Anergy and not clonal ignorance determines the fate of B cells that recognize a physiological autoantigen

Autoantibodies to insulin arise spontaneously in the insulin autoimmune syndrome and in type I diabetes. In addition, administration of insulin to individuals without autoimmune disease routinely results in Abs that bind autologous hormone. These observations and findings in transgenic models of tolerance led to an inference that physiological levels of hormones and growth factors, such as insulin, are not sufficient to induce tolerance in B cells, a state termed clonal ignorance. In contrast, we have discovered that virtually all conventional B cells expressing a low affinity anti-insulin transgene interact with endogenous insulin and are effectively silenced for Ig production and for T cell-dependent immune responses. A fraction of transgenic B cells escapes silencing and functions autonomously to produce insulin Abs that may lower fasting blood sugars similar to an insulin autoimmune syndrome. These B cells have characteristics of a B1-like subset and are depleted by hypotonic peritoneal lysis. These findings question the concept of clonal ignorance and show that physiological concentrations of Ag may effectively silence conventional B cells even when the affinity for autoantigen is low. Self-reactivity may arise in the repertoire because of compartmental differences that govern the fate of B cells and not as a result of true clonal ignorance.     

Intensity of the vaccine-elicited immune response determines tumor clearance.

Tumor Ag-specific vaccines used for cancer immunotherapy can generate specific CD8 responses detectable in PBMCs and in tumor-infiltrating lymphocytes. However, human studies have shown that detection of a systemic vaccine-induced response does not necessarily correlate with the occasional instances of tumor rejection. Because this discrepancy might partially be attributable to the genetic heterogeneity of human cancers, as well as to the immunosuppressive effects of previous treatments, we turned to a mouse model in which these variables could be controlled to determine whether a relationship exists between the strength of vaccine-induced immune responses and tumor rejection. We challenged mice with the beta-galactosidase (beta-gal)-expressing tumor cells, C25.F6, vaccinated them with beta-gal-carrying viral vectors, and used quantitative RT-PCR to measure the vaccine-induced immune response of splenocytes directly ex vivo. We found that the strength of the response increased with increasing doses of beta-gal-carrying vector and/or upon boosting with a heterologous beta-gal-carrying virus. Most importantly, we found that the strength of the detected immune response against this foreign Ag strongly correlated with reduction in the number of lung metastases. The results from this mouse model have major implications for the implementation of tumor vaccines in humans.     

Glycan binding avidity determines the systemic fate of adeno-associated virus type 9

Glycans are key determinants of host range and transmissibility in several pathogens. In the case of adeno-associated viruses (AAV), different carbohydrates serve as cellular receptors in vitro; however, their contributions in vivo are less clear. A particularly interesting example is adeno-associated virus serotype 9 (AAV9), which displays systemic tropism in mice despite low endogenous levels of its primary receptor (galactose) in murine tissues. To understand this further, we studied the effect of modulating glycan binding avidity on the systemic fate of AAV9 in mice. Intravenous administration of recombinant sialidase increased tissue levels of terminally galactosylated glycans in several murine tissues. These conditions altered the systemic tropism of AAV9 into a hepatotropic phenotype, characterized by markedly increased sequestration within the liver sinusoidal endothelium and Kupffer cells. In contrast, an AAV9 mutant with decreased glycan binding avidity displayed a liver-detargeted phenotype. Altering glycan binding avidity also profoundly affected AAV9 persistence in blood circulation. Our results support the notion that high glycan receptor binding avidity appears to impart increased liver tropism, while decreased avidity favors systemic spread of AAV vectors. These findings may not only help predict species-specific differences in tropism for AAV9 on the basis of tissue glycosylation profiles, but also provide a general approach to tailor AAV vectors for systemic or hepatic gene transfer by reengineering capsid-glycan interactions.     

Competition between nuclear localization and secretory signals determines the subcellular fate of a single CUG-initiated form of FGF3.

The presumed open reading frame for mouse FGF3, starting at the most 5' AUG codon, predicts a hydrophobic N-terminus characteristic of a signal peptide for secretion. However, in reticulocyte lysates and transfected COS-1 cells, the full-length Fgf-3 cDNA is translated almost exclusively from an upstream CUG codon. The resultant products are distributed in both the nucleus and the secretory pathway, implying that the single CUG-initiated form of FGF3 has dual fates. By analysing a series of deletion and replacement mutants and by linking parts of FGF3 to a heterologous protein, we show that secretion is mediated by cleavage adjacent to the previously defined signal peptide, whereas nuclear localization is determined primarily by a classical but relatively weak bipartite motif. In the context of FGF3, nuclear localization also requires the N-terminal sequences which lie upstream of the signal peptide. Thus, the subcellular fate of FGF3 is determined by the competing effects of signals for secretion and nuclear localization within the same protein, rather than by alternative initiation or processing.     

