Biomedical subjects
Publications and source records attributed to Brigitte T Huber.
A cell permeable DPP II [also known as DPP2, DPP7, and quiescent cell proline dipeptidase (QPP)] inhibitor has been synthesized. The azabicyclo[3.3.0]octane-based inhibitor is potent and selective and elicits very similar quiescent lymphocyte death to previously characterized inhibitors that are not as selective.
Superantigens are a class of proteins that are derived from microorganisms and have the unique characteristic of stimulating T cells in a TCR Vbeta-specific manner, causing massive T cell proliferation and immune deregulation. For this reason, superantigens have been implicated in the development of multiple diseases. We have previously identified and cloned an EBV-associated superantigen, human endogenous retrovirus (HERV)-K18 envelope protein (Env). This superantigen is transactivated upon IFN-alpha treatment and EBV infection and stimulates human Vbeta13+ T cells. Due to the limited scope of work that can be conducted with human samples and the complexity of HERVs in general, we set out to study the physiological effects of HERV-K18 Env in a murine model. In this report, we demonstrate the superantigen activity of HERV-K18 Env in mice and describe the generation of HERV-K18 transgenics, using a bacterial artificial chromosome as transgenes that allow the faithful reproduction of the expression pattern of this human provirus. From our in vitro and in vivo results we conclude that HERV-K18 Env stimulates Vbeta3+ and Vbeta7+ T cells in mice. The definition of the murine Vbeta specificity and the establishment of a transgenic model will permit the investigation of the role of this superantigen in the life cycle of EBV and its implicated diseases.
EBV, a ubiquitous human herpesvirus, is the causative agent of infectious mononucleosis and is associated with many carcinomas. We have previously shown that the EBV latent genes LMP-1 and LMP-2A (for latent membrane proteins 1 and 2A), transactivate a human endogenous retrovirus (HERV), HERV-K18, in infected B lymphocytes. The envelope (Env) protein of HERV-K18 encodes a superantigen that strongly stimulates a large number of T cells. In this study we report that HERV-K18 env is transactivated even earlier in the infection process, before the establishment of latency; namely, we found that EBV, through its interaction with its cellular receptor CD21, induces the HERV-K18 env gene in resting B lymphocytes. This transactivation is direct and immediate, as up-regulation of transcripts can be detected within 30 min after EBV exposure. Thus, EBV binding to human CD21 on resting B cells triggers the expression of an endogenous superantigen. The biological significance of this superantigen expression for the EBV life cycle is discussed.
Although the causative agent of Lyme disease is definitively known to be the tick-borne spirochete, Borrelia burgdorferi, the etiology of chronic joint inflammation that ensues in a subset of patients remains less well understood. Persistence of arthritis after apparent eradication of the spirochete suggests an autoimmune reaction downstream of the original bacterial infection. We have generated recombinant Ab probes from synovial lesions within affected arthritic joints in an attempt to recapitulate disease-relevant Ag-binding specificities at the site of injury. Using this panel of intra-articular probes, as well as Ab fragments derived from patient peripheral blood, we have identified cytokeratin 10, present in synovial microvascular endothelium, as a target ligand and a putative autoantigen in chronic, antibiotic treatment-resistant Lyme arthritis. Furthermore, there is cross-reactivity between cytokeratin 10 and a prominent B. burgdorferi Ag, outer surface protein A. Release of the self protein in the context of inflammation-induced tissue injury and the resulting in situ response to it could set in motion a feed-forward loop, which amplifies the inflammatory process, thereby rendering it chronic and self-perpetuating, even in the absence of the inciting pathogen.
The identification of the neonatal FcR (FcRn) as an IgG homeostasis regulator has led to research aimed at delineating a role for FcRn in humorally mediated disease. FcRn is a class I-related molecule that prolongs the half-life of serum IgG by preferentially binding IgG at low pH and inhibiting its degradation. Its role in protective immunity to infectious organisms is unknown. We investigated the function of FcRn in the murine model of Lyme arthritis, caused by infection with Borrelia burgdorferi. We infected FcRn(-/-) and wild-type mice with B. burgdorferi and monitored the development of arthritis. Infected FcRn(-/-) mice demonstrated decreased serum levels of anti-B. burgdorferi antibodies and borreliacidal activity. Moreover, these mutant mice developed increased ankle swelling and joint histopathology following infection. Our data suggest that FcRn ameliorates murine Lyme arthritis by preventing the degradation of protective borreliacidal antibodies.
