Direct recognition of hepatocyte-expressed MHC class I alloantigens is required for tolerance induction
Moumita Paul-Heng, Mario Leong, Eithne Cunningham, Daniel L. J. Bunker, Katherine Bremner, Zane Wang, Chuanmin Wang, Szun Szun Tay, Claire McGuffog, Grant J. Logan, Ian E. Alexander, Min Hu, Stephen I. Alexander, Tim D. Sparwasser, Patrick Bertolino, David G. Bowen, G. Alex Bishop, Alexandra Sharland
Moumita Paul-Heng, Mario Leong, Eithne Cunningham, Daniel L. J. Bunker, Katherine Bremner, Zane Wang, Chuanmin Wang, Szun Szun Tay, Claire McGuffog, Grant J. Logan, Ian E. Alexander, Min Hu, Stephen I. Alexander, Tim D. Sparwasser, Patrick Bertolino, David G. Bowen, G. Alex Bishop, Alexandra Sharland
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Research Article Hepatology Transplantation

Direct recognition of hepatocyte-expressed MHC class I alloantigens is required for tolerance induction

Abstract

Adeno-associated viral vector–mediated (AAV-mediated) expression of allogeneic major histocompatibility complex class I (MHC class I) in recipient liver induces donor-specific tolerance in mouse skin transplant models in which a class I allele (H-2Kb or H-2Kd) is mismatched between donor and recipient. Tolerance can be induced in mice primed by prior rejection of a donor-strain skin graft, as well as in naive recipients. Allogeneic MHC class I may be recognized by recipient T cells as an intact molecule (direct recognition) or may be processed and presented as an allogeneic peptide in the context of self-MHC (indirect recognition). The relative contributions of direct and indirect allorecognition to tolerance induction in this setting are unknown. Using hepatocyte-specific AAV vectors encoding WT allogeneic MHC class I molecules, or class I molecules containing a point mutation (D227K) that impedes direct recognition of intact allogeneic MHC class I by CD8+ T cells without hampering the presentation of processed peptides derived from allogeneic MHC class I, we show here that tolerance induction depends upon recognition of intact MHC class I. Indirect recognition alone yielded a modest prolongation of subsequent skin graft survival, attributable to the generation of CD4+ Tregs, but it was not sufficient to induce tolerance.

Authors

Moumita Paul-Heng, Mario Leong, Eithne Cunningham, Daniel L. J. Bunker, Katherine Bremner, Zane Wang, Chuanmin Wang, Szun Szun Tay, Claire McGuffog, Grant J. Logan, Ian E. Alexander, Min Hu, Stephen I. Alexander, Tim D. Sparwasser, Patrick Bertolino, David G. Bowen, G. Alex Bishop, Alexandra Sharland

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Figure 4

The D227K mutation does not impede indirect recognition of Kd by indirectly alloreactive TCR75-RAG T cells.

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The D227K mutation does not impede indirect recognition of Kd by indirec...
(A) C57BL/6 mice were inoculated with 5 × 1011 vgc AAV-Kd or Kd-D227K. B6.Kd mice, which constitutively express the IAb-Kd(54-68) epitope on antigen-presenting cells, were positive-control recipients, while C57BL/6 mice were used as negative controls. CFSE-labeled TCR75-RAG lymphocytes (1 × 107) were transferred to vector-transduced and control mice 7 days after inoculation. After a further 3 days, leucocytes were isolated from the spleen, liver, and liver-draining LN; stained; and examined by flow cytometry (n = 3–4/group). (B) The congenic marker CD45.1 was used, along with CD4, to identify transferred TCR75-RAG cells. TCR75-RAG cells proliferated to a similar extent when transferred to mice transduced with either AAV-Kd (red line) or AAV-Kd-D227K (black line). TCR75-RAG cells transferred into C57BL/6 recipients did not divide (gray filled histogram). In the liver and draining LN, the extent of proliferation was similar to that in positive control B6.Kd mice, while in the spleen, proliferation had progressed further in the positive control than in the transduced mice. Representative histograms are shown (n = 3–4/group). (C and D) In mice inoculated with either AAV-Kd or Kd-D227K, similar patterns of expression of CD69 and PD-1 on TCR75-RAG cells were observed. CD69 surface expression declined with successive cell divisions (C), while PD-1 expression levels increased as division progressed (D). Representative plots from the draining LN (n = 3/group) are shown.