Thomas H. Winkler

The proteasome inhibitor bortezomib depletes plasma cells and protects mice with lupus-like disease from nephritis

Autoantibody-mediated diseases like myasthenia gravis, autoimmune hemolytic anemia and systemic lupus erythematosus represent a therapeutic challenge. In particular, long-lived plasma cells producing autoantibodies resist current therapeutic and experimental approaches. Recently, we showed that the sensitivity of myeloma cells toward proteasome inhibitors directly correlates with their immunoglobulin synthesis rates. Therefore, we hypothesized that normal plasma cells are also hypersensitive to proteasome inhibition owing to their extremely high amount of protein biosynthesis. Here we show that the proteasome inhibitor bortezomib, which is approved for the treatment of multiple myeloma, eliminates both short- and long-lived plasma cells by activation of the terminal unfolded protein response. Treatment with bortezomib depleted plasma cells producing antibodies to double-stranded DNA, eliminated autoantibody production, ameliorated glomerulonephritis and prolonged survival of two mouse strains with lupus-like disease, NZB/W F1 and MRL/lpr mice. Hence, the elimination of autoreactive plasma cells by proteasome inhibitors might represent a new treatment strategy for antibody-mediated diseases.

Siglec-G is a B1 cell-inhibitory receptor that controls expansion and calcium signaling of the B1 cell population.

B1 cells are an important cell population for the production of natural antibodies and for antibacterial immunoglobulin responses. Here we identified the mouse protein Siglec-G as a B1 cell inhibitory receptor. Siglec-G was expressed in a B cell–restricted way, with large amounts present in B1 cells. When overexpressed, Siglec-G inhibited B cell receptor–mediated calcium signaling. Siglec-G-deficient mice had massive expansion of the B1a cell population, which began early in development and was B cell intrinsic. Siglec-G-deficient mice had higher titers of natural IgM antibodies but not a higher penetrance of IgG autoantibodies. Siglec-G-deficient B1 cells showed a strongly enhanced calcium signaling. Our results demonstrate that Siglec-G-dependent negative regulation exists in B1 cells, which may explain the naturally muted signaling response of B1 cells.

Activation of Virus-specific Memory B Cells in the Absence of T Cell Help

Humoral immunity is maintained by long-lived plasma cells, constitutively secreting antibodies, and nonsecreting resting memory B cells that are rapidly reactivated upon antigen encounter. The activation requirements for resting memory B cells, particularly the role of T helper cells, are unclear. To analyze the activation of memory B cells, mice were immunized with human cytomegalovirus, a complex human herpesvirus, and tick-born encephalitis virus, and a simple flavivirus. B cell populations devoid of Ig-secreting plasma cells were adoptively transferred into T and B cell–deficient RAG-1−/− mice. Antigenic stimulation 4–6 d after transfer of B cells resulted in rapid IgG production. The response was long lasting and strictly antigen specific, excluding polyclonal B cell activation. CD4+ T cells were not involved since (a) further depletion of CD4+ T cells in the recipient mice did not alter the antibody response and (b) recipient mice contained no detectable CD4+ T cells 90 d posttransfer. Memory B cells could not be activated by a soluble viral protein without T cell help. Transfer of memory B cells into immunocompetent animals indicated that presence of helper T cells did not enhance the memory B cell response. Therefore, our results indicate that activation of virus-specific memory B cells to secrete IgG is independent of cognate or bystander T cell help.

The EMT-activator Zeb1 is a key factor for cell plasticity and promotes metastasis in pancreatic cancer

Metastasis is the major cause of cancer-associated death. Partial activation of the epithelial-to-mesenchymal transition program (partial EMT) was considered a major driver of tumour progression from initiation to metastasis. However, the role of EMT in promoting metastasis has recently been challenged, in particular concerning effects of the Snail and Twist EMT transcription factors (EMT-TFs) in pancreatic cancer. In contrast, we show here that in the same pancreatic cancer model, driven by Pdx1-cre-mediated activation of mutant Kras and p53 (KPC model), the EMT-TF Zeb1 is a key factor for the formation of precursor lesions, invasion and notably metastasis. Depletion of Zeb1 suppresses stemness, colonization capacity and in particular phenotypic/metabolic plasticity of tumour cells, probably causing the observed in vivo effects. Accordingly, we conclude that different EMT-TFs have complementary subfunctions in driving pancreatic tumour metastasis. Therapeutic strategies should consider these potential specificities of EMT-TFs to target these factors simultaneously.

