CD169+ Macrophages,
Lymph node sentinels.
Rafael
Eduardo
Giuliano
WHOLE MICROORGANISMS
AND/OR LARGE ANTIGENS
Exposure of
uncommon antigens
Capture of these antigens
SOLUBLE
ANTIGENS
Migratory dendritic cells
- VIA FLOW LYMPH =
(LYMPH-BORNE)
Lymph node
Presentation of antigens
Adquired immune response
Nanometre log scale of pathogens
Soluble antigens
Small antigens (<70kD)
Complexed to antibodies
Microbial antigens
Lipids
Dead cell-associated antigens (?)
...how this
presentation occurs
?
Answer: Lymph node-resident APCs
Bachmann & Jennings. 2010 Nat Rev Immuno
MODIFIED
Secondary Lymphoid Organ (SLO)
_ Basic Building Blocks_
Junt et al. 2008 Nat Rev Immuno
Antigen-sampling zone = Subcapsular sinus
Subcapsular macrophages = Metallophilic macrophages = CD169+ macrophages
von Andrian et al. 2003 Nat Rev Immuno
Subcapsular macrophages = Metallophilic macrophages = CD169+ macrophages
Fibroblastic Reticular Cell
Junt et al. 2008 Nat Rev Immuno
CD169+ macrophages as a FILTER
Small antigens (<70kD)
Convencional resident DC (CD8+α)
Complexed to antibodies
Soluble antigens
Microbial antigens
Lipids
CD169+ macrophages
Dead cell-associated antigens (?)
Batista et al. 2009 Nat Rev Immuno
CD169+ = Siglec -1 = Sialoadhesin
Siglec (Sialic acid binding Ig-like Lectin-C)
Sialic acid: generic term for a family of nine-carbon
sugars that are derivatives of neuraminic acid
Involved in the direct recognition of
sialylated glycoconjugates.
Crocker et al. 2001 Trends in Immuno
Expression pattern
of human Siglecs
Crocker et al. 2001 Trends in Immuno
...on the context of an infection
...on the context of a tumor
Defective antigen presentation (Dendritic cells)
Immunosupressive
environment
induced by tumors
Tolerance (T cells)
Major source of tumor antigens:
Dead tumor cells (apoptose)
Dead cell-associated antigens
(Soluble antigens)
Lymph nodes
CD169+ macrophages
Presentation by cross-priming!!!!
Anticancer therapy
Antitumor immunity
Dead cell-associated antigens
(Soluble antigens)
Dmitry Gabrilovich 2004 Nat Rev Immuno
Effects of anticancer therapy on tumor cells
Zitvogel et al. 2008 Nat Rev Immuno
‘Find-me’ and ‘eat-me’ signals and some phagocyte receptors
Ravichandran et al. 2007 Nat Rev Immuno
• Lymph nodes prevent the systemic dissemination of pathogens
• Staging ground of adaptive immune responses;
•How lymph borne virus particles are cleared from afferent lymph
and presented to B cells ?
Eduardo
How virus particles that enter peripheral tissues are
handled within draining lymph nodes?
Multiphoton
Intravital
Microscopy
(MP-IVM)
0’  30’
Popliteal
Lymph
Nodes
UV -VSV  Ultraviolet inactivated
vesicular stomatitis virus
VSV accumulates on the SCS floor
Which are the preferred VSV capturing cells in lymph nodes?
WT - BM
Act (EGFP)
Enhanced GFP in NONhematopoietic cells
VSV are captured by
hematopoietic cells
Which are the VSV capturing leukocytes?
Electron Microscopy – Popliteal lymph
nodes (5min after injection VSV)
VSV selectively bound on the surface of large cells
residing within the SCS or just below de SCS floor.
The VSV retaining cells belong to macrophages population?
Confocal microscopy (30 min after injection UV-VSV)
VSV accumulate rapidly and selectively on macrophages
in the SCS of draining lymph nodes.
What are the consequences of viral capture by SCS macrophages
for virus dissemination and antiviral immunity?
Untreated
CLL treated
*Viable VSV
CLL 
Clodronate
VSV titres 2h/6h after injection
Depletion of CD169+ macrophages
rendered VSV filtration inefficient
How captured VSV is recognized by B cells?
Electron Microscopy –
Popliteal lymph nodes
(30 min after injection VSV)
Viral particles are
presented to B cells within
superficial follicles by
macrophages that extend
across the SCS floor
How the SCS macrophages influence the B cells distribution on
draining lymph nodes?
VSV-IND (Indiana Virus)
VSV-NJ (New Jersey)
+/- CLL
(WT mice)
WT B Cells *** +
VI10YEN B Cells **
MP-IVM
Confocal Microscopy
Specific B Cells rapidly
accumulated below and
within the SCS floor
VI10YEN B Cells  VSV-IND (Indiana
What is the role of the SCS macrophages on the B cell activation?
Surface IgMs on B cell populations on draining lymph node by flow
citometry after VSV-IND injection
No virus
30min
1h
2h
No virus
2h UV-VSV
CLL Treated
Untreated
What is the role of the SCS macrophages on the B cell
migration towards the T/B border?
