The anti-prion activity of cellulose ether (CE) has been reported in rodents, but the mechanism of action is not well understood. As defects in early T-cell development have been reported in Tga20 mice which show only a slight effect of CE administration, we investigated the involvement of immune functions in the CE action. We confirmed an insertion of the prion protein transgene into the pre T-cell antigen receptor α gene of Tga20 mice, and its impaired expression in the thymus and other tissues. The influence of immune suppression on the CE effect was then examined in high CE-responder mice treated with immunosuppressive agents or neonatal thymectomy. As neonatal thymectomy significantly reduced the CE effect, we compared the influence of various T-cell defects in mice with similar genetic backgrounds. The CE effect was increased or unchanged in mice with defects in the αβ T-cell lineage, whereas it was abolished in T-cell receptor δ deficient mice. Further, when other immune defects were examined, the CE effect was reduced in mice with lysosomal trafficking dysfunction, but was unchanged in mice deficient in B-cell differentiation or toll-like receptor 4 signaling. These findings collectively suggest that the mechanism of CE action may involve γδ T cells and lytic granule function, as well as immune factors like natural killer T cells which are lacking in pre T-cell antigen receptor α deficient mice and neonatally thymectomized mice.

Tissue and subcellular localization and its changes upon cell activation of virus-restricting APOBEC3 at protein levels are important to understanding physiological functions of this cytidine deaminase, but have not been thoroughly analyzed in vivo. To precisely follow the possible activation-induced changes in expression levels of APOBEC3 protein in different mouse tissues and cell populations, genome editing was utilized to establish knock-in mice that express APOBEC3 protein with an in-frame FLAG tag. Flow cytometry and immunohistochemical analyses were performed prior to and after an immunological stimulation. Cultured B cells expressed higher levels of APOBEC3 protein than T cells. All differentiation and activation stages of freshly prepared B cells expressed significant levels of APOBEC3 protein, but germinal center cells possessed the highest levels of APOBEC3 protein localized in their cytoplasm. Upon immunological stimulation with sheep red blood cells in vivo, germinal center cells with high levels of APOBEC3 protein expression increased in their number, but FLAG-specific fluorescence intensity in each cell did not change. T cells, even those in germinal centers, did not express significant levels of APOBEC3 protein. Thus, mouse APOBEC3 protein is expressed at distinctively high levels in germinal center B cells. Antigenic stimulation did not affect expression levels of cellular APOBEC3 protein despite increased numbers of germinal center cells.

マウスのフレンドウイルス(FV)感染による赤白血病発症機構については，膜結合型チロシンキナーゼSf-Stk依存性多段階発がん説が主流であったが，我々グループはSf-Stkをもたないマウスでの発症を実証した。その一方で，Sf-Stk非依存性発症機構としてストレス造血が注目されているが，FV感染後ストレス造血が惹起される分子機構は全く不明である。ここではFV誘発赤白血病についてこれまでの知見を総括し，白血病研究におけるFV感染マウスモデルの位置付けを概観しつつ，さらなる研究の意義について考察する。

The aim of this study is to explore a cause-oriented therapy for patients with uterine cervical cancer that expresses erythropoietin (Epo) and its receptor (EpoR). Epo, by binding to EpoR, stimulates the proliferation and differentiation of erythroid progenitor cells into hemoglobin-containing red blood cells. In this study, we report that the HeLa cells in the xenografts expressed epsilon,gamma, and a globins as well as myoglobin (Mb) to produce tetrameric alpha 2 epsilon 2 and alpha 2 gamma 2 and monomeric Mb, most of which were significantly suppressed with an EpoR antagonist EMP9. Western blotting revealed that the EMP9 treatment inhibited the AKT-pAKT, MAPKs-pMAPKs, and STAT5-pSTAT5 signaling pathways. Moreover, the treatment induced apoptosis and suppression of the growth and inhibited the survival through disruption of the harmonized hemoprotein syntheses in the tumor cells concomitant with destruction of vascular nets in the xenografts. Furthermore, macrophages and natural killer (NK) cells with intense HIF-1 alpha expression recruited significantly more in the degenerating foci of the xenografts. These findings were associated with the enhanced expressions of nNOS in the tumor cells and iNOS in macrophages and NK cells in the tumor sites. The treated tumor cells exhibited a substantial number of perforations on the cell surface, which indicates that the tumors were damaged by both the nNOS-induced nitric oxide (NO) production in the tumor cells as well as the iNOS-induced NO production in the innate immune cells. Taken together, these data suggest that HeLa cells constitutively acquire e,. and Mb synthetic capacity for their survival. Therefore, EMP9 treatment might be a cause-oriented and effective therapy for patients with squamous cell carcinoma of the uterine cervix.

Toll-like receptor 7 and Myd88 are required for antiretroviral antibody and germinal center responses, but whether somatic hypermutation and class-switch recombination are required for antiretroviral immunity has not been examined. Mice deficient in activation-induced cytidine deaminase (AID) resisted Friend virus infection, produced virus-neutralizing antibodies, and controlled viremia. Passive transfer demonstrated that immune IgM from AID-deficient mice contributes to Friend virus control in the presence of virus-specific CD4(+) T cells.

In chronic viral infections, persistent antigen presentation causes progressive exhaustion of virus-specific CD8(+) T cells. It has become clear, however, that virus-specific naive CD8(+) T cells newly generated from the thymus can be primed with persisting antigens. In the setting of low antigen density and resolved inflammation, newly primed CD8(+) T cells are preferentially recruited into the functional memory pool. Thus, continual recruitment of naive CD8(+) T cells from the thymus is important for preserving the population of functional memory CD8(+) T cells in chronically infected animals. Friend virus (FV) is the pathogenic murine retrovirus that establishes chronic infection in adult mice, which is bolstered by the profound exhaustion of virus-specific CD8(+) T cells induced during the early phase of infection. Here we show an additional evasion strategy in which FV disseminates efficiently into the thymus, ultimately leading to clonal deletion of thymocytes that are reactive to FV antigens. Owing to the resultant lack of virus-specific recent thymic emigrants, along with the above exhaustion of antigen-experienced peripheral CD8(+) T cells, mice chronically infected with FV fail to establish a functional virus-specific CD8(+) T cell pool, and are highly susceptible to challenge with tumor cells expressing FV-encoded antigen. However, FV-specific naive CD8(+) T cells generated in uninfected mice can be primed and differentiate into functional memory CD8(+) T cells upon their transfer into chronically infected animals. These findings indicate that virus-induced central tolerance that develops during the chronic phase of infection accelerates the accumulation of dysfunctional memory CD8(+) T cells.

