Abstract
Abstract It is the prevailing view that adaptive immune responses are initiated in secondary lymphoid organs. Studies using alymphoplastic mice have shown that secondary lymphoid organs are essential to initiate allograft rejection of skin, heart, and small bowel. The high immunogenicity of lungs is well recognized and allograft rejection remains a major contributing factor to poor outcomes after lung transplantation. We show in this study that alloreactive T cells are initially primed within lung allografts and not in secondary lymphoid organs following transplantation. In contrast to other organs, lungs are acutely rejected in the absence of secondary lymphoid organs. Two-photon microscopy revealed that recipient T cells cluster predominantly around lung-resident, donor-derived CD11c+ cells early after engraftment. These findings demonstrate for the first time that alloimmune responses following lung transplantation are initiated in the graft itself and therefore identify a novel, potentially clinically relevant mechanism of lung allograft rejection.
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📋 Methods
Mice C57BL/6 (B6), C3H/HeJ (C3H), CBA/Ca (CBA), and B6 CD45.1 + mice were purchased from The Jackson Laboratory. B6 CD11c + -enhanced yellow fluorescent protein (EYFP) 3 -positive mice were a gift from M. Nussenzweig (The Rockefeller University, New York, NY). Male B6 aly / aly mice (CLEA Japan) were bred with female C3H mice. Their B6C3F1 aly /+ offspring were intercrossed to produce B6C3F2 mice, which were then screened for expression of the aly mutation of NF- κ B-inducing kinase and genotyped for H-2K k and K b ( 5 ). B6C3F2 aly / aly mice expressing at least one H-2K k allele (B6C3F2 K k/b aly / aly ) were used as donors. Transplantation Lungs and hearts were transplanted in 8- to 10-wk old mice ( 12 ). Graft histology was assessed in a blinded fashion. Adoptive transfers CD90 + T cells were purified from spleens of B6, B6 CD45.1 + , or CBA mice with magnetic beads (Miltenyi Biotec). We injected 10 7 CFSE-labeled B6 CD45.1 + or sorted B6 CD45.1 + CD44 low T cells for flow cytometric studies and 10 7 CellTracker Red CMTPX-labeled CBA or B6 T cells for imaging studies. ELISPOT CD8 + T cells (2 × 10 5 ), purified from spleens of untransplanted B6 aly / aly mice or from lung allografts 7 days after B6C3F2 K k/b aly / aly → splenectomized B6 aly / aly transplantation were cultured with irradiated stimulators (6 × 10 5 ) (syngeneic H-2K b , allogeneic H-2K k , or third party H-2K d ) at a 1:3 ratio in plates coated with capture mouse IFN- γ -specific mAb (BD Biosciences). After 24 h, cells were incubated with biotinylated IFN- γ -specific Ab.
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Mice C57BL/6 (B6), C3H/HeJ (C3H), CBA/Ca (CBA), and B6 CD45.1 + mice were purchased from The Jackson Laboratory. B6 CD11c + -enhanced yellow fluorescent protein (EYFP) 3 -positive mice were a gift from M. Nussenzweig (The Rockefeller University, New York, NY). Male B6 aly / aly mice (CLEA Japan) were bred with female C3H mice. Their B6C3F1 aly /+ offspring were intercrossed to produce B6C3F2 mice, which were then screened for expression of the aly mutation of NF- κ B-inducing kinase and genotyped for H-2K k and K b ( 5 ). B6C3F2 aly / aly mice expressing at least one H-2K k allele (B6C3F2 K k/b aly / aly ) were used as donors. Transplantation Lungs and hearts were transplanted in 8- to 10-wk old mice ( 12 ). Graft histology was assessed in a blinded fashion. Adoptive transfers CD90 + T cells were purified from spleens of B6, B6 CD45.1 + , or CBA mice with magnetic beads (Miltenyi Biotec). We injected 10 7 CFSE-labeled B6 CD45.1 + or sorted B6 CD45.1 + CD44 low T cells for flow cytometric studies and 10 7 CellTracker Red CMTPX-labeled CBA or B6 T cells for imaging studies. ELISPOT CD8 + T cells (2 × 10 5 ), purified from spleens of untransplanted B6 aly / aly mice or from lung allografts 7 days after B6C3F2 K k/b aly / aly → splenectomized B6 aly / aly transplantation were cultured with irradiated stimulators (6 × 10 5 ) (syngeneic H-2K b , allogeneic H-2K k , or third party H-2K d ) at a 1:3 ratio in plates coated with capture mouse IFN- γ -specific mAb (BD Biosciences). After 24 h, cells were incubated with biotinylated IFN- γ -specific Ab.
Flow cytometry
Cells were stained with fluorochrome-labeled anti-CD45.1, anti-CD45.2, anti-CD4, anti-CD8, and anti-CD69 Abs and their isotype controls ( 12 ).
Two-photon microscopy
Tissue was imaged with a custom-built, video-rate two-photon microscope. Fluorescence emission was detected as red, green and blue signal channels. Each plane consisted of an image of 200 × 225 μ m. The z -stacks were acquired by taking 31 sequential steps at 2.5- μ m spacing. We averaged 15 video frames for each z -slice. Tissue volumes (75 × 200 × 225 μ m) containing 11–13 cells were analyzed ( 13 ). Neighboring distributions (cells within a centroid to centroid distance
📊 Figures
FIGURE 1
Lung transplantation in wild-type mice. A , C3H u2192 B6 lung grafts on postoperative day 3. B , Division of adoptively transferred CFSE-labeled CD45.1 + T cells in lung grafts, contralateral lungs (C...
FIGURE 2
T cell clustering in lung grafts and draining lymph nodes. A , B6 CD11c-EYFP + u2192 CBA graft showing adoptively transferred CBA T cells (red), CD11c-EYFP + cells (green), and collagen fibers (blue)....
FIGURE 3
Lung transplantation in the absence of secondary lymphoid organs. A , C3H u2192 splenectomized B6 aly / aly lung grafts on postoperative day 7. B , B6C3F2 K k/b aly / aly u2192 splenectomized B6 aly /...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
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