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⁺CD127^low/−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.

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.

To expand our knowledge of the ontogeny of the T-cell receptor (TCR) repertoire of antigen-specific T-cell subsets, we combined next-generation deep sequencing and single-cell multiplex clonotype analysis to evaluate the diversity and frequency of paired TCRs, their functions and whether clonotypic TCRs are shared among different individuals. Using an HLA-A*02-restricted cytomegalovirus (CMV) pp65-derived immunogenic peptide, we found that the more dominant pp65-specific TCR clonotypes in the blood of healthy donors have higher binding affinities for the CMV peptide and arise from clonotypes that are highly shared among individuals. Interestingly, these highly shared HLA-A*02-restricted CMV-specific TCRs were detected in a CMV-seronegative individual as well as in HLA-A*02-negative donors albeit at lower frequency. More intriguingly, these shared TCR clonotypes were abundant in the stem memory T-cell subset, and TCR diversity of the stem memory T-cell repertoire was significantly lower than in the central memory and effector memory T-cell repertoires. These results suggest that the stem memory T-cell subset may serve as a reservoir of highly shared and highly functional memory T-cells.

Antigen-specific rabbit monoclonal antibodies (RaMoAbs) are useful due to their high specificity and high affinity, and the establishment of a comprehensive and rapid RaMoAb generation system has been highly anticipated. Here, we present a novel system using immunospot array assay on a chip (ISAAC) technology in which we detect and retrieve antigen-specific antibody-secreting cells from the peripheral blood lymphocytes of antigen-immunized rabbits and produce antigen-specific RaMoAbs with 10^–12 M affinity within a time period of only 7 days. We have used this system to efficiently generate RaMoAbs that are specific to a phosphorylated signal-transducing molecule. Our system provides a new method for the comprehensive and rapid production of RaMoAbs, which may contribute to laboratory research and clinical applications.