Anti cxcr3

Isolation of CD8⁺ T cells from HD samples was performed with CD8 magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) according to manufacturer’s instructions. Freshly isolated CD8⁺ T cells, anti-CXCR3 (10 ng/mL, Abcam), or Stattic (10 nM; S7024; Selleck Chem, Houston, TX)-treated CD8⁺ T cells as well as peripheral blood mononuclear cells (PBMCs) from HDs were seeded on the upper chambers of a 5.0-μm pore Transwell. Supernatants of tumor tissue, anti-CXCL10 (100 ng/mL, Abcam), or recombinant human (rh) CXCL10 (Peprotech, Rocky Hill, NJ) were added to the lower chambers. CD8⁺ T cells in the upper chambers were pretreated with rhIL-17A (20 ng/mL), Stattic (10 nM), and/or the supernatants of enriched Th17 cells (5 × 10⁶) with or without anti-IL-17A (10 ng/mL) for 48 h in vitro. Migrated cells in the lower chambers were then counted and analyzed by flow cytometry after 12 h.

Specimens from CRC patients and mice were formalin fixed, sectioned, and embedded into paraffin for immunohistochemistry. Immunofluorescence was performed with frozen samples and freshly fixed cells. The following antibodies were used: anti-CD8 (1:500), anti-CXCL10 (1:300), anti-CD4 (1:300), anti-IL-17A (1:300), and anti-CXCR3 (1:300; all Abcam, Cambridge, UK). Immunohistochemistry and immunofluorescence were performed as described elsewhere [33 (link), 34 (link)]. For immunohistochemistry, three fields of view per sample were imaged. The intensity of immunostaining was considered when analyzing the data. The percentage scoring of immunoreactive cells was as follows: 0 (< 10%), 1 (10–40%), 2 (40–70%), and 3 (> 70%). Staining intensity was visually scored and stratified as follows: 0 (negative), 1 (yellowish), 2 (light brown), and 3 (dark brown). Immunoreactivity scores (IRS) were obtained by multiplying the two items to a total score and ranged from 0 to 9. Protein expression levels were further analyzed by classifying IRS values as low (based on an IRS value ≤ 5) and as high (based on an IRS value > 5).