3.1 The TFH and TFR cells in the germinal center of the spleen and peripheral blood showed a gradual increase in the CIA mouse model.
The CIA model was established by immunizing DBA/1 mice with type II collagen and CFA. The model was successfully established according to the clinical scores (Fig. 1A&B). The levels of TFH and TFR in the spleen and peripheral blood of CIA mice and control mice were analyzed at different time points after the first immunization (week 0). As shown in Fig. 1, at the early stage of modeling (5 weeks), the TFH levels in spleen of CIA mice and the controls were not significantly different, but as the weeks increased, the TFH levels were significantly higher than those of the controls, for both percentages and absolute numbers (Fig. 1C&D). In terms of TFR levels, the result showed that the difference in the early stage of modeling is not large, but as the age increased, TFR levels in spleen of CIA mice were significantly higher than controls (Fig. 1C&D). We studied TFH and TFR levels in peripheral blood and the changes were in accordance with situations in spleens (Fig. 1E). We also compared TFH to TFR ratios and found that there was no significant difference between CIA mice and the controls, suggesting that TFH and TFR Raise in parallel. This part of the results suggests that TFH and TFR levels gradually increase in the germinal center during the process of RA, and the changes in peripheral blood and germinal center TFH and TFR are consistent.
3.2 The spleen and peripheral blood TFH and TFR cells of CIA mice treated with anti-TNFα and anti-IL-1β neutralizing antibodies showed a significant decrease.
We next used neutralizing antibodies of inflammatory cytokines for CIA treatment. The treatment options selected include anti-TNFα + anti-IL1β and anti-IL-1β alone. Because antibodies to different inflammatory factors play different roles, we used two different strategies to treat CIA mice separately, and also aimed to compare the differences in their therapeutic effects. We chose 10w as the termination time point to compare with the CIA mice treated with isotype IgG, because 10-week time point showed highest clinical scores and the level changes of TFH and TFR were also the most significant. We found that the clinical scores were significantly decreased after antibody injection, but there was no significant difference between mice injected with anti-TNFα + anti-IL1β and anti-IL-1β alone (Fig. 2A&B). After using the two regimens, the spleen TFH and TFR of CIA mice showed a significant downward trend, for both (Fig. 2C), while the peripheral blood TFH and TFR also significantly decreased (Fig. 2D). But there is no significant difference between the two different treatment options. We also compared TFH to TFR ratios and found that there was no significant difference among three groups (Fig. 2E). The results showed that TFH and TFR in CIA mice treated with inflammatory factor neutralizing antibodies were significantly reduced, suggesting that the consequence of this anti-inflammatory immunotherapy method is that the TFH/TFR system in mice is consistently reduced.
3.3 TIGIT + and TIGIT + CD226-TFH cells in the spleen of CIA mice were significantly elevated and their levels recovered after inflammatory factor antibody treatment.
To further clarify TFH and TFR subpopulation changes in the germinal center and peripheral blood of CIA mice, the main functionally related marker molecules were analyzed in detail. As shown in Fig. 3A&B, in the TFH cells of the spleen in CIA mice, the expression of T cell immunoglobulin and ITIM domain (TIGIT) was always significantly higher than that of the control group, and the ratio of TIGIT + CD226- subset was also significantly higher than that of the control group. TFH in peripheral blood showed the same trend, but for TFR cells, there was no significant difference (data not shown). It was reported that TIGIT + TFH cell were with a stronger ability to help B cells, so the increase in TFH levels reflects its enhanced function. In terms of treatment, it’ s interesting that after neutralizing antibody treatment, the ratios of TIGIT + and TIGIT + CD226 + in TFH cells were significantly reduced (Fig. 3C).
3.4 The expression of PD-1 and ICOS on the surface of TFH and TFR cells in the spleen of CIA mice was significantly increased.
Considering that CXCR5 alone may not fully represent mouse peripheral TFH and TFR cells, we further studied the changes in the expression levels of PD-1 and ICOS on TFH and TFR cells based on the previous results. We measured PD-1 and ICOS on the surface of TFH and TFR cells in CIA mice. As shown in Fig. 4, PD-1+, ICOS + and PD-1 + ICOS + percentages in CD4 + CXCR5 + FoxP3-TFH cells in the spleen of CIA mice increased significantly compared with the controls. These molecules are considered to indicate functions of TFH cells, so these results suggest that the function of TFH cells in the germinal center of the spleen of CIA mice were increased. In addition, we also found that TFR cells in the spleen of CIA mice also showed a significant increase (Fig. 4), and the changes were consistent with TFH cells.
3.5 Treatment with neutralizing antibodies to inflammatory factors can restore the elevated PD-1 and ICOS expression in spleen TFH and TFR cells of CIA mice.
We further explored the effect of inflammatory factor antibody treatment on PD-1 and ICOS on the surface of TFH and TFR in CIA mice. The results showed that the percentages of PD-1 + and PD-1 + ICOS + cells in TFH cells decreased significantly after treatment, while the percentages of ICOS + cell decreased significantly after treatment with IL-1β antibody alone (Fig. 5). For TFR cells, all the three subsets were decreased significantly after being treated by the two treatment options. And PD-1 + percentages were also lower after treatment with IL-1β antibody alone than treated with anti-TNFα plus anti-IL-1β (Fig. 5). These results suggested that the expression of TFH and TFR marker PD-1 and ICOS were significantly changed after inflammatory factor antibody treatment.
3.6 The ability of TFH to secrete IL-21 and TFR to secrete IL-10 in the spleen of CIA mice is relatively enhanced, and the inflammatory factor neutralizing antibody treatment can restore its secretion ability.
To further study the ability of TFH and TFR to secrete cytokines in the germinal center of CIA mice, we cultured and stimulated lymphocytes and detect the ability of TFH and TFR cells to secrete IL-4, IFNγ, IL-17, IL-21, IL-10 and TNFα. The results are shown in Fig. 6. For TFH cells, the secretion of IL-21 was significantly higher than that of the control group, which suggests that the function of TFH is enhanced in CIA mice. In terms of TFR, the secretion ability of IL-10 in CIA mice is significantly higher than that of the control group. For secretion of other cytokines, there was no difference. In terms of treatment, the levels of IL-21 and IL-10 secreted by TFH and TFR cells after treatment showed a significant decreasing trend, indicating that inflammatory cytokine antibody treatment resulted in a significant decrease in the ability to secrete cytokines for TFH and TFR cells, and its immune function is seriously affected.
3.7 The functions of TFH and TFR cells in the spleen of CIA mice are both enhanced and could be recovered by neutralizing antibodies to inflammatory factors.
In order to directly prove the functional changes of TFH and TFR cells in the spleen germinal center of CIA mice, we used an in vitro co-culture system. TFH cells of different sources were co-cultured with B cells (from CIA mice) to test the function of TFH, and TFR cells of different sources were co-cultured with TFH + B cells (from CIA mice) to test the function of TFR. We measured the functional changes of TFH and TFR in regulating antibody secretion by detecting the percentages of GL7 + IgG1 + B cells in the culture system and the levels of IgG in the supernatant. The results showed that the function of TFH cells derived from CIA mice to help B cells and the function of TFR cells to inhibit TFH cells were both relatively enhanced (Fig. 7A-C).
We also tested the functions of TFH and TFR cells in CIA mice treated with inflammatory cytokine antibodies, and the results showed that the functions of TFH and TFR cells were relatively reduced after antibody treatment (Fig. 7D&E). The difference between the two different treatment options (anti-TNFα + anti-IL1β and anti-IL-1β alone) is not significant enough.