To assess the possible contribution of host immune responses to the exertion of Fv2-associated resistance to Friend virus (FV)-induced disease development, we inoculated C57BL/6 (B6) mice that lacked various subsets of lymphocytes with FV containing no lactate dehydrogenase-elevating virus. Fv2(r) B6 mice lacking CD4(+) T cells developed early polycythemia and fatal erythroleukemia, while B6 mice lacking CD8(+) T cells remained resistant. Erythroid progenitor cells infected with spleen focus-forming virus (SFFV) were eliminated, and no polycythemia was observed in B cell-deficient B6 mice, but they later developed myeloid leukemia associated with oligoclonal integration of ecotropic Friend murine leukemia virus. Additional depletion of natural killer and/or CD8(+) T cells from B cell-deficient B6 mice resulted in the expansion of SFFV proviruses and the development of polycythemia, indicating that SFFV-infected erythroid cells are not only restricted in their growth but are actively eliminated in Fv2r mice through cellular immune responses.

We investigated the role of effector CD8 T cells in the pathogenesis of immune glomerular injury. BALB/c mice are not prone to autoimmune disease, but after 12 immunizations with OVA they developed a variety of autoantibodies and glomerulonephritis accompanied by immune complex (IC) deposition. In these mice, IFN-γ-producing effector CD8 T cells were significantly increased concomitantly with glomerulonephritis. In contrast, after 12 immunizations with keyhole limpet hemocyanin, although autoantibodies appeared, IFN-γ-producing effector CD8 T cells did not develop, and glomerular injury was not induced. In β2-microglobulin-deficient mice lacking CD8 T cells, glomerular injury was not induced after 12 immunizations with OVA, despite massive deposition of IC in the glomeruli. In mice containing a targeted disruption of the exon encoding the membrane-spanning region of the Ig μ-chain (μMT mice), 12 immunizations with OVA induced IFN-γ- producing effector CD8 T cells but not IC deposition or glomerular injury. When CD8 T cells from mice immunized 12 times with OVA were transferred into naive recipients, glomerular injury could be induced, but only when a single injection of OVA was also given simultaneously. Importantly, injection of OVA could be replaced by one injection of the sera from mice that had been fully immunized with OVA. This indicates that deposition of IC is required for effector CD8 T cells to cause immune tissue injury. Thus, in a mouse model of systemic lupus erythematosus, glomerular injury is caused by effector CD8 T cells that recognize Ag presented as IC on the target renal tissue. Copyright © 2013 by The American Association of Immunologists, Inc.

Natural killer (NK) cells function as early effector cells in the innate immune defense against viral infections and also participate in the regulation of normal and malignant hematopoiesis. NK cell activities have been associated with early clearance of viremia in experimental simian immunodeficiency virus and clinical human immunodeficiency virus type 1 (HIV-1) infections. We have previously shown that NK cells function as major cytotoxic effector cells in vaccine-induced immune protection against Friend virus (FV)-induced leukemia, and NK cell depletion totally abrogates the above protective immunity. However, how NK cells recognize retrovirus-infected cells remains largely unclear. The present study demonstrates a correlation between the expression of the products of retinoic acid early transcript-1 (RAE-1) genes in target cells and their susceptibility to killing by NK cells isolated from FV-infected animals. This killing was abrogated by antibodies blocking the NKG2D receptor in vitro. Further, the expression of RAE-1 proteins on erythroblast surfaces increased early after FV inoculation, and administration of an RAE-1-blocking antibody resulted in increased spleen infectious centers and exaggerated pathology, indicating that FV-infected erythroid cells are recognized by NK cells mainly through the NKG2D-RAE-1 interactions in vivo. Enhanced retroviral replication due to host gene-targeting resulted in markedly increased RAE-1 expression in the absence of massive erythroid cell proliferation, indicating a direct role of retroviral replication in RAE-1 upregulation.

Regulators of G protein signaling (RGS) are a multi-functional protein family, which functions in part as GTPase-activating proteins (GAPs) of G protein alpha-subunits to terminate G protein signaling. Previous studies have demonstrated that the Rgs16 transcripts exhibit robust circadian rhythms both in the suprachiasmatic nucleus (SCN), the master circadian light-entrainable oscillator (LEO) of the hypothalamus, and in the liver. To investigate the role of RGS16 in the circadian clock in vivo, we generated two independent transgenic mouse lines using lentiviral vectors expressing short hairpin RNA (shRNA) targeting the Rgs16 mRNA. The knockdown mice demonstrated significantly shorter free-running period of locomotor activity rhythms and reduced total activity as compared to the wild-type siblings. In addition, when feeding was restricted during the daytime, food-entrainable oscillator (FEO)-driven elevated food-anticipatory activity (FAA) observed prior to the scheduled feeding time was significantly attenuated in the knockdown mice. Whereas the restricted feeding phase-advanced the rhythmic expression of the Per2 clock gene in liver and thalamus in the wild-type animals, the above phase shift was not observed in the knockdown mice. This is the first in vivo demonstration that a common regulator of G protein signaling is involved in the two separate, but interactive circadian timing systems, LEO and FEO. The present study also suggests that liver and/or thalamus regulate the food-entrained circadian behavior through G protein-mediated signal transduction pathway(s).

