Initial in vivo experience of pig artery patch transplantation in baboons using mutant MHC (CIITA-DN) pigs

Abstract

Background: In the pig-to-nonimmunosuppressed baboon artery patch model, a graft from an α1,3-galactosyltransferase gene-knockout pig transgenic for human CD46 (GTKO/CD46) induces a significant adaptive immune response (elicited anti-pig antibody response, increase in T cell proliferation on MLR, cellular infiltration of the graft), which is effectively prevented by anti-CD154mAb-based therapy.

Methods: As anti-CD154mAb is currently not clinically applicable, we evaluated whether it could be replaced by CD28/B7 pathway blockade or by blockade of both pathways (using belatacept + anti-CD40mAb [2C10R4]). We further investigated whether a patch from a GTKO/CD46 pig with a mutant human MHC class II transactivator (CIITA-DN) gene would allow reduction in the immunosuppressive therapy administered.

Results: When grafts from GTKO/CD46 pigs were transplanted with blockade of both pathways, a minimal or insignificant adaptive response was documented. When a GTKO/CD46/CIITA-DN graft was transplanted, but no immunosuppressive therapy was administered, a marked adaptive response was documented. In the presence of CD28/B7 pathway blockade (abatacept or belatacept), there was a weak adaptive response that was diminished when compared with that to a GTKO/CD46 graft. Blockade of both pathways prevented an adaptive response.

Conclusion: Although expression of the mutant MHC CIITA-DN gene was associated with a reduced adaptive immune response when immunosuppressive therapy was inadequate, when blockade of both the CD40/CD154 and CD28/B7 pathways was present, the response even to a GTKO/CD46 graft was suppressed. This was confirmed after GTKO/CD46 heart transplantation in baboons.

Keywords: Anti-CD40 monoclonal antibody; Artery patch; CTLA4-Ig; Costimulation blockade; Pig; Xenotransplantation.

Figure 1. Mutant human CIITA-DN suppresses the expression of SLA class II on pAEC

Expression of SLA class II on GTKO/CD46/CIITA-DN pAECs was compared with that on GTKO/CD46 pAECs. The pAECs were activated with porcine interferon-γ (pIFN-γ; 50ng/ml) for 48h. After activation of pAECs from both GTKO/CD46 and GTKO/CD46/CIITA-DN pigs, expression of SLA class I was increased. A small increase in SLA class II expression was observed on GTKO/CD46/CIITA-DN pAECs after activation, but there was a marked increased expression of SLA class II on GTKO/CD46 pAECs. Isotype control (dotted line), before activation (solid line), and after activation (shaded).

Figure 2. Reduction of baboon T cell response to pPBMC associated with suppression of SLA class II expression

(A) Flow cytometry of the CD4⁺ and CD8⁺ responses in representative experiments (% refers to percentage proliferation). There was reduced CD4⁺ and CD8⁺ T cell proliferation to GTKO/CD46/CIITA-DN pPBMC. (B) Mean percentage proliferation of CD4⁺ and CD8⁺ cells. There were significantly lower baboon CD4⁺ and CD8⁺ T cell responses to GTKO/CD46/CIITA-DN pPBMC compared with those to GTKO/CD46 pPBMC (*p<0.05, respectively). The proliferative responses of baboon CD4⁺ and CD8⁺ T cells to autologous PBMC were negligible (not shown). We conclude that the reduced proliferation of CD8⁺ cells was secondary to reduction in CD4⁺ T cell help.

Figure 3. The mean numbers of CD4⁺, CD8⁺ T cells, and CD21⁺ B cells in GTKO or GTKO/CD46 or GTKO/CD46/CIITA-DN pig artery patch recipients

Depletion of CD4⁺ (A) and CD8⁺ (B) T cells in blood of recipient baboons was observed after ATG, but was not seen when no IS was administered. Some depletion of CD21⁺ B cells (C) was observed in some baboons (which we are unable to explain). The mean numbers of CD4⁺, CD8⁺, and CD21⁺ cells in baboons in each group are shown with standard error (SE).

Figure 3. The mean numbers of CD4⁺, CD8⁺ T cells, and CD21⁺ B cells in GTKO or GTKO/CD46 or GTKO/CD46/CIITA-DN pig artery patch recipients

Depletion of CD4⁺ (A) and CD8⁺ (B) T cells in blood of recipient baboons was observed after ATG, but was not seen when no IS was administered. Some depletion of CD21⁺ B cells (C) was observed in some baboons (which we are unable to explain). The mean numbers of CD4⁺, CD8⁺, and CD21⁺ cells in baboons in each group are shown with standard error (SE).

