Development of a humanized mouse model with functional human materno-fetal interface immunity

Abstract

Materno-fetal immunity possesses specialized characteristics to ensure pathogen clearance while maintaining tolerance to the semiallogeneic fetus. Most of our understanding on human materno-fetal immunity is based on conventional rodent models that may not precisely represent human immunological processes owing to the huge evolutionary divergence. Herein, we developed a pregnant human immune system (HIS) mouse model through busulfan preconditioning, which hosts multilineage human immune subset reconstitution at the materno-fetal interface. Human materno-fetal immunity exhibits a tolerogenic feature at the midgestation stage (embryonic day [E] 14.5), and human immune regulatory subsets were detected in the decidua. However, the immune system switches to an inflammatory profile at the late gestation stage (E19). A cell-cell interaction network contributing to the alternations in the human materno-fetal immune atmosphere was revealed based on single-cell RNA-Seq analysis, wherein human macrophages played crucial roles by secreting several immune regulatory mediators. Furthermore, depletion of Treg cells at E2.5 and E5.5 resulted in severe inflammation and fetus rejection. Collectively, these results demonstrate that the pregnant HIS mouse model permits the development of functional human materno-fetal immunity and offers a tool for human materno-fetal immunity investigation to facilitate drug discovery for reproductive disorders.

Keywords: Adaptive immunity; Immunology; Mouse models; Obstetrics/gynecology; Reproductive biology.

Figure 1. Construction of a pregnant HIS mouse model with high levels of human lymphohematopoietic cell reconstitution.

(A–C) Ratios (mean ± SEM) of multilineage human lymphohematopoietic cell reconstitution within human CD45⁺ plus mouse CD45⁺ cell population of HIS mice made by intravenous injection of human CD34⁺ fetal liver cells and transplantation of human fetal thymic tissue under the renal capsule after preconditioning by TBI (A; n = 5), limb local irradiation (B; n = 8), or busulfan treatment (C; n = 19). Percentages of human CD45⁺ leukocytes, CD3⁺ T cells, CD19⁺/CD20⁺ B cells, NK cells, and CD33⁺ myeloid cells in PBMCs at indicated weeks were shown. An unpaired t test was used to analyze the differences in the ratios of human CD45⁺ cells, T cells, and B cells in the PBMCs at week 9 between the TBI and busulfan groups: CD45⁺ cells: P < 0.01; T cells: P < 0.001; B cells: P < 0.0001. (D) The average weight of neonatal mice born to NSG or HIS mice that were mated with BALB/c male mice (busulfan treatment, n = 5). Box plots show the interquartile range, median (line), and minimum and maximum (whiskers). (E) The number of neonatal mice born by NSG or humanized mice that were mated with BALB/c male mice (busulfan treatment, n = 5). (F) The average fetus number (mean ± SEM) of pregnant HIS mice made by mating with NSG males (n = 12) or BALB/c males (n = 13) summarized (busulfan treatment) at E14.5. (G) Ratio of placental junctional to labyrinth zone (JZ/L ratio) in humanized mice (hu-mice) (n = 10) and NSG mice (n = 6) on E14.5. (H) Pictures of placentas and fetuses of a representative HIS mouse euthanized at E14.5 were shown. Statistical analyses were performed using unpaired t test (D–G).

Figure 2. Human immune subset composition in pregnant HIS mice at E14.5.

HIS mice (n = 10) made by the busulfan protocol were mated with BALB/c males and euthanized at E14.5 for analysis. Summarized data about the chimerism (mean ± SEM) of human lymphohematopoietic cells (A) (%huCD45⁺/[%hCD45⁺ + %mCD45⁺]) and ratios (B) of CD3⁺ T cells, CD20⁺ B cells, CD14⁺ macrophages, and CD56⁺ NK cells within human CD45⁺ cells in PBMCs, spleen, and decidua were shown. (C) Summarized results (mean ± SEM; n = 7) of the percentages of CD49a⁺ and CD49a⁺CD103⁺ tissue-resident NK cells in human CD56⁺CD16^– NK cells. (D) Summarized results (mean ± SEM; n = 7) of the percentages of CD56⁺CD16^– NK cells and representative flow cytometric profiles were shown. (E–G) Summarized results (mean ± SEM; n = 7) of the percentages of CD14⁺HLA-DR⁺ human macrophages (E), CD206⁺ M2 macrophages (F), and CD80⁺ M1 macrophages (G) were shown. (H) Summarized results (mean ± SEM; left; n = 5) and representative flow cytometry (FCM) profiles (right) of the ratios of Treg cells. (I) Immunohistochemical (IHC) examination of human CD45⁺ lymphohematopoietic cells (left) and CD4⁺ T cells (right) in decidua. D, decidua. Statistical differences were determined with 1-way ANOVA for multiple-variable comparisons (A and C–H).

