Evidence is available that, in kidney transplantation, ischemic injury causes activation of intragraft dendritic cells (DCs), a key event for T cell alloactivation through the direct pathway. To evaluate whether ischemia impacts on the indirect pathway of antigen presentation we used mouse B6 OVA-DCs, constitutively expressing chicken OVA peptides in the context of class I and class II MHC, and OVA-specific TCR-transgenic B6 syngeneic CD8 (OTI) and CD4 (OTII) T cells as a model of indirect pathway of minor histocompatibility antigen presentation.

Bone marrow derived OVA-DCs were pre-exposed for 4hr to 1% (hypoxia) or 20% O2 (normoxia) and used as stimulators of OTI or OTII T cells for 2 days. The numbers of IFNΓ producing CD8 and CD4 T cells were higher (p<0.05) in response to hypoxic than to normoxic OVA-DCs (OTI 162±26, OTII 72±20 vs OTI 115±23, OTII 36±15 spots/50000 cells, n=4). An even higher increase in IFNΓ producing clones was found in response to hypoxic OVA-DCs exposed to conditioned medium (cm) from hypoxic OVA B6 tubuli vs normoxic OVA-DC exposed to cm from normoxic tubuli (p<0.01).

Similarly, wild-type B6 DCs pre-incubated with OVA protein during hypoxia (plus hypoxic tubular cm) induced higher numbers of IFNΓ producing T cells than OVA-exposed normoxic B6 DCs (plus normoxic tubular cm) (OTI 60±15, OTII 50±10 vs OTI 31±8, OTII 23±6 spots, n=3, p<0.05), indicating that hypoxia augmented the capacity of DCs to present and to cross-present antigens to CD4 and CD8 T cells, respectively.

A similar trend was observed with wild-type non-transgenic B6 T cells as responders. IFNΓ producing T cell clones were detected in B6 T cells stimulated by hypoxic OVA-DCs (plus hypoxic tubular cm, 31±10, n=2) while T cells were poorly stimulated by normoxic OVA-DCs (plus normoxic tubular cm, 13±7, n=2). In-vivo translation of these results was the rejection of OVA kidneys subjected to 25 min cold ischemia and transplanted into B6 syngeneic mice (graft survival: 4±2 days, n=3) while non ischemic OVA kidneys showed graft survival >30 days (n=3). Control syngeneic ischemic B6 kidneys were not rejected as expected (graft survival: >30 days).

Altogether these results suggest that hypoxia activates the indirect pathway of antigen presentation in DCs both by a direct action on DCs and through the release of soluble factors from injured tubuli. Such events eventually translate into an exacerbation of the anti-graft immune response and graft rejection.

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