Clonally Expanded Decidual Effector Regulatory T Cells Increase in Late Gestation of Normal Pregnancy, but Not in Preeclampsia, in Humans

Sayaka Tsuda, Xiaoxin Zhang, Hiroshi Hamana, Tomoko Shima, Akemi Ushijima, Kei Tsuda, Atsushi Muraguchi, Hiroyuki Kishi, Shigeru Saito, Sayaka Tsuda, Xiaoxin Zhang, Hiroshi Hamana, Tomoko Shima, Akemi Ushijima, Kei Tsuda, Atsushi Muraguchi, Hiroyuki Kishi, Shigeru Saito

Abstract

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.

Keywords: T cell repertoire; effector regulatory T cell; human pregnancy; miscarriage; preeclampsia; regulatory T cell.

Figures

Figure 1
Figure 1
Gating strategy to obtain CD4+CD45RA−CD25+CD127low/−effector Treg cells. Lymphocyte in the peripheral blood (upper column) and decidua (lower column) were gated on forward and side scatter parameters. CD3+CD4+ T cells were classified into CD45RA+ naïve T cells and CD45RA− effector T cells. Among CD45RA− effector T cells, CD25+CD127low/− effector Treg cells were single-cell sorted.
Figure 2
Figure 2
TCRβ repertoire analysis of CD4+CD45RA−CD25+CD127low/− effector Treg cells. (A) TCRβ and FoxP3 cDNAs amplified by multiplex one-step RT-PCR were resolved by agarose gel electrophoresis. Each single cell was numbered. NC denotes the cell-free negative control. FoxP3 and TCRβ mRNAs were expressed in 11/11 and 10/11, respectively. (B) Representative data of the TCRβ repertoire of CD4+CD45RA−CD25+CD127low/−effector Treg cells in PBMC and decidua. The TCR repertoires were determined by amino acid and nucleotide sequences of complementarity determining region 3 (CDR3) of TCRβ chain. Each pie chart slice (shaded or closed) indicates clonal T cell population with the same clonotypic TCRβ. An open pie chart slice indicates T cells with unique TCRβ. Frequency of clonal populations among analyzed TCRβ and the Gini coefficients of the TCRβ repertoire were calculated.
Figure 3
Figure 3
TCRβ repertoire and flow cytometric analysis of effector Treg cells in 1st and 3rd trimester decidua and peripheral blood in normal pregnancy. (A) Frequencies of clonal populations among the analyzed TCRβ of effector Treg cells in 1st (n = 9) and 3rd trimester (n = 12) decidua and peripheral blood in normal pregnancies. (B) Gini coefficient of TCRβ repertoire of effector Treg cells. (C) Ratio of CD4+CD45RA−CD25+CD127low/− effector Treg cells per CD4+CD25+CD127low/− total Treg cells. *p from Wilcoxon signed-rank test and **p from Mann–Whitney U-test. Each dot represents one donor, lines indicate median. Deci, decidua; PB, peripheral blood.
Figure 4
Figure 4
Common clonotypic effector Treg cells between PBMC and decidua in 1st and 3rd trimester decidua and peripheral blood in normal pregnancy. Frequencies of common TCRβ in PBMC and decidua among analyzed TCRβ of effector Treg cells.
Figure 5
Figure 5
Shared TCRβ repertoire of decidual effector Treg cells between previous and subsequent pregnancy. (A–C) Frequencies of decidual effector Treg cell clones expressing clonotypic TCRβ with the indicated CDR3 amino acid sequence. The underlined CDR3 sequences were shared in previous and subsequent pregnancies. (A) Case #10 previous pregnancy and case #20 subsequent pregnancy ending in the 3rd trimester with a normal vaginal delivery. (B) Case #13 previous pregnancy and case #17 subsequent pregnancy ending in the 3rd trimester with a normal vaginal delivery. (C) Case #25 previous pregnancy ending in miscarriage with a normal embryo and case #47 subsequent pregnancy ending in miscarriage with an abnormal embryo.
Figure 6
Figure 6
TCRβ repertoire and flow cytometric analysis of decidual effector Treg cells in 1st trimester normal pregnancy and miscarriage. (A) Frequencies of clonal populations among analyzed TCRβ of effector Treg cells in 1st trimester normal pregnancy (n = 9) and miscarriage with abnormal embryo (n = 11) or normal embryo (n = 7). (B) Gini coefficient of TCRβ repertoire of effector Treg cells. (C) Ratio of CD4+CD45RA−CD25+CD127low/− effector Treg cells per CD4+CD25+CD127low/− total Treg cells. *p from Steel's test. Each dot represents one donor; lines indicate median.
Figure 7
Figure 7
TCRβ repertoire and flow cytometric analysis of decidual effector Treg cells in 3rd trimester normal pregnancy and preeclampsia. (A) Frequencies of clonal populations among analyzed TCRβ of effector Treg cells in 3rd trimester normal pregnancy (n = 12) and preeclampsia (n = 7). (B) Gini coefficient of TCRβ repertoire of effector Treg cells. (C) Ratio of CD4+CD45RA−CD25+CD127low/− effector Treg cells per CD4+CD25+CD127low/− total Treg cells are shown. *p from Mann–Whitney U-test. Each dot represents one donor; lines indicate median.

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