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1. Quorum-sensing mechanisms control selection and number of IgM-secreting cells
In humans and mice maintenance of serum IgM levels is critical for the immune system’s function and homeostasis; decreased IgM levels result in diminished protection against bacterial invasion; in contrast, increased IgM titers are often associated with autoimmune manifestations. The mechanisms that control homeostasis of the activated IgM-secreting B cells and whose failure may result in the development of autoimmune hyper-IgM syndromes are, however, largely unknown. We investigated whether control of the number of activated B cells could be achieved if lymphocytes could sense their own density and limit their number accordingly. Many species of bacteria use “quorum-sensing” mechanisms to coordinate their growth rate according to their density. We show in vivo that a “quorum-sensing”-like mechanism, which involves an inhibitory pathway mediated by the FcgRIIB and SHIP, indeed limits the number of activated B cells through the detection of one of their secreted products (IgG). Malfunction of this “quorum-sensing” mechanism may result in uncontrolled B cell activation and autoimmune disease. We studied the fate of mature B cells injected into immune-deficient Rag° hosts. We found that a fraction of the transferred population of LN B cells expanded and persisted for prolonged periods of time. A significant fraction of the surviving B cells express an activated MZ B cell phenotype and about 7-30% of the cells were actively engaged in IgM-secretion. Serum IgM concentrations identical to those of control mice were readily reached in the presence of a reduced number of B cells. These findings confirmed that the numbers of resting and activated B cells are independently regulated, but they did not document the mechanisms that maintain homeostasis of activated B cells. To identify these mechanisms, we investigated whether an established population of B cells could modify the fate of a newly transferred B cell population. We found that with replenishment of the compartment of activated B cells and attainment of normal serum IgM levels, feedback mechanisms regulated the activation of the second population of transferred B cells and, ultimately, IgM production. We investigated the mechanisms of this feedback regulation. We found that contrary to the previous mechanisms described regulating homeostasis, which involve competition for the same niche by cells sharing overlapping survival signals, homeostasis of the innate IgM-secreting B cell pool is achieved when B cell populations are able to monitor the number of activated B cells by detecting their secreted products. Notably, B cell populations are able to assess their density and limit the number of activated IgM-secreting B cells when they sense the levels of secreted IgG via FcgRIIB, a low affinity IgG receptor that is expressed on B cells and acts as a negative regulator of B cell activation by a SHIP-mediated pathway. Such a mechanism is reminiscent of the primordial “quorum-sensing” systems used by some bacteria in which a bacterium senses the accumulation of bacterial signaling metabolites, allowing the bacterium to assess the number of cells present in a population and adapt their growth accordingly. These results reveal a new mechanism of homeostatic regulation, and explain both the development of autoimmune conditions when IgG production is impaired, and the apparent paradox of the beneficial effects of IV Ig therapy in several autoimmune disorders.
2. Biological properties of Memory B cells
Current paradigm defines memory B cells as CD19+IgM-IgG+ cells. In mice populations of CD19+IgM+ expressing AID and able to mount secondary responses have been recently identified. In our studies, we will enlarge the definition of memory B cells to include all B cells that have activated AID transcription (which are YFP+ in AID/YFP Tg mice). Based on our preliminary observations, we postulate that the memory B cell pool contains populations of “central” memory (CD19+IgM+AID+) and “effector” memory (CD19+IgM-AID+) B cells. This is a complete new approach to memory B cell studies. To obtain pure populations of memory B cell we used different experimental protocols. First we immunized SWHELAID/YFP.Rag-/- with OVA-HEL conjugates. Since T cell help was strictly required for the induction of B cell memory, these mice were injected with OVA-specific helper CD4 T cells from OT-II mice transgenic for a TCR that specifically recognizes OVA in the context of MHC I-Ab. Alternatively Rag-deficient hosts were co-transferred with naïve B cells from SWHELAID/YFP.Rag-/- mice and CD4 T cells and immunized with OVA-HEL administered i.p. within 24 hours of cell transfer. We studied B cell and antibody responses to the immunizing HEL. Our results confirm that in the exp protocol used we were able to induce a secondary memory response: a) we characterized the phenotype of the B and T cell populations recovered, b) we detected the presence of anti-HEL IgM and IgG Abs titers in the serum characteristics of a secondary antibody response. We concluded that we were able to generate a population of pure monoclonal memory B cells. We tested whether memory B cell populations are under a strict homeostatic control. We found that independently of the number of naïve HEL-specific B cells present in the host mice (varying from 104 to 107) the number of memory B cell recovered remained the same (at about 106). We are currently investigating whether quorum-sensing mechanism that control the number of activated IgM-secreting B cells also control the number of memory B cells generated in the course of immune responses. As we have now methods to produce pure monoclonal populations of memory B cells we are now finally in position to compare their biological properties. To do that the HEL-specific CD19+IgM+YFP-HEL+ naïve B cells and the CD19+IgM+YFP+HEL+ and CD19+IgM-YFP+HEL+ “central memory and effector memory” B cells generated will be isolated and their functional properties compared, in particular the control of their numbers, their survival requirements, replacement, kinetics and their relative ability to transfer secondary IgG antibody responses and protection after adoptive transfer into new hosts. We will also study the capacity of the immunized mice to mount secondary immune responses and eliminate injected HEL-expressing cells (rate of disappearance of labeled allotype different target HEL-expressing cells transferred from KLK3HEL+ Tg donors) or resist HEL-S.Typhimurium infection (by titrating bacteria cfu). Using Affymetrix micro-arrays we have compared the patterns of gene expression by isolated pure populations of HEL-specific naïve and memory B cells.
