Background: Regulatory T (Treg) cells are necessary for the maintenance of allogenic pregnancy. However, the repertoire of effector Treg cells at the feto-maternal interface in human pregnancy remains unknown. Our objective was to study T cell receptor (TCR) repertoires of Treg cells during pregnancy compared to normal and complicated pregnancies.Methods:Paired samples of peripheral blood and decidua in induced abortion and miscarriage cases were obtained from consenting patients. CD4+CD25+CD127low/−CD45RA− effector Treg cells were single-cell sorted from mononuclear cells. cDNAs of complementarity determining region 3 (CDR3) in TCRβ were amplified from the single cells by RT-PCR and the sequences were analyzed. The TCRβ repertoires were determined by amino acid and nucleotide sequences. Treg cells were classified into clonally expanded and non-expanded populations by CDR3 sequences.Results: We enrolled nine induced abortion cases in the 1st trimester, 12 cases delivered without complications in the 3rd trimester, 11 miscarriages with abnormal chromosomal karyotyped embryo, seven miscarriages with normal chromosomal karyotyped embryo, and seven cases of preeclampsia [median gestational week (interquartile range): 7 (7–9), 39 (38–40), 9 (8–10), 8 (8–10), and 34 (32–37), respectively]. The frequency of clonally expanded populations of effector Treg cells increased in decidua of 3rd trimester cases compared to 1st trimester cases [4.5% (1.4–10.8%) vs. 20.9% (15.4–28.1%), p < 0.001]. Clonally expanded Treg cells were rarely seen in peripheral blood. The ratio of clonally expanded populations of decidual effector Treg cells in miscarriages with abnormal and normal embryos was not significantly different compared with that in 1st trimester normal pregnancy. Interestingly, clonally expanded populations of effector Treg cells decreased in preeclampsia compared with that in 3rd trimester normal pregnancy [9.3% (4.4–14.5%) vs. 20.9% (15.4–28.1%), p = 0.003]. When repertoires in previous pregnancy and subsequent pregnancy were compared, some portions of the repertoire were shared.Conclusion: TCR repertoires of decidual effector Treg cells are skewed in the 3rd trimester of normal pregnancy. Failure of clonal expansion of populations of decidual effector Treg cells might be related to the development of preeclampsia.

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Antigen-specific T cell therapy, or T cell receptor (TCR) gene therapy, is a promising immunotherapy for infectious diseases and cancers. However, a suitable rapid and direct screening system for antigen-specific TCRs is not available. Here, we report an efficient cloning and functional evaluation system to determine the antigen specificity of TCR cDNAs derived from single antigen-specific human T cells within 10 d. Using this system, we cloned and analyzed 380 Epstein-Barr virus-specific TCRs from ten healthy donors with latent Epstein-Barr virus infection and assessed the activity of cytotoxic T lymphocytes (CTLs) carrying these TCRs against antigenic peptide-bearing target cells. We also used this system to clone tumor antigen-specific TCRs from peptide-vaccinated patients with cancer. We obtained 210 tumor-associated antigen-specific TCRs and demonstrated the cytotoxic activity of CTLs carrying these TCRs against peptide-bearing cells. This system may provide a fast and powerful approach for TCR gene therapy for infectious diseases and cancers.

There is currently no clinically effective vaccine against leishmaniasis because of poor understanding of the antigens that elicit dominant T cell immunity. Using proteomics and cellular immunology, we identified a dominant naturally processed peptide (PEPCK335-351) derived from Leishmania glycosomal phosphoenolpyruvate carboxykinase (PEPCK). PEPCK was conserved in all pathogenic Leishmania, expressed in glycosomes of promastigotes and amastigotes, and elicited strong CD4(+) T cell responses in infected mice and humans. I-A(b)-PEPCK335-351 tetramer identified protective Leishmania-specific CD4(+) T cells at a clonal level, which comprised ~20% of all Leishmania-reactive CD4(+) T cells at the peak of infection. PEPCK335-351-specific CD4(+) T cells were oligoclonal in their T cell receptor usage, produced polyfunctional cytokines (interleukin-2, interferon-γ, and tumor necrosis factor), and underwent expansion, effector activities, contraction, and stable maintenance after lesion resolution. Vaccination with PEPCK peptide, DNA expressing full-length PEPCK, or rPEPCK induced strong durable cross-species protection in both resistant and susceptible mice. The effectiveness and durability of protection in vaccinated mice support the development of a broadly cross-species protective vaccine against different forms of leishmaniasis by targeting PEPCK.

CD8 + T cells, the most abundant T cell subset in the decidua, play a critical role in the maintenance of pregnancy. The majority of decidual CD8 + T cells have an effector memory phenotype, while those in the peripheral blood display a naive phenotype. An increased amount of highly differentiated CD8 + T cells in the decidua indicates local antigen stimulation and expansion, albeit these CD8 + T cells are suppressed. In decidual CD8 + T cells, co-inhibitory molecules such as PD-1, TIM-3, LAG-3, and CTLA-4 are upregulated, reflecting the suppression of cytotoxicity. Previous studies established the importance of the PD-1/PD-L1 interaction for feto-maternal tolerance. CD8 + T cells could directly recognize fetal-specific antigens, such as HLA-C, expressed by trophoblasts. However, although fetal-specific CD8 + T cells have been reported, their TCR repertoires have not been identified. In this study, we analyzed the TCR repertoires of effector memory CD8 + T cells (CD8 + EM cells) and naive CD8 + T cells (CD8 + N cells) in the decidua and peripheral blood of women with normal or complicated pregnancy and examined PD-1 expression at a single-cell level to verify whether antigen-specific CD8 + T cells accumulate in the decidua and to identify immunological differences related to the suppression of antigen-specific CD8 + T cells between normal pregnancy, miscarriage, and preeclampsia. We observed that some TCRβ repertoires, which might recognize fetal or placental antigens, were clonally expanded. The population size of clonally expanded CD8 + EM cells was higher in the decidua than in the peripheral blood. CD8 + EM cells began to express PD-1 during the course of normal pregnancy. We found that the total proportion of decidual CD8 + EM cells not expressing PD-1 was increased both in miscarriage and in preeclampsia cases, although a different mechanism was responsible for this increase. The amount of cytotoxic CD8 + EM cells increased in cases of miscarriage, whereas the expression of PD-1 in clonally expanded CD8 + EM cells was downregulated in preeclampsia cases. These results demonstrated that decidual CD8 + EM cells were able to recognize fetal-specific antigens at the feto-maternal interface and could easily induce fetal rejection.