CD11b⁺/Gr-1⁺ myeloid-derived suppressor cells (MDSC) contribute to tumor immune evasion by restraining the activity of CD8⁺ T-cells. Two major MDSC subsets were recently shown to play an equal role ...in MDSC-induced immune dysfunctions: monocytic- and granulocytic-like. We isolated three fractions of MDSC, i.e. CD11b⁺/Gr-1high, CD11b⁺/Gr-1int, and CD11b⁺/Gr-1low populations that were characterized morphologically, phenotypically and functionally in different tumor models. In vitro assays showed that CD11b⁺/Gr-1int cell subset, mainly comprising monocytes and myeloid precursors, was always capable to suppress CD8⁺ T-cell activation, while CD11b⁺/Gr-1high cells, mostly granulocytes, exerted appreciable suppression only in some tumor models and when present in high numbers. The CD11b⁺/Gr-1int but not CD11b⁺/Gr-1high cells were also immunosuppressive in vivo following adoptive transfer. CD11b⁺/Gr-1low cells retained the immunosuppressive potential in most tumor models. Gene silencing experiments indicated that GM-CSF was necessary to induce preferential expansion of both CD11b⁺/Gr-1int and CD11b⁺/Gr-1low subsets in the spleen of tumor-bearing mice and mediate tumor-induced tolerance whereas G-CSF, which preferentially expanded CD11b⁺/Gr-1high cells, did not create such immunosuppressive environment. GM-CSF also acted on granulocyte-macrophage progenitors in the bone marrow inducing local expansion of CD11b⁺/Gr-1low cells. These data unveil a hierarchy of immunoregulatory activity among MDSC subsets that is controlled by tumor-released GM-CSF.
Altered control of T follicular helper (Tfh) cells can lead to generation of autoantibodies and autoimmune manifestations. Signaling pathways that selectively limit pathogenic responses without ...affecting the protective function of Tfh cells are unknown. Here we show that the ATP-gated ionotropic P2X7 receptor restricts the expansion of aberrant Tfh cells and the generation of self-reactive antibodies in experimental murine lupus, but its activity is dispensable for the expansion of antigen-specific Tfh cells during vaccination. P2X7 stimulation promotes caspase-mediated pyroptosis of Tfh cells and controls the development of pathogenic ICOS
IFN-γ-secreting cells. Circulating Tfh cells from patients with systemic lupus erythematosus (SLE) but not primary antiphospholipid syndrome (PAPS), a nonlupus systemic autoimmune disease, were hyporesponsive to P2X7 stimulation and resistant to P2X7-mediated inhibition of cytokine-driven expansion. These data point to the P2X7 receptor as a checkpoint regulator of Tfh cells; thus, restoring P2X7 activity in SLE patients could selectively limit the progressive amplification of pathogenic autoantibodies, which deteriorate patients' conditions.
Dendritic cells (DC) are highly specialized antigen-presenting cells characterized by the ability to prime T-cell responses. Mesenchymal stem cells (MSC) are adult stromal progenitor cells displaying ...immunomodulatory activities including inhibition of DC maturation in vitro. However, the specific impact of MSC on DC functions, upon in vivo administration, has never been elucidated. Here we show that murine MSC impair Toll-like receptor-4 induced activation of DC resulting in the inhibition of cytokines secretion, down-regulation of molecules involved in the migration to the lymph nodes, antigen presentation to CD4+ T cells, and cross-presentation to CD8+ T cells. These effects are associated with the inhibition of phosphorylation of intracellular mitogen-activated protein kinases. Intravenous administration of MSC decreased the number of CCR7 and CD49dβ1 expressing CFSE-labeled DC in the draining lymph nodes and hindered local antigen priming of DO11.10 ovalbumin-specific CD4+ T cells. Upon labeling of DC with technetium-99m hexamethylpropylene amine oxime to follow their in vivo biodistribution, we demonstrated that intravenous injection of MSC blocks, almost instantaneously, the migration of subcutaneously administered ovalbumin-pulsed DC to the draining lymph nodes. These findings indicate that MSC significantly affect DC ability to prime T cells in vivo because of their inability to home to the draining lymph nodes and further confirm MSC potentiality as therapy for immune-mediated diseases.
