Unusual timing of CD127 expression by mouse uterine natural killer cells

Abstract

Decidualization, a progesterone-dependent process that alters endometrial stromal cells at implantation sites in humans and rodents, is accompanied by a highly regulated, NK cell-dominated leukocyte influx into decidual basalis (DB). Whether uNK cells differentiate from uterine progenitor cells is unknown, as are the mechanisms restricting leukocytes to DB. We asked if cells expressing the early NK lineage marker CD127 (IL-7Rα) occurred in mouse decidua. CD127 was absent from gd6.5 decidual lymphoid cells but became expressed by a mature uNK cell subset in gd10.5 DB. DB and transient myometrial structures (MLAp) that ring maternal blood vessels supplying placentae expressed IL-7 and TSLP, the CD127 ligands, but with differing temporal and spatial patterns. UNK cells expressed TSLPR, and study of gd10.5 implantation sites from mice deleted for IL-7, CD127, or TSLPR suggested that IL-7 and its receptor have physiological roles in limiting expansion of immature uNK cells within MLAp, while the TSLP signaling pathway is used in DB to sustain IFN-γ production from a subset of mature uNK cells. Regionalized, dynamic expression of the additional lymphoid organ stromal markers gp38/podoplanin and ER-TR7, but not CD157, were seen by immunohistochemistry in implantation sites, and DB and MLAp contained transcripts for Aire, a tolerance-promoting factor. These observations suggest that CD127(+) NK lineage progenitors are not present in the early postimplantation period of mouse uterus and that decidualized endometrial stroma has key immunoregulatory properties.

Figure 1.. CD127 expression in midsagittal serial sections from gd6.5 to gd12.5 B6 implantation sites.

(A) Immunohistochemistry for CD127 (upper) or DBA-lectin (lower). (B) Immunofluorescence staining of gd10.5 B6 implantation site. Detection was nuclear DNA, DAPI (blue); uNK cells, DBA lectin (green); and CD127 antibody-reactive cells (red). DBA⁺ uNK cells (brown) were present in gd6.5 DB, but none of these or other cells with a lymphoid appearance expressed detectable CD127 (A). Rare stromal cells were CD127⁺ (arrowheads). At gd8.5, the DBA⁺ uNK cell population contained infrequent, weakly stained CD127⁺ cells (arrow). At gd10.5, when uNK cells peak, many strongly stained CD127⁺DBA⁺ uNK cells (arrows) were present in DB. At gd12.5, CD127⁺DBA⁺ uNK cells remained in DB (arrows) and were now detected in MLAp. Trophoblast cells were CD127⁺DBA⁻, as shown for gd10.5 placenta (PL). Gd12.5 fetal liver (FL) was used as a positive control for CD127. Two or more viable implantation sites from each of multiple [–5] females were studied at each gd. L, Residual uterine lumen; SA, spiral artery.

Figure 2.. CD127 expression in different mouse tissues.

(A) Isolated uterine lymphocytes were stained with FITC-conjugated DBA lectin, PE-conjugated CD122 (5H4), PE-Cy5-conjugated CD3 (145-2C11), and APC-Alexa Fluor-conjugated CD127 (A7R34). CD3⁻CD122⁺ NK cells were further gated to analyze their DBA and CD127 expression. Plots representative of three experiments are shown. Percentages of DBA⁺ or CD127⁺ populations among gated CD3⁻CD122⁺ cells are shown. (B) MFI of DBA⁺, CD127⁺ NK (CD3⁻CD122⁺) cells in various tissues. *P < 0.05. (C) Photomicrographs of implantation sites from Rag2^−/−/Il2rg^−/− mice without or with engraftment of normal BM, at different gd. Serial sections were stained with DBA-lectin (brown, left side) or CD127 (brown, right side). WT gd6.5 Rag2^−/−/Il2rg^−/− implantation sites had no DBA⁺ or CD127⁺ uNK cells (i, ii). After engraftment, DBA⁺ uNK cells lacking CD127 expression were present at gd6.5 (iii, iv). At gd12.5, graft-derived DBA⁺ uNK cells populated DB (v), and MLAp (vii). graft-derived CD127⁺ uNK cells were found at gd12.5 in interstitial and intravascular areas of DB (vi, viii); n = 3.

Figure 3.. IL-7 and CD127 in B6 implantation sites.

(A) Real-time PCR analysis of relative Il7 transcript abundance in gd7.5 DB and in gd10.5 DB and MLAp. (B, i–vi) Immunofluorescence colocalization of IL-7 (red) with DBA⁺ uNK cells or CD127⁺ cells (green). IL-7 was detected in DB. CD127 was detected in lateral DB at gd7.5 (iv; inset). Nuclear DNA was detected by DAPI (blue). (C) Real-time PCR analysis of relative abundance of Cd127 (Il7rα) transcripts at gd7.5 in DB and at gd10.5 in DB and MLAp. (A and C) Data are shown as mean ± sd. *P < 0.05.

Figure 4.. TSLP expression in B6 implantation sites.

(A) Relative Tslp expression analyzed by real-time PCR. (B and C) Immunohistochemical detection of TSLP at gd7.5 in DB at lower and higher power revealed that TSLP⁺ decidual cells (green) were not DBA⁺ uNK cells (red). At gd10.5, TSLP reactivity was present in DB and with some colocalization to DBA⁺ uNK cells (D) but barely detectable in MLAp (E). Nuclear DNA detected by DAPI (blue). (F) PCR detection of Tslp and Tslpr expression in homogenates of gd10.5 DB and in FACS-sorted CD3⁻CD122⁺DBA⁺ uNK cells. Data are shown as mean ± sd. *P < 0.05.

Figure 5.. Distribution and IFN-γ reactivity of uNK cells in gd10.5 B6, Il7^−/−, Il7rα^−/−, and Tslpr^−/− mice.

(A) uNK cell enumeration in MLAp and DB. More uNK cells (DBA⁺) were present in the MLAp of Il7^−/−, Il7rα^−/− mice; no differences were present in DB between the strains. (B–I) Immunofluorescent staining was used to identify DBA⁺ uNK cells (red) and IFN-γ (green) in the MLAp and DB. IFN-γ was undetectable in Tslpr^−/− implantation sites. Nuclear DNA was detected by DAPI (blue). Two or more viable implantation sites from each of multiple [–5] females were studied. Data are shown as mean ± sd. *P < 0.05.

Figure 6.. Stromal networks and uNK cells in gd7.5 and gd10.5 B6 implantation sites.

Immunofluorescent reactivity using DBA lectin (red) and gp38/podoplanin (green; A and B) or ER-TR7 (green; C and D) identified decidual stromal regions expressing lymphoid stroma-associated markers and their relationships with uNK cells. The relationships between CD127⁺ cells and the gp38⁺ microenvironment are shown at gd7.5 (E and F) and at gd10.5 (G and H). DAPI (blue) was used to localize cell nuclei. (I) PCR detected transcripts of Aire in adult nonpregnant thymus, DB, and MLAp. H₂O served as the negative control. M, Mesometrial side; AM, antimesometrail side; Em, embryo. Scale bars indicate image magnification. Two or more viable implantation sites from each of multiple [–5] females were studied.