H-2-associated and background genes influence the development of a murine retrovirus-induced immunodeficiency syndrome

Host genetic determinants of resistance or susceptibility to a retrovirus-induced immunodeficiency syndrome, termed MAIDS, were evaluated in Fv-1b mice infected with the mixture of ecotropic, MCF, and defective murine leukemia viruses designated LP-BM5 murine leukemia viruses. Genes of the MHC were shown to exert a major influence on the development of disease and the extent of virus spread in mice infected as adults. Strains bearing the b, f, k, q, r, and s haplotypes were moderately to highly susceptible to MAIDS whereas mice of the d haplotype were the most resistant. Resistance to disease was strongly associated with inhibition of mink cell focus-inducing virus spread and, to a lesser extent, with inhibition of ecotropic virus expression. Mapping studies localizing resistance associated with the d haplotype to H-2Dd were confirmed by the demonstration that B6 mice carrying this gene as a transgene or by recombination were resistant to disease. Penetrance of resistance to disease associated with expression of H-2Dd was markedly influenced by MHC genes mapping to the left of H-2D and by non-MHC loci such that some strains bearing this gene were highly susceptible to MAIDS. The combined effects of MHC and background genes among 40 strains examined yielded a remarkably wide spectrum of disease phenotypes with the onset of advanced disease ranging from 10 wk to 72 wk postinfection. Resistance to disease in moderately to highly resistant strains was shown to develop with age. Unexpectedly, the disease resistant phenotype was found to be a recessive trait.     

Absence of involvement of nitric oxide in LP-BM5-induced immunodeficiency syndrome

Abstract To examine the role of nitric oxide (NO) in murine AIDS (MAIDS) pathogenesis, we determined NO production and inducible NOS (iNOS) mRNA expression in the macrophages of LP-BM5-infected mice, together with the in vivo effects of l -NAME, a competitive inhibitor of NO synthase. LP-BM5 infection induced neither spontaneous nitrite production nor iNOS mRNA expression. No differences in IFNγ + LPS-induced nitrite production or iNOS mRNA expression were observed in macrophages from non-infected or infected mice. Spleen weight, ecotropic MuLV replication, the blood lymphocyte phenotype and proliferative response of splenocytes were not modified by l -NAME. LP-BM5 infection did not increase macrophage NO production and NO production did not appear to protect against LP-BM5-induced immunodeficiency.     

Functional and phenotypic alterations in T cell subsets during the course of MAIDS, a murine retrovirus-induced immunodeficiency syndrome

The functional and phenotypic characteristics of Ly-4(CD4)+ and Ly-2(CD8)+ T cells were studied after induction of murine AIDS with LP-BM5 murine leukemia virus. Assays of spleen cells for their ability to generate in vitro CTL responses to TNP-modified autologous cells (self + x CTL) and to alloantigens (allo CTL) showed that self + x CTL responses were greatly impaired at 3 to 4 wk postinfection and were undetectable thereafter. Allo CTL responses were normal at 3 to 4 wk, but were reduced at 8 to 9 wk and absent at 14 wk postinfection. This sequential loss of self + x and allo CTL responses was related to a selective defect in Ly-4(CD4)+ Th cell function associated with impaired production of IL-2 and deficient proliferative responses to Con A or to soluble Ag. Changes in the functional characteristics of Ly-4(CD4)+ T cells were unrelated to changes in their frequency in spleen, but did correlate with marked alterations in their distribution among four subsets defined by mAb SM3C11 and SM6C10. Assays of CTL responses generated by mixtures of spleen cells from normal and infected mice suggested that active suppression of Ly-4(CD4)+ Th function may contribute to this defect. Studies of Ly-2(CD8)+ T cells showed that infection with LP-BM5 murine leukemia virus also induced a major phenotypic shift in subpopulations defined by their reactivity with mAb 6C10. However, this phenotypic change did not appear to correlate with major functional defects.     

