The H-Y Antigen in Embryonic Stem Cells Causes Rejection in Syngeneic Female Recipients

Abstract

Pluripotent stem cells are promising candidates for cell-based regenerative therapies. To avoid rejection of transplanted cells, several approaches are being pursued to reduce immunogenicity of the cells or modulate the recipient's immune response. These include gene editing to reduce the antigenicity of cell products, immunosuppression of the host, or using major histocompatibility complex-matched cells from cell banks. In this context, we have investigated the antigenicity of H-Y antigens, a class of minor histocompatibility antigens encoded by the Y chromosome, to assess whether the gender of the donor affects the cell's antigenicity. In a murine transplant model, we show that the H-Y antigen in undifferentiated embryonic stem cells (ESCs), as well as ESC-derived endothelial cells, provokes T- and B cell responses in female recipients.

Keywords: Y-chromosome; allograft; immunogenicity; pluripotent stem cells; stem cell therapeutics.

FIG. 1.

Immune responses to mESCs carrying the Y chromosome. In ELISPOT assays, BALB/c female recipients (n = 5) showed higher spot frequencies than BALB/c male recipients (n = 12) for both (a) IFN-γ (Th1) and (b) IL-4 (Th2) activation. (c–f) Only box plots of immune cell subpopulations that show significant differences by the mass cytometry data analysis are shown. First, SPADE was used to gate four major immune populations (CD4⁺ T cells, CD8⁺ T cells, and innate and B cells) based on common lineage markers. Subsequently, clusters obtained by individual graph-based clustering of the major populations were merged and manually annotated into biologically comprehensible subpopulations and statistically compared between both mice groups (Supplementary Fig. S1). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (g) Mean fluorescence intensity of IgM binding to mESCs incubated with recipient serum after 5 days (mean ± SD, 5 female and 12 male animals per group, two-tailed Student's t-test), the background fluorescence in naive mice is shown in gray (mean ± SD, 3 animals per group, Student's t-test). (h) Diagram of Medawar experimental design comparing adult female mice with neonatal (tolerant state) exposure to male mESCs compared to naive female mice. (i). In ELISPOT assays, BALB/c_reinject (n = 8) showed a significantly lower IFN-γ response than BALB/c (n = 12). (j) In mixed lymphocyte reaction, BALB/c_reinject (n = 8) showed significantly lower T cell than BALB/c (n = 12). All results ****P < 0.0001. mESC, mouse embryonic stem cell; SD, standard deviation; ELISPOT, Enzyme-Linked ImmunoSpot; IgM, immunoglobulin M; IFN-γ, interferon-gamma; IL, interleukin.

FIG. 2.

Survival of mESCs carrying the Y chromosome. (a) mESCs grown on a mouse feeder layer show colony formation typical of pluripotent stem cells. (b) G-banding shows a normal karyotype. (c) Polymerase chain reaction or pluripotency markers are positive in mESC, compared to the C57BL/6 fibroblast control. (d) Histology of mESC-sourced teratoma, formed in immunodeficient SCID/beige mice, reveals endoderm, mesoderm, and ectodermal lineage potential. (e) Fluorescence in situ hybridization from teratomas developed in animals from Fig. 2d demonstrates the presence of the H-Y chromosome in the donor cell population used in all in vivo assays. (f) Teratoma formation assays in male BALB/c mice reveals strong proliferative growth of ESCs with teratoma formation in 60% of mice receiving 1 × 10⁵ male ESCs (top panel, n = 6) and in 100% of mice receiving 2.5 × 10⁵ (middle panel, n = 6) and 5 × 10⁵ (bottom panel, n = 5) male mESC. (g) In H-Y mismatched female BALB/c mice, teratoma formation is reduced to 0% of female mice receiving 1 × 10⁵ male mESCs (top panel, n = 6), and 66.6% of mice receiving 2.5 × 10⁵ male mESCs (middle panel), with only the group receiving 5 × 10⁵ (bottom panel) male mESC reaching 100% teratoma formation (n = 6).(h) Positive immunostaining for cell markbrachyury, cytokeratin, and GFAP, indicating the presence of cells from all three germ layers in teratomas developed in animals from Fig. 2f and g. GFAP, glial fibrillary acidic protein.

FIG. 3.

H-Y immunogenicity in ESC-derived ECs. (a) Characterization of mESC-derived ECs showing CD31⁺ and VE cadherin⁺ staining, as well as the formation of tubes in a tube formation assay. (b) Tracking of luciferase labeled male mESC-ECs in vivo shows prolonged survival of cells after subcutaneous injection in male BALB/c recipients (n = 5/group), whereas (c) luciferase labeled male mESC-ECs were rejected within app. Fifty days in female BALB/c (n = 4/group). EC, endothelial cell. VE, vascular endothelial.