Identification of HIV Transmitting CD11c+ Human Epidermal Dendritic Cells
Andrew Harman
The Westmead Institute for Medical Research The University of Sydney, Australia Mononuclear Phagocytes and Sexual Transmission of HIV
Epidermis Turville et al., Blood 2004 Harman et al., J. Immunol 2006 Langerhans cells Harman et al., Blood 2009 Mercier et al., PLoS Pathog 2013 Nasr et al., J. Immunol 2015
CD4 T cells
Dendritic cells cDC1 Paterson et al., Am J Pathol 2002 Hladik et al., Immunity 2007 Ganor et al., Mucosal Immunol 2010 cDC2
tissue resident Dermis / lamina propria monocyte derived macrophage Macrophages macrophage
Monocytes
minimal cleavage Enzymatic Cleavage of Surface Markers partial cleavage complete Trypsin Collagenase Collagenase cleavage Blend F Type IV Collagenase Trypsin (Type IV) HLA-DR
CD45 DR
CD1a - Langerhans
Langerin HLA cells cells markers
CD1c CD45 CD11c CD14
markers CD141
Dermal MNP CD1a CD4 CCR5 CD11c
HIV HIV entry CXCR4 receptors
Botting et al., The Journal of Leukocyte Biology 2017 Identificationof LangerhansUsingCells Trypsin
HLA-DR CD45
CD1a Langerin Identification of Novel Epidermal Mononuclear Phagocytes
Collagenase Digestion **** **** HLA-DR and CD45n 100 very high
o HLA-DR and CD45 low
i
t
a DR
l n = 75 - u 80
p CD1a lower CD1a
CD33 Langerin -
o
HLA
P
live CD3-, CD19- singlets + 60 5 CD11c+ 4 CD33 low/- CD45 Langerin CD11c D
C 40
+
R D
CD1c high - 20
A
L
R1
ε H
MR 0
% Fc CD1c Mannose receptor+ s s s C P C L N D M + w c o 1 CD11c l 1 CD11c Langerin 3 3 D D C C Langerhans Cell CD11c+ DC CD33low MNP
Rachel Botting, PhD Visualisation of Epidermal CD11c+ Dendritic Cells in situ
DAPI Langerin CD11c DIC
Epidermal CD11c+ DC
Epidermal Langerhans cell
dermis Hafsa Rana, PhD Epidermal Mononuclear Phagocyte Morphology
Langerhans Cell
CD33lowMNP
Epidermal CD11c+ DC
Dermal CD11c+ cDC2
10mm
James Fletcher (Haniffa group) Transcriptional Profiling of Epidermal Mononuclear Phagocytes
Epidermis
Langerhans CD33low CD11c+ cell MNP MNP
Dermis
cDC1 cDC2 cDC2 (langerin-) (langerin+)
tissue resident monocyte-derived macrophage macrophage
Epidermal CD11c DCs are probably dermal cDC2 that migrate into the epidermis (Hafsa Rana PhD) Epidermal CD11c+ Dendritic Cells are more Potent APCs than their Dermal CD11c+ cDC2 Counterparts
T cell activation assay
Langerhans Cell CD33low MNP CD11c+ DC CD11c+ cDC2 Epidermal CD11c+ Dendritic Cells are Preferential Targets for HIV
HIV entry receptor Uptake (2 hours) Infection (96 hours)
expression not CCR5 meditated p=0.0176 2 5 0 4 0
* s
1 0 0 s
l
l
l
l s
2 0 0 s
e
e P
P 3 0 TF Strain Z3678M
c
c
N
8 0 N d
d Bal
M
M
e e
(Eric Hunter)
1 5 0
t
t
0
0
c
c
0 0
e 2 0
6 0 e
f
0
f
CD4
0
n
n
0
I 0
I 1 0 0
1
f
1
f
o
r o
4 0 r
.
e 1 0
.
