CD169CD169+ + Macrophages, Macrophages, Lymph node sentinels.Lymph node sentinels.

RafaelRafaelEduardoEduardoGiulianoGiuliano

WHOLE WHOLE MICROORGANISMS MICROORGANISMS

AND/OR LARGE AND/OR LARGE ANTIGENSANTIGENS

Capture of these antigens Capture of these antigens

Migratory dendritic cellsMigratory dendritic cells

Lymph nodeLymph node

Adquired immune responseAdquired immune response

Presentation of antigensPresentation of antigens

Exposure of uncommon antigens

Exposure of uncommon antigens

SOLUBLE SOLUBLE

ANTIGENANTIGENSS

- VIA FLOW - VIA FLOW LYMPH = LYMPH =

(LYMPH-BORNE)(LYMPH-BORNE)

Nanometre log scale of pathogensNanometre log scale of pathogens

Bachmann & Jennings. 2010 Nat Rev ImmunoMODIFIED

Dead cell-associated antigens (?)Dead cell-associated antigens (?)

Soluble antigensSoluble antigensMicrobial antigensMicrobial antigensComplexed to antibodiesComplexed to antibodiesSmall antigens (<70kD)Small antigens (<70kD)

LipidsLipids

?...how this presentation occurs

Answer: Lymph node-resident APCs

Secondary Lymphoid Organ (SLO)Secondary Lymphoid Organ (SLO)_ Basic Building Blocks__ Basic Building Blocks_

Junt et al. 2008 Nat Rev Immuno

Antigen-sampling zone = Antigen-sampling zone = Subcapsular sinusSubcapsular sinus

Subcapsular macrophages = Metallophilic macrophages = CD169Subcapsular macrophages = Metallophilic macrophages = CD169+ + macrophagesmacrophages

von Andrian et al. 2003 Nat Rev Immuno

Junt et al. 2008 Nat Rev ImmunoFibroblastic Reticular Cell

Subcapsular macrophages = Metallophilic macrophages = CD169Subcapsular macrophages = Metallophilic macrophages = CD169+ + macrophagesmacrophagesSubcapsular macrophages = Metallophilic macrophages = CD169Subcapsular macrophages = Metallophilic macrophages = CD169+ + macrophagesmacrophages

CD169CD169+ + macrophages as a FILTERmacrophages as a FILTER

Dead cell-associated antigens (?)Dead cell-associated antigens (?)

Soluble antigensSoluble antigensMicrobial antigensMicrobial antigens

Complexed to antibodiesComplexed to antibodies

Small antigens (<70kD)Small antigens (<70kD)Convencional resident DC (CD8Convencional resident DC (CD8++α)α)

CD169CD169+ + macrophagesmacrophages

Batista et al. 2009 Nat Rev Immuno

LipidsLipids

Crocker et al. 2001 Trends in Immuno

Siglec (Siglec (SSialic acid binding ialic acid binding IgIg-like -like LecLectin-C)tin-C)

Involved in the direct recognition of Involved in the direct recognition of sialylated glycoconjugates.sialylated glycoconjugates.

SSialic acid: generic term for a family of nine-carbonialic acid: generic term for a family of nine-carbonsugars that are derivatives of neuraminic acidsugars that are derivatives of neuraminic acid

CD169CD169++ = Siglec -1 = Sialoadhesin = Siglec -1 = Sialoadhesin

Crocker et al. 2001 Trends in Immuno

Expression pattern Expression pattern of human Siglecsof human Siglecs

...on the context of an infection...on the context of an infection

Major source of tumor antigens: Dead tumor cells (apoptose)

Dead cell-associated antigens (Soluble antigens)

Lymph nodes

CD169+ macrophages

Anticancer therapy

Immunosupressive environment

induced by tumors

Defective antigen presentation (Dendritic cells)

Tolerance (T cells)

Presentation by cross-priming!!!!

...on the context of a tumor...on the context of a tumor

Antitumor immunityAntitumor immunity

Dead cell-associated antigens (Soluble antigens)

Dmitry Gabrilovich 2004 Nat Rev Immuno

Effects of anticancer therapy on tumor cellsEffects 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

Eduardo

•How lymph borne virus particles are cleared from afferent lymph and presented to B cells ?

• Staging ground of adaptive immune responses;

• Lymph nodes prevent the systemic dissemination of pathogens

How virus particles that enter peripheral tissues are handled within draining lymph nodes?

UV -VSV Ultraviolet inactivated vesicular stomatitis virus

Multiphoton Intravital

Microscopy(MP-IVM)

Popliteal Lymph Nodes

0’ 30’

VSV accumulates on the SCS floor

Which are the preferred VSV capturing cells in lymph nodes?

WT - BM

Act (EGFP)

Enhanced GFP in NON-hematopoietic 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?

VSV accumulate rapidly and selectively on macrophages in the SCS of draining lymph nodes.

