoattractant (BLC; CXCL13) was highly and ectopically expressed in aged (NZB × NZW)F1 (BWF1) mice developing lupus nephritis, and that B1 cells were preferentially chemoattracted toward BLC. We demonstrate in this study that B1 cells fail to home to the peritoneal cavity in aged BWF1 mice developing lupus nephritis, and that they are preferentially recruited to the target organs including the kidney, lung, and thymus when injected i.v. In contrast, B1 cells homed to the peritoneal cavity in aged BALB/c mice as effectively as in young mice. Accumulation of B1 cells to the omentum milky spots was also impaired in aged BWF1 mice compared with young mice. CD11bhighF4/80high cells with macrophage morphology were confirmed to be a major cell source for BLC in the peritoneal cavity both in young and aged BWF1 mice. However, the number of BLC-producing peritoneal macrophages was markedly decreased in aged BWF1 mice. These results suggest that the decreased number of BLC-producing peritoneal macrophages together with ectopic high expression of BLC in aged BWF1 mice result in abnormal B1 cell trafficking during the development of murine lupus.

Aberrant B1 cell migration into the thymus results in activation of CD4 T cells through its potent antigen-presenting activity in the development of murine lupus.

B1 cells have different origin and function from conventional B (B2) cells and are considered to be involved in autoantibody production in the development of autoimmune disease. We found that B1 cells preferentially accumulated in the target organs including thymus in aged BWF1 mice, a murine model for systemic lupus erythematosus, and that B lymphocyte chemoattractant (BLC/CXCL13) expression was increased in the thymus before the onset of lupus nephritis, while stromal cell‐derived factor‐1 (SDF‐1/CXCL12) and secondary lymphoid tissue chemokine (SLC/CCL21) expression remained unchanged. Adhesion molecules such as peripheral node addressin (PNAd), ICAM‐1, and VCAM‐1 were also expressed on endothelial cells in the enlarged thymic perivascular space (PVS) in aged BWF1 mice. BLC protein and PNAd were co‐localized on these high‐endothelial‐venules‐like vessels in enlarged PVS. B1 cells expressed higher level of costimulatory molecules and showed a potent antigen‐presenting activity in allogeneic mixed lymphocyte reaction comparable to splenic dendritic cells. Interestingly, B1 cells stimulated proliferation of autologous thymic CD4 T cells in the presence of IL‐2. These results indicate that aberrant B1 cell trafficking into the thymus due to ectopic high expression of BLC may result in an activation of self‐reactive T cells in the development of murine lupus.

GANP-mediated recruitment of activation-induced cytidine deaminase to cell nuclei and to immunoglobulin variable region DNA

AID (activation-induced cytidine deaminase) catalyzes transcription-dependent deamination of C → U in immunoglobulin variable (IgV) regions to initiate somatic hypermutation (SHM) in germinal center B-cells. SHM is essential in generating high affinity antibodies. Here we show that when coexpressed with GANP (germinal center-associated nuclear protein) in COS-7 cells, AID is transported from the cytoplasm and concentrated in the nucleus. GANP forms a complex with AID in cotransfected cells in vivo and in vitro. We have isolated AID mutants that bind with reduced affinity to GANP compared with wild type AID. One of these mutants, AID (D143A) binds GANP with a 10-fold lower affinity compared with wild type AID yet retains substantial C-deamination activity in vitro. Mutant AID (D143A) remains localized predominantly in the cytoplasm when coexpressed with GANP. Exogenous expression of GANP in Ramos B-cells promotes binding of AID to IgV DNA and mRNA and increases SHM frequency. These data suggest that GANP may serve as an essential link required to transport AID to B-cell nuclei and to target AID to actively transcribed IgV regions.

CCR7 mediates the migration of Foxp3+ regulatory T cells to the paracortical areas of peripheral lymph nodes through high endothelial venules.