Antigen presentation determines the fate of the T memory response in vivo after sublethal gamma-irradiation.

The survival of memory T cells is critical to vaccination strategies for infectious diseases and cancer, whereas their elimination may be crucial for treatment of autoimmune states. We examined the consequences of gamma-irradiation, which induces apoptosis of memory T cells in vitro, on the memory response to MHC class I alloantigen in vivo. Sublethal gamma-irradiation of primed mice eliminated accelerated rejection of skin allografts but failed to induce tolerance. Accelerated rejection was restored in irradiated mice by infusion of bone marrow cells expressing the priming alloantigen on immunostimulatory APCs (dendritic cells), whereas the memory response was not restored by infusion of bone marrow cells expressing the priming alloantigen on nonstimulatory APCs (B cells). Strikingly, irradiated mice infused with nonstimulatory bone marrow APCs exhibited long-term survival or tolerance to skin grafts expressing the priming MHC class I alloantigen. The mechanism of tolerance in this setting is explored.     

Mode of targeting to the proteasome determines GFP fate

The ubiquitin–proteasome system is the canonical pathway for protein degradation in eukaryotic cells. GFP is frequently used as a reporter in proteasomal degradation assays. However, there are multiple variants of GFP in use, and these variants have different intrinsic stabilities. Further, there are multiple means by which substrates are targeted to the proteasome, and these differences could also affect the proteasome''s ability to unfold and degrade substrates. Herein we investigate how the fate of GFP variants of differing intrinsic stabilities is determined by the mode of targeting to the proteasome. We compared two targeting systems: linear Ub₄ degrons and the UBL domain from yeast Rad23, both of which are commonly used in degradation experiments. Surprisingly, the UBL degron allows for degradation of the most stable sGFP-containing substrates, whereas the Ub₄ degron does not. Destabilizing the GFP by circular permutation allows degradation with either targeting signal, indicating that domain stability and mode of targeting combine to determine substrate fate. Difficult-to-unfold substrates are released and re-engaged multiple times, with removal of the degradation initiation region providing an alternative clipping pathway that precludes unfolding and degradation; the UBL degron favors degradation of even difficult-to-unfold substrates, whereas the Ub₄ degron favors clipping. Finally, we show that the ubiquitin receptor Rpn13 is primarily responsible for the enhanced ability of the proteasome to degrade stable UBL-tagged substrates. Our results indicate that the choice of targeting method and reporter protein are critical to the design of protein degradation experiments.     

Heterologous expression and epitope mapping of a human small nuclear ribonucleoprotein-associated Sm-B'/B autoantigen.

The U snRNP associated B'/B polypeptides are primary targets of Sm autoantibodies in patients with systemic lupus erythematosus. We have bacterially expressed a Sm-B'/B autoantigen from Raji cells as a fusion with the anthranilate synthase protein from Escherichia coli. The recombinant Sm-B'/B fusion displays comparable immunologic reactivity to the native protein when tested with both monoclonal and polyclonal antibodies. To map Sm-B'/B epitopes, we constructed a series of 12 anthranilate synthase fusions spanning different regions of Sm-B'/B and tested such fusions on immunoblots against a panel of characterized sera. In this manner, we have identified six epitopes, five of which overlap the proline-rich carboxyl-terminus of the protein. Some of these epitopes appear to be conformational. The human sera tested can be divided, according to the epitopes they recognize, into six groups. Finally, we have shown that anti-Sm recognition of the (U1)RNP-specific A protein is attributable to cross-reactivity between the Sm-B'/B and A autoantigens.     

Peripheral tolerance to a nuclear autoantigen: dendritic cells expressing a nuclear autoantigen lead to persistent anergic state of CD4+ autoreactive T cells after proliferation.

It remains unknown why the T cell tolerance to nuclear autoantigens is impaired in systemic autoimmune diseases. To clarify this, we generated transgenic mice expressing OVA mainly in the nuclei (Ld-nOVA mice). When CD4+ T cells from DO11.10 mice expressing a TCR specific for OVA(323-339) were transferred into Ld-nOVA mice, they were rendered anergic, but persisted in vivo for at least 3 mo. These cells expressed CD44(high), CD45RB(low), and were generated after multiple cell divisions, suggesting that anergy is not the result of insufficient proliferative stimuli. Whereas dendritic cells (DCs) from Ld-nOVA (DCs derived from transgenic mice (TgDCs)), which present rather low amount of the self-peptide, efficiently induced proliferation of DO11.10 T cells, divided T cells stimulated in vivo by TgDCs exhibited a lower memory response than T cells stimulated in vitro by peptide-pulsed DCs. Furthermore, we found that repeated transfer of either TgDCs or DCs derived from wild-type mice pulsed with a lower concentration of OVA(323-339) induced a lower response of DO11.10 T cells in Ag-free wild-type recipients than DCs derived from wild-type mice. These results suggest that peripheral tolerance to a nuclear autoantigen is achieved by continuous presentation of the self-peptide by DCs, and that the low expression level of the peptide might also be involved in the induction of hyporesponsiveness.     

