Inhibiting T-Cell-Mediated Rejection of the Porcine Meniscus Through Freeze-Thawing and Downregulating Porcine Xenoreactive Antigen Genes

Abstract

Immune rejection presents a significant challenge in xenogenic meniscal transplantation. Pigs are widely regarded as an advantageous tissue source for such transplants, with porcine GGTA1, CMAH, and B4GALNT2 being among the most common xenoreactive antigen (Ag) genes. While some studies have suggested that allogeneic meniscus (AM) transplants may exhibit immunoprivileged properties, our study observed slight immunological rejection has been observed following contact between human meniscal cells (HMCs) and human peripheral blood mononuclear cells (PBMCs). Given the limited systematic research on immune responses following xenograft meniscus transplantation, we established porcine meniscus transplantation (PMT) models to comprehensively assess the immunogenicity of porcine meniscus (PM) from both innate and adaptive immune perspectives. Our investigations confirmed that PMT beneath the epidermis led to innate cell infiltration into the xenografts and T-cell activation in local lymph nodes. T-cell activation upregulated the interleukin (IL)-17 signaling pathway, disrupting collagen organization and metabolic processes, thereby hindering PM regeneration. Using freeze-thaw treatment on PM alleviated T-cell activation post-transplantation by eliminating xenogenic DNA. In vitro findings demonstrated that gene editing in porcine meniscal cells (PMCs) suppressed human T-cell activation by downregulating the expression of xenoreactive Ag genes. These results suggest that GGTA1/CMAH/B4GALNT2 knockout (KO) pigs hold significant promise for advancing the field of meniscal transplantation.

Keywords: B4GALNT2; CMAH; GGTA1; transplantation; xenogenic meniscus.

Graphical abstract

Figure 1.

Freeze-thaw treatment of PM reduced the content of xenogenic reactive genes. Biochemical characteristics of the specimens of (A) DNA, (B) water content, and (C) collagen in the NM and FM groups (n = 3). Relative mRNA expression of (D) GGTA1, (E) CMAH, and (F) B4GALNT2 in the NM and FM groups, respectively. (G) DAPI staining of longitudinal sections from the NM and FM groups; scale bar: 20 µm. (H) Total number and area of nuclei in each section in the NM and FM groups (n = 3). NS: P > 0.05, *P < 0.05, **P < 0.01 and ***P< 0.001.

Figure 2.

Lymphocytes infiltrated into the transplant border. (A) SEM images of FM and NM; scale bar: 10 µm. (B) The meniscus section was transplanted under the dorsal epidermis, as indicated by the green arrow. (C) The survival curve of the xenografts in the NM and FM groups. (D) H&E staining images of the transplanted NM and FM at D 7 and D 14; the infiltrated lymphocytes were marked with black arrowheads in the images; scale bar: 100 µm. (E) Immunofluorescence staining of the transplanted NM and FM at D 7 and D 14. Blue indicates DAPI, green indicates CD16, and purple indicates CD11b; scale bar: 20 µm. **P < 0.01.

Figure 3.

T-cell activation in the draining lymph node. (A) Flow cytometric quantification of the ratio and representative FVD^negCD3⁺CD69⁺ cells in the sham, NM and FM groups on D 7. Statistical analysis was performed using a two-tailed independent t-test for comparisons between two groups (n = 3). (B) Representative images and statistical analysis of FVD^negCD3⁺CD69⁺ cells in the sham, NM and FM groups on D 14 by flow cytometry (n = 3). (C) Immunofluorescence staining of the transplanted FM and NM, and the skin of the sham group. Blue indicates DAPI, green indicates a-Gal, and red indicates CD207; scale bar: 20 µm. Statistical analysis was performed using a two-tailed independent t-test to compare two groups (n = 6). * P < 0.05, **P < 0.01,***P < 0.001 and ****P < 0.0001.

Figure 4.

PMCs triggered human T-cell activation. (A) Flow cytometric analysis and statistical comparison of the MFI value of untreated and PMC-stimulated naive T-cells. (B) Flow cytometric analysis and statistical comparison of the MFI value of untreated and PMC-stimulated naive B cells. (C) Representative flow cytometry images indicating the CD69 expression level at different time points. (D) Flow cytometric quantification of CD80 expression levels in CD20⁺ cells on D3. (E) Flow cytometric quantification and statistical analysis of CD69 expression levels in CD3⁺ cells according to the stimulated cells in the MLR on D3 (n = 3). (F) IL-2 (left, n = 3) and IFN-γ (right, n = 3) concentrations in PBMC supernatants measured using ELISA. (G) Determination of the expression of CD146, CD90, and HLA-I in HMCs by flow cytometry. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 5.

RNA-seq analysis of tendon between naive and activate T-cell groups. (A) Volcano plot of gene expression (naive versus activated; fold change ≥ 2; q value < 0.05). N, naive; A, activated. (B) Heat map of differentially expressed genes. (C) GO analysis of differentially expressed genes. (D) KEGG analysis of upregulated genes in activated T-cell group. GSEA of the genes associated with cytokine-cytokine receptor interaction (E) and the IL-17 signaling pathway (F). NES, normalized enrichment score; FDR, false discovery rate.

Figure 6.

Downregulation of porcine gene alleviated immune response. (A) Confocal microscopy images of siTF-treated PMCs. Green indicates siRNA and blue indicates DAPI; scale bar: 20 µm. (B) The relative gene expression of GGTA1 (left), CMAH (middle), and B4GALNT2 (right) in the PMCs of the NC and siTF-treated groups. (C) The relative levels of Ag expression of α-Gal (left), Neu5Gc (middle) and Sda (right) in the PMCs of the NC and siTF groups. (D) IL-2 (left, n = 3) and IFN-γ (right, n = 3) concentrations in PBMC supernatants from different MLR groups. (E) Expression levels of CD80 (left, n = 3) in CD20⁺ cells and CD69 (right, n = 3) in CD3⁺ cells. Representative flow cytometry images of CD80 (F) and CD69 (G) in different groups. *P < 0.05, **P < 0.01 and ***P < 0.001.