Prsentation PowerPoint

1
António A. Freitas
Sylvie Garcia Dominique Rueff-Juy Catarina
Leitao Caroline Montaudouin Aude Thiriot Bruno
Zaragoza Marie-Pierre Mailhé Anne Marie Drapier
Fabien Agenes Afonso Almeida Jose Borghans Alix
de la Coste Antoine El Azzi Jaime Franco
Emmanuelle Gaudin
Yi Hao Nicolas Legrand Angela McLean Ninog
Peresse Manuela Rosado Vanesa Sanchez-Guajardo Rob
ert Wildin
2
Fabien Agenes Emmanuelle Gaudin Yi Hao Caroline
Montaudouin Manuela Rosado Angela McLean
3
B cell Homeostasis Mechanisms that control
lymphocyte numbers
4
The I. S. shows a return tendency, due to
density-dependent processes, to approach a
stationary distribution of population densities
usually referred to as Homeostasis.
5
Return after  perturbation 
6
Homeostatic control shapes lymphocyte
repertoires and thus, the patterns of immune
responses
7
Homeostasis of B cell populations
8
Why the number of B cells is what it is?
9
B cell population size

• Rates of B cell production
• Anatomical space
• Resources

10
Repopulation Strategy
B6.Rag2-/- hosts. Lethal irradiation
Purpose to obtain different groups of BM
chimeras with a diminished production of B cells.
11
Bone Marrow Chimeras
Reconstitution 6-8 weeks
Quantify the different B Lymphocyte populations
by Flow cytometry and the serum Igs by ELISA
12
MIXED B6.Ly5a/Rag2-/- BM CHIMERAS
13
Mice with a three fold reduced production of B
cells have the same number of peripheral naïve B
cells as normal mice
14
Can we alter the B cell pool size by modifying
the available peripheral space?
15
PARABIOSIS
Normal mouse
B cell deficient (empty) mouse
In parabiosis the two mice share circulation -
we can study chimerism and numbers of B cells
16
PARABIOSISB cell number after parabiosis between
B6.Ly5a and Rag2-deficient B6.Ly5b B cell
deficient mutant mice.
17
PARABIOSISB cell numbers in triads of one normal
B6.Ly5a and two B-cell deficient mice
18
PARABIOSIS

• The physiological BM B cell production of one
  mouse is sufficient to replenish the peripheral B
  cell pools of two to three mice.

Thus, two different approaches give similar
results.
19
In normal mice there is an excess of daily B cell
production, suggesting the presence of
competition among the newly formed and the
resident cells to survive in the peripheral pools.
Conclusion
20
Competition Criteria

• 1. Different equilibrium population sizes in the
  presence of competitors
• 2. Different population dynamics and
  life-expectancies in the presence of competitors

21
Selection relies not only on the intrinsic
properties of the cell and on its interactions
with antigen, but it is also determined by
theNumber and nature of competing cells.
Lymphocytes by resource consumption can
indirectly modify the fate of other competing
cells.
22
Types of competition

• Interference competition populations may
  interact directly with each other or one
  population can prevent a second population from
  occupying an habitat and from exploiting the
  resources in it.
• Exploitation competition different populations
  have a common need for resources present in
  limited supply.
• In this case competition is directly related to
  level of resources available.

23
Competition occurs when resources are limiting.
24
Can we compare the fate of lymphocyte populations
at equilibrium and during growth?
What happens during early life, when lymphocyte
populations are expanding and the resources are
in excess?
25
Fate of mature B cells transferred into adult or
newborn hosts
26
Fate of B Cells transferred into adult or
neonatal hosts
27
NEONATAL VS. ADULT

(GROWTH VS EQUILIBRIUM)

• In neonatal mice the excess of resources
  determines a permissive environment where B cells
  can survive.
• In the adult, resources become scarce,
  competition starts and only a fraction of the
  injected B cells is able to survive.

28
What happens if the B cells are transferred into
an empty B cell deficient host where the
resources are not being used?
29
Fate of mature B cell populations in B cell
deficient hosts
Rag-/- hosts.
Transfer of mature LN B cells from allotype
different donors
30
Fate of LN B cells transferred into B cell
deficient hosts
31
Changes in B cell phenotype after transfer
32
Conclusion

• Upon transfer into immune-deficient hosts, a
  selected fraction of the transferred B cells
  expands and persists
• However it does not fill up the host, which
  indicates that you require a continuous new B
  cell input to do so and that B cell numbers may
  be also controlled by terminal differentiation.

33
Conclusion
B cell survival is related to resource level
34
RESOURCES

• RESOURCES can be defined as any factor for which
  an increase in its availability leads to the
  increased "per capita" reproductive rates,

through at least some range of its availability.
E.R. Pianka
35
Antigen as a resource

• Monoclonal BCR Tg mice have different B cell
  numbers according to the specificity of their Ig
  receptor.
• B cell numbers are determined by the quantities
  of self-antigen
• in the HEL/anti-HEL Tg model.
• Thus, peripheral B cell numbers may be limited
  by the availability of BCR ligands. This
  indicates indicating that antigen may function
  either as a resource or as a surrogate resource.

36
However, for a diverse B cell population antigen
is never limiting!
37
ANTIGEN

• SURROGATE RESOURCE?
• a resource, not in itself in limited supply,
  which is competed for because of the access it
  provides to some other resource, which is or may
  become limited in supply.

38
Antigen recognition acts as a surrogate resource
and allows B cells usage of BAFF, which is the
limiting survival resource.

