Rmil 21

For administration of rmIL-21, the C57BL/6 mice were inoculated intranasally with 0.5 μg rmIL-21 (PEPROTECH, 5 Cedarbrook Drive, Cranbury, NJ, USA) in 20 μL PBS at 1 day before infection and at days 0, 2, 4, and 6, and the control group was given 20 μL sterile PBS on the same schedule. The mice were euthanized at the designated time points after infection. Lung tissue was isolated and lung single cell was prepared for flow cytometry to detect iNOS.

The in vitro PB differentiation protocol of the previous study was followed with slight modifications (14 (link)). Mice were immunized with NP-CGG either by s.c. or i.p. On day 7 following immunization, B cells ( $ $⁺), Tfh (TCRβ⁺CD4⁺CXCR5⁺PD1⁺FOXP3^–), and Tfr (TCRβ⁺CD4⁺CXCR5⁺PD1⁺FOXP3⁺) were isolated by cell sorter, FACSAria, from spleens or LNs. A total of 5 × 10⁴ of B cells were cultured with or without 3 × 10⁴ Tfh alone or with 1.5 × 10⁴ Tfr. To stimulate T and B cells, anti-CD3 (2 μg/mL) and anti-IgM F(ab’)₂ (5 μg/mL) were included in the culture medium. For the activation of antigen-specific T and B cells, NP-OVA (20 μg/mL) was added instead of anti-IgM. Cells were cultured for 6 days and PB differentiation was investigated by GL-7⁺ intracellular Ig (IgG1, IgM, IgG2, and IgG3) in the live B cells.
For certain experiments, 10 μg/mL of anti–IL-17–neutralizing Abs (Invitrogen, model 1402nAF); 1 μg/mL of anti–IL-21 (Invitrogen, model FFA21); 10 μg/mL mouse IgG1 kappa (Invitrogen, model P3.6.2.8.1); and 1 ng/mL of recombinant mouse (rm) IL-17 (R&D Systems) or 1 ng/mL of rmIL-21 (Peprotech) were added at day 0 of coculture.

Male C57BL/6 mice (5 weeks old) purchased from Shanghai Jihui Co. (Shanghai, China) were bred under the specific pathogen-free (SPF) conditions. The armpit of each mouse was injected subcutaneously with 1 × 10⁷ PANC-2 cells in 200 μL of PBS (pH 7.4). Three days later, the mice in the control group were injected with PBS, and the mice in the three experimental groups were injected with recombinant mouse (rm) CXCL13 (5 μg, Peprotech, Rocky Hill, NJ, USA), rmIL-21 (5 μg, Peprotech) and 1 × 10⁵ Tfh cells, separately, via the tail vein every 3 days and euthanized 2 weeks later. The mice weight and tumor measurements were conducted every two days and the tumor volume (mm³) was estimated as (length × width²)/2. Tumors were harvested and either digested into a single-cell suspension for flow cytometry analysis or fixed in formaldehyde for immunohistochemistry (IHC) of mouse CD8 (1:300; Abcam, Cambridge, MA, USA) and mouse CD19 (1:300; Invitrogen, Waltham, MA, USA). The animal studies were conducted under protocols approved by the Ethics Committee of Fudan University Shanghai Cancer Center (ethic code: FUSCC-IACUC-S20210159, approved at 23 February 2021).