Chronic lymphocytic leukemia (CLL) is unique among malignancies since it represents an accumulation of B-lymphocytes resistant to apoptosis. Several factors are thought to confer this unusual feature to a CLL B-cell. Misbalance between cytoplasmic pro-survival and pro-death molecules, such as Bcl-2, Mcl-1 and alike, appears to be one of the key factors defining B-cell longevity. Autocrine pathways, such as vascular endothelial growth factor-receptor pathway, also contribute to survival. The role of B-cell receptor (BCR) is less straightforward. In the last decade it became clear that CLL does not constitute a uniform disease, but, based on the prevalence of mutations in the BCR heavy chain (IgVH), can be classified into two distinct subgroups. Several molecular markers correlate with IgVH mutations. Some of them, like zeta-chain associated protein kinase, are also involved in BCR signaling and influence cell cycle. Yet the primary pathogenic event leading to increased proliferation and survival in CLL is difficult to ascertain. Molecules involved in BCR signaling pathways and cytoplasmic pro-survival players probably act in concert to confer resistance to apoptosis. In this respect, the role of the B-CLL environment, which includes nurse-like cells and T-cells, cannot be underestimated. Nurse-like cells provide stimuli necessary for perpetuation of life in CLL. On the other hand, abnormal T-cell function, whether it is excessive immunosuppression delivered by regulatory T-cells or insufficient anti-tumor immunity rendered by T-helpers, allows malignant CLL cells to go unnoticed by the cellular immune system.
The primary manifestation of X-linked lymphoproliferative syndrome, caused by a dysfunctional adapter protein, signaling lymphocyte activation molecule-associated protein (SAP), is an excessive T cell response upon EBV infection. Using the SAP-/- mouse as a model system for the human disease, we compared the response of CD8+ T cells from wild-type (wt) and mutant mice to various stimuli. First, we observed that CD8+ T cells from SAP-/- mice proliferate more vigorously than those from wt mice upon CD3/CD28 cross-linking in vitro. Second, we analyzed the consequence of SAP deficiency on CTL effector function and homeostasis. For this purpose, SAP-/- and wt mice were infected with the murine gamma-herpesvirus 68 (MHV-68). At 2 wk postinfection, the level of viral-specific CTL was much higher in mutant than in wt mice, measured both ex vivo and in vivo. In addition, we established that throughout 45 days of MHV-68 infection the frequency of virus-specific CD8+ T cells producing IFN-gamma was significantly higher in SAP-/- mice. Consequently, the level of latent infection by MHV-68 was considerably lower in SAP-/- mice, which indicates that SAP-/- CTL control this infection more efficiently than wt CTL. Finally, we found that the Vbeta4-specific CD8+ T cell expansion triggered by MHV-68 infection is also enhanced and prolonged in SAP-/- mice. Taken together, our data indicate that SAP functions as a negative regulator of CD8+ T cell activation.
More than half of patients with X-linked lympho-proliferative disease, which is caused by a defect in the intracellular adapter protein SH2D1A, suffer from an extreme susceptibility to Epstein-Barr virus. One-third of these patients, however, develop dysgammaglobulenemia without an episode of severe mononucleosis. Here we show that in SH2D1A(-/-) mice, both primary and secondary responses of all Ig subclasses are severely impaired in response to specific antigens. Because germinal centers were absent in SH2D1A(-/-) mice upon primary immunization, and because SH2D1A was detectable in wt germinal center B cells, we examined whether SH2D1A(-/-) B cell functions were impaired. Using the adoptive cotransfer of B lymphocytes from hapten-primed SH2D1A(-/-) mice with CD4(+) T cells from primed wt mice into irradiated wt mice provided evidence that signal transduction events controlled by SH2D1A are essential for B cell activities resulting in antigen specific IgG production. Defects in naive SH2D1A(-/-) B cells became evident upon cotransfer with non-primed wt CD4(+) cells into Rag2(-/-) recipients. Thus, both defective T and B cells exist in the absence of SH2D1A, which may explain the progressive dysgammaglobulinemia in a subset of X-linked lympho-proliferative disease patients without involvement of Epstein-Barr virus.