CD19 signaling pathways play a major role for murine AIDS induction and progression

Infection of genetically susceptible mice with the LP-BM5 mixture of murine leukemia viruses including an etiologic defective virus (BM5def) causes an immunodeficiency syndrome called murine AIDS (MAIDS). The disease is characterized by interactions between B cells and CD4(+) T cells resulting in polyclonal activation of both cell types. It is known that BM5def is expressed at highest levels in B cells and that B cells serve as viral APC. The CD19-CD21 complex and CD22 on the surface of B cells play critical roles as regulators of B cell responses to a variety of stimuli, influencing cell activation, differentiation, and survival. CD19 integrates positive signals induced by B cell receptor ligation by interacting with the protooncogene Vav, which leads to subsequent tyrosine phosphorylation of this molecule. In contrast, CD22 negatively regulates Vav phosphorylation. To analyze the role of CD19, CD21, Vav, and CD22 in MAIDS, we infected mice deficient in CD19, CD21 (CR2), Vav-1, or CD22 with LP-BM5 murine leukemia viruses. Infected CR2(-/-) mice developed MAIDS with a time course and severity indistinguishable from that of wild-type mice. In contrast, CD19 as well as Vav-1 deficiency restricted viral replication and suppressed the development of typical signs of MAIDS including splenomegaly, lymphadenopathy, and hypergammaglobulinemia. Finally, CD22 deficiency was found to accelerate MAIDS development. These results provide novel insights into the B cell signaling pathways required for normal induction and progression of MAIDS.     

Role of a cytotoxic-T-lymphocyte epitope-defined, alternative gag open reading frame in the pathogenesis of a murine retrovirus-induced immunodeficiency syndrome

LP-BM5 murine leukemia virus-infected C57BL/6 mice develop profound immunodeficiency and B-cell lymphomas. The LP-BM5 complex contains a mixture of defective (BM5def) and replication-competent helper viruses among which BM5def is the primary causative agent of disease. The BM5def primary open reading frame (ORF1) encodes the single gag precursor protein (Pr60gag). Our lab has recently demonstrated that a novel immunodominant cytotoxic-T-lymphocyte (CTL) epitope (SYNTGRFPPL) is expressed from a +1-nucleotide translational open reading frame of BM5def during the course of normal retrovirus expression. The SYNTGRFPPL CTL epitope may be generated from either of two initiation methionines present, ORF2a or ORF2b, located downstream of the ORF1 initiation site. This study investigates the role(s) of the alternative ORF2-derived gag protein(s) of BM5def in viral pathogenesis. We have examined the disease-inducing capabilities of mutant viruses in which the translational potential of either the initiating ORF2a or ORF2b AUG has been disrupted. Although these mutated viruses are capable of wild-type ORF1 expression, they are unable to induce disease. Our data strongly suggest the existence of a novel ORF2 product(s) that is required for LP-BM5-induced pathogenesis and have potentially broad implications for other retroviral diseases.     

Impaired calcium mobilization in CD4+ and CD8+ T cells in a retrovirus-induced immunodeficiency syndrome, murine AIDS.

After infection with LP-BM5 murine leukemia viruses, susceptible strains of mice develop a severe and progressive immunodeficiency disease, termed murine AIDS (MAIDS), features of which include markedly impaired T cell response to mitogens or specific Ag stimulation and decreased production of IL-2. Since an elevation of intracellular calcium concentration resulting from binding of Ag to the TCR is associated with IL-2 production, T cells from mice either uninfected or infected with LP-BM5 murine leukemia viruses were examined by a calcium mobilization assay. Both CD4+ and CD8+ T cells from infected mice manifested impaired calcium mobilization responses upon in vitro stimulation with anti-CD3 mAb or Con A. The abnormalities appeared early after virus inoculation and showed no difference in time course between subsets of T cells. Frequencies of prestimulation calcium-positive cells among both CD4+ and CD8+ cells in mice with MAIDS were significantly higher than those for uninfected mice. These abnormalities were associated with presence of the MAIDS-inducing defective virus genome, but were not induced by infection of mice genetically resistant to development of MAIDS or with nonpathogenic helper murine leukemia virus, a virus component that induces high spontaneous proliferation of T cells, even in MAIDS-resistant mice.     

