Immunity, Volume 36

Supplemental Information

Human Epidermal Langerhans Cells Maintain

Immune Homeostasis in Skin by Activating

Skin Resident Regulatory T Cells

Julien Seneschal, Rachael A. Clark, Ahmed Gehad, Clare M. Baecher-Allan, and

Thomas S. Kupper

Supplemental Experimental Procedures

Supporting Figures

Figure S1. Purification of isolated LC, Dermal DC and skin resident memory T cells

Figure S2. Surface molecule expression by isolated LC and Dermal DC

Figure S3. LC are powerful inducers of skin resident memory T cells proliferation.

Figure S4. LC induce proliferation of skin resident memory Treg cells .

Figure S5. Proliferating skin resident memory Treg cells are found close to LC either

in the papillary dermis and in the epidermis

Figure S6. Expansion of skin resident memory T cells, in the presence of C.albicans,

involves MHC class II, CD80/86 ligation, IL-2, IL-15 and partially CD1a

Figure S7. Proliferating skin resident memory Treg cells produce neither IL-17, IFNγ,

nor IL-22, while in the presence of C.albicans, expanded skin resident Tem cells

produce effector cytokines and contain a TCR-biased T cells.

Supporting Table

Table S1. Antibodies used for flow cytometry and immunofluorescence

Figure S1. Purification of isolated LC, Dermal DC and Skin resident T cells. LC,

Dermal DC and T cells were isolated as described in Materials and Methods.

Numbers represent percentage of gated cells.

Figure S2. Surface molecule expression by isolated LC and Dermal DC. Flow

cytometry analysis of surface molecules on epidermal LC and dermal DC (DDC).

HLA-DR+CD207+ cells from epidermis and HLA-DRhiBDCA1+ cells from dermis were

analysed for expression of CD80, CD86, CD83, DC-LAMP. Grey lines indicate

isotype controls. Data are representative of two independent experiments.

Figure S3. LC are powerful inducers of skin resident memory T cells

proliferation. Proliferation of CFSE labeled skin resident memory T cells, stimulated

for 6 days with skin DDC or LC at different ratio. The graph shows means +/- SD.

Data are representative of three independent experiments.

Figure S4. LC induce proliferation of skin resident memory Treg cells. (A) Skin

resident memory T cells proliferating in the presence of autologous LC express

CTLA-4, GITR and are negative for CD69. Flow cytometry analysis of surface

molecules on CD3+ T cells. Proliferative (solid line) and non proliferative (dashed

line) T cells were analyzed for expression of CTLA-4, GITR and CD69. Grey lines

indicate isotype controls. (B) LC and skin resident memory T cells co-cultured for 6

days were stained on a slide using anti-CD1a (red), ant-CD3 (red), anti-Ki67 (red)

and anti-FoxP3 (green) monoclonal antibodies and counterstained with DAPI (blue).

Immunofluorescence analysis on slide shows CD1a positive LC and FoxP3/CD3

double positive skin resident memory Treg cells aggregates. FoxP3+ (green) Treg

cells co-expressing Ki67 (yellow overlay) are found in connection with LC (red).

Figure S5. Proliferating skin resident memory Treg cells are found close to LC

either in the papillary dermis and in the epidermis. (A) Immunofluorescence on

skin section shows expression of FoxP3 (green) and CD25 (red) either in epidermis

(left panel) and in dermis (right panel). Skin resident memory Treg cells expressing

FoxP3, are positive for CD25 resulting in a yellow overlay (B) Left panel,

immunofluorescence on skin section shows expression of FoxP3 (green), nuclear

staining Ki67 (red) and CD3 (red). FoxP3+ skin resident memory Treg cells

expressing Ki67 result in a yellow overlay. Right panel: immunofluorescence, on the

same skin section than left panel, showing interaction between CD1a+ LC (red) and

FoxP3+ skin resident memory Treg cells (green). (C) Upper panel left:

Immunofluorescence shows FoxP3+ (green) and CD3+ (red) skin resident memory

Treg cells, lower panel left: interaction between CD1a+ LC (red) and Foxp3+ skin

resident memory Treg cells (green) within the epidermis. Middle panel: Proliferating

Foxp3+ skin resident memory Treg cells (green) and Ki67+ (red) were found within

the epidermis resulting in a yellow overlay. Right panel: Proliferating Ki67+ Foxp3+

skin resident memory Treg cells interact with CD1a+ LC (red) within the epidermis.

(D) Immunofluorescence shows BDCA1+ Dermal DC (green) located in the papillary

dermis close to FoxP3+ (green) KI67- skin resident memory Treg cells in a resting

state.

Figure S6. Expansion of skin resident memory T cells, in the presence of

C.albicans, involves MHC class II, CD80/86 ligation, IL-2, IL-15 and partially

CD1a. Proliferation of CFSE labeled skin resident memory T cells, stimulated with

autologous LC in the presence of C.albicans. Indicated neutralizing antibodies or

isotype-matched, non-reactive antibodies as control were added. Proliferation of

CD3+ skin resident memory T cells measured by CFSE dilution. Numbers represent

the percent of divided cells for one representative donor. The graph shows means +/-

SD. Data are representative of 4 independent experiments.