e
o
p o
p 5 0 N 2 0 N 0 0
+ w 0 s lo s c C 2 1 L 3 C 1 3 D
%p24+ HIV RNA+ cells RNA+ HIV %p24+ s s CCR5 D D c C C C C L D + c 1 CCR5 Blocking Assay MNP – CD4 T Cell Transfer Assay1 D C
p=0.0286 -ve +ve 1 5
s
l l
1 5 e
p=0.0175 TF Strain Z3678M c
T
1 0
s +
l p=0.0286 (Eric Hunter)
l
P
e
F
c
1 0 G -
T 5
V
I +
A LC CD11c+ DC CDC2
- H
P Bal
F
% G
SSC 0
- 5 V
I + c v 1 o H r HIV1 HIVi + D v
% a Mock HIV HIV + C r miraviroca M + 0 P24 (KC57) PE miraviroc c 1 1 + s w 2 D c C o C C 1 L l 1 3 D D 3 C C D Jake Rhodes, PhD C Langerhans Cell CD33low MNP CD11c+ DC CD11c+ cDC2 Access to Human Anogenital Tissue
extensive collaborations with surgeons at Westmead for access to all human anogenital tissue types
female genital tract male genital tract anorectal tract
Perineum Labia Foreskin
inner outer Anal Canal Vagina Glans
Rectum Cervix Urethra Epidermal CD11c+ Dendritic Cells are Enriched in Anogenital Tissues (ex vivo Flow Cytometry) p=0.0368 p=0.0023 p=0.0003 Langerhans Cell p=0.044 p=0.0018 CD11c+ DC p=0.0008 1 0 0 CD33low MNP
+ 8 0
R
D A
L 6 0
H
+
5 4 0
4
D C
2 0 %
0
Skin Type II Mucosae Epidermal CD11c+ Dendritic Cells are Enriched in Anogenital Tissues (in situ fluorescence microscopy)
1 0 0
+ 8 0 R
Inner foreskinD
A Inner Foreskin
L 6 0
H
+ 80 5 4 0
4 LCs
D C
2 60 CD11cs
2 0
%
m m / 40
0 s
l
l e
C 20
0 s s c C 1 L 1 D dermis C dermis DAPI CD11c
Hafsa Rana, PhD Anogenital Epidermal CD11c+ Dendritic Cells Express Langerin
Live, HLA-DR+, CD45+ singlets , CD11c+ DCs 1 0 0
Abdomen Glans 8 0
+
n i
r 6 0
e
g n
a 4 0
L
10.7% 36.4% %
2 0 CD1a CD1a BV510 0
l n a a s a e i in in s m n i b k n i r n m a k a u g a a Langerin Vioblue s s l e l c o L e e n a u d r r G i V c l o r i a b o e v n A F F r r P a A e e N t n a u n s O I s o F
Anogenital CD11c+ DCs cannot be discerned from LCs unless CD11c staining is included !!! CD11c+ Dendritic Cells are Enriched in Inflamed Tissue
* CD11c+ DC
100 s
l *
l Langerhans Cell
e
C
cells
+ Inner foreskin
+ R
D LCs
- A
L 50
CD45 CD11c+
+
H
+
5
DR
-
4
D
C
%
% HLA % 0 s + s + Healthyc Inflamedc C 1 C 1 L 1 L 1 D D Jake Rhodes, PhD C C Uninflamed Inflamed Epidermal CD11c+ Dendritic Cells Capture HIV in situ
Donor 1 Donor 2 (BaL) (BaL)
40um
Donor 3 (BaL) LC
CD11c+ MNP TF Strain TF Strain (Eric Hunter) (Eric Hunter)
DAPI CD11c HIV RNA Langerin DIC Heeva Baharlou, PhD Overall Summary
Harman et al., J.Immunol 2006
Epidermis x x Langerhans cell x CD11c+ DC / cDC2 x Nasr et al., J. Immunol 2015 Turville et al., Blood 2004 CD33low MNP Harman et al., Blood 2009
CD4 T cells Mercier et al., PLoS Pathog 2013
• Functionally efficient antigen presenting cells Dermis • Are a cDC2 subset • Enriched in anogenital tissues • Sample HIV at the epithelial surface • Functionally inefficient antigen presenting cells • Express higher HIV entry receptor levels (CCR5) • A Langerhans cell precursor? • Support higher level of HIV infection • Do not bind HIV or transfer it CD4 T cells • Most efficient at transfer HIV to CD4 T cells Acknowledgements
HIV and mucosal Immunology group 2017
Tony Cunningham Eric Hunter
Martijn Gosselink Jake Rhodes Toby Plasto Heeva Baharlou Chloe Doyle Kirstie Bertram Andrew Harman Hafsa Rana
Plastic Surgeons Colorectal Surgeons Urologists Jacob Estes Muzlifah Haniffa Tim Papadopoulos Grahame Ctercteko Ralph Cohen Jake Lim Martijn Gosselink Andrew Brooks Laith Barnouti Kenneth Loon Michale Wines Mark Kohout Nimala Pathma-Nathan Rachel Botting Steve Merten Mark Gladman Gavin Sandercoe Alan Meagher Peter Laniewski