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Activity report 2012
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
Previous activity reports
Last update : March 12, 2013
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