Dissecting Serial Immune Response Stages of Chronic Rejection after Murine Orthotopic Lung Transplantation

Purpose “Chronic rejection” (CR) after lung transplantation (LTx) is considered to be a cellular and humoral immune response, accompanied by angiogenesis and fibrosis, clinically denominated as chronic lung allograft dysfunction (CLAD). We aimed to reinvestigate the chronological stages and spatial organization of the immune response of CR. Methods Orthotopic single LTx was performed to create isograft (n=9; C57BL/6N to C57BL/6N) and two allograft (n=16; BALB/c to C57BL/6N) subgroups: 1/ observational group (n=8), with serial sacrifice at week 1, 3, 5 and 8 to evaluate the sequential, spatial and cellular events of CR; and 2/ validation group (week 10; n=8) to assess variation within CR. All mice received daily immunosuppression of 10 mg/kg cyclosporine A and 1.6 mg/kg methylprednisolone. Results In 1 isograft technical failure occurred and 8 had normal lung architecture at week 10. In 3 allografts failure occurred: 2 in the observational, 1 in the validation group. Histological evaluation of allografts allowed reconstruction of CR: STAGE 1: innate inflammation around end-arteries; STAGE 2: innate immune cells around end-arteries and end-venules; STAGE 3: adaptive immune cell organization around end-arteries; innate immune cells around end-venules; pleural infiltration of lymphocytes; STAGE 4: fibrosis around end-arteries with end-arteritis obliterans; adaptive immune cells around the end-venules; pleural lymphatic vessel growth and fibrosis; bronchiolar wall folding and sporadic intraluminal fibrotic plugging. In the validation group, we identified 2 mice in STAGE 2, 2 in STAGE 3 and 3 in STAGE 4. µCT confirmed the progression of severe (stage 4) vs mild (stage 2-3) in lung attenuation (1.05±0.04 vs 1.14±0.01; p=0.002) (Figure) and total and tidal volume. Conclusion CR demonstrated an innate onset, evolving to adaptive organization and fibrosis, starting at end-arteries and then spreading towards venules, pleura and airways. This pattern seems to resemble RAS after human LTx.