Human T cells upregulate CD69 after coculture with xenogeneic genetically-modified pig mesenchymal stromal cells

Abstract

Mesenchymal stromal cells (MSC) obtained from α1,3-galactosyltransferase gene knock-out pigs transgenic for the human complement-regulatory protein CD46 (GTKO/CD46 pMSC) suppress in vitro human anti-pig cellular responses as efficiently as allogeneic human MSC. We investigated the immunoregulatory effects of GTKO/CD46 pMSC on human CD4(+) and CD8(+) T cell proliferation in response to pig aortic endothelial cells (pAEC). pMSC efficiently suppressed T cell proliferation, which was associated with downregulation of granzyme B expression. No induction of CD4(+)CD25(+)Foxp3(hi) regulatory T cells or T cell apoptosis was documented. In correlation with T cell proliferation, CD25 expression was upregulated on T cells in response to pAEC but not to pMSC. In contrast, CD69 expression was upregulated on T cells in response to both pMSC and pAEC, which was associated with a significant increase in the phosphorylation of STAT5. GTKO/CD46 pMSC possibly regulate human T cell responses through modulation of CD69 expression and STAT5 signaling.

Keywords: CD69; GTKO/CD46; GTKO/hCD46; Gal; LAG-3; Mesenchymal stem cells; PBMC; Pig; STAT5; T cells; Xenotransplantation; galactose-α1,3-galactose; lymphocyte activation gene-3; pAEC; pMSC; pSTAT5; peripheral blood mononuclear cells; phosphorylated signal transducer and activator of transcription 5; pig aortic endothelial cells; pig mesenchymal stromal cells; α1,3-galactosyltransferase gene-knockout/human CD46.

Figure 1. Suppression of human CD4⁺ and CD8⁺ T cell proliferation

In MLR, human CD4⁺ (top) or CD8⁺ (bottom) T cell proliferation was measured when these cells were cultured alone (A), cocultured with GTKO/CD46 pMSC (B) or with GTKO pAEC (C) at a responder:stimulator ratio of 1:10. Additionally, GTKO/CD46 pMSC were mixed with GTKO pAEC at 2:1, 1:1, and 1:2 ratios (D, E, and F, respectively). GTKO/CD46 pMSC significantly downregulated the responses of CD4⁺ or CD8⁺ T cells in a dose-dependent manner. Data represent 3 different experiments, and are presented as mean ± SD of triplicates. *p<0.05, **p<0.01.

Figure 2. Downregulation of CD4⁺ granzyme B⁺ and CD8⁺ granzyme B⁺ effector T cells after coculture with pMSC

Purified human CD4⁺ (top) or CD8⁺ (bottom) T cells were separately cultured with (A) pMSC, (B) pAEC, or (C) pAEC + pMSC for 5 days, and then harvested for flow cytometry. Dot plots were boxed for granzyme B⁺ T cells. The percentages of granzyme B⁺ cells in the cocultures of CD4⁺ or CD8⁺ T cells with pAEC alone, pMSC alone, and pAEC + pMSC are indicated.

Figure 3. No induction of CD4⁺CD25⁺Foxp3^hi regulatory T cells after coculture with pMSC

Purified human CD4⁺ T cells were cocultured with pAEC in the presence or absence of pMSC for 5 days, and then harvested for flow cytometry. (A) in the dot plot, CD4⁺CD25⁺ T cells are gated. The percentages of cells expressing FoxP3 in the cocultures of CD4⁺ cells with (B) pAEC alone, (C) pMSC alone, and (D) pAEC + pMSC are indicated.

Figure 4. pMSC do not induce human T cell apoptosis

Human PBMC were cultured alone, with pMSC only, or with pMSC+pAEC for 5 days. PBMC were treated with camptothecin (5μM) for 4h (to induce T cell apoptosis, as a control), and then harvested for flow cytometry. Similar to cells cultured alone, minimal T cells were observed to be apoptotic/dead when either cultured with pMSC only or with pMSC+pAEC.

Figure 5. Up-regulation of expression of CD69 on CD4⁺ and CD8⁺ T cells after coculture with pMSC

Purified human CD4⁺ (A) or CD8⁺ (B) T cells were cultured alone or cocultured with pMSC alone, pAEC alone, or pAEC and pMSC for 5 days, and then harvested for flow cytometry analysis after gating on CD3⁺CD4⁺ or CD3⁺CD8⁺ T cells, to determine the expression of activation markers CD25, CD69, and LAG-3. The percentages of cells positive for each activation marker are indicated. (C) Human PBMC were cocultured with pMSC for 3 and 5 days. CD69 expression was evaluated by flow cytometry as in (A) and (B). Data represent average of four different human PBMC cocultures with pMSC. * p<0.05 in comparison to cells cultured alone. ^# p<0.05 in comparison to cell cocultured for 3 days.