Lyme arthritis is initiated by the tick-borne spirochete, Borrelia burgdorferi. In a subset of patients, symptoms do not resolve in response to standard courses of antibiotics. Chronic joint inflammation may persist despite spirochetal killing, suggesting an autoimmune etiology. The pathogenic mechanisms that sustain chronic Lyme arthritis have not been fully elucidated, although T cells are believed to play a role. The synovial lesion contains elements of a peripheral lymph node, with lymphoid aggregates, plasma cells and follicular dendritic cells. An analysis of activated cells at the site of injury could yield clues regarding the nature of the response and the identity of potential autoantigens. Using laser-capture microdissection, we have isolated plasma cells from the joint tissue of chronic Lyme arthritis patients who underwent synovectomy. Expressed Ig V regions were amplified by RT-PCR. A majority of isolated cells expressed gamma H chains, which is indicative of a class-switched response. There were a large number of nucleotide substitutions from germline, with a higher fraction of replacement mutations in the CDRs, suggesting a process of Ag-driven selection. We have recovered clonal clusters of cells containing identical junctions and V(D)J rearrangements. Sequence analysis reveals a hierarchy of shared somatic mutations between members of a given clone. Intraclonal diversity among plasma cells of close physical proximity points toward an ongoing process of diversification and affinity maturation, possibly driven by the chronic presence of an autoantigen.
A panel of CD4 T-cell clones was isolated from synovial fluid by single cell flow cytometry from a patient with treatment-resistant Lyme arthritis using a DRB1*0401 major histocompatibility complex (MHC) class II tetramer covalently loaded with outer surface protein A (OspA) peptide164-175, an immunodominant epitope of Borrelia burgdorferi. Sequencing of the T-cell receptors of the OspA reactive clones showed significant skewing of the T-cell receptor repertoire. Of the 101 T-cell clones sequenced, 81 possessed TCR beta chains that were present in at least one other clone isolated. Complete sequencing of both alpha and beta chains of a subset of clones showed that at least two distinct T-cell clones were expanded in vivo. Binding studies using a panel of Ala-substituted peptide ligands were performed to determine potential MHC binding sites of the OspAp164-175 to DRB1*0401. In addition, T-cell clones were tested functionally for their reactivity to the wild-type peptide as well as to altered peptide ligands (APLs) and peptide libraries based on the OspA epitope in order to determine the TCR contact residues and the stringency in T cell recognition. We are among the first to define the characteristics of TCR usage of T cells isolated from an inflamed immune compartment in an individual with an autoimmune disease potentially triggered by a microbial antigen.
Lyme disease is a tick-transmitted inflammatory disorder, caused by the spirochete Borrelia burgdorferi (Bb). Recent discoveries cast new light on Bb dissemination and the ensuing pathogenesis of inflammation. Although the strong proinflammatory Bb lipoproteins have been implicated in the induction of inflammation, they do not seem to act exclusively through Toll-like receptor (TLR) engagement. In fact, mice that are deficient for MyD88, a component of the TLR signaling pathway, manifest similar or increased recruitment of cells into Bb-infected tissues. By contrast, the absence of the chemokine receptor CXCR2 results in reduced inflammation. Overall, these findings highlight the complexity of Lyme disease pathogenesis and identify chemokine pathways as novel therapeutic targets for the control of Bb-induced inflammation.