Immunotherapy of murine retrovirus-induced acquired immunodeficiency by CD4 T regulatory cell depletion and PD-1 blockade

LP-BM5 retrovirus induces a complex disease featuring an acquired immunodeficiency syndrome termed murine AIDS (MAIDS) in susceptible strains of mice, such as C57BL/6 (B6). CD4 T helper effector cells are required for MAIDS induction and progression of viral pathogenesis. CD8 T cells are not needed for viral pathogenesis, but rather, are essential for protection from disease in resistant strains, such as BALB/c. We have discovered an immunodominant cytolytic T lymphocyte (CTL) epitope encoded in a previously unrecognized LP-BM5 retroviral alternative (+1 nucleotide [nt]) gag translational open reading frame. CTLs specific for this cryptic gag epitope are the basis of protection from LP-BM5-induced immunodeficiency in BALB/c mice, and the inability of B6 mice to mount an anti-gag CTL response appears critical to the initiation and progression of LP-BM5-induced MAIDS. However, uninfected B6 mice primed by LP-BM5-induced tumors can generate CTL responses to an LP-BM5 retrovirus infection-associated epitope(s) that is especially prevalent on such MAIDS tumor cells, indicating the potential to mount a protective CD8 T-cell response. Here, we utilized this LP-BM5 retrovirus-induced disease system to test whether modulation of normal immune down-regulatory mechanisms can alter retroviral pathogenesis. Thus, following in vivo depletion of CD4 T regulatory (Treg) cells and/or selective interruption of PD-1 negative signaling in the CD8 T-cell compartment, retroviral pathogenesis was significantly decreased, with the combined treatment of CD4 Treg cell depletion and PD-1 blockade working in a synergistic fashion to substantially reduce the induction of MAIDS.     

CD40-associated TRAF 6 signaling is required for disease induction in a retrovirus-induced murine immunodeficiency

LP-BM5 retrovirus-infected C57BL/6 mice develop splenomegaly, lymphadenopathy, hypergammaglobulinemia, and immunodeficiency; thus, this disease has been named mouse AIDS. In this syndrome, CD154/CD40 interactions are required for but do not mediate disease by upregulation of CD80 or CD86. We report here that there is nonetheless a necessity for CD40 signaling competence, specifically an intact tumor necrosis factor receptor-associated factor 6 (TRAF 6) binding site.     

Expression of interleukin-1 receptors and their role in interleukin-1 actions in murine microglial cells

Interleukin (IL)-1 is an important mediator of acute brain injury and inflammation, and has been implicated in chronic neurodegeneration. The main source of IL-1 in the CNS is microglial cells, which have also been suggested as targets for its action. However, no data exist demonstrating expression of IL-1 receptors [IL-1 type-I receptor (IL-1RI), IL-1 type-II receptor (IL-1RII) and IL-1 receptor accessory protein (IL-1RAcP)] on microglia. In the present study we investigated whether microglia express IL-1 receptors and whether they present target or modulatory properties for IL-1 actions. RT-PCR analysis demonstrated lower expression of IL-1RI and higher expression of IL-1RII mRNAs in mouse microglial cultures compared with mixed glial or pure astrocyte cultures. Bacterial lipopolysaccharide (LPS) caused increased expression of IL-1RI, IL-1RII and IL-1RAcP mRNAs, induced the release of IL-1beta, IL-6 and prostaglandin-E2 (PGE2), and activated nuclear factor kappaB (NF-kappaB) and the mitogen-activated protein kinases (MAPKs) p38, and extracellular signal-regulated protein kinase (ERK1/2), but not c-Jun N-terminal kinase (JNK) in microglial cultures. In comparison, IL-1beta induced the release of PGE2, IL-6 and activated NF-kappaB, p38, JNK and ERK1/2 in mixed glial cultures, but failed to induce any of these responses in microglial cell cultures. IL-1beta also failed to affect LPS-primed microglial cells. Interestingly, a neutralizing antibody to IL-1RII significantly increased the concentration of IL-1beta in the medium of LPS-treated microglia and exacerbated the IL-1beta-induced IL-6 release in mixed glia, providing the first evidence that microglial IL-1RII regulates IL-1beta actions by binding excess levels of this cytokine during brain inflammation.     

