Rotaviruses are the leading cause of severe diarrheal disease in young children. Intestinal mucosal IgA responses play a critical role in protective immunity against rotavirus reinfection. Rotaviruses consist of three concentric capsid layers surrounding a genome of 11 segments of double-stranded RNA. The outer layer proteins, VP4 and VP7, which are responsible for viral attachment and entry, are targets for protective neutralizing antibodies. However, IgA mAb’s directed against the intermediate capsid protein VP6, which do not neutralize the virus, have also been shown to protect mice from rotavirus infection and clear chronic infection in SCID mice. We investigated whether the anti-VP6 IgA (7D9) mAb could inhibit rotavirus replication inside epithelial cells and found that 7D9 acted at an early stage of infection to neutralize rotavirus following antibody lipofection. Using electron cryomicroscopy, we determined the three-dimensional structure of the virus-antibody complex. The attachment of 7D9 IgA to VP6 introduces a conformational change in the VP6 trimer, rendering the particle transcriptionally incompetent and preventing the elongation of initiated transcripts. Based on these observations, we suggest that anti-VP6 IgA antibodies confers protection in vivo by inhibiting viral transcription at the start of the intracellular phase of the viral replication cycle.
Ningguo Feng, Jeffrey A. Lawton, Joana Gilbert, Nelly Kuklin, Phuoc Vo, B.V. Venkataram Prasad, Harry B. Greenberg
While the pathologic mechanisms responsible for organ-specific tissue damage in primary biliary cirrhosis (PBC) remain an enigma, it has been suggested that the pathology is mediated by autoreactive T cells infiltrating the intrahepatic bile ducts. Previously, we have documented that there is 100-fold enrichment in the frequency of CD4+ autoreactive T cells in the liver that are specific for peptides encoded by the E2 components of the pyruvate dehydrogenase complexes (PDC-E2). We have also recently characterized the first MHC class I–restricted epitope for PDC-E2, namely amino acid 159–167, a region very similar to the epitope recognized by MHC class II–restricted CD4+ cells and by autoantibodies. The effector functions of these PDC-E2159-167–specific CD8+ cytotoxic T lymphocytes (CTLs) are not well understood. We have taken advantage of tetramer technology and report herein that there is tenfold increase in the frequency of PDC-E2159-167–specific CTLs in the liver as compared with the blood in PBC. In addition, the precursor frequency of the CTLs in blood was significantly higher in early-stage PBC. Of interest was the fact that, upon stimulation with the peptide, the response of PDC-E2159-167 tetramer-positive cells is heterogeneous with respect to IFN-γ synthesis. These data, we believe for the first time, document the enrichment of autoantigen-specific CD8+ T cells in the PBC liver, suggesting that CD8+ T cells play a significant role in the immunopathogenesis of PBC.
Hiroto Kita, Shuji Matsumura, Xiao-Song He, Aftab A. Ansari, Zhe-Xiong Lian, Judy Van de Water, Ross L. Coppel, Marshall M. Kaplan, M. Eric Gershwin
Older bone marrow transplantation (BMT) recipients are at heightened risk for acute graft-versus-host disease (GVHD) after allogeneic BMT, but the causes of this association are poorly understood. Using well-characterized murine BMT models we have explored the mechanisms of increased GVHD in older mice. GVHD mortality, morbidity, and pathologic and biochemical indices were all worse in old recipients. Donor T cell responses were significantly increased in old recipients both in vivo and in vitro when stimulated by antigen-presenting cells (APCs) from old mice, which also secreted more TNF-α and IL-12 after LPS stimulation. In a B6 → B6D2F1 model, CD4+ donor T cells but not CD8+ T cells mediated more severe GVHD in old mice. We confirmed the role of aged APCs in GVHD using B6D2F1 BM chimeras created with either old or young BM. Four months after chimera creation, allogeneic BMT from B6 donors caused significantly worse GVHD in old BM chimeras. APCs from these mice also stimulated greater responses from allogeneic cells in vitro. These data demonstrate a hitherto unsuspected mechanism of amplified donor T cell responses by aged allogeneic host APCs that increases acute GVHD in aged recipients in this BMT model.