Several host genes control retroviral replication and pathogenesis through the regulation of immune responses to viral antigens. The Rfv3 gene influences the persistence of viremia and production of virus-neutralizing antibodies in mice infected with Friend mouse retrovirus complex (FV). This locus has been mapped within a narrow segment of mouse chromosome 15 harboring the APOBEC3 and BAFF-R loci, both of which show functional polymorphisms among different strains of mice. The exon 5-lacking product of the APOBEC3 allele expressed in FV-resistant C57BL/6 (B6) mice directly restricts viral replication, and mice lacking the B6-derived APOBEC3 exhibit exaggerated pathology and reduced production of neutralizing antibodies. However, the mechanisms by which the polymorphisms at the APOBEC3 locus affect the production of neutralizing antibodies remain unclear. Here we show that the APOBEC3 genotypes do not directly affect the B-cell repertoire, and mice lacking B6-derived APOBEC3 still produce FV-neutralizing antibodies in the presence of primed T helper cells. Instead, higher viral loads at a very early stage of FV infection caused by either a lack of the B6-derived APOBEC3 or a lack of the wild-type BAFF-R resulted in slower production of neutralizing antibodies. Indeed, B cells were hyperactivated soon after infection in the APOBEC3- or BAFFR-deficient mice. In contrast to mice deficient in the B6-derived APOBEC3, which cleared viremia by 4 weeks after FV infection, mice lacking the functional BAFF-R allele exhibited sustained viremia, indicating that the polymorphisms at the BAFF-R locus may better explain the Rfv3-defining phenotype of persistent viremia.

During chronic viral infection, persistent exposure to viral Ags leads to the overexpression of multiple inhibitory cell-surface receptors that cause CD8(+) T cell exhaustion. The severity of exhaustion correlates directly with the level of infection and the number and intensity of inhibitory receptors expressed, and it correlates inversely with the ability to respond to the blockade of inhibitory pathways. Friend virus (FV) is a murine retrovirus complex that induces acute high-level viremia, followed by persistent infection and leukemia development, when inoculated into immunocompetent adult mice. In this article, we provide conclusive evidence that FV infection results in the generation of virus-specific effector CD8(+) T cells that are terminally exhausted. Acute FV-induced disease is characterized by a rapid increase in the number of virus-infected erythroblasts, leading to massive splenomegaly. Most of the expanded erythroblasts strongly express programmed death ligand-1 and MHC class I, thereby creating a highly tolerogenic environment. Consequently, FV-specific effector CD8(+) T cells uniformly express multiple inhibitory receptors, such as programmed cell death 1 (PD-1), T cell Ig domain and mucin domain 3 (Tim-3), lymphocyte activation gene-3, and CTLA-4, rapidly become nonresponsive to restimulation and are no longer reinvigorated by combined in vivo blockade of PD-1 and Tim-3 during the memory phase. However, combined blockade of PD-1 and Tim-3 during the priming/differentiation phase rescued FV-specific CD8(+) T cells from becoming terminally exhausted, resulting in improved CD8(+) T cell functionality and virus control. These results highlight FV's unique ability to evade virus-specific CD8(+) T cell responses and the importance of an early prophylactic approach for preventing terminal exhaustion of CD8(+) T cells. The Journal of Immunology, 2010, 184: 4696-4707.

Several members of the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like complex 3 (APOBEC3) family in primates act as potent inhibitors of retroviral replication. However, lentiviruses have evolved mechanisms to specifically evade host APOBEC3. Likewise, murine leukemia viruses ( MuLV) exclude mouse APOBEC3 from the virions and cleave virion-incorporated APOBEC3. Although the betaretrovirus mouse mammary tumor virus has been shown to be susceptible to mouse APOBEC3, it is not known if APOBEC3 has a physiological role in restricting more widely distributed and long-coevolved mouse gammaretroviruses. The pathogenicity of Friend MuLV (F-MuLV) is influenced by several host genes: some directly restrict the cell entry or integration of the virus, while others influence the host immune responses. Among the latter, the Rfv3 gene has been mapped to chromosome 15 in the vicinity of the APOBEC3 locus. Here we have shown that polymorphisms at the mouse APOBEC3 locus indeed influence F-MuLV replication and pathogenesis: the APOBEC3 alleles of F-MuLV-resistant C57BL/6 and -susceptible BALB/c mice differ in their sequences and expression levels in the hematopoietic tissues and in their abilities to restrict F-MuLV replication both in vitro and in vivo. Furthermore, upon infection with the pathogenic Friend virus complex, (BALB/c x C57BL/6) F 1 mice displayed an exacerbated erythroid cell proliferation when the mice carried a targeted disruption of the C57BL/6-derived APOBEC3 allele. These results indicate, for the first time, that mouse APOBEC3 is a physiologically functioning restriction factor to mouse gammaretroviruses.

Several host genes control retroviral replication and pathogenesis. These include genes that directly affect the replication of retroviruses in target cells and those that control the host immune responses to the viral. antigens. Host genetic factors that affect retroviratal reptication and immune responses to the viratal antigens have been best studied in mouse models of Friend leukemia virus (FV) infection. Several genes Located within the major histocompatibitity comptex (MHC), along with a separate gene not Linked to the MHC, influence the host immune responses to FV antigens. The tatter, the Rfv3, regutates the production of virus-neutratizing antibodies, and thus affects the duration of viremia. T-cell responses to the viral epitopes are controlled by MHC class I and class 11 genotypes, and both CD8(+) and CD4(+) T-cells are required for spontaneous immune resistance to FV infection. When CD4(+) T-helper cells are efficiently primed with a viral epitope, however, CD8(+) T-cells are not required for immune protection against FV infection, white B cells are absolutely required. There are individuals who possess human immunodeficiency virus type I (HIV-1)-reactive IgA antibodies in their mucosal. secretions and show strong T-cell responses to HIV-1 antigens, even though they are negative for HIV-1 genome and HIV-1-reactive serum IgG. These HIV-1-exposed but uninfected individuals rarely possess resistance-associated alleles at known AIDS-restricting loci such as CCR5 Delta 32. Recent genetic analyses have indicated that a Large proportion of such exposed but uninfected individuals may share a common genetic background.(c) 2008 Elsevier Ltd. All rights reserved.

