Canto 1

The following antibodies were obtained from BD Biosciences (San Diego, CA, USA): anti-CD3-APC (145-2C11), anti-cKit-Pe-Cy7 (2B8), and anti CD11b-PE (M1/70). For Lineage depletion these markers were used: CD3 (145-2C11), CD4 (L3T4), CD8 (53-6.7), CD11b (M1/70), Gr1 (RB6-8C5), B220 (Ra3-6B2), Ter119 (Ly76) and Nk1.1 (PK136) biotin and subsequently were stained with streptavidin eFluor 450 (48-4317) from eBioscience (Vienna, Austria). The following antibodies were also purchased from eBioscience: Ly5.1-PE-Cy7 (A20), Ly5.2 Alexa Fluor 780 (104), B220 PE-Cy7 (RA3-6B2), Gr1 eFluor 450 (RB6-8C5) and Sca1 PE-Cy7 (D7). Cells were stained in fluorescence activated cell sorter (FACS) buffer (PBS, 2% bovine serum albumin, 0.1% sodium azide) for 30 min at 4 °C. Ultimately, cells were washed and measured either on a Canto I, or an Aria (BD Biosciences) FACS. For FL LSK, Mac1 was precluded from the lineage gate, as FL LSK express Mac1.^{45 (link)} For apoptosis analysis, E14 FL cells were stained with 7AAD/AnnexinV kit (BD Bioscience) in combination with LSK staining. For proliferation analysis E14 FL cells were stained with PE mouse anti-Ki67 set (BD Pharmingen, San Diego, CA, USA) in combination with LSK staining or for cell cycle analysis with an adapted protocol for combined LSK and propidium iodide staining.^{46 (link)} Data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

All binding and uptake experiments were performed at 4°C or 37°C for 45 minutes. Mean fluorescence intensity was measured by flow cytometry. When quantifying, MFI from non-transfected parental cells was subtracted.
For binding and uptake assays, 300.19 pre-B cells stably transfected with receptors (and GFP in a T2A system), or parental, were incubated with the indicated concentrations of chemokines (all labeled in AF647 or Dy649) for 45 minutes. Cells were washed and analyzed by FACS (Fortessa, BD). The MFI of GFP, which is proportional to the level of receptor expressed (45 (link)), was used for normalization.
Direct binding was measured by FACS incubating 300.19 cells expressing GPR182 T2A GFP with increasing concentrations of fluorescently labeled chemokine. Competition binding and scavenging was measured by FACS incubating 300.19 cells expressing GPR182-GFP with a fixed amount (3 nM) of fluorescently labeled chemokine and increasing concentrations of unlabeled chemokine. After 45 minutes cells were washed and analyzed by FACS (Canto I, BD).

Cytometric 13-multiplex cytokine bead arrays (CBA) were used to quantify IL-23, IL-1α, IFN-γ, TNF-α, CCL2 (MCP-1), IL-12p70, IL-1β, IL-10, IL-6, IL-27, IL-17A, IFN-β, GM-CSF (Biolegend) in culture samples. The determinations were performed in duplicate according to the manufacturer’s instructions. Samples were run on a CANTO I or FORTESSA (BD Biosciences) cytometer. The calibration curves were above r² 0.97.

A 13-multiplex cytometric CBA (IL1β, IFNα2, IFNγ, TNFα, MCP1/CCL2, IL6, IL8/CXCL8, IL10, IL12p70, IL17A, IL18, IL23 and IL33: Biolegend, CA, USA), and human IgG isotype bead array (IgG1, IgG2, IgG3 and IgG4: Biolegend) were used to quantify the cytokines and IgG isotypes in NLF samples. The determinations were performed in duplicate according to the manufacturer´s instructions. Samples were run on a CANTO I (BD Biosciences, San José, CA, USA) cytometer and the calibration curves were above r² 0.97.

All binding and uptake experiments were performed at 4°C or 37°C, respectively, for 45 minutes. Mean fluorescence intensity (MFI) was measured by flow cytometry. MFI was normalized to parental cells.
For binding and uptake assays, 300.19 pre-B cells stably transfected with receptors (and GFP in a T2A system), or parental, were incubated with 300 nM chimeric chemokines (all labeled in AF647 or Dy649) for 45 minutes. Cells were washed and analyzed by FACS (Fortessa, BD). The MFI of GFP, which is proportional to the level of receptor expressed [58 (link)], was used to normalize.
Displacement binding was measured by FACS by incubating 300.19 cells expressing GPR182-GFP with a fixed amount (5 nM) of fluorescently labeled (chimeric) chemokine and increasing concentrations of unlabeled (chimeric) chemokine. After 45 minutes, cells were washed and analyzed by FACS (Canto I, BD).

Ipsilateral submandibular lymph nodes were considered draining lymph nodes of the eye and were harvested eight weeks post corneal transplantation. Organs were mechanically disrupted, pressed through 40 µM strainer and subjected to Red blood cell lysis buffer. Cell suspensions were washed with Flow cytometry Buffer containing 1% FBS and 20 µM HEPES.
Cell were stained extracellularly for CD3 (Pe-Cy7, Biolegend, Koblenz, Germany), CD4 (APC, eBioscience, Darmstadt, Germany), CD8 (APC-Cy7, Biolegend, Koblenz, Germany), CD11c (PE, Biolegend, Koblenz, Germany), CD11b (Pe-Cy7 Biolegend, Koblenz, Germany) and CD45 (APC, Biolegend, Koblenz, Germany) for 30 min on ice. Samples were then fixed and permabilized using eBioscience Fix/Perm kit and stained for Foxp3 (FITC, eBioscience, Darmstadt, Germany). Data were acquired by Millipore (Guava Incyte, Darmstadt, Germany) or Canto I (BD Bioscience, Heidelberg, Germany) and analyzed using FlowJo (v.10, Ashland, Oregon, USA).