Figure 3. Human immune profile alternation in the decidua and spleen of pregnant HIS mice at E14.5 and E19.

Human CD45⁺ cells were purified from the spleen and decidua of pregnant HIS mice made by mating with BALB/c males at mid and late gestation and examined by scRNA-Seq (mother N = 1 per time point; E14.5, n = 6 decidua, pooled; E19, n = 5 decidua, pooled). Each sample represents a single pregnant mouse. Decidua from the same mouse were pooled together. (A and B) Workflow diagram (A) and the t-distributed stochastic neighbor embedding (t-SNE) plots of main cell types (B) were shown. GEMs, gel beads in emulsion; DC, dendritic cells; M0, M0 macrophages; M1, M1 macrophages; M2, M2 macrophages; NKp, NK proliferative cells. (C) Expression of representative markers for different cell clusters are plotted onto the t-SNE map. Color key from gray to purple indicates relative expression levels from low to high. (i) CD79A (B cells), (ii) CD3D (T cells), (iii) HDC (basophils), (iv) FKBP11 (plasmablasts), (v) LILRA4 (DCs), (vi) SPARC (progenitor cells), (vii) GZMM (CD16⁺ NK), (viii) XCL2 (CD56⁺ NK), (ix) TYMS (NKp), (x) S100A8 (monocytes), (xi) C15orf48 (macrophages), (xii) LYZ (M1), and (xiii) FCER1G (M2). (D) The cell-cell interaction profiles at E14.5 decidua and E19 decidua made by CellPhoneDB were shown. (E and F) Heatmaps show average count of the genes annotated as cytokines (E) and chemokine receptors (F, upper panel) and chemokine ligands (F, lower panel) in different cell types.

Figure 4. TCR repertoire and immune profile transition of human dTreg cells at E14.5 and E19.

(A) Quantification of percentage of T cells per clone size. (B) Venn diagrams showing the number of TCR clonotypes in dTreg, sTreg, dTconv, and sTconv cells. (C) Ratio of Helios⁺ cells in CD4⁺CD25⁺Foxp3⁺ Treg cells in decidua and spleen at E19 (n = 4). Significance was determined using paired t test. (D) Representative flow cytometric profiles of Helios expression in decidual and splenic Treg cells. (E) Violin plots showing the expression of IKZF2 in tTreg and iTreg cells. (F) Volcano plot of differential gene expression between dTreg cells at E19 and E14.5. (G) Heatmaps show genes related to Treg activation/differentiation (left panel), tissue resident (middle panel), and transcription factors (right panel). (H) Pseudotime trajectories of Treg, EM CD4⁺ T, and naive CD4⁺ T cells in decidua and spleen were analyzed using Monocle 2.

Figure 5. Inhibition of human Treg cells causes pregnancy termination and fetal rejection in pregnant HIS mice.

Treg cell inhibitory function examination. (A) Summarized data (n = 3; mean ± SEM) of the immune-inhibitory effect of human dTreg or sTreg cells on human sTconv cell proliferation in the presence of 30 Gy–irradiated BALB/c splenic cells. Representative FCM profiles of Tconv cell proliferation. Treg/Tconv = 1:16, BALB/c stimulator cells/Tconv = 1:2. Statistical differences were determined with 1-way ANOVA for multiple-variable comparisons. (B) Schematic outline of the experimental design. Anti-CD25 mAb (basiliximab; n = 6) or equal volume of PBS (n = 5) was i.p. injected into pregnant HIS mice (200 μg/mouse) made by mating with BALB/c males at E2.5 and E5.5, and the mice were euthanized at E14.5. Percentages (mean ± SEM, C) and representative FCM profiles (D) of Treg cells after anti-CD25 mAb injection were shown. Statistical analyses were performed using unpaired t test. (E) The number of implantation sites of pregnant HIS mice treated with PBS (n = 5) or basiliximab (n = 6). Statistical differences were calculated with 2-way ANOVA. (F) Representative photos of the uteri harvested from HIS mice treated with basiliximab (upper panel) or PBS (lower panel).

Figure 6. Inhibition of human Treg cells causes an inflammatory response in pregnant HIS mice.

(A) Concentration (mean ± SEM) of human TNF- α, IFN-γ, IL-17A, granzyme B, perforin, IL-10, IL-4, granzyme A, granulysin, IL-6, and IL-2 in mouse serum. Statistical differences were determined with 1-way ANOVA for multiple-variable comparisons. NP, nonpregnant HIS mice. (B) Representative H&E images of the decidua in PBS group (left) and the resorbed embryos collected from the basiliximab treatment group (right). D, decidua; Jz, junctional zone; L, labyrinth zone. (C) IHC examination of the placenta (PBS) or uterus (basiliximab treatment) section stained with anti-human CD45 and CD4 antibodies. Scale bars for each picture are shown at lower right corner.