3. The homeostasis of the IL-2 producing T cells.
We have shown that the interactions between the CD4+CD25+ regulatory T cells and naïve CD25-CD4+ T cells are of major relevance for the establishment of peripheral CD4 T cell homeostasis. We demonstrated that the IL-2Ra is an absolute requirement for the generation of the regulatory cells. The expression of the high-affinity IL-2Ra endows these cells with the capacity to explore the IL-2 resource, which ensures their peripheral survival, while keeping their number tied to the number of CD4+ T cells that produce IL-2. The indexing of CD4+CD25+Foxp3+ Treg cells to the number of activated IL-2-producing CD4+ T cells may constitute a feedback mechanism that controls T cell expansion during immune responses, thus preventing autoimmune or lymphoproliferative diseases. These results indicated that the number of IL-2-producing cells is relevant for regulatory T cells homeostasis as they may control their maintenance in the peripheral pools. These findings suggest that a quorum-sensing feedback loop, where the IL-2 produced by T cell sub-population is detected by a sub-population of CD4 Treg cells expressing the high-affinity IL-2Ra-chain that controls the number of total CD4 T cells. That is to say: overall CD4 T cell populations adapt their behavior according to the detection of the quantities of IL-2 produced. We are currently investigating the properties and homeostasis of IL-2 producing (IL-2p) T cells. To track IL-2 producing (IL-2p) T cells we will use IL-2/GFP knock-in/knock-out and IL-2/GFP transgenic mice where GFP is expressed under the control of regulatory regions of the IL-2 locus. In these IL-2/GFP mice, T cells transiently express GFP under the control of the IL-2 promoter and are easily detectable by flow cytometry. We have found that the number of IL-2p cells is controlled by the level of IL-2 and show a strict correlation with the number of Treg cells. We believe that, regulation of T cell activation occurs as T cells register the results of their activation by detecting the IL-2 they have made. Il-2 induces expansion of the Treg cell population that control activation and expansion of overall CD4 T cells. Malfunction of this “quorum-sensing” mechanism may result in uncontrolled T cell activation and autoimmune disease.
4. Establishment of new models of humanized mice (Sylvie Garcia).
The use of immunocompromized??RAG-/- or? SCID) mice deficient for the ?g?chain? of IL-2R (gc-/-) and thus deprived of NK cells represented an important progress to the creation of human/mouse chimeras to study human immune cell functions in vivo. Although immune reconstitution of the gc-/- hosts by human hematopoietic progenitors was observed, the number of human T cells recovered was overall poor and their MHC restriction undefined (murine versus human MHC). To improve both thymus selection and peripheral T cell survival, and ensuring correct human MHC restriction, we have derived RAG-/- gc-/- hosts in which we replaced the murine MHC Class I and class II molecules by human HLA-A2 Class I and HLA-DR1 Class II molecules. In addition, we have transgenized our CH1-2 mice with the human SIRPA gene under the control of the c-fms promoter, which drives specific gene expression in mono/macrophages and dendritic cells. CH1-2 SIRPA+ reconstituted chimera support the development of a complete human immune system able to mount HLA-restricted human T cell response upon vaccination.
keywords : Lymphocyte homeostasis; Immunological memory; Immune regulation; Autoimmunity; B cells; T cells; Regulatory T cells; IgM-secreting cells; IgG; FcgRIIB; IL-2; IL-2-producing T cells