Rainer Ordemann, Raymond Hutchinson, Jeffrey Friedman, Steven J. Burakoff, Pavan Reddy, Ulrich Duffner, Thomas M. Braun, Chen Liu, Takanori Teshima, James L.M. Ferrara
Heterozygosity for C1 inhibitor (C1INH) deficiency results in hereditary angioedema. Disruption of the C1INH gene by gene trapping enabled the generation of homozygous- and heterozygous-deficient mice. Mating of heterozygous-deficient mice resulted in the expected 1:2:1 ratio of wild-type, heterozygous, and homozygous-deficient offspring. C1INH-deficient mice showed no obvious phenotypic abnormality. However, following injection with Evans blue dye, both homozygous and heterozygous C1INH-deficient mice revealed increased vascular permeability in comparison with wild-type littermates. This increased vascular permeability was reversed by treatment with intravenous human C1INH, with a Kunitz domain plasma kallikrein inhibitor (DX88), and with a bradykinin type 2 receptor (Bk2R) antagonist (Hoe140). In addition, treatment of the C1INH-deficient mice with an angiotensin-converting enzyme inhibitor (captopril) increased the vascular permeability. Mice with deficiency of both C1INH and Bk2R demonstrated diminished vascular permeability in comparison with C1INH-deficient, Bk2R-sufficient mice. These data support the hypothesis that angioedema is mediated by bradykinin via Bk2R.
Eun D. Han, Ryan C. MacFarlane, Aideen N. Mulligan, Jennifer Scafidi, Alvin E. Davis III
The vitamin D receptor (VDR) is a transcription factor that mediates the actions of its ligand, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], which can promote monocyte/macrophage differentiation and inhibit proliferation and cytokine production by activated T lymphocytes. In this study, VDR knockout (KO) mice were used to investigate the possible role of VDR in hematopoiesis. The relative number of red and white peripheral blood cells and the percentage of bone marrow macrophages did not differ between VDR KO and wild-type mice. 12-O-tetradecanoylphorbol-13-acetate, but not 1,25(OH)2D3, induced differentiation of bone marrow-committed myeloid stem cells from VDR KO mice to monocytes/macrophages. Production of IL-18, a Th1-promoting cytokine, was reduced in macrophages from these mice. Antigen-stimulated spleen cells from VDR KO mice showed an impaired Th1 cell response and had decreased expression of STAT4, a Th1 cell transcription factor. These results demonstrate the absolute requirement of VDR for 1,25(OH)2D3-induced monocyte/macrophage differentiation but show that monocyte/macrophage differentiation can occur in the absence of this receptor. The observed reduction in Th1 population in these mutant mice may be explained by a loss of macrophage IL-18 production or a suppression of STAT4 expression by activated splenocytes.
James O’Kelly, Junichi Hisatake, Yasako Hisatake, June Bishop, Anthony Norman, H. Phillip Koeffler
T cells leave the thymus at a specific time during differentiation and do not return despite elaboration of known T cell chemoattractants by thymic stroma. We observed differentiation stage–restricted egress of thymocytes from an artificial thymus in which vascular structures or hemodynamics could not have been playing a role. Hypothesizing that active movement of cells away from a thymic product may be responsible, we demonstrated selective reduction in emigration from primary thymus by inhibitors of active movement down a concentration gradient (chemofugetaxis). Immature intrathymic precursors were insensitive to an emigration signal, whereas mature thymocytes and peripheral blood T cells were sensitive. Thymic stroma was noted to elaborate at least two proteins capable of inducing emigration, one of which was stromal cell–derived factor-1. Thymic emigration is mediated, at least in part, by specific fugetaxis-inducing factors to which only mature cells respond.
Mark C. Poznansky, Ivona T. Olszak, Richard H. Evans, Zhengyu Wang, Russell B. Foxall, Douglas P. Olson, Kathryn Weibrecht, Andrew D. Luster, David T. Scadden
Susceptibility to myasthenia gravis (MG) is positively linked to expression of HLA-DQ8 and DR3 molecules and negatively linked to expression of the DQ6 molecule. To elucidate the molecular basis of this association, we have induced experimental autoimmune MG (EAMG) in mice transgenic for HLA-DQ8, DQ6, and DR3, and in DQ8×DQ6 and DQ8×DR3 F1 transgenic mice, by immunization with human acetylcholine receptor (H-AChR) in CFA. Mice expressing transgenes for one or both of the HLA class II molecules positively associated with MG (DQ8 and DR3) developed EAMG. T cells from DQ8 transgenic mice responded well to three cytoplasmic peptide sequences of H-AChR (α320-337, α304-322, and α419-437), of which the response to α320-337 was the most intense. DR3 transgenic mice also responded to this sequence very strongly. H-AChR– and α320-337 peptide–specific lymphocyte responses were restricted by HLA class II molecules. Disease resistance in DQ6 transgenic mice was associated with reduced synthesis of anti-AChR IgG, IgG2b, and IgG2c Ab’s and reduced IL-2 and IFN-γ secretion by H-AChR– and peptide α320-337–specific lymphocytes. Finally, we show that DQ8 imparts susceptibility to EAMG and responsiveness to an epitope within the sequence α320-337 as a dominant trait.