Chronic lymphocytic leukaemia (CLL) is a unique malignancy where quiescent B cells accumulate in the peripheral blood. Since clinical outcomes in CLL are very heterogeneous, it is of utmost importance to correctly assess the disease prognosis in each individual case. Recently, it has been shown that high ZAP-70 [Zeta-chain (T-cell receptor) associated protein kinase (70 kDa)] expression level strongly correlates with lack of IgV(H) mutations and poor prognosis in B-CLL. As CLL malignant cells are arrested in G(0), we investigated whether Dipeptidyl Peptidase 2 (DPP2), a serine protease that plays a key role in keeping cells in the quiescent state, is involved in cell-cycle control in CLL. We have previously shown that specific inhibition of DPP2 results in apoptosis of normal lymphocytes. In this study, cell apoptosis experiments were conducted in 38 patients with B-CLL. Two distinct subsets of B-CLL were identified, susceptible and resistant to DPP2-inhibition-induced apoptosis. If resistant to apoptosis (42.1%), the CLL cells have higher expression of ZAP-70 and exhibit a worse prognosis, such as shorter treatment-free time period. Thus, resistance vs. susceptibility to DPP2-inhibiton induced apoptosis can be employed as a novel prognostic factor in CLL.
CD18 hypomorph mice expressing reduced levels of the common beta2 integrin chain develop aggravated Lyme carditis, compared to that developed by wild-type (WT) mice, upon infection with the spirochete Borrelia burgdorferi. The enhancement of Lyme carditis in these mice is characterized by increased macrophage infiltration, correlating with augmented expression of the monocyte/macrophage chemoattractant protein 1 (MCP-1). The lack of CD18 results in the deficiency of all beta2 integrins, i.e., CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1/CR3), CD11c/CD18 (p150,95/CR4), and CD11d/CD18. To determine the roles of the various beta2 integrins in controlling the development of aggravated Lyme carditis, disease induction was analyzed in CD11a-/-, CD11b-/-, and CD11c-/- mice. CD11a-/- and CD11c-/- mice, but not CD11b-/- mice, developed aggravated Lyme carditis after exposure to B. burgdorferi. Similarly to CD18 hypomorph mice, CD11c-/- mice expressed higher levels of MCP-1, compared to both WT and CD11a-/- mice, as determined by in vitro analysis of MCP-1 secretion by bone marrow-derived dendritic cells and in vivo analysis of MCP-1 mRNA expression in B. burgdorferi-infected hearts. On the other hand, CD11a deficiency was associated with heightened heart B. burgdorferi burden relative to that of WT mice. Overall, our results suggest that the increased severity of Lyme carditis in CD18 hypomorph mice is caused by deficiency in CD11a or CD11c, possibly via different mechanisms.
Infection of C57BL/6 (B6) mice with the Lyme disease spirochete Borrelia burgdorferi can result in development of arthritis and carditis. B. burgdorferi induces expression of beta2/CD18 integrins, adhesion molecules that mediate the firm adhesion of leukocytes to the endothelium necessary for cellular extravasation during inflammation. The important role of beta2/CD18 integrins during extravasation suggests that these molecules play a role in the development of Lyme arthritis and carditis. The dependency of these inflammatory processes on the beta2 integrins was investigated in CD18 hypomorph mice, which express low levels of CD18. The results indicate that CD18 deficiency did not abrogate development of Lyme arthritis or carditis. Moreover, it resulted in increased severity of Lyme carditis. B. burgdorferi-infected CD18 hypomorph mice showed an increased macrophage infiltration of the heart, while they produced lower levels of borreliacidal anti-B. burgdorferi antibodies compared to wild-type mice. In accordance with these results, we demonstrate that dendritic cells from CD18 hypomorph mice secrete higher levels of monocyte/macrophage chemoattractant protein 1 (MCP-1/CCL2) in response to B. burgdorferi. Similarly, we show by real-time PCR that B. burgdorferi-infected hearts from CD18 hypomorph mice express increased levels of MCP-1 RNA compared to wild-type mice. Overall, our results indicate that beta2 integrin deficiency does not abrogate B. burgdorferi-induced inflammation; rather, it results in increased recruitment of macrophages into the B. burgdorferi-infected heart, likely due to the increased expression of MCP-1 in this tissue. Thus, beta2 integrins may play a regulatory role in B. burgdorferi-induced inflammation beyond mediating adhesion of leukocytes to the endothelium.