Thymus‐derived forkhead box p3+ naturally occurring regulatory T cells (nTreg) are thought to circulate throughout the body to maintain peripheral immunological self‐tolerance through interactions with dendritic cells (DCs), resulting in regulation of conventional T cells. However, the chemokine receptors, which are putatively involved in the in vivo migration of nTreg, have not been fully established. Here, we demonstrated that lymph node nTreg preferentially migrated to the paracortical area of lymph nodes after adoptive transfer, where they were observed to make contact frequently with CD8α+ DCs and CD8α− CD11b− DCs. This migration of nTreg to the paracortical areas was impaired severely when cells were prepared from CCR7‐deficient mice. However, to some extent, CCR7‐independent migration of nTreg in such CCR7‐deficient mice was also observed, but this occurred mainly in the medullary high endothelial venules. Taken together, these data provide the evidence that CCR7 mediates nTreg migration to the paracortical areas of lymph nodes under steady‐state conditions; however, CCR7‐independent migration also takes place in the medulla.

Intervention of MAdCAM-1 or fractalkine alleviates graft-versus-host reaction associated intestinal injury while preserving graft-versus-tumor effects.

Coincidence of the beneficial graft‐vs.‐tumor (GVT) effects and the detrimental graft‐vs.‐host disease (GVHD) remains the major obstacle against the widespread use of allogeneic bone marrow transplantation (BMT) as tumor immunotherapy. We here demonstrate that intervention of MAdCAM‐1 (mucosal vascular addressin cell adhesion molecule‐1) or fractalkine/CX3CL1 after the expansion of allo‐reactive donor CD8 T cells selectively inhibits the recruitment of effector donor CD8 T cells to the intestine and alleviates the graft‐vs.‐host reaction (GVHR) associated intestinal injury without impairing GVT effects. In a nonirradiated acute GVHD model, donor CD8 T cells up‐regulate the expression of intestinal homing receptor α4β7 and chemokine receptors CXCR6 and CX3CR1, as they differentiate into effector cells and subsequently infiltrate into the intestine. Administration of anti‐MAdCAM‐1 antibody or anti‐fractalkine antibody, even after the expansion of alloreactive donor CD8 T cells, selectively reduced the intestine‐infiltrating donor CD8 T cells and the intestinal crypt cell apoptosis without affecting the induction of donor derived anti‐host CTL or the infiltration of donor CD8 T cells in the hepatic tumor. Moreover, in a clinically relevant GVHD model with myeloablative conditioning, these antibodies significantly improved the survival and loss of weight without impairing the beneficial GVT effects. Thus, interruption of α4β7‐MAdCAM‐1 or CX3CR1‐fractalkine interactions in the late phase of GVHD would be a novel therapeutic approach for the separation of GVT effects from GVHR‐associated intestinal injury.

Prior Immunization with Severe Acute Respiratory Syndrome (SARS)-Associated Coronavirus (SARS-CoV) Nucleocapsid Protein Causes Severe Pneumonia in Mice Infected with SARS-CoV

The details of the mechanism by which severe acute respiratory syndrome-associated coronavirus (SARS-CoV) causes severe pneumonia are unclear. We investigated the immune responses and pathologies of SARS-CoV-infected BALB/c mice that were immunized intradermally with recombinant vaccinia virus (VV) that expressed either the SARS-CoV spike (S) protein (LC16m8rVV-S) or simultaneously all the structural proteins, including the nucleocapsid (N), membrane (M), envelope (E), and S proteins (LC16m8rVV-NMES) 7–8 wk before intranasal SARS-CoV infection. The LC16m8rVV-NMES-immunized group exhibited as severe pneumonia as the control groups, although LC16m8rVV-NMES significantly decreased the pulmonary SARS-CoV titer to the same extent as LC16m8rVV-S. To identify the cause of the exacerbated pneumonia, BALB/c mice were immunized with recombinant VV that expressed the individual structural proteins of SARS-CoV (LC16mOrVV-N, -M, -E, -S) with or without LC16mOrVV-S (i.e., LC16mOrVV-N, LC16mOrVV-M, LC16mOrVV-E, or LC16mOrVV-S alone or LC16mOrVV-N + LC16mOrVV-S, LC16mOrVV-M + LC16mOrVV-S, or LC16mOrVV-E + LC16mOrVV-S), and infected with SARS-CoV more than 4 wk later. Both LC16mOrVV-N-immunized mice and LC16mOrVV-N + LC16mOrVV-S-immunized mice exhibited severe pneumonia. Furthermore, LC16mOrVV-N-immunized mice upon infection exhibited significant up-regulation of both Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-5) cytokines and down-regulation of anti-inflammatory cytokines (IL-10, TGF-β), resulting in robust infiltration of neutrophils, eosinophils, and lymphocytes into the lung, as well as thickening of the alveolar epithelium. These results suggest that an excessive host immune response against the nucleocapsid protein of SARS-CoV is involved in severe pneumonia caused by SARS-CoV infection. These findings increase our understanding of the pathogenesis of SARS.