• B cells occupy Niches, which can be represented
  as multi-dimensional spaces (n1, n2, n3, n4, n5,
  n?) defined by the different resources that
  affect survival and proliferation of a particular
  cell type.

39
Homeostasis of B cell populations
40
What about the number of activated IgM-secreting
cells?
41
MIXED B6.Ly5a/Rag2-/- BM CHIMERAS
100
?
?
?
Number of spleen B cells (x10-6)
?
?
?
10
0
2
1
3
5
4
Number of Ly5a BM pre-B cells (x10-6)
42
MIXED Ly5a / mMT BM CHIMERAS
100
l
l
l
l
l
l
l
l
l
l
Number of IgM-secreting cells (x10-6)
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
10
5
0
1
2
3
4
5
Number of Ly5a BM pre-B cells (x10-6)
43
MIXED Ly5a / mMT BM CHIMERAS
2500
1000
l
l
l
l
l
l
l
l
l
l
l
Serum IgM levels (mg/ml)
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
100
0
1
2
3
4
5
Number of Ly5a BM pre-B cells (x10-6)
44

• The number of IgM-secreting B cells is
  independent of the peripheral B cell pool size
  and of the number of BM pre-B cells.
• suggesting an autonomous homeostatic regulation
  of the IgM-secreting B cells.

45
What happens to the IgM-secreting B cell
compartment after B cell transfer into empty
hosts?
46
Fate of LN B cells transferred into B cell
deficient hosts
Days after transfer
47
The number of IgM-secreting B cells is
independent of the number of B cells transferred
and of the number of total B cells present in the
hosts.
Conclusion
Suggesting again an autonomous homeostatic
regulation of the IgM-secreting B cells.
48
Conclusion

• The independent homeostatic regulation of the
  resting and activated B cell compartments implies
  an hierarchical organization of the immune system
  in which the first priority is the maintenance of
  normal serum IgM levels.
• It provides an efficient mechanism to ensure both
  a first natural barrier of protection and a
  maximum of repertoire diversity.

49
How is the number of IgM-secreting cells
controlled?
50
Sequential transfer of mature LN B cells
Rag2-/- hosts.
51
Sequential transfer of mature LN B cells
52
What is the distribution of both the first and
the second population among the hosts resting
and activated B cell pools?
53
Distribution of the 1st (Ly5.1-) and the 2nd
(Ly5.1) B cell population among the resting
(IgDbrightIgMlow) and activated (IgDlowIgMbright)
host B cells
54
Feedback inhibition of IgM secretion
55
Conclusion

• Mature B cells transferred into an B cell
  deficient host, are activated, persist, and can
  feedback regulate the state of activation of a
  second population of transferred B cells
• First come, First served

56
Conclusions
The number of peripheral B cells is not
determined by the rates of BM production, but
controlled in the peripheral compartments. The
number of resting and activated IgM-secreting
cells have independent homeostatic
controls. IgM-secreting B cells exert an active
feedback control preventing the entry of new B
cells into the activated B cell compartment.
57
Is feedback regulation mediated bycompetitive
pre-emption (interference) by activated B
cells?Or/and byB cell products Igs?
58
The IgMs-/- mouse
The absence of secreted forms of IgM may allow to
determine its role in feedback regulation of IgM
production
59
Sequential transfer of mature LN B cells
Rag2-/- hosts.
60
Feedback inhibition of IgM secretion
IgMs produced by 2nd population
61
Conclusion
Feedback control of terminal B cell
differentiation into IgM-secretion is not serum
IgM dependent but determined by the number of
activated B cells.
62
Can the feedback regulation be mediated by
secreted IgG?
63
Sequential transfer of mature LN B cellsusing T
cell deficient mice
Rag2-/- hosts.
64
No IgG in absence of T cells
65
No inhibition in absence of T cells and IgG
66
Sequential transfer of mature LN B cellsusing
FcgRII-/- deficient mice
Rag2-/- hosts.
67
Using FcgRII deficient mice
Groups 1) 2) 3) 4) 5)
1st population (µa/Ly5.1) 5x106 B cells of total
LN B6 IgHa Ly5.1 B6 IgHa Ly5.1 // B6 IgHa
Ly5.1 //
2nd population (µb/Ly5.2) 5x106 B cells of total
LN // Fc?RII-/- Fc?RII-/- B6 IgHb Ly5.2 B6 IgHb
Ly5.2
6 weeks later
8 weeks later
68
IgM concentrations in the serum
10000
p0,045
1000
IgMb concentration (µg/ml)
100
10
1
WT Fc?-/-
WT
1st (IgHa) 2nd (IgHa)
WT
WT WT
Fc?-/-
69
IgGs produced by the first B cell population
inhibit the activation and IgM production by the
second B cell population.This inhibition is
mediated by the FcgRII expressed by the second
population of B cells. (what causes aggregation
of these receptors?)These findings suggest a
role for IgGs and FcRs in the control of the
number of activated IgM-secreting cells.
70
Quorum sensing mechanism?

• B cells are able to sense some of their own
  products (IgG), which then act to prevent further
  B cell activation.
• This process helps to control the number of
  activated B cells.
• Does this process also occurs for resting B cells
  or other lymphocyte populations?
• What are the molecules involved?

71
The immune system is a set of rules rather than a
cohort of cells.
72
Acknowledgements
Michael Ehrenstein, London, UK Marc Daeron,
IP, France Pierre Bhruns, IP France
Fabien Agenes Emmanuelle Gaudin Yi Hao Angela
McLean Caroline Montaudouin Manuela
Rosado Marie-Pierre Mailhé