Hyperthermia (HT), in combination with other conventional therapeutic modalities, has become a promising approach in cancer therapy. In addition to heat-induced apoptosis, an augmented immunological effect is considered to be a benefit of hyperthermic treatment over chemo- or radiotherapy. Here, we investigated the effect of regional HT targeting the liver on immune cells, especially T cells and antigen-presenting cells, which are important in recognizing and eliminating tumor cells and pathogens such as viruses. In healthy volunteers exposed to such regional HT, both CD4(+) and CD8(+) T cells that express an activation marker CD69 increased transiently at I h post-treatment, with a subsequent decrease to base levels at 6 It after the treatment. At 24 h post-treatment, the percentage of CD69-positive cells significantly increased again but only among CD8(+) T cells. IFN-gamma production from PHA-stimulated peripheral blood mononuclear cells was gradually and significantly increased in the 2 days following the heating procedure, peaking at 36 h post-treatment. Furthermore, we found marked increases in plasma levels of IL-1 beta and IL-6 starting at 24 h post-treatment. With regard to the number of each leukocyte subpopulation, a transient and dramatic decrease in the number of a subset of monocytes, CD14(+) CD16(-) cells, was observed at 1 h after the hyperthermic treatment, suggesting that the regional HT aimed at the liver may have influenced the extravasation of blood monocytes. No significant changes in T-cell activities or monocyte counts were observed in the volunteers exposed to heating of the lungs or the legs. These results suggest that heating of the liver may efficiently induce cellular immune responses to liver cancers.

MILL (MHC class I-like located near the leukocyte receptor complex) is a family of MHC class I-like molecules encoded outside the MHC, which displays the highest sequence similarity to human MICA/B molecules among known class I molecules. In the present study, we show that the two members of the mouse MILL family, MILL1 and MILL2, are GPI-anchored glycoproteins associated with beta(2)-microglobulin (beta(2)m) and that cell surface expression of MILL1 or MILL2 does not require functional TAP molecules. MILL1 and MILL2 molecules expressed in bacteria could be refolded in the presence of beta(2)m, without adding any peptides. Hence, neither MILL1 nor MILL2 is likely to be involved in the presentation of peptides. Immunohistochemical analysis revealed that MILL1 is expressed in a subpopulation of thymic medullary epithelial cells and a restricted region of inner root sheaths in hair follicles. The present study provides additional evidence that MILL is a class I family distinct from MICA/B.

CD8(+) CTLs and virus-neutralizing antibodies have been associated with spontaneous and vaccine-induced immune control of retroviral infections. We previously showed that a single immunization with an env gene-encoded CD4(+) T cell epitope protected mice against fatal Friend retrovirus infection. Here, we analyzed immune cell components required for the peptide-induced anti-retroviral protection. Mice lacking CD8(+) T cells were nevertheless protected against Friend virus infection, while mice lacking B cells were not. Virus-producing cells both in the spleen and bone marrow decreased rapidly in their number and became undetectable by 4 weeks after infection in the majority of the peptide-immunized animals even in the absence of CD8(+) T cells. In the vaccinated animals the production and class switching of virus-neutralizing and anti-leukemia cell antibodies were facilitated; however, virus-induced erythroid cell expansion was suppressed before neutralizing antibodies became detectable in the serum. Further, the numbers of virus-producing cells in the spleen and bone marrow in the early stage of the infection were smaller in the peptide-immunized than in unimmunized control mice in the absence of B cells. Thus, peptide immunization facilitates both early cellular and late humoral immune responses that lead to the effective control of the retrovirus-induced disease, but CD8(+) T cells are not crucial for the elimination of virus-infected cells in the peptide-primed animals.

Objectives—We tested the effect of a clinically applicable dose of moxibustion on adjuvant-induced arthritis (AIA) of rat, an experimental model of rheumatoid arthritis.
Methods—Male Lewis rats were inoculated with Mycobacterium butyricum suspended in paraffin oil into the right hind paw to induce arthritis. Moxibustion (60°C) was applied to the right hind limb point, Tsu-san-Li (ST36), twice a week for 4 consecutive weeks. The efficacy of the above treatment was determined by the measurements of paw swelling, arthritic score. The effects of moxibustion upon immune and inflammatory responses were analyzed by enumerating peripheral blood leukocyte subsets. The data were analyzed with Mann-Whitney U-test between the experimental and control groups.
Results—Moxibustion significantly suppressed paw swelling in the systemic phase, but not in the acute phase, of arthritis. Moxibustion also significantly suppressed the increase in T lymphocyte numbers in the late acute phase and that of neutroplils in the systemic phase.
Conclusion—After the treatment with moxibustion, significant alterations were observed in the numbers of peripheral blood leukocyte subsets in AIA, along with the amelioration of clinical signs. These observations suggest that suppression of AIA with moxibustion may be mediated through the suppression of proliferating number of T-cell and acceleration of decrease in number of neutrophils in the peripheral blood.