Human bone marrow multipotent mesenchymal stromal cells are progenitor cells that can be expanded in vitro and differentiate into various cells of mesodermal origin. They contribute to the bone ...marrow reticular niche, where mature B cells and long-lived plasma cells are maintained. Multipotent mesenchymal stromal cells were recently shown to modulate T- and B-cell proliferation and differentiation, dendritic cell maturation, and natural killer activity. These immunoregulatory properties encouraged a possible use of these cells to modulate autoimmune responses in humans. We studied the influence of bone marrow mesenchymal stem cells on highly purified B-cell subsets isolated from healthy donors and total B cells from pediatric systemic lupus erythematosus patients. Bone marrow mesenchymal stem cells promoted proliferation and differentiation into immunoglobulin-secreting cells of transitional and naive B cells stimulated with an agonist of Toll-like receptor 9, in the absence of B cell receptor triggering. They strongly enhanced proliferation and differentiation into plasma cells of memory B-cell populations. A similar effect was observed in response to polyclonal stimulation of B cells isolated from pediatric patients with systemic lupus erythematosus. This study casts important questions on bone marrow mesenchymal stem cells as a therapeutic tool in autoimmune diseases in which B-cell activation is crucially implicated in the pathogenesis of the disease.
Production of antibodies can last for a lifetime, through mechanisms that remain poorly understood. Here, we show that human memory B lymphocytes proliferate and differentiate into plasma cells in ...response to polyclonal stimuli, such as bystander T cell help and CpG DNA. Furthermore, plasma cells secreting antibodies to recall antigens are produced in vivo at levels proportional to the frequency of specific memory B cells, even several years after antigenic stimulation. Although antigen boosting leads to a transient increase in specific antibody levels, ongoing polyclonal activation of memory B cells offers a means to maintain serological memory for a human lifetime.
Because ethical restrictions limit in vivo studies of the human hematolymphoid system, substitute human to small animal xenotransplantation models have been employed. Existing models, however, ...sustain only limited development and maintenance of human lymphoid cells and rarely produce immune responses. Here we show that intrahepatic injection of CD34+human cord blood cells into conditioned newborn$Rag2^{-/-}\gamma_{c}^{-/-}$mice leads to de novo development of B, T, and dendritic cells; formation of structured primary and secondary lymphoid organs; and production of functional immune responses. This provides a valuable model to study development and function of the human adaptive immune system in vivo.
Background T helper 17 cells (TH-17) represent a lineage of effector T cells critical in host defence and autoimmunity. In both mouse and human IL-1β has been indicated as a key cytokine for the ...commitment to TH-17 cells. Cryopyrin-associated periodic syndromes (CAPS) are a group of inflammatory diseases associated with mutations of the NLRP3 gene encoding the inflammasome component cryopyrin. In this work we asked whether the deregulated secretion of IL-1β secondary to mutations characterizing these patients could affect the IL-23/IL-17 axis. Methodology/Principal Findings A total of 11 CAPS, 26 systemic onset juvenile idiopathic arthritis (SoJIA) patients and 20 healthy controls were analyzed. Serum levels of IL-17 and IL-6 serum were assessed by ELISA assay. Frequency of TH17 cells was quantified upon staphylococcus enterotoxin B (SEB) stimulation. Secretion of IL-1β, IL-23 and IL-6 by monocyte derived dendritic cells (MoDCs), were quantified by ELISA assay. A total of 8 CAPS and 11 SoJIA patients were also analysed before and after treatment with IL-1β blockade. Untreated CAPS patients showed significantly increased IL-17 serum levels as well as a higher frequency of TH17 compared to control subjects. On the contrary, SoJIA patients displayed a frequency of TH17 similar to normal donors, but were found to have significantly increased serum level of IL-6 when compared to CAPS patients or healthy donors. Remarkably, decreased IL-17 serum levels and TH17 frequency were observed in CAPS patients following in vivo IL-1β blockade. On the same line, MoDCs from CAPS patients exhibited enhanced secretion of IL-1β and IL-23 upon TLRs stimulation, with a reduction after anti-IL-1 treatment. Conclusion/Significance These findings further support the central role of IL-1β in the differentiation of TH17 in human inflammatory conditions.
