Lung transplant acceptance is facilitated by early events in the graft and is associated with lymphoid neogenesis

Abstract

Early immune responses are important in shaping long-term outcomes of human lung transplants. To examine the role of early immune responses in lung rejection and acceptance, we developed a method to retransplant mouse lungs. Retransplantation into T-cell-deficient hosts showed that for lungs and hearts alloimmune responses occurring within 72 h of transplantation are reversible. In contrast to hearts, a 72-h period of immunosuppression with costimulation blockade in primary allogeneic recipients suffices to prevent rejection of lungs upon retransplantation into untreated allogeneic hosts. Long-term lung acceptance is associated with induction of bronchus-associated lymphoid tissue, where Foxp3(+) cells accumulate and recipient T cells interact with CD11c(+) dendritic cells. Acceptance of retransplanted lung allografts is abrogated by treatment of immunosuppressed primary recipients with anti-CD25 antibodies. Thus, events contributing to lung transplant acceptance are established early in the graft and induction of bronchus-associated lymphoid tissue can be associated with an immune quiescent state.

Figure 1

Mouse lung retransplants. (a) Schematic outlining retransplantation of organ graft “G” into secondary recipient after initial transplantation into primary host. Gross appearance and histology (H&E) (100x) of B6 lung grafts (Tx), initially transplanted into syngeneic B6 host for 72 hours and then retransplanted into B6 mouse shown 7 days (b, c) (n=4) and 30 days (d, e) (n=4) after retransplantation.

Figure 2

Lung retransplants into wildtype donor strain. Histological appearance (100x) of B6 lung grafts, initially transplanted into CBA recipient for 72 hours and then retransplanted into (a) B6 or (b) B6 CD45.1⁺ mouse, respectively, shown 7 days after retransplantation. (c) Flow cytometric analysis depicting IFN-γ production of graft-infiltrating CD4⁺ and CD8⁺ T cells for transplants shown in (b). Virtually all graft-infiltrating T cells (CD90.2) are derived from the secondary recipient (CD45.1). Results are representative of at least 3 transplants in each group.

Figure 3

Early alloimmune responses and lung graft rejection. Histological appearance (100x) of B6 lung grafts, initially transplanted into CBA host for 72 hours and then retransplanted into nude mouse, shown (a) 7 and (b) 30 days after retransplantation. (c) Histological appearance of B6 lung grafts, initially transplanted into CBA host for 72 hours and then retransplanted into secondary CBA recipient shown 30 days after retransplantation. (d) Rejection scores (ISHLT grade A) for transplants shown in (a) (n=5), (b) (n=3) and (c) (n=6). (** p < 0.01)

Figure 4

Early alloimmunity and cardiac graft rejection. Histological appearance (100x) of (a) B6 hearts transplanted into CBA host (shown at day 12 at time of cessation of heart beat) (n=4), (b) B6 cardiac grafts, initially transplanted into CBA mouse for 72 hours and then retransplanted into nude host (sacrificed at 30 days, at which point heart was beating strongly) (n=4) and (c) B6 hearts, initially transplanted into CBA mouse for 72 hours and then retransplanted into secondary CBA recipient (n=4). (d) Graft survival curves for groups shown in (a), (b) and (c), p<0.01 for (a) vs. (b) and (c) vs. (b).

Figure 5

Early responses and lung allograft acceptance. Histological appearance (100x) of B6 grafts 30 days after transplantation into (a) untreated (n=4) or (b) immunosuppressed (MR1 250 μg i.p. day 0, CTLA4-Ig 200 μg i.p. day 2) CBA hosts (n=6). Histological appearance of B6 lung grafts, initially transplanted into immunosuppressed CBA hosts for (c) 30 days (n=3) or (d) 72 hours (n=6) and then retransplanted into untreated CBA mouse shown 30 days after retransplantation. (e) Rejection scores (ISHLT grade A) for transplants shown in (a), (b), (c) and (d) (** p<0.01)

