1. CX 3 CR1 + CD CD common macrophage/DC precursors and the role of CX 3 CR1 in their response to infl ammation
Cedric Auff ray , Darin K. Fogg , Emilie Narni-Mancinelli , Brigitte Senechal , Celine Trouillet , Noah Saederup , Julia Leemput , Karine Bigot , Laura Campisi , Marc Abitbol , Thierry Molina Israel Charo , David A. Hume , Ana Cumano , Gregoire Lauvau , and Frederic Geissmann

2. Mononuclear phagocyte system
Monocytes, macrophages, and DCs
Play major roles in development, inflammation and antipathogen defenses
Highly heterogeneous in phenotype, tissue distribution and function

3. DCs Specialized APCs (antigen-presenting cell )
Recognize, acquire, process and present antigens to T cells for the induction of an antigen-specific immune response
Widely distributed in the body (lymphoid and non-lymphoid tissues)
GM-CSF: Granulocyte/macrophage colony-stimulating factor
IL-3: Interleukin-3
FLT3L: fms-related tyrosine kinase 3 ligand

4. CMP Common myeloid progenitor
GMP Granulocyte/macrophage progenitor
MDP Macrophage/DC progenitor
CDP Common DC precursor
PDC plasmacytoid DC
Differentiation of the macrophage/DC progenitor and origin of macrophage and DC subsets

5. Experiment Design Test the role of CX 3 CR1 for the recruitment of
monocytes in the spleen
Analyze mouse BM Lin- precursors by FACS
competitive
adoptive transfer model
Adoptive Transfer
Test the role of CX 3 CR1 for the monocyte survival and MDP survival& proliferation
Investigate the differentiation potential in vivo of MDP
The role of CX 3 CR1 in the differentiation of mononuclear phagocytes
The differentiation potential of MDP

6. MDP and CDP share expression of CX 3 CR1, CD115, and FLT3
Results
Analysis of mouse BM Lin- precursors by FACS
BM cells
from Cx3 cr1 gfp/+ mice
CDP and MDP are overlapping populations;
CDP: Lin- CD117 low CD115 + CD135 + CX3CR1+
MDP: Lin- CD117 int CD115 + CD135 + CX3CR1+
MDP and CDP share expression of CX 3 CR1, CD115, and FLT3

7. PDCA1+ cells express CD11b- CD11c int B220 intCX 3CR1 + splenocytes
PDCs (CD11cint CD11b- B220 int CX3CR1+ )
Cx 3 cr1 gfp/+ reporter
splenocytes
cDCs
day 7
pDCs
Donor
Recipient
Adoptive transfer
PDCA1+ cells express CD11b- CD11c int B220 intCX 3CR1 + splenocytes

8. MDP gave rise to PDCs, cDCs and monocyte/macrophages
Investigate the differentiation potential in vivo of MDP
Cx 3 cr1 gfp/+ reporter
splenocytes
Donor
Recipient
PDCs (Lin-CD11cint CD11b-CX3CR1+)
cDCs (Lin-CD11chigh CD11b-CX3CR1-)
cDCs (Lin-CD11cint CD11b+CX3CR1+)
Monocyte (Lin-CD11c- CD11b+CX3CR1+)
PDCA1: pDC specific antibody
MDP gave rise to PDCs, cDCs and monocyte/macrophages

9. Test the effects of the AFS98 anti-CD115 antibody on the differentiation of MDP
Cocktail +/- AFS98
BM cells
from Cx3cr1gfp/+ mice
Sorting
AFS98: block CSF-1R
Proliferative response of MDP to CSF-1 in vitro
Addition of AFS98
to the antibody cocktail for cell sorting
(-)
Differentiation of MDP into DCs or monocytes in vivo

10. Investigate the role of CX 3 CR1 in DC and monocyte development from MDP
Competitive adoptive transfer
R4:NK1.1-, CD3-, CD19-, Ly6G- , CX 3 CR1 highF4/80int, Gr1 +
CX 3 CR1 is dispensable for MDP development in the BM
CX 3 CR1 is important for the development of CD11b + CD11c - monocytes