OBJECTIVE: To investigate the presence of a host-encoded superantigen as possible etiologic factor in pediatric rheumatic disease. We measured the expression and the ability of interferon-alpha (IFN-alpha) to induce the human endogenous retrovirus HERV-K18 superantigen in juvenile rheumatoid arthritis (JRA) and pediatric systemic lupus erythematosus (SLE). METHODS: Expression levels of HERV-K18 were measured in peripheral blood or synovial fluid mononuclear cells (SFMC) from 13 patients with JRA, 11 pediatric SLE patients, and 24 healthy controls, by semiquantitative reverse transcription-polymerase chain reaction, comparing 18S ribosomal transcripts as endogenous standard. IFN-alpha induction was tested by pretreatment of samples with 2000 U/ml. RESULTS: HERV-K18 expression was significantly elevated in peripheral blood from patients with JRA (mean ratio of HERV-K18 to 18S ribosomal transcripts 2.456, SD 2.122; p = 0.014), but not patients with SLE (mean 0.997, SD 0.579; p = 0.258), compared to controls (mean 0.749, SD 0.598). HERV-K18 transcripts were detected in SFMC of 7/7 JRA patients. IFN-alpha induced HERV-K18 strongly in JRA (mean fold induction = 8.934, SD 15.556) and controls (mean 8.270, SD 6.609), but weakly in SLE (mean 2.432, SD 2.219; p = 0.009). HERV-K18 levels were found to be independent of previously determined modifiers of expression, including Epstein-Barr virus infection, IFN-alpha levels, or the percentage of B cells in peripheral blood. CONCLUSION: HERV-K18 superantigen levels were elevated in JRA patients, but not pediatric patients with SLE, suggesting a possible mechanism for autoimmunity in the former group by superantigen stimulation of autoreactive T cells.
The antigenic component of a common Lyme disease vaccine is recombinant outer surface protein A (rOspA) of Borrelia burgdorferi (Bb), the causative agent of Lyme disease. Coincidentally, patients with chronic, treatment-resistant Lyme arthritis develop an immune response against OspA, whereas those with acute Lyme disease usually do not. Treatment-resistant Lyme arthritis occurs in a subset of Lyme arthritis patients and is linked to HLA.DRB1*0401 (DR4) and related alleles. Recent work from our laboratory identified T cell crossreactivity between epitopes of OspA and lymphocyte function-associated antigen 1alpha(L) chain (LFA-1alpha(L)) in these patients. We generated a form of rOspA, FTK-OspA, in which the LFA-1alpha(L)/rOspA crossreactive T cell epitope was mutated to reduce the possible risk of autoimmunity in genetically susceptible individuals. FTK-OspA did not stimulate human or mouse DR4-restricted, WT-OspA-specific T cells, whereas it did stimulate antibody responses specific for WT-OspA that were similar to mice vaccinated WT-OspA. We show here that the protective efficacy of FTK-OspA is indistinguishable from that of WT-OspA in vaccination trials, as both C3H/HeJ and BALB/c FTK-OspA-vaccinated mice were protected from Bb infection. These data demonstrate that this rOspA-derived vaccine lacking the predicted cross-reactive T cell epitope, but retaining the capacity to elicit antibodies against infection, is effective in generating protective immunity.
Superantigens are microbial proteins that strongly stimulate T cells. We described previously that the Epstein-Barr virus (EBV) transactivates a superantigen encoded by the human endogenous retrovirus, HERV-K18. We now report that the transactivation is dependent upon the EBV latent cycle proteins. Moreover, LMP-2A is sufficient for induction of HERV-K18 superantigen activity.
Bim is a pro-apoptotic member of the Bcl-2 protein family. Bim has three isoforms, EL, L, and S, of which the EL form is the least cytotoxic. We show here that Bim is serine phosphorylated in lymphocytes, predominantly on the EL form. Withdrawal of IL-2 from IL-2-dependent T lymphocytes or culture of thymocytes leads to reduced Bim phosphorylation and apoptosis induction. This decrease in Bim phosphorylation occurs when most cells in culture are still viable, indicating that reduction of Bim phosphorylation may be an early event in apoptosis signaling of lymphocytes.