SARS-CoV spike protein-expressing recombinant vaccinia virus efficiently induces neutralizing antibodies in rabbits pre-immunized with vaccinia virus.

Abstract A vaccine for severe acute respiratory syndrome (SARS) is being intensively pursued against its re-emergence. We generated a SARS coronavirus (SARS-CoV) spike protein-expressing recombinant vaccinia virus (RVV-S) using highly attenuated strain LC16m8. Intradermal administration of RVV-S into rabbits induced neutralizing (NT) antibodies against SARS-CoV 1 week after administration and the NT titer reached 1:1000 after boost immunization with RVV-S. Significantly, NT antibodies against SARS-CoV were induced by administration of RVV-S to rabbits that had been pre-immunized with LC16m8. RVV-S can induce NT antibodies against SARS-CoV despite the presence of NT antibodies against VV. These results suggest that RVV-S may be a powerful SARS vaccine, including in patients previously immunized with the smallpox vaccine.

Breakdown of Mucosal Immunity in the Gut and Resultant Systemic Sensitization by Oral Antigens in a Murine Model for Systemic Lupus Erythematosus

Secreted IgA plays a pivotal role in the mucosal immunity to maintain the front line of body defense. We found that the level of fecal IgA was dramatically decreased in aged (NZB × NZW)F1 (BWF1) mice developing lupus nephritis, whereas levels in similarly aged New Zealand Black (NZB) and New Zealand White (NZW) mice remained unchanged compared with young mice. The number of cells obtained from Peyer’s patches was markedly decreased in aged BWF1 mice. Aged BWF1 mice showed increased susceptibility to pathogenic bacterial infection. Furthermore, oral administration of OVA failed to inhibit secondary IgG response induced by systemic immunization, suggesting defective oral tolerance in aged BWF1 mice. A significant amount of orally administered OVA was incorporated directly into the intestinal lamina propria in aged BWF1 mice whereas it was mainly localized in subepithelial domes and interfollicular region in Peyer’s patches in young mice. T cells obtained from renal and pulmonary lymph nodes of aged BWF1 mice that had been orally administered with OVA showed an Ag-specific T cell proliferation, whereas those from young BWF1, aged NZB, and aged NZW mice did not. Interestingly, aerosol exposure to OVA of aged BWF1 mice, which had been orally administered with the same Ag, provoked an eosinophil infiltration in the lung. These results demonstrate that mucosal immunity in the gut is impaired and oral Ags induce systemic sensitization instead of oral tolerance in the development of murine lupus.

Critical Role of Pcid2 in B Cell Survival through the Regulation of MAD2 Expression

The mitotic checkpoint is essential for maintaining genomic stability in differentiating B cells undergoing genetic alterations of the Ig gene. In this study, using real-time RT-PCR and in situ RNA hybridization, we demonstrated that MAD2 mRNA export is selectively regulated by Pcid2/Thp1. Pcid2 small interfering RNA induced a cell-cycle abnormality with increased apoptosis and polyploidy, as previously observed in MAD2-knockdown cells. Pcid2 small interfering RNA reduced MAD2 expression, but not the expression of other cell-cycle checkpoint proteins, such as MAD1 and BUBR1, or the cell-cycle–associated proteins, cyclin A, cyclin B1, and cyclin-dependent kinase 1. In mouse B lineage cells, Pcid2 transcripts appeared in a stage-dependent manner at high levels in bone marrow pre-B and immature B cells, and in spleen transitional 1 and follicular B cells, but at lower levels in pro-B, transitional 2, and marginal zone B cells, suggesting a stage-dependent requirement for MAD2 regulation. Cd19-cre–derived targeting of the Pcid2 gene induced a mature B cell deficiency in mice. These findings indicate that Pcid2 is essential for B cell survival through the regulation of MAD2 expression during B cell differentiation.