Current paradigms of peripheral B cell selection suggest that autoreactive B cells are controlled by clonal deletion, anergy, and developmental arrest. We report that changes to the human antibody repertoire likely resulting from these mechanisms both for a well-characterized autoreactivity from antibodies encoded by the VH4-34 gene and for other hallmarks of an autoreactive repertoire are apparent mainly for class-switched B cells and not for IgM germinal center, IgM memory, or IgM plasma cells. Other possible indicators of autoreactivity found selected with immunoglobulin class include JH6 gene segment usage, increased frequency of B cells with long third hypervariable regions, and distal Jκ gene segment bias. Of particular interest is the finding that B cells with these same characteristics are selected into the lineage of B cells that have undergone the unusual class switch from constant region Cμ to Cδ (Cδ-CS). The Cδ-CS population also displays an increased frequency of charged amino acids localized to the complementarity-determining regions, further suggesting autoreactivity, and evidence is presented that these B cells had undergone extensive receptor editing. Thus, the Cδ-CS lineage may be a “sink” for B cells harboring autoreactive specificities in normal humans. A model for a new tolerizing mechanism that could account for the Cδ-CS lineage is presented.
Nai-Ying Zheng, Kenneth Wilson, Xiaojian Wang, Angela Boston, Grant Kolar, Stephen M. Jackson, Yong-Jun Liu, Virginia Pascual, J. Donald Capra, Patrick C. Wilson
The ability of autoreactive T cells to provoke autoimmune disease is well documented. The finding that immunization with attenuated autoreactive T cells (T cell vaccination, or TCV) can induce T cell–dependent inhibition of autoimmune responses has opened the possibility that regulatory T cells may be harnessed to inhibit autoimmune disease. Progress in the clinical application of TCV, however, has been slow, in part because the underlying mechanism has remained clouded in uncertainty. We have investigated the molecular basis of TCV-induced disease resistance in two murine models of autoimmunity: herpes simplex virus-1 (KOS strain)–induced herpes stromal keratitis and murine autoimmune diabetes in non-obese diabetic (NOD) mice. We find that the therapeutic effects of TCV depend on activation of suppressive CD8 cells that specifically recognize Qa-1–bound peptides expressed by autoreactive CD4 cells. We clarify the molecular interaction between Qa-1 and self peptides that generates biologically active ligands capable of both inducing suppressive CD8 cells and targeting them to autoreactive CD4 cells. These studies suggest that vaccination with peptide-pulsed cells bearing the human equivalent of murine Qa-1 (HLA-E) may represent a convenient and effective clinical approach to cellular therapy of autoimmune disease.
Vily Panoutsakopoulou, Katharina M. Huster, Nami McCarty, Evan Feinberg, Rijian Wang, Kai W. Wucherpfennig, Harvey Cantor
Studies of B cell antigen receptors (BCRs) expressed by leukemic lymphocytes from patients with B cell chronic lymphocytic leukemia (B-CLL) suggest that B lymphocytes with some level of BCR structural restriction become transformed. While analyzing rearranged VHDJH and VLJL genes of 25 non–IgM-producing B-CLL cases, we found five IgG+ cases that display strikingly similar BCRs (use of the same H- and L-chain V gene segments with unique, shared heavy chain third complementarity-determining region [HCDR3] and light chain third complementarity-determining region [LCDR3] motifs). These H- and L-chain characteristics were not identified in other B-CLL cases or in normal B lymphocytes whose sequences are available in the public databases. Three-dimensional modeling studies suggest that these BCRs could bind the same antigenic epitope. The structural features of the B-CLL BCRs resemble those of mAb’s reactive with carbohydrate determinants of bacterial capsules or viral coats and with certain autoantigens. These findings suggest that the B lymphocytes that gave rise to these IgG+ B-CLL cells were selected for this unique BCR structure. This selection could have occurred because the precursors of the B-CLL cells were chosen for their antigen-binding capabilities by antigen(s) of restricted nature and structure, or because the precursors derived from a B cell subpopulation with limited BCR heterogeneity, or both.
Fabio Ghiotto, Franco Fais, Angelo Valetto, Emilia Albesiano, Shiori Hashimoto, Mariella Dono, Hideyuki Ikematsu, Steven L. Allen, Jonathan Kolitz, Kanti R. Rai, Marco Nardini, Anna Tramontano, Manlio Ferrarini, Nicholas Chiorazzi
Regulatory T (TR) cells consist of phenotypically and functionally distinct CD4+ and CD8+ T cell subsets engaged both in maintaining self-tolerance and in preventing anti–non-self effector responses (microbial, tumor, transplant, and so on) that may be harmful to the host. Here we propose that the proinflammatory function of virus-specific memory effector CCR7–CD8+ T cells, which are massively recruited in the liver, are inefficient (in terms of IFN-γ production) in patients with chronic hepatitis C virus (HCV) infection because of the concomitant presence of virus-specific CCR7–CD8+ TR cells producing considerable amounts of IL-10. These CD8+ TR cells are antigen specific, as they can be stimulated by HCV epitopes and suppress T cell responses that are in turn restored by the addition of neutralizing anti–IL-10. This study provides for the first time to our knowledge direct evidence of the existence of virus-specific CD8+ TR cells that infiltrate the livers of patients with chronic HCV infection, identifies IL-10 as a soluble inhibitory factor mediating suppression, and suggests that these cells play a pivotal role in controlling hepatic effector CD8+ T cell responses.