Recent studies have demonstrated an essential role of Gag-specific CD4(+) T-cell responses for viral control in individuals infected with human immunodeficiency virus type 1. However, little is known about epitope specificities and functional roles of the Gag-specific helper T-cell responses in terms of vaccine-induced protection against a pathogenic retroviral challenge. We have previously demonstrated that immunization with Friend murine leukemia virus (F-MuLV) Gag proteins protects mice against the fatal Friend retrovirus (FV) infection. We report here the structure of a protective T helper cell (Th) epitope, (I)VTWEAIAVDPPP, identified in the p15 (MA) region of F-MuLV Gag. In mice immunized with the Th epitope-harboring peptide or a vaccinia virus-expressed native full-length MA protein, FV-induced early splenomegaly regressed rapidly. In these mice, FV-infected cells were eliminated within 4 weeks and the production of virus-neutralizing antibodies was induced rapidly after FV challenge, resulting in strong protection against the virus infection. Interestingly, mice immunized with the whole NIA mounted strong CD4+ T-cell responses to the identified Th epitope, whereas mice immunized with mutant MA proteins that were not bound to the plasma membrane failed to mount efficient CD4+ T-cell responses, despite the presence of the Th epitope. These mutant MA proteins also failed to induce strong protection against FV challenge. These data indicate the importance of the properly processible NIA molecule for CD4+ T-cell priming and for the resultant induction of an effective immune response against retrovirus infections.

The B cell adaptor containing src homology 2 domain (BASH; also termed BLNK or SLP-65), is crucial for B cell antigen receptor (BCR)-mediated activation, proliferation, and differentiation of B cells. BCR-mediated tyrosine-phosphorylation of BASH creates binding sites for signaling effectors such as phospholipase C gamma (PLC gamma )2 and Vav, while the function of its COOH-terminal src homology 2 domain is unknown. We have now identified hematopoietic progenitor kinase (HPK)1, a STE20-related serine/threonine kinase, as a protein that inducibly interacts with the BASH SH2 domain. BCR, ligation induced rapid tyrosine-phosphorylation of HPK1 mainly by Syk and Lyn, resulting in its association with BASH and catalytic activation. BCR-mediated activation of HPK1 was impaired in Syk- or BASH-deficient B cells. The functional SH2 domain of BASH and Tyr-379 within HPK1 which we identified as a Syk-phosphorylation site were both necessary for interaction of both proteins and efficient HPK1 activation after BCR stimulation. Furthermore, HPK1 augmented, whereas its kinase-dead mutant inhibited I kappaB kinase beta (IKK beta) activation by BCR, engagement. These results reveal a novel BCR signaling path-way leading to the activation of HPK1 and subsequently IKK beta, in which BASH recruits tyrosine-phosphorylated HPK1 into the BCR signaling complex.

B cell antigen receptor signals development, activation, proliferation, or apoptosis of B cells depending on their condition, and its proper signaling is critical for activation and homeostasis of the immune system. The B cell-restricted adaptor protein BASH (also termed BLNK/SLP-65) is rapidly phosphorylated by the tyrosine kinase Syk after BCR ligation and binds to various signaling proteins. BASH structurally resembles SLP-76, which is essential for T cell development and T cell receptor signaling. To evaluate the role for BASH in B cell development and function in vivo, we disrupted BASH alleles in embryonic stem cells by means of homologous recombination and used these cells to complement lymphocyte-incompetent blastocysts from RAG2-deficient mice. In the resultant chimeric mice, T cell development was apparently normal, but B cell development was impaired, and a normally rare population of large preB cells expressing preB cell receptor dominated in the bone marrow in place of small preB cells, although they were mostly noncycling. In addition, the mature B cell populations in the periphery and the bone marrow profoundly decreased in size, as did B-l cells in the peritoneal cavity, and serum lg was severely reduced. The BASH-deficient B cells scarcely proliferated or up-regulated B7-2 in response to BCR ligation and poorly proliferated upon CD40 ligation or lipopolysaccharide stimulation. This phenotype indicates that BASH is critical for preB cell receptor signaling inducing proliferation of large preB cells and the following differentiation. for peripheral B cell maturation, and for BCR signaling inducing activation/proliferation of B cells.

The purpose of this study was to determine the mechanism of the local cytokine-mediated immune response in the skin of chickens. The incorporation of 3H-thymidine into spleen T lymphocytes from 9- to IO-week-old chickens was augmented by the addition of epidermal tissue culture supernatant (ESN) from Ii-day-old embryos. The colony formation of neonatal chicken bone marrow cells in methyl-cellulose medium was also significantly increased by addition of ESN. When axonal outgrowth in matrigel was investigated, the embryonal sympathetic ganglion was found to grow axons outwards towards the epidermal tissue specimens. The above results suggest that chicken epidermal cells (probably keratinocytes) produce T-cell growth factor (corresponding to IL-1), colony-stimulating factor for macrophages (M-CSF) and granulocytes (G-CSF), and nerve growth factor (NGF).

The bursa of Fabricius is a gut-associated lymphoid organ that is essential for the generation of a diversified B cell repertoire in the chicken, We describe here a: novel gene preferentially expressed in bursal R cells. The gene encodes an 85-kDa protein, designated-BASH (B cell adaptor containing SH2 domain), that contains N-terminal acidic domains with SH2 domain-binding phosphotyrosine-based moths, a proline-rich domain, and a C-terminal SH2 domain, BASH shows a substantial sequence similarity to SLP-76, an adaptor protein functioning in TCR-signal transduction, BASH becomes tyrosin-phosphorylated with the B cell Ag receptor (BCR) cross-link or by coexpression with Syk and Lyrr and associates with signaling molecules including Syk and a putative chicken Shc homologue, Overexpression of BASH results in suppression of the NF-AT activation induced by BCR-cross-linking. These findings suggest that BASH is involved in BCR-mediated signal transduction and could play a critical role in B cell development in the bursa.

Stimulation of the B cell Ag receptor (BCR) induces activation of tyrosine kinases such as Lyn and Syk, phosphorylation and activation of multiple signaling components, and eventually, the expression of several genes including c-myc. Syk is required for activation of phospholipase C-γ2 and the subsequent phosphatidylinositol hydrolysis, leading to protein kinase C (PKC) activation and intracellular Ca2+ increase. In contrast, the function of Lyn remains obscure. Here, we report that BCR-mediated induction of c-myc promoter activity and of PKC activity, but not the expression level of functional PKC, was markedly augmented in Lyn-deficient chicken B cells. This enhancement was reversed to the level of wild-type cells by the expression of exogenous Lyn of kinase-inactive form. These results indicate that Lyn inhibits BCR-mediated activation of a large portion of PKC isozymes in a kinase-independent fashion. This finding reveals a novel role of Lyn in negative regulation of BCR signaling.

