6-week-old female BALB/c mice were purchased from Janvier Laboratory (Le Genest Saint Isle, France) and experiments were conducted in a conventional facility (C75-14-05). Transgenic female Fra-2 (B6.Cg-Tg(H2-K-Fosl2,EGFP)13Wag) mice were bred in a SPF facility (C75-14-02). All mice were housed in ventilated cages with sterile food and water ad libitum. Animals received humane care in compliance with the guidelines implemented at our institution (INSERM and University Paris Descartes).
ALPN-101 molecule and pharmacological treatment
ALPN-101 (acazicolcept; ICOSL vIgD-Fc), provided by Alpine Immune Sciences (AIS) (Seattle, WA), is a dual human ICOS/CD28 inhibitor Fc fusion protein (31). ALPN-101 (produced at KBI Biopharma, Durham NC) and Fc control protein (produced at AIS) were diluted in PBS and injected intraperitoneally twice a week at molar-matched doses of 400 µg/mouse and 267 µg/mouse, respectively. The mouse dosing regimen was identified from prior mouse pharmacokinetic/pharmacodynamic studies as one that provided adequate exposure and disease modifying activity in multiple mouse models of autoimmunity and inflammation. However, this dosing regimen would not be used directly to predict human regimens due to species- and disease-related differences in multiple factors including target abundance, binding, and clearance.
HOCL induction of dermal fibrosis and ALPN-101 treatment
Dermal fibrosis was induced in six-week-old BALB/c mice according to the protocol described by Servettaz et al (32). A total of 400 µL hypochlorous acid (HOCl) solution was prepared extemporaneously by adding NaClO (9.6% as active chlorine) to KH2PO4 solution (100 mM, pH: 6.2), usually using a 1:100 ratio. The correct amount of NaClO was adjusted to obtain the desired HOCl concentration, defined by the absorbance of the mixture at 292 nm (optical density between 0.7 and 0.9). 200 µL of HOCl solution was injected intradermally into each shaved flank of the mice using a 27-gauge needle, 5 days a week for 6 weeks. Control mice were injected intradermally with 200 µL of sterilized phosphate-buffer saline (PBS) into each shaved flank. 100 µL of ALPN-101 or Fc control dosing solutions were injected intraperitoneally twice a week during the 6 weeks of HOCl-treatment. Mice were divided into the following groups : PBS (n=6), HOCL + Fc control (n=8), and HOCL + ALPN-101 (n=8). Mice were euthanized by cervical dislocation after 6 weeks of treatment (Supplementary Figure 1). This experiment has been carried out once.
Fra-2 transgenic mice and ALPN-101 treatment
Transgenic mice expressing the Fra-2 transgene under the control of ubiquitous major histocompatibility complex class I antigen H-2Kb promoter develop microangiopathy, systemic inflammation, lung fibrosis, and pulmonary hypertension (33). These features follow a similar temporal sequence as observed in human SSc. In the lungs, perivascular inflammatory infiltrates and vascular remodeling appear at the 12th week of age and are followed by fibrosis development at 15th week of age (34). Fra-2 transgenic mice display severe vascular remodeling of pulmonary arteries leading to their intimal thickening and, in the worst case, to obliteration of vessels (35). Two groups of Fra-2 transgenic female mice were treated starting at 12 weeks of age with intraperitoneal injections of ALPN-101 (n=11) or Fc control (n=8) twice a week, for a total of 6 weeks. Mice were euthanized by exsanguination after right ventricular systolic pressure (RVSP) measurement at 18 weeks of age (Supplementary Figure 2). This experiment has been carried out twice.
ALPN-101 serum measurement
The concentration of ALPN-101 was measured in serum samples collected 24 hours after the 8th or 13th dose in the HOCL model, or after the 10th and 13th dose in the Fra-2 Tg model, using an ELISA method developed at Alpine Immune Sciences. ALPN-101 was captured by Fc-specific donkey anti-human IgG antibody (Jackson ImmunoResearch), immobilized onto a 96-well microtiter plate and detected with F(ab’)2 fragment, Fc-specific donkey anti-huIgG:HRP (Jackson ImmunoResearch). A calibration curve was generated for each assay plate using SoftMax Pro data acquisition and analysis software (version 7.1, Molecular Devices).