Daniele Accapezzato, Vittorio Francavilla, Marino Paroli, Marco Casciaro, Lucia Valeria Chircu, Agostino Cividini, Sergio Abrignani, Mario U. Mondelli, Vincenzo Barnaba
The prevailing paradigm is that in human rheumatoid arthritis (RA), the accumulation of monocytes and T cells in the joint, mediated in part by such CC chemokine receptors (CCRs) as CCR2 and CCR5, respectively, plays a central role in disease pathogenesis. To further validate this paradigm, we conducted proof-of-principle studies and tested the hypothesis that gene inactivation of Ccr2 or Ccr5 will ameliorate experimental RA. Contrary to our expectations, we found that in two well-established murine models of experimental RA, CCR2 expression in the hematopoietic cell compartment served as a negative regulator of autoantibody production as well as arthritic disease onset, severity, and resolution. In contrast, the RA phenotype in Ccr5-null mice was similar to that of WT mice. Remarkably, the collagen-induced arthritis phenotype of Ccr2–/– mice mimicked closely that of severe human RA, including production of rheumatoid factor, enhanced T cell production, and monocyte/macrophage accumulation in the joints. Our findings demonstrate an essential protective role of CCR2 expression in RA, indicate the existence of alternative receptors responsible for monocyte/macrophage accumulation to inflamed joints, and emphasize the need to clarify carefully the complex effects of the chemokine system in RA before they can be considered as therapeutic targets.
Marlon P. Quinones, Sunil K. Ahuja, Fabio Jimenez, Jason Schaefer, Edgar Garavito, Arun Rao, George Chenaux, Robert L. Reddick, William A. Kuziel, Seema S. Ahuja
While much experimental data shows that vaccination efficiently inhibits a subsequent challenge by a transplantable tumor, its ability to inhibit the progress of autochthonous preneoplastic lesions is virtually unknown. In this article, we show that a combined DNA and cell vaccine persistently inhibits such lesions in a murine HER-2/neu mammary carcinogenesis model. At 10 weeks of age, all of the ten mammary gland samples from HER-2/neu–transgenic mice displayed foci of hyperplasia that progressed to invasive tumors. Vaccination with plasmids coding for the transmembrane and extracellular domain of rat p185neu followed by a boost with rp185neu+ allogeneic cells secreting IFN-γ kept 48% of mice tumor free. At 22 weeks, their mammary glands were indistinguishable from those of 10-week-old untreated mice. Furthermore, the transcription patterns of the two sets of glands coincided. Of the 12,000 genes analyzed, 17 were differentially expressed and related to the antibody response. The use of B cell knockout mice as well as the concordance of morphologic and gene expression data demonstrated that the Ab response is the main mechanism facilitating tumor growth arrest. This finding suggests that a new way can be found to secure the immunologic control of the progression of HER-2/neu preneoplastic lesions.
Elena Quaglino, Simona Rolla, Manuela Iezzi, Michela Spadaro, Piero Musiani, Carla De Giovanni, Pier Luigi Lollini, Stefania Lanzardo, Guido Forni, Remo Sanges, Stefania Crispi, Pasquale De Luca, Raffaele Calogero, Federica Cavallo
IκB proteins play an important role in regulating NF-κB induction following a diverse range of environmental injuries. Studies evaluating IκBβ knock-in mice (AKBI), in which the IκBα gene is replaced by the IκBβ cDNA, have uncovered divergent properties of IκBα and IκBβ that influence their ability to activate hepatic NF-κB and subsequent downstream proinflammatory processes in a stimulus-specific manner. While AKBI mice demonstrated identical levels of hepatic NF-κB activation in response to endotoxin, a significantly reduced level of hepatic NF-κB activation was observed in AKBI mice after liver ischemia/reperfusion (I/R) injury. This reduced level of NF-κB activation in AKBI mice after liver I/R also correlated with decreased induction of serum TNF-α, reduced hepatic inflammation, and increased survival. In contrast, no differences in any of these indicators were observed between AKBI mice and WT littermates after a lethal injection of LPS. Molecular studies suggest that the specificity of IκBα, but not IκBβ, to properly regulate NF-κB induction during the acute phase of I/R injury is due to injury context–specific activation of c-Src and subsequent tyrosine phosphorylation of IκBα on Tyr42. These results demonstrate that IκBα and IκBβ play unique injury context–specific roles in activating NF-κB–mediated proinflammatory responses and suggest that strategies aimed at inhibiting IκBα gene expression may be of potential therapeutic benefit in hepatic I/R injury.