Perivascular cells expressing annexin A5 define a novel mesenchymal stem cell-like population with the capacity to differentiate into multiple mesenchymal lineages

The annexin A5 gene (Anxa5) was recently found to be expressed in the developing and adult vascular system as well as the skeletal system. In this paper, the expression of an Anxa5-lacZ fusion gene was used to define the onset of expression in the vasculature and to characterize these Anxa5-lacZ-expressing vasculature-associated cells. After blastocyst implantation, Anxa5-lacZ-positive cells were first detected in extra-embryonic tissues and in angioblast progenitors forming the primary vascular plexus. Later, expression is highly restricted to perivascular cells in most blood vessels resembling pericytes or vascular smooth muscle cells. Viable Anxa5-lacZ+ perivascular cells were isolated from embryos as well as adult brain meninges by specific staining with fluorescent X-gal substrates and cell-sorting. These purified lacZ+ cells specifically express known markers of pericytes, but also markers characteristic for stem cell populations. In vitro and in vivo differentiation experiments show that this cell pool expresses early markers of chondrogenesis, is capable of forming a calcified matrix and differentiates into adipocytes. Hence, Anxa5 expression in perivascular cells from mouse defines a novel population of cells with a distinct developmental potential.

CD22 x Siglec-G double-deficient mice have massively increased B1 cell numbers and develop systemic autoimmunity.

CD22 and Siglec-G are inhibitory coreceptors for BCR-mediated signaling. Although CD22-deficient mice show increased calcium signaling in their conventional B2 cells and a quite normal B cell maturation, Siglec-G–deficient mice have increased calcium mobilization just in B1 cells and show a large expansion of the B1 cell population. Neither CD22-deficient, nor Siglec-G–deficient mice on a pure C57BL/6 or BALB/c background, respectively, develop autoimmunity. Using Siglec-G × CD22 double-deficient mice, we addressed whether Siglec-G and CD22 have redundant functions. Siglec-G × CD22 double-deficient mice show elevated calcium responses in both B1 cells and B2 cells, increased serum IgM levels and an enlarged population of B1 cells. The enlargement of B1 cell numbers is even higher than in Siglecg−/− mice. This expansion seems to happen at the expense of B2 cells, which are reduced in absolute cell numbers, but show an activated phenotype. Furthermore, Siglec-G × CD22 double-deficient mice show a diminished immune response to both thymus-dependent and thymus-independent type II Ags. In contrast, B cells from Siglec-G × CD22 double-deficient mice exhibit a hyperproliferative response to stimulation with several TLR ligands. Aged Siglec-G × CD22 double-deficient mice spontaneously develop anti-DNA and antinuclear autoantibodies. These resulted in a moderate form of immune complex glomerulonephritis. These results show that Siglec-G and CD22 have partly compensatory functions and together are crucial in maintaining the B cell tolerance.

B Cell Repertoire Analysis Identifies New Antigenic Domains on Glycoprotein B of Human Cytomegalovirus which Are Target of Neutralizing Antibodies

Human cytomegalovirus (HCMV), a herpesvirus, is a ubiquitously distributed pathogen that causes severe disease in immunosuppressed patients and infected newborns. Efforts are underway to prepare effective subunit vaccines and therapies including antiviral antibodies. However, current vaccine efforts are hampered by the lack of information on protective immune responses against HCMV. Characterizing the B-cell response in healthy infected individuals could aid in the design of optimal vaccines and therapeutic antibodies. To address this problem, we determined, for the first time, the B-cell repertoire against glycoprotein B (gB) of HCMV in different healthy HCMV seropositive individuals in an unbiased fashion. HCMV gB represents a dominant viral antigenic determinant for induction of neutralizing antibodies during infection and is also a component in several experimental HCMV vaccines currently being tested in humans. Our findings have revealed that the vast majority (>90%) of gB-specific antibodies secreted from B-cell clones do not have virus neutralizing activity. Most neutralizing antibodies were found to bind to epitopes not located within the previously characterized antigenic domains (AD) of gB. To map the target structures of these neutralizing antibodies, we generated a 3D model of HCMV gB and used it to identify surface exposed protein domains. Two protein domains were found to be targeted by the majority of neutralizing antibodies. Domain I, located between amino acids (aa) 133–343 of gB and domain II, a discontinuous domain, built from residues 121–132 and 344–438. Analysis of a larger panel of human sera from HCMV seropositive individuals revealed positivity rates of >50% against domain I and >90% against domain II, respectively. In accordance with previous nomenclature the domains were designated AD-4 (Dom II) and AD-5 (Dom I), respectively. Collectively, these data will contribute to optimal vaccine design and development of antibodies effective in passive immunization.

