New concepts of immune modulation in xenotransplantation

Abstract

The shortage of human organs for transplantation has focused research on the possibility of transplanting pig organs into humans. Many factors contribute to the failure of a pig organ graft in a primate. A rapid innate immune response (natural anti-pig antibody, complement activation, and an innate cellular response; e.g., neutrophils, monocytes, macrophages, and natural killer cells) is followed by an adaptive immune response, although T-cell infiltration of the graft has rarely been reported. Other factors (e.g., coagulation dysregulation and inflammation) appear to play a significantly greater role than in allotransplantation. The immune responses to a pig xenograft cannot therefore be controlled simply by suppression of T-cell activity. Before xenotransplantation can be introduced successfully into the clinic, the problems of the innate, coagulopathic, and inflammatory responses will have to be overcome, most likely by the transplantation of organs from genetically engineered pigs. Many of the genetic manipulations aimed at protecting against these responses also reduce the adaptive response. The T-cell and elicited antibody responses can be prevented by the biological and/or pharmacologic agents currently available, in particular, by costimulation blockade-based regimens. The exogenous immunosuppressive regimen may be significantly reduced by the presence of a graft from a pig transgenic for a mutant (human) class II transactivator gene, resulting in down-regulation of swine leukocyte antigen class II expression, or from a pig with "local" vascular endothelial cell expression of an immunosuppressive gene (e.g., CTLA4-Ig). The immunomodulatory efficacy of regulatory T cells or mesenchymal stromal cells has been demonstrated in vitro but not yet in vivo.

Figure 1. Increased human PBMC and CD4⁺T cell proliferation in response to thrombin-activated porcine aortic endothelial cells (pAEC)

GTKO pAEC were activated using thrombin (40U/mL), pIFN-γ (40U/mL), or hIFN-γ (200U/mL), for 24h. The human PBMC (top) and CD4⁺T cell (bottom) proliferative responses to pIFN-γ- and thrombin-activated GTKO pAEC were significantly higher than to non-activated pAEC. (Stimulator:responder ratios of 1:10 and 1:20.) Data are representative of three different experiments. (*p<0.01, **p<0.001 in comparison to non-activated pAEC). (Reproduced from Ezzelarab C, et al, Xenotransplantation 2012; 19:311-316 [33] with permission.)

Figure 2. Thrombin does not upregulate SLA class I or II expression on GTKO porcine aortic endothelial cells (pAEC)

GTKO pAEC were activated using thrombin (40U/mL), pIFN-γ (40U/mL), or hIFN-γ (200U/mL) for 24h. SLA class II expression was upregulated only after pIFN-γ activation, but not after thrombin or hIFN-γ activation. There was no change in SLA class I expression after activation. Data are representative of three different experiments. (Reproduced from Ezzelarab C, et al, Xenotransplantation 2012; 19:311-316 [33] with permission).

Figure 3. Proliferative response of human T cells to wild-type (WT) and GTKO porcine aortic endothelial cells (pAEC)

A: The proliferative response of human CD4⁺T cells (n=3) to WT and GTKO pAEC before and after activation by pIFN-γ (left). The response was significantly less to GTKO pAEC before (p<0.001) and after (p<0.01) activation. Additionally, MLRs were harvested on 3 consecutive days (4, 5 and 6) where CD4⁺T cell proliferation was consistently lower in response to GTKO pAEC (right). B: The proliferative response of human CD8⁺T cells (n=3) to WT and GTKO pAEC before and after activation by pIFN-γ (left). The response was significantly less to GTKO pAEC before (p<0.001) and after (p<0.05) activation. Additionally, MLRs were harvested on 3 consecutive days (4, 5 and 6) where CD8⁺T cell proliferation was consistently lower in response to GTKO pAEC (right). In both A and B, ³H incorporation values are presented as CPM. Data represent the mean (+/- SEM) and are representative of three different experiments. (Reproduced from Wilhite T, et al, Xenotransplantation 2012; 19:56-63 [54] with permission).

Figure 3. Proliferative response of human T cells to wild-type (WT) and GTKO porcine aortic endothelial cells (pAEC)

A: The proliferative response of human CD4⁺T cells (n=3) to WT and GTKO pAEC before and after activation by pIFN-γ (left). The response was significantly less to GTKO pAEC before (p<0.001) and after (p<0.01) activation. Additionally, MLRs were harvested on 3 consecutive days (4, 5 and 6) where CD4⁺T cell proliferation was consistently lower in response to GTKO pAEC (right). B: The proliferative response of human CD8⁺T cells (n=3) to WT and GTKO pAEC before and after activation by pIFN-γ (left). The response was significantly less to GTKO pAEC before (p<0.001) and after (p<0.05) activation. Additionally, MLRs were harvested on 3 consecutive days (4, 5 and 6) where CD8⁺T cell proliferation was consistently lower in response to GTKO pAEC (right). In both A and B, ³H incorporation values are presented as CPM. Data represent the mean (+/- SEM) and are representative of three different experiments. (Reproduced from Wilhite T, et al, Xenotransplantation 2012; 19:56-63 [54] with permission).

Figure 4

(A) Significant down-regulation of SLA class II expression on aortic endothelial cells from GTKO/CD46/CIITA-DN pigs The expression of SLA class II DR and DQ on GTKO/CD46/CIITA-DN porcine aortic endothelial cells (pAECs) was compared with those on GTKO/CD46 pAECs. pAECs were activated with pIFN-γ (50ng/mL) for 48h. The pAECs were stained with specific anti-SLA DR or DQ mAbs. Isotype control (dotted line), quiescent (solid line), and activated pAECs (gray filled). Expression of SLA class II on quiescent pAECs was undetectable or minimal on both GTKO/CD46 and GTKO/CD46/CIITA-DN pAECs. However, expression of SLA class II on GTKO/CD46 pAECs was significantly up-regulated when the pAECs were activated with pIFN-γ for 48h. In contrast, up-regulation of expression of SLA class II on GTKO/CD46/CIITA-DN pAECs was minimal. (B) Significant reduction of the hCD4⁺T cell response to CIITA-DN cells hCD4⁺T cells were co-cultured with PBMCs from WT, GTKO/CD46, and GTKO/CD46/CIITA-DN pigs for 6 days. The responses of hCD4⁺T cells were measured by ³H-thymidine incorporation. As a negative control, hCD4⁺T cells were cultured with medium only (spontaneous) or autologus PBMCs (auto). There was a significantly lower hCD4⁺T cell response to GTKO/CD46/CIITA-DN pig PBMCs than to either WT or GTKO/CD46 pig PBMCs (**p<0.01).