Apoptosis by CD95 (Fas)-dependent and -independent mechanisms in Peyer's patch lymphocytes in murine retrovirus-induced immunodeficiency syndrome.

CD95 (Fas)/CD95 ligand (CD95 L)-mediated apoptosis is thought to be involved in the delayed progression of murine AIDS (MAIDS) induced by LP-BM5 murine leukemia virus (MuLV). We show evidence of apoptosis in lymphocytes of Peyer's patches (PP) at the early stage of MAIDS. Both T and B cells in PP expressed CD95 at the early stage of MAIDS and decreased in number thereafter. The decrease in T cells was not evident in CD95-mutated lpr mice with MAIDS, suggesting that CD95/CD95 L interaction is involved in the apoptosis of T cells in PP during the course of MAIDS. On the other hand, the number of B cells was also decreased in PP of lpr mice with MAIDS. The proliferative ability of B cells in PP of MAIDS mice in response to immunoglobulin M cross-linking or lipopolysaccharide was severely impaired, while the B cells normally proliferated in response to anti-CD40 monoclonal antibody. These findings imply that aberrantly activated B cells in PP undergo apoptosis independently of the CD95/CD95 L system during the course of infection with MAIDS virus.     

Phagocytosis, a potential mechanism for myeloid-derived suppressor cell regulation of CD8+ T cell function mediated through programmed cell death-1 and programmed cell death-1 ligand interaction

CD8(+) T cells become exhausted, inducing cell surface protein programmed cell death-1 (PD-1) as chronic virus diseases or tumors progress, but underlying mechanisms of this are unclear. We previously showed that M-CSF is important for developing tolerogenic dendritic cells (DCs) from human CD14(+) monocytes. In this article, we identify M-CSF-derived DCs (M-DCs) after stimulation with IL-10 as myeloid-derived suppressor cells with additional tolerogenic activities to CD8(+) T cells. IL-10 increased PD-1 ligand expression on M-DC, and IL-10-stimulated M-DCs (M-DC/IL-10) induced expression of PD-1 on, and apoptosis of, CD8(+) T cells and phagocytosed CD8(+) T cells. Enhanced phagocytic activity of M-DC/IL-10 required IFN-γ, which further increased PD-1 ligand and PD-2 ligand expression on M-DC/IL-10. IFN-γ-stimulated M-DC/IL-10 cells were phenotypically macrophage-like cells with little or no expression of CD86, a costimulatory molecule, but with high expression levels of CD14, CD200R, and CD80. No phagocytic activity was detected with GM-CSF-derived DCs. We propose that phagocytosis by IFN-γ-stimulated M-DC/IL-10 cells, which may be DCs or, alternatively, a unique subset of macrophages, may be a mechanism by which IFN-γ-producing CD8(+) T cells are tolerized after type 1 immune responses to chronic virus or tumor, and that IFN-γ links effector CD8(+) T cells to their phagocytic clearance.     

Evolution of B cell lineage lymphomas in mice with a retrovirus-induced immunodeficiency syndrome, MAIDS

Mice infected with LP-BM5 murine leukemia virus develop lymphadenopathy, splenomegaly, hypergammaglobulinemia, and profound immunosuppression associated with enhanced susceptibility to infection. In this study, molecular genetic analyses of spleen and lymph node cells from infected mice showed the early course of disease was associated with polyclonal proliferations of both B and T cells but that by 12 wk oligoclonal expansions of B or T cells could be detected. When near death, the mice were killed and almost all exhibited clonally restricted populations of B cells, and continuous cultures of B lineage cells were established from three of 19 mice. Histologically, lymph nodes with polyclonal lymphoproliferative lesions were indistinguishable from nodes with clonally restricted populations of cells. However, aggressive immunoblastic lymphomas of characteristic morphology were seen in nonlymphoid organs, particularly in the brain. The demonstration of terminal B cell lymphomas in murine AIDS extends the similarities between this syndrome and AIDS in humans.     