Figure 6

Early responses and cardiac allograft survival. Histological appearance (100x) of (a) B6 hearts transplanted into immunosuppressed (MR1 250 μg i.p. day 0, CTLA4-Ig 200 μg i.p. day 2) CBA host (sacrificed at 30 days, at which point heart was beating strongly) (n=4) and (b) B6 cardiac grafts, initially transplanted into immunosuppressed CBA mouse for 72 hours and then retransplanted into untreated CBA host (shown at day 11 at time of cessation of heart beat) (n=4). (c) Graft survival curves for groups shown in (a) and (b), p<0.01. No statistically significant differences were observed between retransplants depicted here and B6 → CBA cardiac transplants shown in Figure 4 (p=0.28).

Figure 7

Lymphoid aggregates in accepted lung allografts. Immunostaining (100x) for CD3, B220, PNAd, CCL21 for B6 grafts 30 days after transplantation into immunosuppressed CBA host (a–e) or B6 lung grafts, initially transplanted into immunosuppressed CBA host for 72 hours and then retransplanted into untreated CBA mouse 30 days after retransplantation (f–j).

Figure 8

Two-photon images of dendritic cells in accepted Balb/c lung grafts 30 days after transplantation into immunosuppressed (MR1 250 μg i.p. day 0, CTLA4-Ig 200 μg i.p. day 2) B6 CD11c⁺ EYFP recipients. Dendritic cells (a) in BALT and (b) adjacent graft tissue. (c) Neighboring and (d) volumetric analysis of dendritic cells within BALT and adjacent lung. Two-photon images of quantum dot (655-nm)-labeled vessels (red) within areas of (e) BALT, (f) adjacent lung and (g) cervical lymph node. (h) Quantitative analysis of tortuosity index of vessels at indicated sites. (*p<0.05, **p<0.005) Scale bar = 60 μm.

Figure 9

Intravital two-photon imaging of dynamic T cell behavior within induced BALT in accepted Balb/c lung grafts 30 days after transplantation into immunosuppressed (MR1 250 μg i.p. day 0, CTLA4-Ig 200 μg i.p. day 2) B6 CD11c⁺ EYFP recipients. (a) Time-lapse behavior of adoptively transferred CMTMR-labeled B6 T cells in dendritic cell clusters with tracks indicating representative T cells. Images are individual frames from a continuous time-lapse movie. Relative time is displayed in hr:min:sec. (b) Number of extravasated T cells within BALT and adjacent graft tissue. Plots of (c) average track speed (μm/min) and (d) meandering index of extravasated T cells within BALT. Meandering index was calculated by dividing the distance a cell traveled from its starting point by the track length. (e) Individual T lymphocytes were tracked and the cell displacement squared (μm²) vs. time (min) shows a strong linear correlation indicative of random cell migration. n=35, *p<0.05, Scale bar = 60 μm.

Figure 10

Regulatory T cells in accepted lung grafts. (a) Flow cytometric analysis of Foxp3-expressing CD4⁺ T cells in (left) lungs derived from naïve B6 mice (3.01% ± 0.46 (s.e.m.)) and (right) B6 lung allografts 30 days after transplantation into MR1- and CTLA4-Ig-treated CBA mice (21.77% ± 3.55) (n=3 each) (b) Immunofluorescent staining of Foxp3⁺ cells in BALT of Balb/c lung grafts 30 days after transplantation into MR1- and CTLA4-Ig-treated B6 CD11c-EYFP recipient. Foxp3-expressing cells are red, CD11c⁺ dendritic cells are green and nucleated cells are blue (DAPI). (c) Histological appearance and (d) rejection score (ISHLT A) for B6 lung grafts that were initially transplanted into immunosuppressed (MR1, CTLA4-Ig) CBA recipient that also received PC61 and then retransplanted into nonimmunosuppressed CBA host shown 30 days after retransplantation (n=4).