Immunological and inflammatory mapping of vascularized composite allograft rejection processes in a rat model

<div><p>Background</p><p>Hand and face vascularized composite allotransplantation (VCA) is an evolving and challenging field with great opportunities. During VCA, massive surgical damage is inflicted on both donor and recipient tissues, which may contribute to the high VCA rejection rates. To segregate between the damage-induced and rejection phase of post-VCA responses, we compared responses occurring up to 5 days following syngeneic versus allogeneic vascularized groin flap transplantations, culminating in transplant acceptance or rejection, respectively.</p><p>Methods</p><p>The immune response elicited upon transplantation of a syngeneic versus allogeneic vascularized groin flap was compared at Post-operative days 2 or 5 by histology, immunohistochemistry and by broad-scope gene and protein analyses using quantitative real-time PCR and Multiplex respectively.</p><p>Results</p><p>Immune cell infiltration began at the donor-recipient interface and paralleled expression of a large group of wound healing-associated genes in both allografts and syngrafts. By day 5 post-transplantation, cell infiltration spread over the entire allograft but remained confined to the wound site in the syngraft. This shift correlated with upregulation of IL-18, INFg, CXCL9, 10 and 11, CCL2, CCL5, CX3CL1 and IL-10 in the allograft only, suggesting their role in the induction of the anti-alloantigen adaptive immune response.</p><p>Conclusions</p><p>High resemblance between the cues governing VCA and solid organ rejection was observed. Despite this high resemblance we describe also, for the first time, a damage induced inflammatory component in VCA rejection as immune cell infiltration into the graft initiated at the surgical damage site spreading to the entire allograft only at late stage rejection. We speculate that the highly inflammatory setting created by the unique surgical damage during VCA may enhance acute allograft rejection.</p></div

Inflammation in syngrafts remains concentrated at the graft-recipient interface through POD 5.

<p>Samples from the syngraft-recipient interface were removed on POD 2 (A) or POD 5(B) and paraffin sections were stained with H&E. A panoramic view made by stitching images, is presented. Insets of the original images are displayed to provide a higher image resolution of selected regions. The area of the syngraft is surrounded by a black line.</p

Immunohistochemical quantification of cell infiltration into allografts, syngrafts and normal skin (NS).

<p>Samples (n = 3–5 rats per group) from the graft-recipient interface of a syngraft, allograft or from NS were removed at the indicated times and paraffin sections were stained with anti CD68, CD8 or CD4 antibodies followed by hematoxylin counterstaining. Stained cells were counted in 10 fields and the average stained cells per field is displayed in the graphs. * P<0.05 and ** P<0.01.</p

Animals used in the study.

<p>Animals used in the study.</p

Comparison of the expression changes of an array of immune modulators between syngrafts and allograft from POD 2 to POD 5.

<p>Samples (n = 3 per group) from the graft-recipient intersection of syngrafts, allografts or NS were removed at on POD 2 and POD 5. RNA was extracted and gene expression analysis was performed by qRTPCR. (A) Comparison of gene expression levels on POD 2 versus POD 5, in both syngrafts and allografts. Genes displaying a similar gene expression pattern on POD 2 and POD5, in syngrafts (I) and allografts (II). Genes displaying a different gene expression pattern on POD 2 and POD 5, in syngrafts (III) and allografts (IV). (B) Comparison of the fold-changes in gene expression levels, with respect to NS, in syngrafts versus allografts. Genes displaying either nonsignificant (I) or a statistically significant (II) difference in expression between syngraft and allograft. * P<0/05 and ** P<0.01.</p

Acute allograft rejection is clinically manifested in the vascularized groin flap model between POD 5 and 6.

<p>Monitoring of the groin flaps post op. (A) Syngraft at POD 2, 7 and 90. Note the regular non edematous surface good perfusion with no discoloration at POD 2 and 7 and complete healing with only the different direction of hair growth to indicate the syngraft location. (B) Allograft at POD 2, 5, 6, 7. Note that the POD 2 allograft already shows early signs of inflammation, edema, red color indicating hyperemia. POD 5 and 6 allograft shows clinical signs of rejection, patches of different colors indicating necrosis and extremely swollen and hard on palpation. POD 7 allograft is completely rejected and non-viable.</p

Grouping of the examined genes according to their expression in the syngeneic and allogeneic grafts.

<p>Grouping of the examined genes according to their expression in the syngeneic and allogeneic grafts.</p

Comparison of the RNA expression of an array of immune modulators of syngeneic or allogeneic grafts 5 days post transplantation with normal (uninflamed) skin (NS).

<p>Comparison of the RNA expression of an array of immune modulators of syngeneic or allogeneic grafts 5 days post transplantation with normal (uninflamed) skin (NS).</p

Schematic summary of the genes that control the wound healing and the anti-alloantigen components of VCA rejection.

<p>(A) Genes upregulated in both syngraft and allografts suggested to participate in the wound healing response following transplantation. (B) A schematic representation of VCA rejection process, as determined by the genes specifically upregulated during allograft rejection in the current study and in solid allograft rejection in earlier studies. (C) Th2 and Th17 phenotypes were not upregulated compared to NS and seemingly play no role in VCA rejection.</p

Allograft immune rejection initiates at the donor-recipient interface and then spreads to the entire allograft.

<p>Samples from the allograft-recipient interface were removed on POD 2 (A) or POD 5 (B) and paraffin sections were stained with hematoxylin-eosin (H&E). A panoramic view made by stitching images, is presented. Insets of the original images are displayed to provide a higher image resolution of selected regions. (C) An enlarged view of the inflamed zone demonstrates the high granulocyte content amongst infiltrating leukocytes.</p