Chenguang Fan, Qiang Li, Yulong Zhang, Xiaoming Liu, Meihui Luo, Duane Abbott, Weihong Zhou, John F. Engelhardt
Vanin-1 is a membrane-anchored pantetheinase highly expressed in the gut and liver. It hydrolyzes pantetheine to pantothenic acid (vitamin B5) and the low-molecular-weight thiol cysteamine. The latter is believed to be a key regulating factor of several essential metabolic pathways, acting through sulfhydryl-disulfide exchange reactions between sulfhydryl groups of the enzymes and the oxidized form, cystamine. Its physiological importance remains to be elucidated, however. To explore this point, we developed Vanin-1–deficient mice that lack free cysteamine. We examined the susceptibility of deficient mice to intestinal inflammation, either acute (NSAID administration) or chronic (Schistosoma infection). We found that Vanin-1–/– mice better controlled inflammatory reaction and intestinal injury in both experiments. This protection was associated with increased γ-glutamylcysteine synthetase activity and increased stores of reduced glutathione, as well as reduced inflammatory cell activation in inflamed tissues. Oral administration of cystamine reversed all aspects of the deficient phenotype. These findings suggest that one cysteamine function is to upregulate inflammation. Consequently, the pantetheinase activity of Vanin-1 molecule could be a target for a new anti-inflammatory strategy.
Florent Martin, Marie-France Penet, Fabrice Malergue, Hubert Lepidi, Alain Dessein, Franck Galland, Max de Reggi, Philippe Naquet, Bouchra Gharib
Glucocorticoids have potent immunosuppressive properties, but their effects are often modulated by the conditions prevailing in the local immune milieu. In this study we determined whether the action of glucocorticoids is influenced by the degree of signaling during T cell activation. We found that dexamethasone (Dex) effectively suppressed T cell receptor–induced (TCR-induced) proliferation of naive CD4+ T cells, through a mechanism involving downregulation of c-Fos expression and inhibition of activator protein-1 (AP-1), nuclear factor of activated T cells (NF-AT), and NF-κB transcriptional activity. However, enhancement of TCR signaling by CD28- or IL-2–mediated costimulation abrogated the suppressive effect of Dex on c-Fos expression and AP-1 function and restored cellular proliferation. The amount of signaling through the MAPK pathway was critical in determining the effect of Dex on T cell activation. In particular, costimulatory signaling via MAPK kinase (MEK) and extracellular signal–regulated kinase (ERK) was essential for the development of T cell resistance to Dex. Selective blockade of MEK/ERK signal transduction abolished the costimulation-induced resistance. In contrast, transmission of IL-2 signals via STAT5 and CD28 signals via NF-κB remained inhibited by Dex. These results imply that the immune system, by regulating the degree of local costimulation through MEK/ERK, can modify the effect of glucocorticoids on T cells. Moreover, these findings suggest that MAPK inhibitors may offer a therapeutic solution for glucocorticoid resistance.
Daphne C. Tsitoura, Paul B. Rothman
Sepsis is a common, life-threatening disease for which there is little treatment. The cysteine protease dipeptidyl peptidase I (DPPI) activates granule-associated serine proteases, several of which play important roles in host responses to bacterial infection. To examine DPPI’s role in sepsis, we compared DPPI–/– and DPPI+/+ mice using the cecal ligation and puncture (CLP) model of septic peritonitis, finding that DPPI–/– mice are far more likely to survive sepsis. Outcomes of CLP in mice lacking mast cell DPPI reveal that the absence of DPPI in mast cells, rather than in other cell types, is responsible for the survival advantage. Among several cytokines surveyed in peritoneal fluid and serum, IL-6 is highly and differentially expressed in DPPI–/– mice compared with DPPI+/+ mice. Remarkably, deleting IL-6 expression in DPPI–/– mice eliminates the survival advantage. The increase in IL-6 in septic DPPI–/– mice, which appears to protect these mice from death, may be related to reduced DPPI-mediated activation of mast cell tryptase and other peptidases, which we show cleave IL-6 in vitro. These results indicate that mast cell DPPI harms the septic host and that DPPI is a novel potential therapeutic target for treatment of sepsis.
Jon Mallen–St. Clair, Christine T.N. Pham, S. Armando Villalta, George H. Caughey, Paul J. Wolters