Tobias Scheel

Eosinophils are required for the maintenance of plasma cells in the bone marrow

Plasma cells are of crucial importance for long-term immune protection. It is thought that long-lived plasma cells survive in specialized niches in the bone marrow. Here we demonstrate that bone marrow eosinophils localized together with plasma cells and were the key providers of plasma cell survival factors. In vitro, eosinophils supported the survival of plasma cells by secreting the proliferation-inducing ligand APRIL and interleukin-6 (IL-6). In eosinophil-deficient mice, plasma cell numbers were much lower in the bone marrow both at steady state and after immunization. Reconstitution experiments showed that eosinophils were crucial for the retention of plasma cells in the bone marrow. Moreover, depletion of eosinophils induced apoptosis in long-lived bone marrow plasma cells. Our findings demonstrate that the long-term maintenance of plasma cells in the bone marrow requires eosinophils.

V-region gene analysis of locally defined synovial B and plasma cells reveals selected B cell expansion and accumulation of plasma cell clones in rheumatoid arthritis

OBJECTIVE To elucidate the development of synovial tissue-specific B cell immune responses, the clonality of individual naive B cells, memory B cells, and plasma cells and their organization and histologic localization in the inflamed tissue were investigated in patients with rheumatoid arthritis (RA). METHODS B and plasma cells were isolated by laser capture microdissection (LCM) from the synovial tissue of patients with RA. In addition, single naive B cells, memory B cells, and plasma cells were sorted from synovial tissue cell suspensions. RNA was extracted from the cells, and Ig VH genes were amplified, cloned, and sequenced. RESULTS Both LCM and single cell sorting analyses showed that naive and memory B cells infiltrated the RA synovial tissue. Comparison of the V-gene repertoire of B and plasma cells suggested that synovial plasma cells were generated, by and large, from locally activated B cells, indicating that a selected population of memory B cells differentiates into large plasma cell clones that then accumulate in the inflamed tissue. Clonally related plasma cells were isolated from separate and distinct localized areas of the tissue, suggesting that the newly generated plasma cells have a high migratory capacity. CONCLUSION These results support the idea of a continuous activation of selected B cell clones, and hence a massive accumulation of plasma cells, in RA synovial tissue. As B cells and their secreted antibodies are an important factor in controlling inflammatory processes, patients with RA displaying intensive synovial tissue lymphocytic infiltrations might benefit from B cell depletion therapy. Early treatment will prevent accumulation of pathogenic plasma cells.

Repression of Cyclic Adenosine Monophosphate Upregulation Disarms and Expands Human Regulatory T Cells

The main molecular mechanism of human regulatory T cell (Treg)-mediated suppression has not been elucidated. We show in this study that cAMP represents a key regulator of human Treg function. Repression of cAMP production by inhibition of adenylate cyclase activity or augmentation of cAMP degradation through ectopic expression of a cAMP-degrading phosphodiesterase greatly reduces the suppressive activity of human Treg in vitro and in a humanized mouse model in vivo. Notably, cAMP repression additionally abrogates the anergic state of human Treg, accompanied by nuclear translocation of NFATc1 and induction of its short isoform NFATc1/αA. Treg expanded under cAMP repression, however, do not convert into effector T cells and regain their anergic state and suppressive activity upon proliferation. Together, these findings reveal the cAMP pathway as an attractive target for clinical intervention with Treg function.

Manifestation of Spontaneous and Early Autoimmune Gastritis in CCR7-Deficient Mice

Autoimmune gastritis is a common autoimmune disorder characterized by chronic inflammatory cell infiltrates, atrophy of the corpus and fundus, and the occurrence of autoantibodies to parietal cell antigen. In CCR7-deficient mice, autoimmune gastritis developed spontaneously and was accompanied by metaplasia of the gastric mucosa and by the formation of tertiary lymphoid organs at gastric mucosal sites. T cells of CCR7-deficient mice showed an activated phenotype in the gastric mucosa, mesenteric lymph nodes, and peripheral blood. In addition, elevated serum IgG levels specific to gastric parietal cell antigen were detected. Because the role of organized lymphocytic aggregates at this inflammatory site is not completely understood, we first analyzed the cellular requirements for the formation of these structures. Autoreactive CD4(+) T cells were pivotal for tertiary lymphoid follicle formation, most likely in cooperation with dendritic cells, macrophages, and B cells. Second, we analyzed the necessity of secondary lymph nodes and tertiary lymphoid organs for the development of autoimmune gastritis using CCR7 single- and CCR7/lymphotoxin α double-deficient mice. Strikingly, manifestation of autoimmune gastritis was observed in the absence of secondary lymph nodes and preceded the development of tertiary lymphoid organs. Taken together, these findings identify an inflammatory process where gastric autoreactive T cells independent of organized tertiary lymphoid organs and classic lymph nodes can induce and maintain autoimmune gastritis.