Figure 4. Recipient baboon mean cellular responses to GTKO or GTKO/CD46 or GTKO/CD46/CIITA-DN pig artery patch grafts *

When a CTLA4-Ig (abatacept or belatacept)-based regimen was administered (Group 2A), CD4⁺ (A) and CD8⁺ (B) T cell proliferative responses were seen on MLR, as well as in the single baboon in Group 1 that received no IS. It was only when anti-CD40mAb+belatacept were administered together that no T cell proliferative response was documented (Groups 1B and 2B). The CD4⁺ and CD8⁺ T cellular proliferative responses were less to a GTKO/CD46/CIITA-DN graft (Group 2B) than to a GTKO/CD46 graft (Group 1B) (statistical analyses were not carried out as numbers too small). The assays were carried out pre-transplantation (pre-Tx) and during the last week of each study (post-Tx) (see Table 1). We could not detect any relationship between the result of the MLR and the day of the assay, i.e., day 21–28 or 48 (C and D). (*We do not show the MLR data in Group 1A because we used a different method of measuring the MLR in Group 1A from that in other groups.

Figure 4. Recipient baboon mean cellular responses to GTKO or GTKO/CD46 or GTKO/CD46/CIITA-DN pig artery patch grafts *

When a CTLA4-Ig (abatacept or belatacept)-based regimen was administered (Group 2A), CD4⁺ (A) and CD8⁺ (B) T cell proliferative responses were seen on MLR, as well as in the single baboon in Group 1 that received no IS. It was only when anti-CD40mAb+belatacept were administered together that no T cell proliferative response was documented (Groups 1B and 2B). The CD4⁺ and CD8⁺ T cellular proliferative responses were less to a GTKO/CD46/CIITA-DN graft (Group 2B) than to a GTKO/CD46 graft (Group 1B) (statistical analyses were not carried out as numbers too small). The assays were carried out pre-transplantation (pre-Tx) and during the last week of each study (post-Tx) (see Table 1). We could not detect any relationship between the result of the MLR and the day of the assay, i.e., day 21–28 or 48 (C and D). (*We do not show the MLR data in Group 1A because we used a different method of measuring the MLR in Group 1A from that in other groups.

Figure 5. Recipient baboon mean IgM (A) and IgG (B) antibody responses to GTKO or GTKO/CD46 or GTKO/CD46/CIITA-DN pig artery patch grafts

Left panel shows the mean IgM and IgG antibody responses throughout the course of each experiment. Right panel shows the percentage change in IgM and IgG antibody responses at the end of each experiment. The single baboon with no IS developed anti-pig IgM (A) and IgG (B) antibody responses. In abatacept- or belatacept-based regimens (Groups 1A and 2A), the antibody response was greatly attenuated, though there remained a modest IgG response; the IgG response in Group 2A was significantly reduced when compared with that in Group 1A (p<0.05), suggesting a beneficial effect of the CIITA-DN mutation. With the anti-CD40mAb+belatacept-based regimen, an elicited IgG antibody response was minimal or absent (Groups 1B or 2B, respectively),

Figure 6. Histopathology of GTKO or GTKO/CD46 or GTKO/CD46/CIITA-DN pig artery patch grafts at euthanasia

(A) At euthanasia on day 28, the GTKO/CD46/CIITA-DN pig artery patch graft in the baboon with no IS showed a massive full-thickness infiltrate (eosinophilic, lymphocytic, and monocytic). (B) The pig grafts in Groups 1A and 2A showed a mild infiltration with mixed inflammatory cells (lymphocytes and monocytes). Cellular infiltration in the GTKO/CD46/CIITA-DN graft was absent in Group 2B (day 28) (C), and minimal in Group 1B (day 28 or 48) (in which a GTKO/CD46 graft was transplanted) (D). No obvious differences were observed in the grafts, whether the experiments were terminated on day 28 or 48. (Magnification x20)

Figure 6. Histopathology of GTKO or GTKO/CD46 or GTKO/CD46/CIITA-DN pig artery patch grafts at euthanasia

(A) At euthanasia on day 28, the GTKO/CD46/CIITA-DN pig artery patch graft in the baboon with no IS showed a massive full-thickness infiltrate (eosinophilic, lymphocytic, and monocytic). (B) The pig grafts in Groups 1A and 2A showed a mild infiltration with mixed inflammatory cells (lymphocytes and monocytes). Cellular infiltration in the GTKO/CD46/CIITA-DN graft was absent in Group 2B (day 28) (C), and minimal in Group 1B (day 28 or 48) (in which a GTKO/CD46 graft was transplanted) (D). No obvious differences were observed in the grafts, whether the experiments were terminated on day 28 or 48. (Magnification x20)