Characterization of a cell cycle-dependent nuclear autoantigen

Analysis of reactivity to nuclear antigens in autoimmune sera revealed a serum that produced a previously undescribed cell cycle-dependent immunofluorescence staining pattern. By indirect immunofluorescence using HEp-2 cells as substrate, the serum generated a speckled and nucleolar pleomorphic staining pattern. This characteristic immunofluorescence pattern was detected in different cell lines from various species indicating that the antigen was highly conserved. This serum immunoprecipitated a 85 kDa protein using an extract from [³⁵S]-labeled HeLa cells. Indirect immunofluorescence of proliferating mouse 3T3 cells displayed the characteristic pleomorphic staining which was not observed in serum-starved cells. Resting human and mouse peripheral blood lymphocytes were negative in immunofluorescence while mitogen-stimulated lymphocytes were positive. Germinal centers of mice two weeks after immunization with 2-phenyl-oxazolone showed speckled immunofluorescence staining in the dark zones whereas unimmunized mice were completely negative. Cell synchronization experiments showed a characteristic sequence of locations of the antigen during the cell cycle. In G₁, cells were completely negative. In late G₁, G₁/S and S phase, speckles were visible. In early G₂, speckles were visible, and later in G₂, the nucleoli were positive. During mitosis chromosomes were stained. Further characterization of this antibody specificity and cloning of cDNA are in progress.     

Epithelial entry rather than the ensuing systemic immune response determines the pathogenicity of two Salmonella enterica serovar Typhimurium strains in a mouse model

Most studies of Salmonella enterica serovar Typhimurium infection focus only on the pathogenicity of one strain. We investigated whether differences in pathogenicity of two wild-type S. Typhimurium strains; DT120 and SL1344, were related to gut invasion or the resulting immune response.Oral administration of a ten-fold lower number of SL1344 (10⁶ CFU) as compared to DT120 (10⁷ CFU) resulted in higher bacterial counts in liver and lymph nodes, and led to massive neutrophil infiltration of the spleen, while DT120 administration did not. In contrast, administration of the same dose (10³ CFU) of the two strains intravenously resulted in the same levels of bacteria and neutrophils in spleen and bone marrow. Oral administration of SL1344 led to an increase in neutrophil apoptosis in both spleen and the bone marrow and four out of five mice died before Day 8, while in DT120 mice, no increase in neutrophil apoptosis was observed and all mice survived until Day 8. This study reveals that two wild-type S. Typhimurium strains, despite evoking highly comparable immune responses upon intravenous injection, exhibit diverse pathogenicity in mice and thus suggests that differences in their invasiveness and survival during gut passage determines the success of the ensuing immune response.     

Altered humoral immune response to sheep red blood cells by sheep erythrocyte-soluble hemolysate.

Adult C3H/He mice were rendered unresponsive to a primary injection of sheep red blood cells (SRBC) by pretreatment with sheep hemolysate supernatant (SHS) or subfractions of SHS isolated by column chromatography. The following effects of SHS on the immune response were observed: SHS did not kill antigen-reactive cells, it did not prevent the release of antibody by cells actively synthesizing and secreting antibody, and SHS-induced tolerance was not inhibited or abrogated by methods which terminate or abolish tolerance. In addition, cell-mediated responses were not affected in animals whose humoral responses were suppressed; however, the secondary plaque-forming cell (PFC) response was enhanced by SHS treatment. SDS gel electrophoresis revealed SHS to contain several proteins ranging from 12,000 to approximately 500,000 daltons.     

Tissue communication in a systemic immune response of Drosophila

Several signaling pathways, including the JAK/STAT and Toll pathways, are known to activate blood cells (hemocytes) in Drosophila melanogaster larvae. They are believed to regulate the immune response against infections by parasitoid wasps, such as Leptopilina boulardi, but how these pathways control the hemocytes is not well understood. Here, we discuss the recent discovery that both muscles and fat body take an active part in this response. Parasitoid wasp infection induces Upd2 and Upd3 secretion from hemocytes, leading to JAK/STAT activation mainly in hemocytes and in skeletal muscles. JAK/STAT activation in muscles, but not in hemocytes, is required for an efficient encapsulation of wasp eggs. This suggests that Upd2 and Upd3 are important cytokines, coordinating different tissues for the cellular immune response in Drosophila. In the fat body, Toll signaling initiates a systemic response in which hemocytes are mobilized and activated hemocytes (lamellocytes) are generated. However, the contribution of Toll signaling to the defense against wasps is limited, probably because the wasps inject inhibitors that prevent the activation of the Toll pathway. In conclusion, parasite infection induces a systemic response in Drosophila larvae involving major organ systems and probably the physiology of the entire organism.     