Prevalence and Gene Characteristics of Antibodies with Cofactor-induced HIV-1 Specificity

Background: Normal immune repertoires have a fraction of cofactor-binding antibodies. Results: Heme exposure results in acquisition of reactivity to gp120 HIV-1 in 24% of antibodies in a seronegative immune repertoire; these antibodies have less mutated variable regions. Conclusion: Human immune repertoires contain high frequency of antibodies with cofactor-induced antigen specificities. Significance: This work enhances understanding of molecular features of heme-sensitive Abs and their contribution to diversification of the immune repertoires. The healthy immune repertoire contains a fraction of antibodies that bind to various biologically relevant cofactors, including heme. Interaction of heme with some antibodies results in induction of new antigen binding specificities and acquisition of binding polyreactivity. In vivo, extracellular heme is released as a result of hemolysis or tissue damage; hence the post-translational acquisition of novel antigen specificities might play an important role in the diversification of the immunoglobulin repertoire and host defense. Here, we demonstrate that seronegative immune repertoires contain antibodies that gain reactivity to HIV-1 gp120 upon exposure to heme. Furthermore, a panel of human recombinant antibodies was cloned from different B cell subpopulations, and the prevalence of antibodies with cofactor-induced specificity for gp120 was determined. Our data reveal that upon exposure to heme, ∼24% of antibodies acquired binding specificity for divergent strains of HIV-1 gp120. Sequence analyses reveal that heme-sensitive antibodies do not differ in their repertoire of variable region genes and in most of the molecular features of their antigen-binding sites from antibodies that do not change their antigen binding specificity. However, antibodies with cofactor-induced gp120 specificity possess significantly lower numbers of somatic mutations in their variable region genes. This study contributes to the understanding of the significance of cofactor-binding antibodies in immunoglobulin repertoires and of the influence that the tissue microenvironment might have in shaping adaptive immune responses.

A cryptic polyreactive antibody recognizes distinct clades of HIV-1 glycoprotein 120 by an identical binding mechanism.

Background: Polyreactive antibodies can efficiently neutralize HIV. Results: Heme induces polyreactivity of a human antibody, which recognizes distinct gp120 clades by an identical binding mechanism. Conclusion: Antigen-binding promiscuity of a polyreactive antibody allows recognition of antigen with high molecular heterogeneity. Significance: Characterization of the interaction of polyreactive antibodies with envelope proteins of HIV reveals novel strategies for control of the virus. Polyreactive antibodies play an important role for neutralization of human immunodeficiency virus (HIV). In addition to intrinsic polyreactive antibodies, the immune system of healthy individuals contains antibodies with cryptic polyreactivity. These antibodies acquire promiscuous antigen binding potential post-translationally, after exposure to various redox-active substances such as reactive oxygen species, iron ions, and heme. Here, we characterized the interaction of a prototypic human antibody that acquires binding potential to glycoprotein (gp) 120 after exposure to heme. The kinetic and thermodynamic analyses of interaction of the polyreactive antibody with distinct clades of gp120 demonstrated that the antigen-binding promiscuity of the antibody compensates for the molecular heterogeneity of the target antigen. Thus, the polyreactive antibody recognized divergent gp120 clades with similar values of the binding kinetics and quantitatively identical changes in the activation thermodynamic parameters. Moreover, this antibody utilized the same type of noncovalent forces for formation of complexes with gp120. In contrast, HIV-1-neutralizing antibodies isolated from HIV-1-infected individuals, F425 B4a1 and b12, demonstrated different binding behavior upon interaction with distinct variants of gp120. This study contributes to a better understanding of the physiological role and binding mechanism of antibodies with cryptic polyreactivity. Moreover, this study might be of relevance for understanding the basic aspects of HIV-1 interaction with human antibodies.

