Cd4 clone 4sm95

Sectioning and Staining: After harvesting, tumors were immediately fixed overnight in 10% neutral-buffered formalin. Fixed tumors were embedded in paraffin and sections were cut at a thickness of 5 μm. Full-section slides of tumor tissues were stained using Opal multiplex 6-plex kits, according to the manufacturer’s protocol (PerkinElmer), for DAPI, Epcam (polyclonal; Abcam, 1:100 dilution), CD3 (clone SP7; Spring Biosciences; 1:100 dilution), CD8 (clone 4SM15; ThermoFisher; 1:500), CD4 (clone 4SM95; eBioscience, 1:50), Foxp3 (polyclonal; ThermoFisher, 1:500), and Granzyme B (polyclonal; Abcam, 1:200). Single color controls and an unstained slide were also included.
Multispectral imaging and analysis: Multispectral image capture was done at 20X magnification using Vectra (PerkinElmer, Hopkinton, MA). Images were analyzed using inForm software version 2.4.1 (PerkinElmer) as previously described [31 (link)]. Further details are presented in the Supplementary Materials.

To detect infiltrated immune cells, paw sections (3.5 µm) were fixed with acetone and denatured with 100, 95, and 75% ethanol for 15 s separately. For antigen retrieval, sections were heated in Tris-EDTA retrieval buffer (10 mM Tris Base, 1 mM EDTA solution, 0.05% Tween 20, pH 9.0) in a water bath at 98°C for 20 min. After cooling down at RT for 30 min, sections were incubated with antibodies against F4/80 (clone CI:A3-1; Abcam), GR-1 (clone RB6-8C5; R&D), CD4 (clone 4SM95; Thermo Fisher), CD8 (clone 4SM15; Thermo Fisher), and CD19 (clone 6OMP31; Thermo Fisher) at 4°C overnight for detecting macrophages, neutrophils, CD4+ T cells, CD8+ T cells, and B cells respectively. Next, sections were washed with PBS and incubated with anti-Rat IgG (Alexa Fluor 488 conjugated; Thermo Fisher) secondary antibody. Cell nucleus was counterstained with Hoechst 33342 (Thermo Fisher), and sections were visualized using a fluorescence microscope (Olympus BX61). Quantification of fluorescence intensity was performed by count function in the software of CellSens dimension (Olympus). Briefly, five different regions in arthritis paw from DTHA mice and normal paw from untreated mice with strongest fluorescence intensity were selected for each sample. Then the average value of fluorescence intensity was calculated from these five regions and used for the next statistical analysis.

The antibodies used in the study are summarized as follows: CD4 (clone 4SM95; number 14-9766-82), CD8 (clone 4SM15; number 14-0808-82), CD49b/Integrin Subunit Alpha 2 (ITGA2) (clone DX5; number 14-5971-85), forkhead box P3 (FOXP3) (clone FJK-16s; number 14-5773-82), inducible nitric oxide synthase/nitric oxide synthase 2 (number PA1-036), arginase 1 (ARG1; number PA5-29645), pan-cytokeratin (number PA1-27114), CD68 (clone FA-11; number 14-0681-82), and F4/80 (clone BM8; number 14-4801-82) were purchased from ThermoFisher Scientific (Waltham, MA). TP53 (clone POE316A/E9; number MABE1808) and tubulin (number T9026) were from MilliporeSigma (Burlington, MA). HPV16 E6 (number orb10837) for Western blot analysis and HPV16 E7 (number orb10573) for immunofluorescence were from Biorbyt LLC (St. Louis, MO). HPV16 E6 (clone C1P5; number NB100-2729) for immunofluorescence, HPV16 E7 (clone TVG 701Y; number NB100-2768) for Western blot analysis, and bromodeoxyuridine (BrdU; clone BU1/75; number NB500169) were from Novus Biologicals (Centennial, CO).