b The number of live fetuses per uterus from pregnant CBA/J females following treatment with the indicated blocking antibodies. cells was associated with miscarriage. These findings underscored the important roles of the CTLA-4 and Tim-3 pathways in regulating dCD4+T cells function and maintaining normal pregnancy. Our study also emphasized the importance of careful consideration of reproductive safety when choosing immune checkpoint blockade therapies in real world clinical care. Introduction T cell activation following antigen recognition requires a secondary co-stimulatory signal, which can be either positive or unfavorable. Treatment with neutralizing antibodies that target inhibitory signals, or checkpoint blockade to enhance immune responses, has been proven as a promising therapeutic strategy for a variety of cancers and chronic viral infections1. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed death 1 (PD-1), and T-cell immunoglobulin mucin-3 (Tim-3) are the major targetable co-inhibitory receptors on T cells. The development of these immunotherapy agents has increased since the first approval of anti-CTLA-4 therapy (ipilimumab) by the United States Food and Drug Administration for melanoma in 20112. Despite their success, the single use of currently approved antibodies was effective in only 20C30% of patients3. Currently, combination approaches against different targets seem to be effective for favorable clinical outcomes4. For example, CTLA-4 had a role in both early and late stages of T cell activation and was mainly expressed on T cells residing in lymph nodes5, while Tim-3 could exert its function by regulating cell apoptosis6, so the combination of anti-CTLA-4 and anti-Tim-3 could restore the greatest degree of T cell function. During normal pregnancy, the semi-allogeneic fetus has the capacity to avoid immune attack by the maternal immune system, and the placenta is regarded as AURKB a pseudo-malignant type of tissue7. Impaired tolerance induction or excessive (2-Hydroxypropyl)-β-cyclodextrin inflammation can lead to severe pregnancy complications such as recurrent spontaneous abortion (RSA), pre-eclampsia, or preterm delivery8. T cells, particularly CD4+T cells, seem to play a pivotal role in inducing and maintaining maternal-fetal tolerance. Driven by a set of transcriptional regulators and cytokines, naive CD4+T helper (Th) cells are able to differentiate into distinct subsets, including Th1, Th2, Th17, and Treg cells9. Treg growth and a polarization toward Th2 bias in the maternal immune response have long been considered the main mechanisms of inducing tolerance toward the fetus8. Women who experienced RSA exhibited a marked Th1 bias10. The expression of the Th1-type cytokine TNF- was observed in decidual tissues from failing human pregnancies, and this cytokine was shown to lead to the fetal loss in mice8. (2-Hydroxypropyl)-β-cyclodextrin A lower IL-10 to IFN- ratio was associated with abnormal pregnancy outcome in mice, and pregnancy outcomes were improved when Treg cells were transferred from the maternal-fetal interface11. Given the similarities between a tumor and a fetus, the effects of checkpoint blockade around the reproductive system and the role of co-signaling molecules in maternal-fetal immunity need to be explored. A second anti-CTLA-4 monoclonal antibody (mAb), tremelimumab, displayed (2-Hydroxypropyl)-β-cyclodextrin activity in early phase studies12. One anti-Tim-3 mAb (MBG453) was also being investigated in phase I-II clinical trial in patients with advanced malignancies; however, no clinical results have yet been reported13. In the present study, efficacy studies of anti-CTLA-4 and anti-Tim-3 were first done in mouse pregnancy models, and then the expression and function of CTLA-4/Tim-3 on CD4+T cells during normal pregnancy and miscarriage were explored. The current data demonstrates that combined blockade of the CTLA-4 and Tim-3 pathways results in an increased fetal loss in an experimental mouse pregnancy model by altering the function of decidual CD4+T (dCD4+T) cells. Furthermore, the co-expression of CTLA-4 and Tim-3 on dCD4+T cells is usually important in Th2 bias and Treg growth at the maternal-fetal interface, thereby, maintaining a normal pregnancy. Results Effects of dual blockade of CTLA-4 and Tim-3 on mouse pregnancy In the first assay, we examined pregnant CBA/J females challenged with CTLA4- and/or Tim-3-blocking antibody. Treatment with either blocking antibody caused a higher rate of embryo resorption (data not shown), decreased growth in body weight (Fig.?1a), and reduction in the number (2-Hydroxypropyl)-β-cyclodextrin of live fetuses per uterus (Fig.?1b). Furthermore, dual blockade of the CTLA4- and Tim-3 pathways had a combined effect, leading to the greatest susceptibility to fetal loss (Fig.?1a, b). These data indicated that CTLA4- and Tim-3-blocking antibody had some side effects around the fertility of mice. Open in a separate windows Fig. 1 Effects of anti-CTLA-4 or/and Tim-3 antibody during early pregnancy.a The weight of pregnant CBA/J females treated with isotype IgG, anti-CTLA-4, anti-Tim-3 antibody, both antibodies i.p. at doses of 500, 250, and 250?mg at days 4.5, 6.5, and 8.5, respectively. b The number of live fetuses per uterus from pregnant CBA/J females following treatment with the.