The programmed death-1 ligand 1:B7-1 pathway restrains diabetogenic effector T cells in vivo

Programmed death-1 ligand 1 (PD-L1) is a coinhibitory molecule that negatively regulates multiple tolerance checkpoints. In the NOD mouse model, PD-L1 regulates the development of diabetes. PD-L1 has two binding partners, programmed death-1 and B7-1, but the significance of the PD-L1:B7-1 interaction in regulating self-reactive T cell responses is not yet clear. To investigate this issue in NOD mice, we have compared the effects of two anti-PD-L1 Abs that have different blocking activities. Anti-PD-L1 mAb 10F.2H11 sterically and functionally blocks only PD-L1:B7-1 interactions, whereas anti-PD-L1 mAb 10F.9G2 blocks both PD-L1:B7-1 and PD-L1:programmed death-1 interactions. Both Abs had potent, yet distinct effects in accelerating diabetes in NOD mice: the single-blocker 10F.2H11 mAb was more effective at precipitating diabetes in older (13-wk-old) than in younger (6- to 7-wk-old) mice, whereas the dual-blocker 10F.9G2 mAb rapidly induced diabetes in NOD mice of both ages. Similarly, 10F.2H11 accelerated diabetes in recipients of T cells from diabetic, but not prediabetic mice, whereas 10F.9G2 was effective in both settings. Both anti-PD-L1 mAbs precipitated diabetes in adoptive transfer models of CD4(+) and CD8(+) T cell-driven diabetes. Taken together, these data demonstrate that the PD-L1:B7-1 pathway inhibits potentially pathogenic self-reactive effector CD4(+) and CD8(+) T cell responses in vivo, and suggest that the immunoinhibitory functions of this pathway may be particularly important during the later phases of diabetogenesis.     

Adoptive transfer of polyclonal and cloned cytolytic T lymphocytes (CTL) specific for mouse AIDS-associated tumors is effective in preserving CTL responses: a measure of protection against LP-BM5 retrovirus-induced immunodeficiency.

Cytolytic T lymphocytes (CTL) can be raised against C57BL/6 B-cell lymphomas from mice with LP-BM5 murine leukemia virus-induced AIDS (MAIDS). Adoptive transfer of polyclonal anti-MAIDS tumor CTL or two CTL clones specific for the B6-1710 MAIDS lymphoma caused preservation of major histocompatibility complex-restricted and allogeneic CTL responses, which may be interpreted as indices of protection from LP-BM5 murine leukemia virus-induced immunodeficiency.     

程序性死亡-1蛋白及其配体与移植免疫耐受相关性研究进展

通过对B7超家族分子中新热点:程序性死亡-1蛋白(PD-1)及其配体(PD-L1和PD-L2)的相关信息及其在免疫应答调控中的作用,以及其在心脏移植、肝脏移植、异体角膜移植、肾移植等领域的移植免疫耐受研究的结果认为,PD-1信号通路的增强已经成为各移植领域寻求增强免疫耐受的重要新途径,对其机制的进一步研究,将在临床器官移植领域拥有广阔的应用前景。     

Cytolytic T lymphocytes specific for tumors and infected cells from mice with a retrovirus-induced immunodeficiency syndrome.

LP-BM5 retrovirus complex-infected C57BL/6 mice develop immunodeficiency, somewhat analogous to AIDS, termed murine AIDS (MAIDS). After secondary stimulation with syngeneic B-cell lymphomas from LP-BM5-infected mice, C57BL/6 mice produced vigorous CD8+ cytotoxic T lymphocytes specific for MAIDS-associated tumors. An anti-LP-BM5 specificity was suggested because spleen and lymph node cells from LP-BM5-infected mice served as target cells in competition assays, and cells from LP-BM5, but not ecotropic, virus-infected mice functioned as secondary in vitro stimulators to generate cytotoxic T lymphocytes to MAIDS tumors.     