The function of polysaccharide (PSE) extracted from pine seed shells as a immunopotentiator was investigated The phagocytosis and chemotaxis of mouse peritoneal macrophages (MP) were augmented by oral administration of PSE. The incorporation of H-3-thymidine by Con A-stimulated mouse splenic cells mas significantly intensified by administration of PSE but the adherent-cells (MP)-eliminated splenic lymphocytes was nor increased. It is shown that the existence of activated MP is needed for the activation (proliferation) of T lymphocytes. The responsiveness of mouse T cells to alloantigen was also augmented by administration of PSE. The number of anti-SRBC plaque-forming cells in the spleen of mouse and chicken was also significantly increased. The results indicate that MP are first activated by oral administration of PSE and that the activation of T cells is then initiated indirectly by humoral factors (cytokines) produced by activated MP.

This study was designed to evaluate the significance of the Harderian gland (HG) in the chicken IgAproduction system by investigating the influence of KG on the increase of surface IgA (sIgA)-positive cells in other lymphoid organs and also the possibility of migration of HG lymphocytes into other organs. Non-contact culture of splenic or caecal tonsil (CT) lymphocytes with HG whole cells did not enhance the expression of sIgA. The experiment of HG lymphocyte transfer showed that the transferred HG lymphocytes migrated mainly into the CT in the 3-week-old recipient, and mainly into the CT and bursa of Fabricius (BF) in the 6-week-old recipient It was also found that the transferred sIgA-positive HG lymphocytes migrated selectively into CT in both 3-week-old and G-week-old recipients. These results indicated that chicken HG plays a central role not only in governing the local immunity in the eyes and respiratory tract but also as an organ which supplies precursory IgA-producing cells involved in local immunity in the intestine.

The mechanism of the suppression of lymphocyte proliferation by peritoneal macrophages (MPs) in chickens and the presence of a suppressive substance in the culture supernatant of MPs were studied. The results showed that gelatin- and pepton-induced peritoneal MPs had an inhibitory accessory function against blastogenesis of mitogen-stimulated splenic lymphocytes and that resident peritoneal MPs had an enhanced accessory function. The suppressive factor produced by gelatin-induced peritoneal MPs is a protein having a pI of 3.6 and a molecular weight of 64,000 and it is physically and chemically a relatively stable substance. Moreover, it has no cytotoxic effect against lymphocytes and it was verified that the suppressor in the culture supernatant of MPs was not prostaglandin E(2) or alpha-acid glycoprotein. Copyright (C) 1996 Elsevier Science Ltd.

To examine the role of T cell subpopulations in an acute graft-versus-host (GVH) reaction. gamma delta T cells and alpha beta T cells expressing one or the two prototypic V beta gene families were negatively isolated from adult blood samples and injected into allogeneic chick embryos, CD4(+) alpha beta T cells expressing either V beta 1 or V beta 2 receptors were equally capable of inducing acute GVH reactions. consistent with the idea that alpha beta T cell alloreactivity is determined by CDR3 variability. By themselves. the gamma delta T cells were incapable of inducing GVH reactions. However. host gamma delta T cells were recruited into the donor alpha beta T cell-initiated lesions. where they were activated and induced to proliferate. The data suggest that gamma delta T cells may play a secondary role in GVH reactions.

By polymerase chain reaction (PCR) using a pair of primers specific for Salmonella phoE gene a 365-bp specific gene fragment could be amplified from yolk of infertile eggs and dead-in-shell chicken embryos, and from environmental samples. Out of 45 dead-in-shell embryo samples, 20 (44.4%) were found positive for Salmonella DNA by PCR compared to 11 (24.4%) by bacteria isolation. Salmonella DNA could also be detected from infertile eggs, chicken faeces, floor litter and chick fluff, which incidence was higher than that by bacteria isolation.

The mechanism of accumulation of surface immunoglobulin A (sIgA) bearing cells in chicken Harderian gland (HG) was examined. Almost no sIgA-bearing cells were identified in HG of 1.5-week-old chickens. In 3.5-week-old chickens, however, 46.4% of the HG lymphocytes were sIgA-bearing cells. When bursa of Fabricius (BF) lymphocytes were transferred into recipients, BF lymphocytes migrated into the HG but the number of these lymphocytes was extremely small. In addition, no correlation was found between the surface immunoglobulin class of BF lymphocytes that migrated into HG and the migration. HG lymphocytes were co-cultured with HG whole cells (which contained 50-60% configurationally epithelial cells) in a non-contacting manner for 80 h using an Intercell culture vessel. The results showed a significant increase in the ratio of sIgA-bearing cells in HG lymphocytes compared with the control which was cultured without placing HG whole cells in the external well. B lymphocytes that migrated into HG seemed to undergo specific differentiation in situ into sIgA-bearing cells owing to microenvironmental factors, probably humoral factors excreted from HG epithelial cells, and subsequently undergo proliferation to result in accumulation of sIgA-bearing cells in HG tissue.

TLR7 is expressed on several types of cell including dendritic cells and B cells. In this study, we analyzed the requirement of B-cell intrinsic TLR7 for the protection of retrovirus-induced leukemia. Mice lacking of B cell-intrinsic TLR7 produced virus-neutralizing antibodies as efficiently as the wild-type mice by priming of T helper cells, and rapid elimination of exogenous retroviruses were observed. Nevertheless,the B cell-intrinsic TLR7 lacking mice later died of leukemia. These results suggest that B cell-intrinsic TLR7 is essential to regulate the appearance of endogenous and/or recombinant retroviruses even when the virus-specific Th cells are efficiently activated.