VSV-IND
WT mice
WT B Cells *** OR
VI10YEN B Cells **
+ /- CLL
6h
Confocal micrograph
Even without SCS
macrophages, B cells are
eventually activated by VSVderived antigens, although less
efficiently
Conclusion
Capture of lymph borne viruses and guide them to
presentation and activation of B cell
Aim: To identify how APCs in the lymph node (LN) internalize and
crosspresent soluble antigens (dead tumor cells) to CD8+ T cells.
Immunization with dead tumor cells activates
antitumor immunity?
OT-I T cell CFSE labeled
i.v.
OVA-expressing
dead cell
WT
64hr
EG7 cells - Xray
WT
10 days
s.c. /right
flank
Proliferation FACS
live EG7 cells /
left flank
tumor
volumes
ndLN
dLN
This immunization serves as na effective tumor
“vaccination” by activation of specific CTL
What is the contribution of migratory DCs to
the delivery of cellular antigens?
Hind leg foot
pads
PBS or
CFA or
Dead Cell
(apoptotic tumor cells)
12 -18hr
Violet light
30hr
CD11c+ DCs
of dLN
Kaede mice
(Tg)
Migratory DCs do not participate in the delivery of dead
cells-associated antigens
Kaede - photoconvertible fluorescence protein
How antigens reach the lymph node?
Until 4
days
24hr
PKH26- labeled
dead cells (EG7)
s.c. /foot
pads
3, 6, 9, 18, hr and 4 days
dLN
Cell corpses traveled to the draining LNs shortly after the
injection via lymphatic flow and were trapped in the sinus
by CD169+ MΦ
Which cells phagocytosed the apoptotic cell
corpses on dLN?
PKH26-labeled dead cells
(EG7)
24hr
Sorting FACS (dLN)
ndLN
dLN
Predominantly residents / non-migratory CD169+ MΦ
CD169+ MΦ depletion can change antitumor
immunity?
WT
EG7-Xray
day 14
s.c. /right
flank
EG7 live cells /
left flank
tumor
volumes
CD-169-DTR
Mice depleted of CD169+
MΦ could no longer
reject viable tumor cells
Tumor degradation in vivo might be controled
by CD169+ MΦ?
EG7 /right flank
WT
day 7
oxaliplatin
day 12
dLN
OVA
IFN-γ ELISA
CD-169-DTR
Both vaccination with tumor
cells killed ex vivo and by
degradation of established
tumor in vivo is controled by
CD169+ MΦ
Are CD169+ MΦ required to antigen-specific T
cell proliferation?
WT
CFSE-labeled OT-I
T cell
i.v.
s.c. foot
pads
dead cell-OVA or
CFA-OVA
dLN proliferation
CD-169-DTR
CD169+ MΦ are essential
for the crosspresentation of
dead cell-associated
antigens
What is the efficacy of vaccination in these
mice?
WT
OT-I T cell
EG7-Xray / 14 days
right flank
EG7 live cells /
left foot pads
2 days
dLN CD8+ T cell
CD-169-DTR
To a efficacy of vaccination
and priming of T CD8+
cells, CD169+ MΦ are
essentially necessary
How is the crosspresentation of APCs in vitro?
WT
24hr
CD-169-DTR
dead cell-OVA
dLN APCs
OT-I T cells
(magnetic beads)
42hr
72hr
proliferation
IFN-γ ELISA
Dependent of CD169+ MΦ in the LN sinus
How is the crosspresentation of APCs in vitro?
Dependent of CD11c+ CD169+ MΦ in the LN sinus
What is the localization of CD169+ MΦ?
CD169+ localized in the sinus, CD11c+ in the T cell zone,
CD169+ CD11c+ in the cortical and paracortical sinus
Are CD11c+ and CD11c- CD169+ MΦ
differents in function?
CD11c+ CD169+ MΦ
CD11c- CD169+ MΦ
LPS
CpG
Two CD169+ MΦ subsets have distinct cytokine production
profiles
What is the molecular mechanism of apoptotic
cell phagocytosis by CD11c+ CD169+ MΦ?
WT
W3 dead
cell-OVA
PKH26
D89E or
E1E2PT
24hr
dLN APCs Citometry
i.v.
CFSE-labeled OT-I
T cell
s.c. foot
pads
dead cell-OVA
with PBS, D89E or
E1E2PT
dLN proliferation
CD169+ MΦ phagocytose dead cells in a PS-dependent
manner
D89E and E1E2PT  milk fat globule-EGF-factor 8 (MFG-E8) mutants
Conclusion - Paper
Highlights
► Dead tumor cells in periphery accumulate in the draining lymph node sinus;
► CD169+ macrophages phagocytose and crosspresent dead cell-associated antigens;
► CD169+ macrophage-depleted mice fail to crossprime tumor-specific CD8 T cells;
► CD169+ macrophages link tumor cell death and induction of antitumor immunity.
Conclusion - Journal
TUMOR
INFECTION
“Soluble” antigens are
drained to lymph nodes
CD169+ Macrophages sinus take this antigens and
generate a humoral and cellular immune response!