1. TY101 abrogates TNF-induced proliferation of Treg cells
Previously, we and others reported that, in the presence of low levels of IL-2, TNF can preferentially stimulate the proliferation of Tregs present in cultured CD4 cells . We thus used this assay to examine the effect of an anti-TNFR2 antibody TY101 on TNF-induced Treg proliferation. To this end, MACS-purified mouse CD4 T cells were cultured with IL-2 and stimulated with TNF. As expected, TNF treatment increased the proportion of Foxp3-expressing Tregs as well as the absolute number of Tregs in the cultured CD4 cells by 51.7%and 150%, respectively (Fig 1A-C, p<0.01). Treatment with TY101 (10 µg/ml) alone did not change the proportion of Tregs in CD4 cells cultured with IL-2 alone, while it completely blocked the expansion of Tregs induced by TNF (Fig 1A-C, p<0.01). Therefore, just like M861 , TY101 is an antagonistic anti-mouse TNFR2 antibody that inhibited TNF-induced proliferative expansion of Tregs.
2. TY101 treatment increases the death of Treg cells in the presence of Dex (Dexamethasone)
It was reported that anti-TNFR2 antibody was able to induce the death of Tregs in purified CD4 T cells [21, 22], we thus used LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit to assess the effect of TY101 on Treg viability. However, TY101 alone did not affect Treg viability (Fig S1A-B).To further determine the role of TNF-TNFR2 axis on Treg viability, we examined the effect of TY101 on dexamethasone (Dex)-induce the death of Treg cells. To this end, lymphocytes from normal mice were cultured with Dex (1×10-8 M), or Dex plus IL-2 (10 ng/ml), or Dex plus IL-2 and TNF (20 ng/ml), or Dex plus IL-2, TNF and TY101 (10 µg/ml) or IL-2 alone for 72 hours. The result showed that dexamethasone alone markedly reduced the proportion of Foxp3-expressing Tregs and a decreased viability of Tregs (Fig 2A-B) and (Fig 2C-D). IL-2 alone partially maintained the survival of Tregs, while addition of TNF was able to almost completely abrogated Treg death induced by Dex (Fig 2C-D). Furthermore, TNF treatment resulted in a marked increase of proportion of Foxp3+ Tregs in the cultured CD4 cells (Fig 2A-B), which was attributable to the proliferative expansion of Tregs even in the presence of Dex as we reported previously[23, 24]. In compare with IL-2 treated group, IL-2 plus TNF treated group can further increase Treg viability (from 77.2% to 91.5%). These effects of TNF on the expansion of Tregs and on the survival of Tregs was largely inhibited by the treatment of TY101 (Fig 2C-D).
3. TNFR2 signaling dampens death induction by Dex on Tregs
To further clarify if the effect of TY101 on Treg survival was mediated by the blockade of TNFR2, we determined the role of TNFR2 on Treg survival. To this end, lymphocytes were isolated from wide type mice or from TNFR2-/- mice. The cells were cultured with IL-2, with or without TNF, in the presence or absence of Dex. The viability of Tregs was determined by LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit, by gating on Foxp3+ Tregs. The result show that, when cells were cultured in IL-2 alone, the viability of both WT Tregs and TNFR2-deficient Tregs were comparable(Fig S2 A-B). Thus, genetic ablation of TNFR2 did not enhance the death of Treg cells in the presense of IL-2 alone. Nevertheless, TNF-TNFR2 interaction markedly protected the death of Tregs induced by Dex, as evidenced by the fact that TNF markedly enhanced proportion and viablility of Dex-treated WT Tregs (p<0.001), but not Dex-treated TNFR2-deficient Tregs (P>0.05, Fig 3A-D). Interestingly, TNF treatment did not enhance the viability of WT Teffs (Fig S3A-B), which could be attributable to the relatively lower TNFR2 expression on Teffs.
To verify the in-vivo effect of TNFR2 expression on Treg viability, Dex (5 mg/kg/d) was i.p. injected into the WT or TNFR2-/- mice for three times as described previously . One day after the last treatment, the mice were sacrificed and the ratio and number of CD4+Foxp3+ Tregs in the spleen and LNs were analyzed by flow cytometry. The results showed the absolute number of Treg cells decreased significantly after the Dex-treated WT mice and TNFR2-/- mice (p<0.001, Fig 3F-G). The absolute number of Treg cells decreased by 50.3% (Fig 3F) in TNFR2-/- mice group. In contrast, the percentage of Tregs decreased by 30.7% in WT mice ( Fig 3G). There results indicated TNFR2 deficient Treg were more susceptible to DEX-induced cell death in vivo (Fig 3H). Therefore, the both in-vivo and in-vitro data support the pro-survival effect of TNFR2.
4. TY101 potently inhibits the growth of EG7 lymphoma in mice
To investigate whether TY101 enhance antitumor responses as previsouly reported [16, 25], we next examined the in vivo anti-tumor effect of TY101 in the mouse EG7 lymphoma model. As shown in the schematic diagram in Fig. 4A, C57BL/6 mice were inoculated subcutaneously with EG7 lymphoma. Treatment was started on day 7 after tumor inoculation (tumor diameter ~10 mm). TY101 or isotype matched IgG control was injected into the tumor bearing mice (200 µg, i.p.) twice a week for up to 5 times. One day after the last treatment (Day 24), the mice were sacrificed. The results show that the administration of TY101 markedly inhibited the growth of EG7 lymphoma (p<0.001, Fig. 4B-C), and complete regression of EG7 lymphoma was observed in 60% of the mice at the end of the experiment (50 days after tumor inoculation, FIG 4D). In contrast, the tumor grew rapidly in the mice treated with control IgG, and all mice died from the tumor burden by day 30 (FIG 4D).
To investigate whether TY101 could induce long term tumor specific immunity, treated mice with completely tumor regression were re-inoculated with EG7 lymphoma into the right flank 8 weeks after becoming tumor-free. In comparison, irrelevant B16-F10 melanoma cells were inoculated into the left flank. As another control, normal mice were also inoculated with EG7 lymphoma into the right flank and B16-F10 melanoma into the left flank in the same manner. None of the mice with complete regression of tumor developed EG7 lymphoma (0/3), while all of them developed B16-F10 melanoma (3/3). In contrast, all the normal control mice developed both B16-F10 melanoma and EG7 lymphoma (3/3), FIG 4E). Thus, this result clearly indicated that TY101 treatment induced long-term tumor specific immune memory.
5. TY101 treatment promote tumor infiltrating Treg cell death and enhance the antitumor responses
To determine whether TNFR2 antagonistic antibody elicit antitumor immune response by promoting intratumoral Treg cell death, we started the treatment after the tumor size reach about 10 mm in diameter, then the tumor-infiltrating Tregs were analyzed by flow cytometry. As shown in Fig 5A-B, over 95% of Treg cells were viable in IgG-treated control group, while TY101 sigificantly reduced the Treg viability in tumors (From 97.2% to 77.6%). Moreover, administration of TY101 decreased proportion of tumor infiltrating CD4+ Foxp3+ T cells by about 50% in mouse EG7-OVA tumor (FIG 5C-D). The absolute number of tumor-infiltrating Tregs also decreased significantly in the TY101 treated mice group (FIG 5E), which was accompanied with a significant reduction of TNFR2 expression on Tregs as well (FIG 5F-G). Percentage of IFN-gamma expressed CD8 T cells were increased by about 50% as compare with control (FIG 5H-I). Moreover, the ratio of CD8 T cells to Treg was significantly increased after treatment with TY101 (FIG 5J). Therefore, the TY101 could enhance the antitumor immune response by reducing intratumoral Treg viability.