Evidence for interleukin-1-independent stimulation of interleukin-12 and down-regulation by interleukin-10 in Helicobacter pylori-infected murine dendritic cells deficient in the interleukin-1 receptor

Helicobacter pylori infection is characterized by infiltration of cells of the immune system, including dendritic cells, into the gastric mucosa. During chronic inflammation with Helicobacter pylori infection, a variety of cytokines are secreted into the mucosa, including interleukin-1beta (IL-1beta). The role of IL-1 in H. pylori infection was investigated using bone-marrow-derived dendritic cells from wild-type and IL-1 receptor-deficient (IL-1R-/-) mice. Dendritic cells were incubated with H. pylori at a multiplicity of infection of 10 and 100, and cytokine production evaluated. Helicobacter pylori SS1, H. pylori SD4, and an isogenic cagE mutant of SD4 stimulated IL-12, IL-6, IL-1beta, IL-10, and tumor necrosis factor-alpha at comparable levels in dendritic cells from both wild-type and IL-1R-/- mice. IL-10 production required the higher inoculum, while IL-12 was decreased at this bacterial load. Pretreatment of dendritic cells with an antibody to IL-10 resulted in an increased production of IL-12, confirming the down-regulation of IL-12 by IL-10. cagE was required for maximum stimulation of IL-12 by H. pylori. We speculate that the down-regulation of IL-12 by IL-10 at the higher multiplicity of infection represents the modulation of the host inflammatory response in vivo by H. pylori when the bacterial load is high, allowing for persistent colonization of the gastric mucosa.     

Mitochondria-associated hexokinases play a role in the control of programmed cell death in Nicotiana benthamiana

Recent findings suggest a pivotal role for mitochondria-associated hexokinase in the regulation of apoptosis in animal cells. In this study, virus-induced gene silencing (VIGS) of a hexokinase-encoding Hxk1 caused necrotic lesions on leaves, abnormal leaf morphology, and retarded plant growth in Nicotiana benthamiana. Hxk1 was associated with the mitochondria, and this association required the N-terminal membrane anchor. VIGS of Hxk1 reduced the cellular glucose-phosphorylating activity to approximately 31% of control levels without changing the fructose-phosphorylating activity and did not alter hexose phosphate content severely. The affected cells showed programmed cell death (PCD) morphological markers, including nuclear condensation and DNA fragmentation. Similar to animal cell apoptosis, cytochrome c was released into the cytosol and caspase-9- and caspase-3-like proteolytic activities were strongly induced. Furthermore, based on flow cytometry, Arabidopsis thaliana plants overexpressing Arabidopsis HXK1 and HXK2, both of which are predominantly associated with mitochondria, exhibited enhanced resistance to H(2)O(2)- and alpha-picolinic acid-induced PCD. Finally, the addition of recombinant Hxk1 to mitochondria-enriched fractions prevented H(2)O(2)/clotrimazole-induced cytochrome c release and loss of mitochondrial membrane potential. Together, these results show that hexokinase critically regulates the execution of PCD in plant cells, suggesting a link between glucose metabolism and apoptosis.     

Regulatory role of endogenous interleukin-10 in cutaneous inflammatory response of murine wound healing.

This study investigated the role of endogenous interleukin (IL)-10 in cutaneous wound healing. Both IL-10 mRNA and protein were detectable in murine incised wounds for 10 days after injury. The IL-10 protein level peaked 3 h after incision, returned to the normal level by 24 h, but increased again to another peak at 72 h. In situ hybridization studies and immunostaining revealed that epidermal cells and infiltrating mononuclear cells were the major source of IL-10. Neutralizing antibody studies demonstrated that IL-10 inhibited the infiltration of neutrophils and macrophages toward the site of injury. IL-10 also inhibited overexpression of C-C chemokines (monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha) and proinflammatory cytokines (IL-1beta, IL-6, tumor necrosis factor-alpha) in vivo. These results suggest that IL-10 may play an important regulatory role in the phase-specific infiltration of neutrophils and macrophages as well as the cytokine production in the inflammatory response of cutaneous wound healing.