Wide-type male C57BL/6 (H2b, 8–12 weeks) and BALB/c (H2d, 8-12 weeks) mice were purchased from Vital River Laboratories (Beijing, China). Animals were allowed free access to water and standard chow. Animal experiments were approved by the Institutional Ethics committee for Research on Animals. The use and care of animals were in accordance with the Institutional Animal Care and Use Committee guidelines.

Monoclonal αIL-21R was obtained from BioXcell (Lebanon, NH, USA), diluted with PBS, and added to the cell culture medium at 10 μg/mL. For in vivo experiments, mice were treated with intraperitoneal injections of 1 mg/kg αIL-21R or isotype starting on the day of transplantation and three times per week for three weeks.

Single cell suspensions were prepared from the spleens of C57BL/6 mice. Naive CD4⁺ T cells were isolated by negative selection, using biotinylated antibodies directed against markers of non-naive CD4⁺ T cells (CD8, CD11b, CD11c, CD19, CD24, CD25, CD44, CD45R, CD49b, TCRγ/δ, and TER119) and streptavidin-coated magnetic particles (Stemcell Technologies, Vancouver, BC, Canada). Cells were used at a purity of > 95%.

Naive CD4⁺ T cells were cultured in 96-well flat-bottom plates (2.0 x 10⁵ per well) suspended in 0.5 mL RPMI 1640 media supplemented with 10% fetal calf serum, 200 mM L-glutamine, 100 U/mL penicillin/streptomycin and 5 × 10^-5 M 2-mercaptoethanol at 37°C in a 95%/5% air/CO₂ atmosphere with saturating humidity. The wells were coated with 8 μg/mL anti-mouse α-CD3, and 2 μg/mL soluble anti-mouse α-CD28 was added to the media. The Tfh cell differentiation procedure was modified from Gao et al. (19) T cell-depleted splenocytes were stimulated with lipopolysaccharide (LPS) for 24 h and co-cultured with naive CD4⁺ T cells (1:10) to stimulate Tfh cell differentiation. IL-6 (100ng/mL) and IL-21 (50ng/mL) were added to the cell culture. For Treg differentiation, a cocktail containing IL-2, TGF-β, and all-trans retinoic acid (Immunocult; Stemcell Technologies, Vancouver, BC, Canada) was added to cell culture. After 5 days, the cells were collected, and analyzed by flow cytometry. To determine the absolute number of viable lymphocytes, Trypan blue staining was performed and live cells were counted using a hemocytometer.

Single cell suspensions were prepared from the spleens of recipient mice. Cells were labeled for surface and intracellular markers using fluorescence-labeled anti-mouse α-CD4, α-CXCR5, α-ICOS, α-PD1, α-FOXP3, α-GL7, and α-Fas antibodies (eBioscience, CA, USA). Intracellular FOXP3 staining was performed using a commercially available staining kit that included permeabilization solution and buffer (eBioscience). Flow cytometric measurements were performed on a FACSAria III (BD Bioscience, CA, USA) and data were analyzed using FlowJo (FlowJo Software, OR, USA). For proper gating, apoptotic cells were excluded, and the samples were compared with isotype controls, fluorescence minus one (FMO)-stained, permeabilized, and unpermeabilized unstained cells. For the carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen, CA, USA) cell proliferation assay, cells were labeled with 1 µM CFSE. The CFSE dilution was measured by flow cytometry.

Fully mismatched skin transplants were performed as described previously (20). In detail, full-thickness skin grafts (1 cm²) were obtained from the tails of BALB/c mice and engrafted onto the dorsolateral thoracic walls of C57BL/6 mice. Graft rejection was monitored daily and necrosis > 90% indicated graft rejection.

To analysis the germinal center, paraffin sections of spleen were deparaffinized, microwaved, immersed in 0.01 M citrate buffer (pH 6.0) for 20 min, washed with PBS, and incubated at room temperature for 30 minutes with biotinylated peanut agglutinin (PNA). After washing, tissues were incubated with peroxidase for 30 minutes. Reaction signals were developed using 3, 3’ diaminobenzidine (DAB; Dako, Denmark).

For DSA detection, serum was diluted (1:18) and incubated with 10⁶ BALB/c splenocytes for 1 h at 4°C. Next, the cells were washed and incubated with anti-IgG and anti-IgM antibodies (Jackson immunoResearch, PA, USA) for 30 min. The mean fluorescence intensity of the cells was measured by flow cytometry.