Regulation of autoantibody activity by the IL-23-TH17 axis determines the onset of autoimmune disease

The checkpoints and mechanisms that contribute to autoantibody-driven disease are as yet incompletely understood. Here we identified the axis of interleukin 23 (IL-23) and the TH17 subset of helper T cells as a decisive factor that controlled the intrinsic inflammatory activity of autoantibodies and triggered the clinical onset of autoimmune arthritis. By instructing B cells in an IL-22- and IL-21-dependent manner, TH17 cells regulated the expression of β-galactoside α2,6-sialyltransferase 1 in newly differentiating antibody-producing cells and determined the glycosylation profile and activity of immunoglobulin G (IgG) produced by the plasma cells that subsequently emerged. Asymptomatic humans with rheumatoid arthritis (RA)-specific autoantibodies showed identical changes in the activity and glycosylation of autoreactive IgG antibodies before shifting to the inflammatory phase of RA; thus, our results identify an IL-23–TH17 cell–dependent pathway that controls autoantibody activity and unmasks a preexisting breach in immunotolerance.

Surface μ Heavy Chain Signals Down-Regulation of the V(D)J-Recombinase Machinery in the Absence of Surrogate Light Chain Components

Early B cell development is characterized by stepwise, ordered rearrangement of the immunoglobulin (Ig) heavy (HC) and light (LC) chain genes. Only one of the two alleles of these genes is used to produce a receptor, a phenomenon referred to as allelic exclusion. It has been suggested that pre–B cell receptor (pre-BCR) signals are responsible for down-regulation of the VDJH-recombinase machinery (Rag1, Rag2, and terminal deoxynucleotidyl transferase [TdT]), thereby preventing further rearrangement on the second HC allele. Using a mouse model, we show that expression of an inducible μHC transgene in Rag2−/− pro–B cells induces down-regulation of the following: (a) TdT protein, (b) a transgenic green fluorescent protein reporter reflecting endogenous Rag2 expression, and (c) Rag1 primary transcripts. Similar effects were also observed in the absence of surrogate LC (SLC) components, but not in the absence of the signaling subunit Ig-α. Furthermore, in wild-type mice and in mice lacking either λ5, VpreB1/2, or the entire SLC, the TdT protein is down-regulated in μHC+LC− pre–B cells. Surprisingly, μHC without LC is expressed on the surface of pro–/pre–B cells from λ5−/−, VpreB1−/−VpreB2−/−, and SLC−/− mice. Thus, SLC or LC is not required for μHC cell surface expression and signaling in these cells. Therefore, these findings offer an explanation for the occurrence of HC allelic exclusion in mice lacking SLC components.

Passive Immunization Reduces Murine Cytomegalovirus-Induced Brain Pathology in Newborn Mice

ABSTRACT Human cytomegalovirus (HCMV) is the most frequent cause of congenital viral infections in humans and frequently leads to long-term central nervous system (CNS) abnormalities that include learning disabilities, microcephaly, and hearing loss. The pathogenesis of the CNS infection has not been fully elucidated and may arise as a result of direct damage of CMV-infected neurons or indirectly secondary to inflammatory response to infection. We used a recently established model of mouse CMV (MCMV) infection in newborn mice to analyze the contribution of humoral immunity to virus clearance from the brain. In brains of MCMV-infected newborn mice treated with immune serum, the titer of infectious virus was reduced below detection limit, whereas in the brains of mice receiving control (nonimmune) serum significant amounts of virus were recovered. Moreover, histopathological and immunohistological analyses revealed significantly less CNS inflammation in mice treated with immune serum. Treatment with MCMV-specific monoclonal antibodies also resulted in the reduction of virus titer in the brain. Recipients of control serum or irrelevant antibodies had more viral foci, marked mononuclear cell infiltrates, and prominent glial nodules in their brains than mice treated with immune serum or MCMV-specific antibodies. In conclusion, our data indicate that virus-specific antibodies have a protective role in the development of CNS pathology in MCMV-infected newborn mice, suggesting that antiviral antibodies may be an important component of protective immunological responses during CMV infection of the developing CNS.