Stable IL-2 Decision Making by Endogenous c-Fos Amounts in Peripheral Memory T-helper Cells

Background: The precise fine-tuning of IL-2 expression is crucial for the immune system. Results: Endogenous expression levels of c-Fos and NFATc2, but not of c-Jun and NF-κBp65, are limiting for IL-2 decision making. Conclusion: Variation in the physiological c-Fos expression leads to diversity and robustness in IL-2 response within the population. Significance: The approach used is beneficial for uncovering basic mechanisms during gene regulation. The cytokine IL-2 performs opposite functions supporting efficient immune responses and playing a key role in peripheral tolerance. Therefore, precise fine-tuning of IL-2 expression is crucial for adjusting the immune response. Combining transcription factor analysis at the single cell and the single nucleus level using flow cytometry with statistical analysis, we showed that physiological differences in the expression levels of c-Fos and NFATc2, but not of c-Jun and NF-κBp65, are limiting for the decision whether IL-2 is expressed in a strongly activated human memory T-helper (Th) cell. Variation in the expression of c-Fos leads to substantial diversity of IL-2 expression in ∼40% of the memory Th cells. The remaining cells exhibit an equally high c-Fos expression level, thereby ensuring robustness in IL-2 response within the population. These findings reveal how memory Th cells benefit from regulated variation in transcription factor expression to achieve a certain stability and variability of cytokine expression in a controlled manner.

IL-2 Expression in Activated Human Memory FOXP3+ Cells Critically Depends on the Cellular Levels of FOXP3 as Well as of Four Transcription Factors of T Cell Activation

The human CD4+FOXP3+ T cell population is heterogeneous and consists of various subpopulations which remain poorly defined. Anergy and suppression are two main functional characteristics of FOXP3+Treg cells. We used the anergic behavior of FOXP3+Treg cells for a better discrimination and characterization of such subpopulations. We compared IL-2-expressing with IL-2-non-expressing cells within the memory FOXP3+ T cell population. In contrast to IL-2-non-expressing FOXP3+ cells, IL-2-expressing FOXP3+ cells exhibit intermediate characteristics of Treg and Th cells concerning the Treg cell markers CD25, GITR, and Helios. Besides lower levels of FOXP3, they also have higher levels of the transcription factors NFATc2, c-Fos, NF-κBp65, and c-Jun. An approach combining flow cytometric measurements with statistical interpretation for quantitative transcription factor analysis suggests that the physiological expression levels not only of FOXP3 but also of NFATc2, c-Jun, c-Fos, and NF-κBp65 are limiting for the decision whether IL-2 is expressed or not in activated peripheral human memory FOXP3+ cells. These findings demonstrate that concomitant high levels of NFATc2, c-Jun, c-Fos, and NF-κBp65 lead in addition to potential IL-2 expression in those FOXP3+ cells with low levels of FOXP3. We hypothesize that not only the level of FOXP3 expression but also the amounts of the four transcription factors studied represent determining factors for the anergic phenotype of FOXP3+ Treg cells.

Adequate immune response ensured by binary IL-2 and graded CD25 expression in a murine transfer model

The IL-2/IL-2Ralpha (CD25) axis is of central importance for the interplay of effector and regulatory T cells. Nevertheless, the question how different antigen loads are translated into appropriate IL-2 production to ensure adequate responses against pathogens remains largely unexplored. Here we find that at single cell level, IL-2 is binary (digital) and CD25 is graded expressed whereas at population level both parameters show graded expression correlating with the antigen amount. Combining in vivo data with a mathematical model we demonstrate that only this binary IL-2 expression ensures a wide linear antigen response range for Teff and Treg cells under real spatiotemporal conditions. Furthermore, at low antigen concentrations binary IL-2 expression safeguards by its spatial distribution selective STAT5 activation only of closely adjacent Treg cells regardless of their antigen specificity. These data show that the mode of IL-2 secretion is critical to tailor the adaptive immune response to the antigen amount. DOI: http://dx.doi.org/10.7554/eLife.20616.001

Neutralization of Japanese Encephalitis Virus by heme-induced broadly reactive human monoclonal antibody.

Geographical expansion and re-emerging new genotypes of the Japanese encephalitis virus (JEV) require the development of novel therapeutic approaches. Here, we studied a non-conventional approach for antibody therapy and show that, upon exposure to heme, a fraction of natural human immunoglobulins acquires high-affinity reactivity with the antigenic domain-III of JEV E glycoprotein. These JEV-reactive antibodies exhibited neutralizing activity against recently dominant JEV genotypes. This study opens new therapeutic options for Japanese encephalitis.