Clinical follow-up of Fra-2 mice
Fra-2 transgenic mice developed a disease phenotype requiring their clinical follow-up. Monitoring included weighing the mice once a week for the duration of the experiment. All the mice were scored individually using body weight change and observation of their physical appearance and behavior. Mice received a clinical score of 0 to 3, with 0 = normal ; 1 = weight loss <10%, lack of grooming and behavior minor modifications ; 2 = weight loss between 10-15%, alopecia and skin lesions, reduced mobility, Raynaud’s syndrome ; 3 = weight loss > 20%, ruffled fur, hunched posture, lethargy. If mice reached a clinical score of 3 before the end of the experiment, they were euthanized to respect the 3R rule.
Skin thickness measurement of HOCL-treated mice
Skin thickness (expressed in millimeters) was assessed using a caliper to measure the dermal thickness of the shaved backs of the mice. The measurement was performed once a week until the end of the experiment.
Collagen content was measured in a 3-mm punch from the back skin of HOCL-treated mice or from lung biopsies (right lobes) of Fra-2 mice using Sircol® soluble collagen assay (Biocolor, UK) according to the manufacturer’s instructions. Collagen content was determined from the slope of the standard curve calculated using known collagen concentrations.
Immunohistochemistry and immunofluorescence
Paraffin-embedded sections of dorsal skin were obtained from (1) PBS/, HOCL/Fc control- and HOCL/ALPN-101-treated mice, and (2) healthy or lesional skin biopsies obtained from healthy human controls or SSc patients. After antigen retrieval, blocking and tissue permeabilization with PBS + 0.25% Triton X-100, mouse skin sections were incubated with the following primary antibodies diluted in PBS+0.5% BSA overnight at 4°C : rat anti-CD3 (Abcam, clone RM0027-3B19, dilution : 1/50), rabbit anti-CD68 (Abcam, Polyclonal, dilution : 1/250), rat anti-CD20 (Abcam, clone GOT214A, dilution : 1/100), rat anti-Ly6G (BD Biosciences, Clone 1A8, dilution : 1/500), and rabbit anti-alpha-SMA (Abcam, Clone E184, dilution : 1/250). Then, slides were incubated with the following secondary antibodies diluted in PBS + 1% BSA for one hour at RT : goat anti-rabbit (Pierce, dilution : 1/200) and goat anti-rat (Invitrogen, dilution : 1/500 for Ly6G and CD20 ; 1/150 for CD3). Visualization was performed with Dako Liquid DAB+Substrate Chromogen System (Agilent Technologies), slides were counterstained with hematoxylin (ThermoFisher) and mounted using aqueous mounting medium (Merck Millipore). For human skin sections, the following primary antibodies diluted in PBS+0.5% BSA were incubated overnight at 4°C : anti-ICOS (Abcam goat polyclonal, dilution : 1/130) and anti-CD3 (Abcam Clone CD3-12, dilution : 1/250). Then, slides were incubated with the following secondary antibodies diluted in PBS+1% BSA for one hour at RT : anti-goat AF594 (ThermoFisher, dilution 1/200) and anti-rat AF488 (Thermofisher, dilution 1/200). Slides were mounted using Vectashield® mounting medium with DAPI (Vector Laboratories, UK). Analysis of the immunostaining was performed using the Lamina Multilabel Slide Scanner (Perkin Elmer, USA). Slide staining analysis was performed with CaseViewer software (version 2.4).
Histopathologic assessment of dermal fibrosis in HOCL-treated mice and fibrosing alveolitis in the Fra-2 model
Fixed 6-mm skin punch biopsies from HOCL-treated mice or left lung from Fra-2 mice were embedded in paraffin. A 4-µm thick tissue section was stained with hematoxylin, eosin, and saffron. Slides were scanned with the Lamina Multilabel Slide Scanner. For HOCL skin sections, dermal thickness was evaluated at 100-fold magnification by measuring the distance between the epidermal-dermal junction and the dermal-subcutaneous fat junction at five sites on skin sections by two independent blinded examiners with the CaseViewer software (version 2.4). The mean of the 10 values obtained by the two examiners was calculated for each skin section. For Fra-2 Tg lung sections, the severity of fibrosing alveolitis was semi-quantitatively assessed by examining the entire slide, by two examiners blinded to the treatment. The grading criteria were as follows: 0 = normal lung ; 1 = Minimal fibrous thickening of alveolar or bronchioalveolar walls ; 2-3 = moderate thickening of walls without obvious damage to lung architecture ; 4-5 = Increased fibrosis with definite damage to lung structure and formation of fibrous bands or small fibrous masses ; 6-7 = Severe distortion of structure and large fibrous areas and 8 = Total fibrous obliteration (36).