TLR7 is expressed on several types of cell including dendritic cells and B cells. In this study, we analyzed roles of B cell-intrinsic TLR7 signaling on humoral immune responses against retrovirus infection. We found that primary B cell responses to retroviral infection required B cell-intrinsic TLR7 signaling, whereas preexisting virus-specific memory Th cells elicited the B cell responses without TLR7 signaling. Moreover, we showed that in the presence of virus-specific memory Th cells, the lack of TLR7 signaling in B cells facilitated anti-virus antibody responses and led to delayed progression of virus-induced leukemia.

APOBEC3 is a DNA mutator that restricts retrovirus replication, and its allelic differences have been associated with kinetics of the production of virus-neutralizing antibodies. We wished to elucidate how APOBEC3 affects antibody production. CD8+ T cells were dispensable while CD4+ T cells were crucial for the elimination of virus-infected erythroid cells. In the absence of B-lymphocytes, infected erythroid cells were eliminated but retrovirus replication persisted in myeloid cells. Friend retrovirus also infected the thymus, and thymic expression of the viral antigens lead to negative selection of virus-specif T cells. Further, mice lacking activation-induced cytidine deaminase nevertheless produced neutralizing antibodies, and non-mutated IgM conferred resistance to infection in the presence of virus antigen-primed CD4+ T cells. Thus, APOBEC3 seems to interfere with virus-induced derangement of CD4+ T-cell functions that are crucial for the production of neutralizing antibodies.

Mouse APOBEC3, a host factor that restricts the replication of exogenous retrovirus, is abundantly expressed in virus-resistant C57BL/6 (B6) mice. We have found that loss of APOBEC3 causes spontaneous mammary carcinoma and T cell lymphomas in B6 mice. The transcripts of endogenous mouse mammary tumor virus are abundant in the tumor cells prepared from APOBEC3-deficient mice. These results suggest that APOBEC3 contributes to the control of activated endogenous retrovirus.

Polymorphisms at the mouse APOBEC3(mA3) gene locus have been associated with different susceptibilities to infection with mouse leukemia viruses. In virus-resistant C57BL/6(B6) mice, mA3 transcripts and protein are more abundant than those in susceptible BALB/c mice, and these strains of mice also express mA3 transcripts with different splicing patterns. B6 mice express predominantly the exon 5-deficient transcript, which confer more efficient translation than the full-length transcript. By employing in vitro splicing assays using genomic DNA clones, we identified two critical determinants that regulate exon 5 inclusion into mA3 transcripts : the number of TCCT repeats upstream of exon 5 and the newly identified single nucleotide polymorphism within exon 5 located 12 bases upstream of the exon 5/intron 5 boundary.

Addition of the anti-GD1b antibodies from rabbits affected with sensory ataxic neuropathy to cultured rabbit dorsal root ganglia neurons caused morphological changes in the neuritis. Anti-GM1 antibodies from patients with axonal type of Guillain-Barre syndrome(GBS) directly influence the integrity of the lipid rafts and alter the signal transduction in the cells. Antibodies against LM1, a glycolipid localized in human peripheral nerve myelin, and such ganglioside complexes as LM1/GM1 and LM1/GD1b were detected in sera from patients with demyelinating type of GBS and chronic inflammatory demyelinating polyneuropathy.

Despite multiple and repeated exposures to HIV-1, some individuals possess no detectable HIV genome and show T-cell memory responses to the viral antigens. HIV-1-reactive mucosal IgA detected in such uninfected individuals suggests their possible immune resistance against HIV acquisition. However, attempts to associate the above HIV-1-exposed but uninfected status with known resistance factors, including the CCR5A32 mutation, have been unsuccessful. Based on the mapping in chromosome 15 of a gene controlling the production of virus-neutralizing antibodies in a mouse model of retrovirus infection, we have reported a strong association between genotypes at marker loci within the syntenic chromosome 22q13.1 region and a putative dominant locus conferring strong anti-HIV-1 immune responses. In this project, we have compared expression levels of all the genes located within the above chromosomal segment utilizing DNA microarrays, and have revealed a higher induction of two particular genes in HIV antigen-stimulated peripheral blood mononuclear cells only among the exposed but uninfected, but not in the HIV-infected, individuals. Further, extensive genotyping of single nucleotide polymorphisms (SNPs) and genomic sequencing of the exposed but uninfected and HIV-infected individuals have indicated 1) a significant accumulation of a particular genotype at multiple and mutually linked SNP loci surrounding the above highly induced genes, 2) strong linkage disequilibrium between SNP genotypes across the above two gene loci observed only among the exposed but uninfected individuals, and 3) the presence of possibly functional genetic changes in the putative enhancer element located between the above two genes. Further, the expression of the DNA mutater APOBEC3G genes, whose locus is located in the chromosome 22 in close linkage with the above 22q13.1 segment, was significantly higher among the exposed but uninfected than in HIV-1-infected individuals, and its expression became much higher in CD14^+ monocytes than in other cell-types after the stimulation with interferon-α. Thus, genetically determined resistance against HIV-1 acquisition observed in the HIV-1-exposed but uninfected individuals may be explained, at least partially, by the above observed higher induction of chromosome 22 genes including APOBEC3G.

Despite multiple and repeated exprosures to HIV-1, some individuals possess no detectable HIV genome and show T-cell memory responses to the viral antigens. HIV-1-reactive mucosal IgA detected in such uninfected individuals suggests their possible immune resistance against HIV. We tested if the above HIV-1-exprosed but uninfected status was associated with genetic markers other than a homozygous deletion of the CCR5 gene. Based on our mapping in chromosoma 15 of a gene controlling the production of neutralizing antibodies in a mouse retrovirus infection, we genotyped 42 HIV-1-exposed but uninfected Italians at polymorphic loci in the syntenic segment of human chromosome 22, and compared them with 49 HIV-1-infected and 47 uninfected healthy control individuals by a closed testing procedure. A significant association was found between chromosome 22q12-13 genotypes and a putative dominant locus conferring anti-HIV-1 immune responses in the exposed but uninfected individuals. Distributions of linkage disequilibrium across chromosome 22 also differed between the exposed but uninfected and two other phenotypic groups. We next stimulated the peripheral blood mononuclear cells from HIV-1-exposed but uninfected and HIV-1-infected individuals with a mixture of HIV-1 gag and env antigens, and compared the expression of all the genes and open reading frames located in the above candidate region using a DNA microarray technique. Significant induction of several genes at 6 hours after the antigenic stimulation was observed only in the exposed but uninfected individuals. Further linkage of a single nucleotide polymorphism located in one candidate gene with the genotype of the D22S423 marker was observed only in the exposed but uninfected individuals. These data indicated the presence of a new genetic factor associated with the HIV-1-exposed but uninfected status.