Confocal microscopy (30 min after injection UV-VSV)

What are the consequences of viral capture by SCS macrophages for virus dissemination and antiviral immunity?

VSV titres 2h/6h after injection

Depletion of CD169+ macrophages rendered VSV filtration inefficient CLL

Clodronate liposomes

*Viable VSV

UntreatedCLL treated

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

MP-IVMConfocal Microscopy

(WT mice)WT B Cells *** +

VI10YEN B Cells **

Specific B Cells rapidly accumulated below and

within the SCS floor

VI10YEN B Cells VSV-IND (Indiana Virus)

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 virus30min1h2h

No virus2h UV-VSV

CLL TreatedUntreated

What is the role of the SCS macrophages on the B cell migration towards the T/B border?

WT miceWT B Cells *** ORVI10YEN B Cells **

+ /- CLL

VSV-IND

Confocal micrograph

6h

Even without SCS macrophages, B cells are

eventually activated by VSV-derived 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

This immunization serves as na effective tumor “vaccination” by activation of specific CTL

This immunization serves as na effective tumor “vaccination” by activation of specific CTL

64hr

OVA-expressing dead cell

Proliferation FACS

i.v.

ndLN

dLN

WT

EG7 cells - Xray

s.c. /right flank

10 days live EG7 cells / left flank

tumor volumes

WT

What is the contribution of migratory DCs to the delivery of cellular antigens?

Kaede mice (Tg)

PBS orCFA orDead Cell (apoptotic tumor cells)

Hind leg foot pads

Violet light12 -18hr 30hr CD11c+ DCs

of dLN

Kaede - photoconvertible fluorescence protein

Migratory DCs do not participate in the delivery of dead cells-associated antigens

Migratory DCs do not participate in the delivery of dead cells-associated antigens

How antigens reach the lymph node?

PKH26- labeled dead cells (EG7)

3, 6, 9, 18, hr and 4 daysdLN

s.c. /foot pads

Until 4 days

24hr

Cell corpses traveled to the draining LNs shortly after the injection via lymphatic flow and were trapped in the sinus

by CD169+ MΦ

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?

24hrSorting FACS (dLN)PKH26-labeled dead cells

(EG7)

ndLN

dLN

Predominantly residents / non-migratory CD169+ MΦPredominantly residents / non-migratory CD169+ MΦ

CD169+ MΦ depletion can change antitumor immunity?

Mice depleted of CD169+ MΦ could no longer

reject viable tumor cells

Mice depleted of CD169+ MΦ could no longer

reject viable tumor cells

day 14

EG7-Xray

s.c. /right flank

EG7 live cells / left flank

tumor volumes

WT

CD-169-DTR

Tumor degradation in vivo might be controled by CD169+ MΦ?

Both vaccination with tumor cells killed ex vivo and by degradation of established

tumor in vivo is controled by CD169+ MΦ

Both vaccination with tumor cells killed ex vivo and by degradation of established

tumor in vivo is controled by CD169+ MΦ

day 7 oxaliplatin day 12 dLN OVA IFN-γ ELISA

EG7 /right flankWT

CD-169-DTR

Are CD169+ MΦ required to antigen-specific T cell proliferation?

CFSE-labeled OT-I T celli.v.

dead cell-OVA or CFA-OVA

s.c. foot pads

dLN proliferation

CD169+ MΦ are essential for the crosspresentation of

dead cell-associated antigens

CD169+ MΦ are essential for the crosspresentation of

dead cell-associated antigens

WT

CD-169-DTR

What is the efficacy of vaccination in these mice?

dLN CD8+ T cell

OT-I T cell

EG7-Xray / right flank

EG7 live cells / left foot pads

To a efficacy of vaccination and priming of T CD8+ cells, CD169+ MΦ are essentially necessary

To a efficacy of vaccination and priming of T CD8+ cells, CD169+ MΦ are essentially necessary

2 days14 days

WT

CD-169-DTR

How is the crosspresentation of APCs in vitro?

CD-169-DTR dead cell-OVA

WT

24hr dLN APCs (magnetic beads) proliferation

OT-I T cells

42hr

72hr IFN-γ ELISA

Dependent of CD169+ MΦ in the LN sinusDependent of CD169+ MΦ in the LN sinus

How is the crosspresentation of APCs in vitro?

Dependent of CD11c+ CD169+ MΦ in the LN sinusDependent 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

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

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 orE1E2PT

24hrdLN APCs Citometry

D89E and E1E2PT milk fat globule-EGF-factor 8 (MFG-E8) mutants

CFSE-labeled OT-I T cell

i.v.

dead cell-OVA with PBS, D89E or

E1E2PT

s.c. foot pads

dLN proliferation

CD169+ MΦ phagocytose dead cells in a PS-dependent manner

CD169+ MΦ phagocytose dead cells in a PS-dependent manner

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!