Supplementary Figure 1
CCR10 is expressed by nasal mucosal plasma cells.
Nasal mucosal tissue biopsies (n=2) were incubated with collagenase, and stained with the following fluorochrome-conjugated antibodies: CD3-FITC (clone UCHT1, Diatec), CD27-PE (clone O323, Biolegend), CCR10 APC (clone 314305, R&D Systems) and CD138-PE/Cy7 (clone DL101, eBioscience). Plasma cells were visualized as CD3-CD27+ (left) and CD138+, revealing a distinct population expressing CCR10 (right).
Supplementary Figure 2
Rapid intracellular Ca-flux in CCR3+ T cells following CCL28-stimulation.
(A) Expression of CCR3 (clone 61828, R&D Systems) and CCR10 (clone 314305, R&D Systems) on T cells purified from PBMCs using a Dynabeads Untouched Human T cells kit (Life technologies) before (left) and after depletion (right) of CCR10+ cells using anti-CCR10 (clone 1B5, Millenium Pharmaceuticals) followed by Dynabeads Sheep anti-Mouse beads (Life technologies). (B) CCR10-depleted T cells were loaded with the Fluo-3 calcium indicator (Life Technologies), and stained for CCR3. The kinetics of cytoplasmic Ca2+ release was measured in CCR3+ and CCR3- T cells by FACS. An immediate and transient rise of intracellular Ca2+ (arrow) was detected in CCR3+ cells following the addition of 300nM rhCCL28 (R&D Systems). No increase in intracellular Ca2+ was detected in CCR3- cells. Gates were set based on irrelevant isotype controls, and similar results were found in 2 independent experiments.
Supplementary Figure 3
(A) T cells expressing CCR4, CCR7 and CCR6 constitute minor T cell fractions in healthy nasal mucosa.
Nasal mucosal tissue biopsies (n=3) incubated with collagenase were stained for 30 min at 4°C with unconjugated anti-CCR4 (clone 1G1, Millenium Pharmaceuticals) followed by biotin-conjugated goat anti-mouse IgG1 (Southern Biotech). After blocking with mouse IgG (Sigma-Aldrich), cells were stained with the follwing fluorochrome-conjugated antibodies: CD3-APC (clone SK7, BD Bioscience) and CD4 APC/Cy7 (Clone OKT4, Biolegend). In parallel stainings, CCR7-PE (clone 150503, R&D Systems), CCR6-APC (clone R6H1, eBioscience), CD4-BV605 (clone OKT4, Biolegend) and CD3-PE/Cy7 (clone SK7, Biolegend) were used.
(B) Nasal mucosal CCR3+ CD4+ T cells are homogenously positive for T-bet, and partially CXCR3+. Biopsies (n=3) were treated as above, and stained with CCR3-APC (clone 61828, R&D Systems), CXCR3-A488 (clone G025H7, Biolegend), CD4-BV605 (clone OKT4, Biolegend) and CD3-PE/Cy7 (clone SK7, Biolegend). Following fixation and permeabilization (Foxp3 staining buffer set, eBioscience), cells were stained with T-bet-PE (clone 4B10, eBioscience). Gates were set based on irrelevant isotype controls (blue histograms).