Huan Yang, Elzbieta Goluszko, Chella David, David K. Okita, Bianca Conti-Fine, Teh-sheng Chan, Mathilde A. Poussin, Premkumar Christadoss
Insulin-dependent type 1 diabetes is an autoimmune disease mediated by T lymphocytes recognizing pancreatic islet cell antigens. Glutamic acid decarboxylase 65 (GAD65) appears to be an important autoantigen in the disease. However, T cells from both patients with type 1 diabetes and healthy subjects vigorously proliferate in response to GAD65 stimulation ex vivo, leading us to postulate that the critical event in the onset of human diabetes is the activation of autoreactive T cells. Thus, we investigated whether GAD65-reactive T cells in patients with diabetes functioned as previously activated memory T cells, no longer requiring a second, costimulatory signal for clonal expansion. We found that in patients with new-onset type 1 diabetes, GAD65-reactive T cells were strikingly less dependent on CD28 and B7-1 costimulation to enter into cell cycle and proliferate than were equivalent cells derived from healthy controls. We hypothesize that these autoreactive T cells have been activated in vivo and have differentiated into memory cells, suggesting a pathogenic role in type 1 diabetes. In addition, we observed different effects with selective blockade of either B7-1 or B7-2 molecules; B7-1 appears to deliver a negative signal by engaging CTLA-4, while B7-2 engagement of CD28 upregulates T cell proliferation and cytokine secretion.
Vissia Viglietta, Sally C. Kent, Tihamer Orban, David A. Hafler
Chemokines are involved in recruitment and activation of hematopoietic cells in sites of infection and inflammation. The M3 gene of the γ-herpesvirus γHV68 encodes an abundant secreted protein that binds CC chemokines with high affinity. We report here that this gene is essential for efficient induction of lethal meningitis by γHV68. An M3 mutant γHV68 (γHV68-M3.stop) was 100-fold less virulent than wild-type or marker rescue control (γHV68-M3.MR) viruses after intracerebral inoculation. After intracerebral inoculation, γHV68-M3.stop grew to lower titers than γHV68 or γHV68-M3.MR in the brain but spread to and grew normally in the spleen and lung. Expression of several CC chemokines was significantly induced in the CNS by γHV68 infection. Consistent with M3 acting by blockade of CC chemokine action, γHV68 induced a neutrophilic meningeal inflammatory infiltrate, while γHV68-M3.stop induced an infiltrate in which lymphocytes and macrophages predominated. In contrast to the important role of M3 in lethal meningitis, M3 was not required for establishment or reactivation from latent infection or induction of chronic arteritis. These data suggest a role for chemokines in the protection of the nervous system from viral infection and that the M3 protein acts in a tissue-specific fashion during acute but not chronic γHV68 infection to limit CC chemokine–induced inflammatory responses.
Victor van Berkel, Beth Levine, Sharookh B. Kapadia, James E. Goldman, Samuel H. Speck, Herbert W. Virgin IV
Vitiligo is a common depigmenting disorder resulting from the loss of melanocytes in the skin. The pathogenesis of the disease remains obscure, although autoimmune mechanisms are thought to be involved. Indeed, autoantibodies and autoreactive T lymphocytes that target melanocytes have been reported in some vitiligo patients. The objective of this study was to identify pigment cell antigens that are recognized by autoantibodies in vitiligo. Using IgG from vitiligo patients to screen a melanocyte cDNA phage-display library, we identified the melanin-concentrating hormone receptor 1 (MCHR1) as a novel autoantigen related to this disorder. Immunoreactivity against the receptor was demonstrated in vitiligo patient sera by using radiobinding assays. Among sera from healthy controls and from patients with autoimmune disease, none exhibited immunoreactivity to MCHR1, indicating a high disease specificity for Ab’s against the receptor. Inhibition of MCH binding to its receptor by IgG from vitiligo patients was also shown.
E. Helen Kemp, Elizabeth A. Waterman, Brian E. Hawes, Kim O’Neill, Raju V.S.R.K. Gottumukkala, David J. Gawkrodger, Anthony P. Weetman, Philip F. Watson