Nonlinear microscopy and second harmony generation (SHG) processing
A 2-photon Leica SP8 DIVE FLIM (Leica Microsystems GmbH, Wetzlar, Germany) was used for lung and skin tissue imaging. Two lasers at 1040 and 880 nm wavelength were used to generate second harmonic (SHG) and two-photon-excited fluorescence (TPEF) signals, collected by a Leica Microsystems HCX IRAPO 25×/0.95 W objective and two external detectors. Microscopy was performed on 16 µm-thick blank blades of sliced lungs or skin. Five samples of each slice were taken. The SHG score was established by comparing the area occupied by the collagen relative to the sample surface. Image processing and analysis (thresholding and SHG scoring) were performed using ImageJ homemade routine as previously described (37).
Right ventricular systolic pressure (RVSP) measurement in Fra-2 mice
RVSP was assessed in unventilated mice under isoflurane anesthesia (1.5-2.5%, 2L O2/min) using a closed chest technique by introducing a catheter (1.4-F catheter; Millar Instruments Inc., Houston, TX) into the jugular vein and directing it to the right ventricle. After RVSP measurement, blood was collected by direct cardiac puncture leading to mouse sacrifice. The heart and lungs were removed and flushed with 5 mL of buffered saline at 37°C. The left lung was fixed in paraformaldehyde 4%. For 10 Fra-2 Tg mice (4 Fc control- and 6 ALPN-101-treated), one lobe of the right lung was collected to perform FACS analysis and other lobes were immediately snap-frozen in liquid nitrogen and kept at -80°C.
Spleen and lung cell isolation for flow cytometry staining
Flow cytometry staining was performed on 4 spleen/lung Fc control-treated mice and on 6 spleen/lung ALPN-101-treated mice. Spleens were collected and crushed on a 70 µm cell strainer. Red cells were removed with ACK Lysing Buffer (Thermofisher). 1x106 cells were collected for flow cytometry staining. One lung lobe was cut in small pieces and incubated in PBS 10% FBS + Collagenase II (1mg/mL, StemCell Technologies) and DNase I (0.1mg/mL, StemCell Technologies) for 1 hour at 37°C. After mechanical dissociation with vortexing, cell suspensions were passed through a 70 µm cell strainer. Red blood cells were removed with ACK Lysing Buffer (ThermoFisher). A Percoll (Sigma) density gradient was generated by resuspending cells in a 40% Percoll solution and adding an 80% Percoll solution below the 40% solution. The cell ring was collected, and the total lung cell suspension was used for flow cytometry staining.
Spleen and lung cells were incubated with Zombie Dye UV (Biolegend) for 15 minutes at room temperature. Fc receptors were blocked with the TruStain FcX™ (anti-mouse CD16/32) Antibody (Clone 93, Biolegend) for 5 minutes on ice. Cells were first incubated with an anti-CD62L-APC/Cy7 antibody (Clone MEL-14, Biolegend) for 20 minutes on ice. Second, the following antibody mix was incubated with cells for 30 minutes on ice : anti-CD4 BV711 (Clone GK1.5), anti-CD19-AlexaFluor700 (Clone 6D5), anti-CD3-BV510 (Clone 17A2), anti-CD44-BV605 (Clone IM7), anti-CD28-BV421 (Clone 37.51), anti-CD69-BV650 (Clone H1.2F3), anti-PD-1-PE/Dazzle 594 (Clone 29F.1A12), anti-human IgG Fc-PE (clone M1310G05), anti-ICOS-APC (Clone 7E.17G9) purchased from Biolegend and anti-CD8-BUV737 (Clone 53-6.7) purchased from BD Biosciences. Stained cells were fixed in PBS 2% PFA. Data acquisition was performed on a BD LSR Fortessa Cytometer and data were analysed with FlowJo Software (version 10.7.2).
ICOS measurement in human serums
ICOS protein was quantified by ELISA in the serum of 161 patients with SSc and 38 healthy age- and sex-matched volunteers using the Human ICOS (CD278) ELISA Kit (ThermoFisher Scientific™) according to the manufacturer’s instructions.
All data analysis were performed using GraphPad Prism 9 Software. Human data were presented as mean with standard deviation (SD) and analyzed with Student’s t-test. Mouse data were presented as median with ranges and analyzed by Mann-Whitney Test. Correlation data were analyzed with Spearman’s correlation test. A p value of less than 0.05 was considered statistically significant.