Differences between groups were analyzed using unpaired Student’s t-tests. Graft survival was compared using log-rank tests. Results with p < 0.05 were considered statistically significant (*p<0.05; **p<0.01; ***p<0.001).

To test the overall effect of αIL-21R on CD4⁺ T cells, we first investigated whether this monoclonal antibody induced proliferation or apoptosis in the unspecific Th0 condition. As shown by the CFSE proliferation assay, CD4⁺ T cells treated with αIL-21R or isotype control showed comparable proliferating cell rates ( Figure 1A ). Moreover, there was no significant difference the number of in live cells, dead cells, or apoptotic cells between the groups. These results indicate that αIL-21R is not cytotoxic and is not expected to kill a large number of cells or inhibit cell proliferation ( Figure 1B ).

Both Tfh and Tfr cells expressed IL-21R. However, IL-21 promoted Tfh differentiation and inhibited the expression of FOXP3, the signature transcription factor of Tfr and Treg cells. Furthermore, αIL-21R decreased the Tfh/Tfr ratio by inhibiting Tfh differentiation while blocking the IL-21 induced inhibition of Tfr cells. To assess the effect of αIL-21R on Tfh/Tfr ratio, we individually examined the impact of αIL-21R on Tfh and Treg differentiation. Under Tfh differentiation conditions, the frequency of Tfh cells reduced significantly by αIL-21R treatment ( Figure 2A ). Since a well-established method for Tfr differentiation is lacking, we observed the effect of αIL-21R on Tregs. As shown in Figure 2B , IL-21 reduced the frequency of Treg cells, and this effect was reversed by αIL-21R treatment ( Figure 2B ). Notably, αIL-21R treatment alone did not alter Treg differentiation (data not shown). Taken together, these data suggest that αIL-21R treatment can modify the Tfh/Tfr balance.

A skin transplantation model was used to investigate the effect of αIL-21R treatment on the Tfh/Tfr ratio in the context of alloimmune responses. Mice that received fully mismatched skin allografts were treated with αIL-21R or isotype control for 14 days after surgery and their splenocytes were analyzed. We observed that αIL-21R treatment did not reduce the frequency of CD4⁺CXCR5⁺ T cells ( Figure 3A ). However, a significant decrease was observed in the frequency of CD4⁺CXCR5⁺ ICOS⁺PD⁺ Tfh cells ( Figure 3B ). Meanwhile, αIL-21R increased the frequency of CD4⁺CXCR5⁺FOXP3⁺ Tfr cells ( Figure 3C ). The Tfh/Tfr ratio was calculated as the absolute number of CD4⁺CXCR5⁺PD1⁺ICOS⁺ and CD4⁺CXCR5⁺FOXP3⁺ cells. The results showed that αIL-21R significantly decreased the Tfh/Tfr ratio ( Figure 3D ). Notably, the absolute number of Tfh cells was lower in the group treated with αIL-21R, although the results was statistically insignificant ( Figure 3D ).

To assess whether the in vivo decrease in Tfh/Tfr ratio mediated by αIL-21R inhibited the antibody response, we treated skin transplantation recipients with αIL-21R or control IgG and analyzed the GC response after 14 days. Flow cytometry analysis revealed a significant decrease in the frequency of GC B cells (B220⁺GL7⁺Fas⁺ cells) in mice treated with αIL-21R ( Figure 4A ). In line with this finding, PNA staining confirmed that αIL-21R inhibited GC formation ( Figure 4B ). Finally, we investigated the effect of αIL-21R on dnDSA generation by monitoring the levels of donor-specific IgG and IgM for 5-weeks ( Figure 4C ). As previously reported, IgG levels in the control group continued to increase and peaked at 3-weeks post-surgery, after which, the IgG level decreased, and remained at a stable level. No significant difference was observed between the treatment and control groups during the first 2-weeks. However, the difference appeared at 2-weeks as αIL-21R treatment blocked increasing IgG levels, and even caused them to decrease. At 3-weeks after surgery, IgG levels in the αIL-21R treatment group were significantly lower than in the control group. Although no significant difference was observed afterward, the αIL-21R group showed an overall lower IgG level compared to the control group; no significant difference was observed in IgM levels between the groups ( Figure 4C ).