11. CX 3 CR1 may be selectively involved in the development, recruitment, proliferation, and/or survival of CD11b + CD11c- monocytes in spleen

12. CX3CL1 expression and recruitment of monocytes to the spleen
Test the role of CX 3 CR1 for the recruitment of monocytes in the spleen
Spleen CX 3 CL1 –producing cells
periphery of the B cell follicle
CX3CL1: CX3CR1 ligand
CX3CL1 expression and recruitment of monocytes to the spleen

13. Test the role of CX 3 CR1 for the recruitment of monocytes in the spleen
Adoptive transfer of monocytes
monocytes
splenocyte
CX 3 CR1-deficient monocytes are only one tenth as efficient as control at accumulating in the spleen

14. Test the role of CX 3 CR1 for the recruitment of monocytes in the spleen
Lm infection
Lm infection
3×105Lm, 24h
7×105Lm, 24h
7×103Lm, 48h
CX 3 CR1 is dispensable for the recruitment of monocytes in the spleen in the steady state
Accumulationt?
Survival or proliferation of monocytes?
Proliferation?
Survival, proliferation or differentialtion of MDP?

15. Test the role of CX 3 CR1 for the recruitment of monocytes in the spleen
monocytes from the BM and spleen of infected mice
the apoptosis rate
3×103Lm, 24h
104Lm, 24h
DNA Content Analysis
(propidium iodide staining)
Early control of Lm growth in the spleen
Accumulation of spleen monocytes in infection is not a result of their proliferation in spleen

16. CX 3 CR1 does not significantly affect the proliferation and differentiation potential of MDP
CX 3 CR1 is important for the recruitment of monocytes to the spleen and during acute inflammation, and for the early control of bacteria growth
Effector functions: TNF- secretion, iNOS expression,and reactive oxygen production ?

17. Investigate functions of spleen CD115 + Gr1 + /TipDCs
Lin- CD11c low CD11b + Gr1 + /Ly6C + spleen monocytes from Cx 3 cr1 gfp/+ reporter mice
TipDCs: TNF/iNOS-producing dendritic cells
iNOS: inductible nitric oxide synthase
ROIs: reactive oxygen intermediates
Production of TNF- , iNOS, and ROI
CD115 + Gr1 + monocytes recruited to the spleen during Lm infection differentiate into effector cells (TipDCs)

18. Localization of CD115 + Gr1 + /TipDCs in spleen
B cell follicles
INOS+ cells (CD11b + Ly6C +)
T cells
24 h after infection with 10 5 WT Lm
Localization of CD115 + Gr1 + /TipDCs in spleen
(perifollicular area and T cell area)

19. Test the role of CX 3 CR1 for the recruitment of
monocytes in the spleen
Analyze mouse BM Lin- precursors by FACS
competitive
adoptive transfer model
MDP and CDP share a similar phenotype
(CX 3 CR1+CD135 + CD115)
CX 3 CR1-deficient MDPs have a decreased potential
to give rise to spleen monocytes
Investigate the differentiation potential of MDP
Adoptive Transfer
Test the role of CX 3 CR1 for the monocyte survival and MDP survival& proliferation
MDPs give rise to monocytes, cDCs, and PDCs
CX 3 CR1-/-
the recruitment into the spleen of monocytes
during acute infection
the efficiency of bacterial clearance
affect the development
MDPs give rise to blood CD115 + Gr1 + monocytes,
which are recruited to spleen during infection via CX 3 CR1

20. Conclusion MDPs can give rise to PDCs, cDCs and monocytes
MDPs and CDPs share a similar surface phenotype
(Lin- IL7Ra- CD117 int CD135 + CD115 + CX 3 CR1 +)
CX 3 CR1 was redundant for MDP development, survival, homing to the spleen, and differentiation in vivo and in vitro
CX 3 CR1 are important role in Gr1 + monocyte recruitment to the spleen during inflammation
MDPs give rise to blood CD115 + Gr1 + monocytes, and expressed effector functions that are important for Lm elimination.
CX 3 CR1 mediating the recruitment of CD115 +Gr1 + monocytes in the spleen during acute inflammation, possibly through adhesion to the capillary endothelial cells of the marginal zone of the spleen

25. CX3CR1