Antibodies targeting IL-17A or its receptor, IL-17RA, are approved to treat psoriasis and are being evaluated for other autoimmune conditions. Conversely, IL-17 signaling is critical for immunity to ...opportunistic mucosal infections caused by the commensal fungus Candida albicans, as mice and humans lacking the IL-17R experience chronic mucosal candidiasis. IL-17A, IL-17F, and IL-17AF bind the IL-17RA-IL-17RC heterodimeric complex and deliver qualitatively similar signals through the adaptor Act1. Here, we used a mouse model of acute oropharyngeal candidiasis to assess the impact of blocking IL-17 family cytokines compared with specific IL-17 cytokine gene knockout mice. Anti-IL-17A antibodies, which neutralize IL-17A and IL-17AF, caused elevated oral fungal loads, whereas anti-IL-17AF and anti-IL-17F antibodies did not. Notably, there was a cooperative effect of blocking IL-17A, IL-17AF, and IL-17F together. Termination of anti-IL-17A treatment was associated with rapid C. albicans clearance. IL-17F-deficient mice were fully resistant to oropharyngeal candidiasis, consistent with antibody blockade. However, IL-17A-deficient mice had lower fungal burdens than anti-IL-17A-treated mice. Act1-deficient mice were much more susceptible to oropharyngeal candidiasis than anti-IL-17A antibody-treated mice, yet anti-IL-17A and anti-IL-17RA treatment caused equivalent susceptibilities. Based on microarray analyses of the oral mucosa during infection, only a limited number of genes were associated with oropharyngeal candidiasis susceptibility. In sum, we conclude that IL-17A is the main cytokine mediator of immunity in murine oropharyngeal candidiasis, but a cooperative relationship among IL-17A, IL-17AF, and IL-17F exists in vivo. Susceptibility displays the following hierarchy: IL-17RA- or Act1-deficiency > anti-IL-17A + anti-IL-17F antibodies > anti-IL-17A or anti-IL-17RA antibodies > IL-17A deficiency.
Passive serotherapy can confer immediate protection against microbial infection, but methods to rapidly generate human neutralizing monoclonal antibodies are not yet available. We have developed an ...improved method for Epstein-Barr virus transformation of human B cells. We used this method to analyze the memory repertoire of a patient who recovered from severe acute respiratory syndrome coronavirus (SARS-CoV) infection and to isolate monoclonal antibodies specific for different viral proteins, including 35 antibodies with in vitro neutralizing activity ranging from 10(-8)M to 10(-11)M. One such antibody confers protection in vivo in a mouse model of SARS-CoV infection. These results show that it is possible to interrogate the memory repertoire of immune donors to rapidly and efficiently isolate neutralizing antibodies that have been selected in the course of natural infection.
A main feature of Wiskott-Aldrich syndrome (WAS) is increased susceptibility to autoimmunity. A key contribution of B cells to development of these complications has been demonstrated through studies ...of samples from affected individuals and mouse models of the disease, but the role of the WAS protein (WASp) in controlling peripheral tolerance has not been specifically explored. Here we show that B cell responses remain T cell dependent in constitutive WASp-deficient mice, whereas selective WASp deletion in germinal center B cells (GCBs) is sufficient to induce broad development of self-reactive antibodies and kidney pathology, pointing to loss of germinal center tolerance as a primary cause leading to autoimmunity. Mechanistically, we show that WASp is upregulated in GCBs and regulates apoptosis and plasma cell differentiation in the germinal center and that the somatic hypermutation-derived diversification is the basis of autoantibody development.
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•WASp is highly expressed in germinal center B cells, suggesting its relevance in situ•WASp-defective mouse germinal center B cells are sufficient to induce autoimmunity•WASp-defective germinal center B cells show lower apoptosis rate•WASp-defective B cells acquire self-reactivity during the germinal center reaction
Descatoire et al. show that deletion of Wiskott-Aldrich syndrome (WAS) protein from germinal center B cells leads to reduced B cell apoptosis and broad autoimmunity that is dependent on B cell diversification, pointing to a critical role of WASp in the tolerance mechanisms at play in the germinal center reaction.
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