ウイルス感染防御においては、CD8陽性細胞傷害性Tリンパ球(CTL)がウイルス産生細胞を破壊し、中和抗体が感染の拡大とウイルス粒子除去に関与すると信じられている。このため、ワクチン開発に当たって、CTLとウイルス中和抗体の誘導が重要な指標とされることが多い。 我々は、免疫系の完成した成体マウスへの接種により致死性赤白血病を誘発するフレンドレトロウイルス(FV)を用い、FVに感受性の高い(BALB/c × C57BL/6)F_1マウスに、β2-ミクログロブリン遺伝子欠損をホモ接合で導入して、CD8陽性細胞を欠く系統を育成、これと野生型F_1マウス、及びBリンパ球を欠く免疫グロブリン遺伝子膜貫通エクソン欠損マウスを用いて、ペプチドワクチンによる感染防御実験を行った。 ウイルス被膜タンパク質上のCD4陽性Tリンパ球認識エピトープを単独で含む合成ペプチドで、一度だけ免疫することにまり、野生型マウスの80%以上でFV誘発白血病が予防できた。驚くべきことに、CD8陽性Tリンパ球欠損F_1マウスでも、その約70%がペプチドワクチン免疫によりFVに抵抗性となり、感染後の脾腫発症・死亡率は、免疫した野生型マウスと有意差がなかった。しかし、Bリンパ球欠損マウスでは、ペプチドワクチンで白血病死を防止できなかった。 ペプチド免疫マウスでは、CD8陽性Tリンパ球非存在下でも、FV感染4週間後までに骨髄および脾からウイルス産生細胞が排除された。一方、Bリンパ球欠損マウスでは、感染初期のウイルス産生細胞数増加は抑制されたが、感染2週間目以降はワクチンの効果が見られなかった。 以上から、CD4陽性Tリンパ球認識抗原エピトープを用いてフレンド白血病ウイルスに対する感染防御を誘導する系に関しては、CD8陽性細胞傷害性Tリンパ球は感染防御に必須ではないことが明らかとなった。

1) One of the mouse retroviral gag gene products, the N-terminal MA protein, contains multiple T-lymphocyte epitopes recognized by CD4-positive helper cells. One of these T-helper cell epitopes was mapped within amino acid residues 62 and 76, and another epitope was localized between residues 119 and 138 in the MA protein. 2) CD4-positive T cells specifically reacting to the 62-76 epitope produced a large amount of IL-4, while T cells reactive to the epitope within 119-138 did not. 3) To identify the antigenic structures that are required for protection against retroviral infection, various truncated forms of MA were expressed in a newly modified vaccinia virus vector, and mice were immunized with the resultant recombinant vaccinia viruses. All the truncated forms of MA lost the ability to protect mice, while the entire MA was effective. To elucidate if the truncation in the N-terminal portion affected the effectiveness of the whole MA due to the lack of the N-terminal residue required for protein myristylation, Gly at residue 2 was replaced with Ala in a mutant MA. This mutant MA localized in the cell nuclei, instead of cell membrane where myristylated proteins normally accumulate, and at the same time the vaccinia virus expressing this mutant MA lost its ability to protect mice against retroviral infection. 4) Thus, myristylation of MA is necessary for its proper immunogenicity, and when myristylated, the Th2 epitope is not necessary, but an epitope in the C-terminal portion is required for protective efficacy of MA.

B細胞抗原受容体(BCR)が抗原により架橋されると、Syk,Lynなどのチロシンキナーゼ(PTK)が速やかに活性化され、それらの基質となる分子群をリン酸化する。その下流では、PLC_γやRas,RacのGTPaseなどがセカンドメッセンジャーとしての役割を担い、BCRからのシグナルを核へと伝達し、最終的に細胞の運命が決定される。我々は、PTKとその基質分子の橋渡し的役割を担うリンカー分子が、その細胞運命決定へのシグナル伝達を制御している可能性があると考え、BCR下流のリンカー分子であるHS1ならびにBASHの機能解析を行なってきた。 HS1は、通常でも、そのSH3ドメイン介した制御機構により核細胞質間をシャトルしている分子であり、抗原受容体架橋後はリン酸化されたHS1が速やかに核内に存在することを明らかした。またHS1はBtk基質の一つであり、BCR架橋によりBtkとリン酸化されたHS1が細胞質内で会合すること、その会合はHS1結合蛋白であるBP3依存的である結果を得た。また、HS1欠損マウスにおいて、PLC_γのリン酸化レベルが低下していることから、HS1はBtkと結合し、BtkによるPLC_γの活性化に関与している可能性が示唆された(投稿準備中)。BASHに関しては、BASH欠損マウスでのB細胞機能解析を行なったところ、BASH欠損によりB細胞の初期分化異常ならびに末梢B細胞の抗原反応性の顕著な低下が認められた。すなわち、BASHはpre-B cell receptorおよびBCRの直下に位置し、受容体下流の複数のシグナル経路を制御するリンカー分子であることが示唆された(J.Immunol.161(1998)、Proc.Natl.Acad.Sci.USA.(in press))。今後、これら分子の機能をさらに追求することで,BCR刺激によるリンパ球運命決定の制御機構を解明したいと考えている。