Tolerance of Lung Allografts Achieved in Nonhuman Primates via Mixed Hematopoietic Chimerism

Abstract

While the induction of transient mixed chimerism has tolerized MHC-mismatched renal grafts in nonhuman primates and patients, this approach has not been successful for more immunogenic organs. Here, we describe a modified delayed-tolerance-induction protocol resulting in three out of four monkeys achieving long-term lung allograft survival without ongoing immunosuppression. Two of the tolerant monkeys displayed stable mixed lymphoid chimerism, and the other showed transient chimerism. Serial biopsies and post-mortem specimens from the tolerant monkeys revealed no signs of chronic rejection. The tolerant recipients also exhibited T cell unresponsiveness and a lack of alloantibody. This is the first report of durable mixed chimerism and successful tolerance induction of MHC-mismatched lungs in primates.

Keywords: Animal models; basic (laboratory) research/science; lung transplantation/pulmonology; nonhuman primate; tolerance; translational research/science.

Figure 1. Delayed tolerance induction protocol

The sequence and timing of the protocol interventions are depicted relative to the timing of LTx and BMT. The lung allografts were maintained with conventional triple-drug immunosuppression for 4 months, followed by BMT using a nonmyeloablative conditioning regimen. After BMT, the recipients were treated with short courses of anti-CD154 mAb and anti-IL-6 receptor mAb, a single dose of anti-CD8 mAb, along with a 28-day course of CsA. After this, no further immunosuppression was given.

Figure 2. MHC haplotypes of donor–recipient pairs

The MHC class I (A and B loci) and II (DP, DO, and DR loci) genes expressed by each of the recipient monkey (M2411, M4012, M912, and M4711) and its respective donor are shown and color-coded to highlight allelic differences.

Figure 3. Evaluation of donor hematopoietic chimerism in transplanted monkeys

The frequencies of different donor-derived leukocyte subsets, including lymphocytes, monocytes and granulocytes were assessed in each lung-transplanted monkey, at different time points after bone marrow transplant (BMT). Fresh leukocytes in the host peripheral blood were stained with mAb against HLA class I Bw6 expressed on donor cells, but not on recipient cells. The frequencies of Bw6 positive cells were measured by FACS. Dead cells were excluded by propidium iodide staining.M2411 andM4012 exhibited high levels (>20%) of durable lymphoid chimerism, as well as long-lasting myeloid chimerism. M912 showed multilineage chimerism, although it was transient (75 days). No donor chimerism was developed in M4711.

Figure 4. Pathology and chest radiograph of lung recipients

Shown are photomicrographs (H & E staining) of lung biopsies performed at the time of euthanasia and chest radiographs obtained at indicated time points from each monkey. The chest radiographs of M2411, M912, M4012 displayed well-aerated lung allografts in the left thoracic space. M4711 showed diffuse infiltration of the transplanted lung on Day 294 post-lung Tx (Day 176 post-BMT). No signs of rejection were seen in the lung graft biopsy of M2411, M912, and M4012. The lung allograft in M4711 exhibited findings of severe acute cellular rejection, vasculitis, and OB on Day 306 post-lung Tx (Day 188 post-BMT). BMT, bone marrow transplantation; H & E, hematoxylin and eosin; OB, obliterative bronchiolitis; Tx, transplant.

Figure 5. Nature of donor lymphocytes persisting in stable chimeras

The phenotype and frequency of different donor lymphocyte subsets were evaluated by FACS in the peripheral blood of the lung transplant recipients exhibiting durable chimerism after donor bone marrow cell infusion (M4012 and M 2411). The results are expressed as percentages of T cells, B cells, CD8⁺ B cells and recent thymic emigrants (CD31⁺ CD45RA⁺), among donor lymphocytes.

Figure 6. Detection of anti-donor antibodies in transplanted monkeys

Serum samples from each transplanted monkey were collected serially throughout the experiment. The presence of antidonor antibodies in the sera (1/20 dilution) was assessed by FACS as described in the Materials and Methods section and is expressed as mean channel fluorescence (MFI). Three monkeys (M2411, M4012, M912; lungs accepted) had no significant levels of allospecific antibodies throughout the experimental periods, whereas one animal (M4711; lung rejected) developed anti-donor antibody after lung transplantation and subsequent BMT. BMT, bone marrow transplant.

Figure 7. Donor-specific T cell pro-inflammatory immunity

ELISPOT data is presented for M4012, M2411, and M912 showing the frequencies of memory T cells (among 10⁶ peripheral blood T cells) producing IFN-gamma or IL-17 after in vitro culture with donor or third-party irradiated antigen presenting cells (PBMCs) (responder/stimulator ratio was 1:1) for 48 h (mixed lymphocyte reaction). The numbers of spots recorded with autologous APCs or media were <8 spots/million T cells.

Figure 8. Acceptance of donor skin

Full-thickness skins from a lung donor and an allogeneic third-party were grafted on Day 679 post-BMT in M2411. Photograph (left panel) and photomicrograph (right panel; H & E) were obtained 134 days after grafting. Donor skin was elastic and displayed intact appearance all the time through the observational course while the third-party skin rejected in 20 days. Histology of the donor skin showed intact epidermis in a well-preserved skin architecture. Scale bar, 100µm. BMT, bone marrow transplantation; H & E, hematoxylin and eosin.