Figure 5. Up-regulation of expression of CD69 on CD4⁺ and CD8⁺ T cells after coculture with pMSC

Purified human CD4⁺ (A) or CD8⁺ (B) T cells were cultured alone or cocultured with pMSC alone, pAEC alone, or pAEC and pMSC for 5 days, and then harvested for flow cytometry analysis after gating on CD3⁺CD4⁺ or CD3⁺CD8⁺ T cells, to determine the expression of activation markers CD25, CD69, and LAG-3. The percentages of cells positive for each activation marker are indicated. (C) Human PBMC were cocultured with pMSC for 3 and 5 days. CD69 expression was evaluated by flow cytometry as in (A) and (B). Data represent average of four different human PBMC cocultures with pMSC. * p<0.05 in comparison to cells cultured alone. ^# p<0.05 in comparison to cell cocultured for 3 days.

Figure 5. Up-regulation of expression of CD69 on CD4⁺ and CD8⁺ T cells after coculture with pMSC

Purified human CD4⁺ (A) or CD8⁺ (B) T cells were cultured alone or cocultured with pMSC alone, pAEC alone, or pAEC and pMSC for 5 days, and then harvested for flow cytometry analysis after gating on CD3⁺CD4⁺ or CD3⁺CD8⁺ T cells, to determine the expression of activation markers CD25, CD69, and LAG-3. The percentages of cells positive for each activation marker are indicated. (C) Human PBMC were cocultured with pMSC for 3 and 5 days. CD69 expression was evaluated by flow cytometry as in (A) and (B). Data represent average of four different human PBMC cocultures with pMSC. * p<0.05 in comparison to cells cultured alone. ^# p<0.05 in comparison to cell cocultured for 3 days.

Figure 6. Phosphorylation of STAT5 in CD4⁺ and CD8⁺ T cells after coculture with pMSC

(A) STAT5 and pSTAT5 were measured in cell lysates obtained from pMSC alone, human CD4⁺ (left) and CD8⁺ (right) T cells alone, or after coculture with pMSC. Arrows indicate pSTAT5. Blots shown are representative of 3 independent experiments with similar results. β-actin served as the loading control. (B) Gel analysis was done using gray-scale image. The extent of T cell activation is indicated by the ratio of pSTAT5/STAT5. Compared with T cells cultured alone, after co-culture with GTKO/CD46 pMSC, CD4⁺ and CD8⁺ T cells demonstrated significantly higher activity ratios. *p<0.05. (C) pSTAT5 expression was evaluated by flow cytometry. Histograms (left) represent the expression of pSTAT5 before (dashed line) and after (straight line) coculture with pMSC. Cells were gated on CD3⁺CD4⁺ or CD3⁺CD8⁺ T cells. Gray indicates isotype. The mean MFI (mean fluorescence intensity) of four different human PBMC cocultures with pMSC (right) demonstrating significantly higher pSTAT5 following pMSC coculture; *p<0.05.

Figure 6. Phosphorylation of STAT5 in CD4⁺ and CD8⁺ T cells after coculture with pMSC

(A) STAT5 and pSTAT5 were measured in cell lysates obtained from pMSC alone, human CD4⁺ (left) and CD8⁺ (right) T cells alone, or after coculture with pMSC. Arrows indicate pSTAT5. Blots shown are representative of 3 independent experiments with similar results. β-actin served as the loading control. (B) Gel analysis was done using gray-scale image. The extent of T cell activation is indicated by the ratio of pSTAT5/STAT5. Compared with T cells cultured alone, after co-culture with GTKO/CD46 pMSC, CD4⁺ and CD8⁺ T cells demonstrated significantly higher activity ratios. *p<0.05. (C) pSTAT5 expression was evaluated by flow cytometry. Histograms (left) represent the expression of pSTAT5 before (dashed line) and after (straight line) coculture with pMSC. Cells were gated on CD3⁺CD4⁺ or CD3⁺CD8⁺ T cells. Gray indicates isotype. The mean MFI (mean fluorescence intensity) of four different human PBMC cocultures with pMSC (right) demonstrating significantly higher pSTAT5 following pMSC coculture; *p<0.05.