Quantitative immunofluorescence immunohistochemistry
In our previous studies examining the early effects of ischaemia–reperfusion and surgical injury in a non-recovery transplant model, significant effects were observed at 8 h after reperfusion, but the magnitude of the effects was less than interpig variation at baseline . Here we report the first detailed analysis of the acute and delayed mucosal immunological response to orthotopic laryngeal transplantation in a recovery model over the first week. The animals were fully matched at the MHC locus; minor histocompatability mismatches were unlikely to cause acute rejection. Donor and recipient had been housed together prior to the surgery; therefore we expected few differences between animals prior to transplantation. However, donor larynges expressed marginally more macrophage-related molecules than those they were replacing (Table 3). We hypothesize that this was due to the challenge, to the recipient, of a previous anaesthetic/laryngeal intubation performed 2–3 days previously .
Prior to the onset of cold ischaemia, there was a slight drop in expression of molecules associated with myeloid cells in the supraglottis and lymphocytic antigens in the supraglottis, but no change in the trachea. These small differences might be attributed to intra-operative blood loss. At the time of reperfusion there was no clear pattern to the minor changes observed, suggesting no hyperacute effect of this challenge on laryngeal immunological architecture. The length of cold ischaemia in this study (340 min) is between that in the only two reported human laryngeal transplants: 10 h  and 4 h (Birchall; personal communication).
At 48 h, we observed a small decrease in expression of MHC-II and lymphocyte-associated molecules, while there was a small increase in other myeloid-associated molecules. This is consistent with the observations of Friedman et al.  using heterotopic grafts in rats, where separate labelling of donor and recipient cells suggested depletion of donor leucocytes from the graft before the appearance of recipient cells.
An increase in some, but not all, myeloid-associated molecules was observed in the subglottis, supraglottis and trachea at 1 week after transplantation. In the subglottis there was a highly significant increase in areas expressing the combination of CD163+CD172+MHC-II- and a small increase in cells co-expressing CD163+CD172+MHC-II+ molecules. Previous studies in pigs have suggested that CD163+CD172+ cells in blood up-regulate surface MHC-II and differentiate effectively into dendritic cells  and the MHC-II- and MHC-II+ subsets may represent monocytes/macrophages and immature dendritic cells, respectively . The similarity to the results of Friedman et al.  is striking. In both allograft and fully matched rat models, these authors found a significant increase in OX62+ cells, both with and without MHC-II co-expression, between days 5–7: again the strongest effect was seen in the subglottis. The proportion of OX62+ cells expressing MHC-II was low (25%), suggesting that the majority were either immature dendritic cells or other cells expressing the integrin receptor, such as macrophages . Friedman et al.'s observations extended to 9 days, by which time expression of all antigens was falling towards baseline. The increases in myeloid-associated molecules we observed in our orthotopic pig model may also be transient. Similarly, we observed a rise in expression of CD14 molecules [in pigs, the lipopolysaccharide (LPS) receptor]; we saw an increase in number of CD14+CD16-MHC-II- cells and CD14+CD16+MHC-II- cells, but not in cells expressing MHC-II. We interpret this as further evidence that the population we are observing is primarily macrophages. Inspection of the images confirmed that the increase in CD14 staining was not localized to epithelium, endothelium or fibroblasts, all of which can also express this molecule , confirming that these were leucocyte changes.
In the absence of any obvious damage to the grafts at functional, macroscopic, microscopic and vascular levels, we hypothesize that the increase in myeloid cells in the subglottis at 1 week may be a response to a (possibly transient) change in mucosal flora. In support of this, colonization experiments in pigs have shown similar changes in bowel  and laryngeal [28,29] mucosa in response to bacterial challenge.
The observation that expression of CD25 in the absence of either CD4 or CD8 or CD45RC increased, but expression co-localized with CD4 or CD8 did not increase, indicates that the increase in CD25 was associated with a CD4-CD8-CD45RC- cell. Although CD25+CD4-CD8- cells occur in pigs, particularly in young animals as used here (Haverson, July 2008, personal communication), these are unlikely to be conventional T cells in which CD45RC indicates a particular state of differentiation. We hypothesize that unconventional cells expressing CD25 may be responsible for the subsequent recruitment of the myeloid populations seen in laryngeal transplants at 1 week in pig and rat. Alternatively, the CD25+ increase at 1 week could be associated with increased expression of the antigen on monocytes or natural killer (NK) cells.
The study of recipient trachea (proximal to the transplant) at the time of implantation and at death found no significant differences. The few differences that were observed between trachea from donors prior to implantation and recipient trachea at death may have been biased by the fact that the donor animals had slightly higher levels of expression of macrophage-associated molecules before surgery. This suggests that laryngeal transplantation surgery does not alter the immunological architecture of the host airway beyond the confines of the graft, at least in the first week after surgery. The apparent absence of changes in a smaller number of tonsillar biopsies supports this assertion.
TCR-β spectratyping  is a technique that uses the variation in TCR-β CDR3 length as a surrogate measure of diversity of the T cell repertoire, and has been used to provide proxy measures of changes in the T cell repertoire occurring during transplantation, autoimmunity and inflammation. We have recently developed pig TCR Vβ spectratyping assays based upon published porcine Vβ sequences [17–19]. In this study we applied these assays to tissue biopsies from one transplantion experiment to establish if spectratype analysis is a feasible technique for use in our transplant model, and to determine if changes in T cell repertoires within laryngeal mucosal biopsies can be detected. Our results show that spectratype analysis can be performed successfully on laryngeal mucosal biopsies and has the potential to provide a better understanding of changes in the mucosal immunology of the grafted tissue. The donor and recipient laryngeal biopsy Vβ repertoires were initially distinct, consistent with interindividual variation . Interestingly, post-transplantation, the Vβ repertoire of the donor laryngeal samples became more similar to that of the recipient larynx. As only a single transplant pair was analysed, firm conclusions cannot be drawn and further studies are required to determine if this observation is a consistent phenomenon in other laryngeal transplants. It is also possible that the differences in the T cell repertoires in the donor larynx may represent variation in biopsy sampling. Broadly, however, the results would support the hypothesis that between 0 and 7 days post-transplantation the donor larynx is gradually repopulated with recipient T cells, whose repertoire is representative of that normally associated with laryngeal mucosa in the recipient. These are likely to represent a pre-exisiting, laryngeal-homing T cell population, indicating normal recirculation of T cells through the graft. We hypothesize further that rejection will be associated with infiltration by T cells with characteristic repertoires. Spectratyping studies of grafts between mismatched animals would help to explore this question.