Figure S7. Proliferating skin resident memory Treg cells produce neither IL-17,

IFNγ, nor IL-22, while in the presence of C.albicans, expanded skin resident

Tem cells produce effector cytokines and contain TCR-biased T cells. (A)

CFSE labeled skin resident memory T cells co-cultured with autologous LC in the

presence or the absence of C.albicans were isolated and re-stimulated with PMA,

Ionomycin in the presence of Brefeldin during 4h and subsequently analyzed for the

production of indicated cytokines by flow cytometry. Numbers in quadrant indicate

percentage of gated cells in each. Data are representative of 4 independent

experiments. (B) T cells were isolated at day 0 or at day 6 from co-culture with

autologous LC in the presence or the absence of C.albicans and analyzed for the

percentage of cells expressing the indicated V expression by flow cytometry. Data

are representative of two independent donors.

Table S1: Antibodies used for flow cytometry and immunofluorescence

Antigen-Fluorophor

Manufacturer Clone Isotype Dilution

CD1a-PE BD Pharmingen HI149 IgG1 1:33

CD1a In house OKT6 IgG1 1:100

CD1a DAKO 010 IgG1 Ready to use

CD1c-FITC Ancell M241 IgG1 1:100

CD1c Novus Biologicals

NBP1-72101 IgG1 10μg/ml

CD86-Purified BD Pharmingen 2331 (FUN-1) IgG1 1:100

CD80-Purified BD Pharmingen L307.4 IgG1 1:100

HLA DR-DP-DQ-Purified

BD Pharmingen Tu39 IgG2a 1:100

HLA DR BD Pharmingen L243 (G46-6) IgG2a 1 :100

HLA DP abcam B7/21 IgG3 1 :100

HLA DQ abcam SPV-L3 IgG2a 1 :100

HLA A,B,C-Purified

Biolegend W6/32 IgG2a 1:100

Anti-human IL-2 Antibody

R&D Systems 5334 IgG1 5μg/ml

Anti-human IL-15Rα Antibody

R&D Systems Goat IgG 5ug/ml

CD3-PerCP BD Pharmingen SK7 IgG1 1:20

CD3 DAKO F7.2.38 IgG1 1:25

CD4-PE BD Pharmingen RPA-T4 IgG1 1:20

CD8-APC BD Pharmingen RPA-T8 IgG1 1:20

FOXP3-APC eBioscience PCH101 IgG2a 1:50

FOXP3-Purified

eBioscience PCH101 IgG2a 1:25

CD25-APC BD Pharmingen M-A251 IgG1 1:50

CD127-PerCP BD Pharmingen HIL-7R-M21 IgG1 1:50

Ki67 DAKO C8/144B IgG1 Ready to use

Alexa Fluor® 488 goat anti-mouse IgG (H+L)

Invitrogen 1:2000

Alexa Fluor® 488 donkey anti-rat IgG (H+L)

Invitrogen 1:2000

Alexa Fluor® 594 goat anti-mouse IgG (H+L)

Invitrogen 1:2000

IFNγ-APC BD Pharmingen B27 IgG1 1:50

IL-17A-PE eBioscience eBio64DEC17 IgG1 1:20

IL-22 APC R&D 142928 IgG1 1:20

Supplemental Experimental Procedures

Immunofluorescence

immunofluorescence study in Figure S2, cells (105) were allowed to adhere for 10

min at RT onto poly-L-lysine-coated slides. Then cells were fixed by dipping slides in

PBS containing 4% formaldehyde for 15 min. Slides were rinsed in cool running

water and washed in Tris-buffered saline, pH 7.4, before incubation with primary

antibodies and then incubated appropriate secondary antibodies: Alexa Fluor® 488-

conjugated anti-mouse or rat or Alex-Fluor® 594-conjugated anti-mouse. Additional

negative controls were performed by adding isotype mouse antibodies as primary

antibodies. After subsequent washing, the sections were mounted with Prolong Gold

antifade reagent with DAPI (Invitrogen) and covered with cover slip.

Immunofluoresecence reactivity was viewed on an Olympus BX51/52 system

microscope coupled to a Cytovision System (applied Imaging).

Cytokine Analysis

Skin resident T cells (5X104/well) were stimulated with autologous LC (2.5X104/well)

in 96-well plates for 6 days in the presence or the absence of heat-killed C.albicans

(5X104). On day 6, proliferating T cells were re-stimulated with 50ng/ml PMA and 750

ng/ml ionomycin (Sigma) for 4h in the presence of 10μg/ml Brefeldin A. Cells were

stained for surface markers, fixed, permeabilized, stained with anticytokine

antibodies, and examined by flow cytometry.

TCR diversity analysis of T cells

T cells were isolated after 6 days of co-culture with LC and examined by flow

cytometry for Vβ expression and CD3. Vβ staining was performed using the IOtest

Beta Mark TCR Vβ Repertoire kit (Beckman Coulter) as per manufacturer’s

instructions.