Role of self carriers in the immune response and tolerance. VIII. Suppression of the MOPC-104E idiotypic response by idiotype-modified self, but not by dextran-modified self

We have investigated the suppression of the anti-dextran B1355S immune response using our model of modified self. The anti-dextran response is idiotypically well defined in BALB/c mice. This system enables us to examine the contribution of various predominant idiotypes to the antibody response under conditions of suppression by antigen or by idiotype-specific suppressor cells. Our results demonstrate that the total anti-dextran response can be inhibited by pretreatment of animals with dextran-coupled syngeneic spleen cells; however, the representation of major idiotypes constituting this response are not reduced in percentage. In contrast, pretreatment of mice with MOPC-104E-coupled spleen cells leads to a specific suppression of the private IdI-104E idiotype. The total anti-dextran response remains unchanged, as well as proportions of other major idiotypes known (IdI-588 and IdX). This suppression is mediated by Thy-1.2+, Lyt-2.2+ T cells, as demonstrated by adoptive transfer assays. This system will allow the molecular dissection of the regulation of an idiotypically well-defined system for the suppression by either antigen- or idiotype-specific suppressor T cells.     

Altered expression level of Escherichia coli proteins in response to treatment with the antifouling agent zosteric acid sodium salt

Zosteric acid sodium salt is a powerful antifouling agent. However, the mode of its antifouling action has not yet been fully elucidated. Whole cell proteome of Escherichia coli was analysed to study the different protein patterns expressed by the surface-exposed planktonic cells without and with sublethal concentrations of the zosteric acid sodium salt. Proteomic analysis revealed that at least 27 proteins showed a significant (19 upregulated and 8 downregulated, P?相似文献   

The spliceosomal autoantigen heterogeneous nuclear ribonucleoprotein A2 (hnRNP-A2) is a major T cell autoantigen in patients with systemic lupus erythematosus

A hallmark of systemic lupus erythematosus (SLE) is the appearance of autoantibodies to nuclear antigens, including autoantibodies directed to the heterogeneous nuclear ribonucleoprotein A2 (hnRNP-A2), which occur in 20% to 30% of SLE patients as well as in animal models of this disease. To investigate the underlying cellular reactivity and to gain further insight into the nature and potential pathogenic role of this autoimmune response we characterized the T cell reactivity against hnRNP-A2 in patients with SLE in comparison to healthy controls. Cellular proliferation of peripheral blood T cells to hnRNP-A2 was determined by [3H]thymidine incorporation and T cell clones (TCCs) specific for hnRNP-A2 were grown by limiting dilution cloning; IFNγ, IL-4 and IL-10 in culture supernatants were measured by ELISA. Bioactivity of culture supernatants was determined by incubation of anti-CD3/anti-CD28 stimulated peripheral blood CD4+ T cells with supernatants of TCCs. Stimulation assays performed with peripheral blood mononuclear cells of 35 SLE patients and 21 healthy controls revealed pronounced proliferative responses in 66% of SLE patients and in 24% of the controls, which were significantly higher in SLE patients (p < 0.00002). Furthermore, hnRNP-A2 specific TCCs generated from SLE patients (n = 22) contained a relatively high proportion of CD8+ clones and mostly lacked CD28 expression, in contrast to TCCs derived from healthy controls (n = 12). All CD4+ TCCs of patients and all control TCCs secreted IFNγ and no IL-4. In contrast, CD8+ TCCs of patients secreted very little IFNγ, while production of IL-10 did not significantly differ from other T cell subsets. Interestingly, all CD8+ clones producing IL-10 in large excess over IFNγ lacked expression of CD28. Functional assays showed a stimulatory effect of the supernatants derived from these CD8+CD28- hnRNP-A2 specific TCCs that was similar to that of CD4+CD28+ clones. Taken together, the pronounced peripheral T cell reactivity to hnRNP-A2 observed in the majority of SLE patients and the distinct phenotype of patient-derived CD8+ TCCs suggest a role for these T cells in the pathogenesis of SLE.     

Hormonal regulation of the immune response. I. Induction of an immune response in vitro with lymphoid cells from mice exposed to acute systemic stress

The effect of exposure of mice to stress, by acceleration and ether anesthesia, on in vitro immune responsiveness of their spleen or peritoneal cells, has been studied in different mouse strains. Stress 6, 16, or 24 hr prior to stimulation of explanted lymphoid cells with SRBC in vitro leads to suppression of immune reactivity, whereas a time interval of only 15 min resulted in impaired or normal or even enhanced production of PFC's.