Activity report 2013

 

The scientific activity of the UBPL concerned studies on lymphocyte homeostasis, selection and regulation. The following themes were pursued during this last year:

 


1- The homeostasis of the population of activated IgM-secreting B cells.


2- Generation of homogeneous populations of memory B cells


3- The homeostasis of the IL-2 producing T cells.


4- The role of IL-15 in IBD


The unit has also engaged in the development of a new model of humanized mice (mice bearing a human immune system).

 

1. Quorum-sensing mechanisms control selection and number of IgM-secreting cells


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 largely unknown. We investigated mechanisms controlling the number of activated B cells. 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. Thus we demonstrated 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.

 

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(A) « Quorum sensing »: The presence of soluble IgG and the ability of the B cells to detect its levels are crucial to the homeostasis of the immune system. “Quorum sensing”: The IgG secreted by activated B cells is detected (sensed) by the inhibitory FcgRIIB expressed by B cells that prevents further B cell activation. That is to say: overall B cell populations adapt their behavior according to the sensing of the quantities of IgG produced. (B) Failure of “Quorum sensing” by defective sensor molecule: The inability to detect soluble IgG because of defects in the FcgRIIB expression (in FcgRIIB-/- mice) or signaling (in SHIP1-/- mice) leads to hyper IgM syndromes and autoimmune disease. (C) Failure of “Quorum sensing” by absence of the sensed molecule IgG also leads to hyper IgM syndromes and autoimmune pathology.

 

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.


Many species of bacteria use quorum sensing to sense the amounts of secreted metabolites and adapt their growth according to their population density. We asked whether similar mechanisms would operate in lymphocyte homeostasis. We investigated the regulation of the size of Interleukin-2-producing CD4+ T-cell (IL-2p) pool using different IL-2-reporter mice. We found that in the absence of either IL-2 or regulatory CD4+ T-cells (Treg) the number of IL-2p-cells increases. Administration of IL-2 decreases the number of cells of the IL-2p-cell subset and pertinently, abrogates their ability to produce IL-2 upon in vivo cognate stimulation, while increasing Treg-cell numbers. We propose that control of the IL-2p-cell numbers occurs via a quorum-sensing-like feedback loop where the produced IL-2 is sensed by both the activated CD4+ T-cell pool and by Treg-cells, which reciprocally regulate cells of the IL-2p-cell subset. In conclusion, IL-2 acts as a self-regulatory circuit integrating the homeostasis of activated and regulatory T cells as CD4+ T-cells restrain their growth by monitoring IL-2 levels thereby preventing uncontrolled responses and autoimmunity.

 

4. The role of IL-15 in IBD.


Using IL-15-deficient, double knockout mice (IL-15koRAGko) we observed that adoptive transfers of wild type CD4+ T cells into these mice resulted in accelerated manifestation of IBD symptoms, i.e. remarkable body weight loss, rectal bleeding and diarrhea compared to IL-15 competent RAGko mice. Detailed characterization of CD4+ T cells transferred into IL-15koRAGko mice showed that, these cells undergo activation and massive proliferation in secondary lymphoid tissue (spleen and local lymph nodes) and subsequently accumulate in the colon. The predominate cytokine secreted by CD4+ T cells in colitic IL-15-deficient mice is IL-17, which is in line with observations made for human Coeliac Disease patients. Thus, IL-15 may negatively regulate the induction of effector IL-17-producing cells previously demonstrated to play a key role in IBD, like Th17 cells and innate lymphoid cells. Interestingly, our pilot experiments suggest more severe destruction of the colon mucosa upon IBD in case disease-mediating CD4+ T cells were devoid of IL-15 receptor.


Initiation of IBD symptoms in IL-15koRAGko mice is even more rapid when the transferred CD4+ T cell population was previously depleted from regulatory T cells (Tregs), which suggests a possible contribution of these cells to development of IBD in IL-15- deficient mice. Taken together, our preliminary data imply a beneficial, anti-inflammatory role of IL-15 in the course of IBD because the absence of this cytokine significantly accelerated the development of IBD. Interestingly, in independent experiments we observed that in the absence of IL-6, the deleterious effects of IL-15 deficiency could be neutralized, which suggests a possible functional link between these two cytokines and may represent a valuable insight for the development of a future cytokine therapy. On the basis of our data we aim to earnestly characterize the anti-inflammatory role of IL-15 in IBD on cellular and functional levels. Additionally, we will concentrate on the role of Tregs in development of IBD in IL-15 deficient mice. Our preliminary data suggest that the frequency of Tregs (CD4+CD25+Foxp3+) significantly decreases in colitic IL-15koRAGko, which addresses a question of unbalanced Treg function in colitis developed upon IL-15 deficiency.

 

5. Human hematopoietic reconstitution and HLA-restricted responses in alymphoid mice.


We generated a new humanized mouse model using alymphoid hosts to study human leucocyte antigen (HLA)-restricted immune responses. For this purpose we enforced the expression of human SIRPα by murine phagocytes in murine major histocompatibility complex (MHC)-deficient HLA-transgenic alymphoid hosts, an approach that allowed the immune reconstitution of non-permissive mice upon human hematopoietic stem cell (HSC) engraftment. We showed that these new mouse/human chimeras were able to generate HLA restricted immune responses to immunization.

Updated on 27/01/2014

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News

Publication of Book "Tractus Immuno-Logicus : a brief history of the Immune System" by Pr Antonio Freitas (eds. Landes Bioscience)

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Lymphocyte Population Biology
Institut Pasteur        
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