Evidence for the presence of multilineage chimerism and progenitors of donor dendritic cells in the peripheral blood of bone marrow-augmented organ transplant recipients

Abstract

We have postulated that the donor leukocyte microchimerism plays a seminal role in the acceptance of allografts by inducing and perpetuating variable degree of donor-specific nonreactivity in long-surviving organ recipients. Limited information is available, however, concerning the phenotype and function of these chimeric cells in humans. The unequivocal presence of donor dendritic cells (DCs), a prominent lineage in the microchimerism observed in rodents and clinical organ recipients, was difficult to demonstrate in bone marrow (BM)-augmented organ transplant recipients. This enigma was resolved by the recent description of a method for propagating circulating human DCs from their progenitors by culture in a medium enriched with granulocyte-macrophage colony-stimulating factor and interleukin 4, a condition known to inhibit outgrowth of monocytes, thus providing a selective growth advantage to committed progenitors of the myeloid lineage. Cells from BM-augmented organ recipients and normal control subjects harvested from 12- to 14-day cultures exhibited dendritic morphology and potent allostimulatory capacity. Using appropriate primers, the presence of donor DNA was verified by polymerase chain reaction within the lineage(null)/class II(bright) sorted DC. Phenotypic analysis of cultured DCs from BM-augmented patients, unlike that of controls, exhibited a marked down-regulation of B7-1 (CD80) while retaining normal levels of expression of B7-2 (CD86) cell surface molecules. The presence of donor DNA was also confirmed by polymerase chain reaction in individually sorted lineage+ (T, B, and NK) cells and macrophages, suggesting that the chimerism in BM-augmented patients is multilineage. The presence of progenitors of donor DCs in the peripheral blood of BM-augmented patients further substantiates the already convincing evidence of stem cell engraftment.

Figure 1

Sorting for HLA-DR^bright/lineage^null cells using cultured metrizamide-fractionated putative DC generated using PBMCs obtained from a male→female kidney + BM recipient 345 days after transplantation. The sorting gates (C) were set for HLA-DR^bright but lineage^null population, and subsequent to low-speed sorting, the cells were reanalyzed for purity (D). Only cells exhibiting ~100% purity were used for EM studies and for phenotyping. The presence of donor DNA was confirmed by PCR analysis (D) using primers specific for SRY. For flow cytometric analysis. unstained and cells stained with fluorochrome-conjugated irrelevant isotype-matched mAbs were used as controls. PBMCs obtained from normal healthy males and females served as positive and negative controls. respectively, for PCR analysis. PGE, percentage gated events: SSC, side scatter channel: FSC, forward scatter channel.

Figure 2

Determination of expression of costimulatory molecules on cultured/sorted DC generated from the PBMCs obtained from a liver + BM recipient 467 days after transplantation. Spontaneously released cells cultured in rhGM-CSF- and rhIL-4-enriched medium were purified further by flotation on metrizamide columns. The harvested low-density cells were stained with a cocktail of PE-conjugated lineage-specific (CD3/CD14/CD22/CD56) and FITC-conjugated anti-HLA-DR mAb. Lineage^null/HLA class II^bright cells were sorted (C) using an Epics Elite flow cytometer (Coulter) and reanalyzed (D) to determine their purity. Sorted cells were then subjected to staining with PE-conjugated mAbs against CD80 (B7.1; E), CD86 (B7.2; F), and CDla (G). Unstained cells and cells stained with fluorochrome-conjugated irrelevant isotype-matched mAbs were used as controls. SSC, side scatter channel; FSC, forward scatter channel; PGE, percentage gated events.

Figure 3

Allostimulatory capacity of γ-irradiated cultured DC was determined using a primary MLR assay. For the method used to generate DC, refer to legend of Figure 1. PBMCs were obtained from two liver + BM recipients 781 days (A) and 467 days (B) after transplantation. A constant number (10⁵ cells/well) of autologous (□) and allogeneic (○) PBMCs and allogeneic purified naive T cells (■) were used as responders. In addition to cultured DC (1), splenocytes obtained from the cadaveric donor (2) and the PBMCs of recipients (3) were also used as stimulators. The results are expressed as the mean [³H]thynudine incorporation in triplicate cultures.

Figure 4

Analysis of the ultrastructure of the cultured/sorted (lineage^null/HLA class II^bright) DC obtained from the PBMCs of a BM-augmented liver allograft recipient 390 days after transplantation. All cells examined exhibited typical “organelle-deficient” cytoplasmic veils (arrow) with sparse secretory granules. The mitochondrial content varied between cells; some had low (A) but others very high (B) numbers. As is evident, the cytoplasmic development was also highly variable in the cells examined. Ultrastructure was determined using a JEOL 1210 (Peabody, MA) transmission electron microscope (magnification, × 4500).

Figure 5

Establishment of the multilineage nature of chimerism in the peripheral blood of a BM-augmented lung allograft recipient 475 days after transplantation. The orthogonal and side scatter profiles were used to establish analysis gates for lymphocyte (II) and monocyte (III) subpopulations. Within this population, the presence of cells staining with either donor (D)- or recipient (C)-specific mAbs was determined. Unstained and cells stained with fluorochrome-conjugated, irrelevant, isotype-matched mAbs (B) were used as controls. SSC, side scatter channel; FSC, forward scatter channel; PGE, percentage gated events.

Figure 6

The detection of donor DNA by PCR in lineage-positive cells sorted from PBMCs obtained from a male→female liver + BM organ recipient 402 days after transplantation. Scatter profile was used to establish gates specific for the lymphoid (A) and myeloid (E) populations. Subsequently, the purity of sorted T (B), B (C), and NK (D) cells and monocyte/macrophages (F) was determined by reanalysis. Within the sorted population, the presence of donor DNA was confirmed by PCR analysis using primers specific for the SRY. For flow cytometry, unstained and cells stained with fluorochrome-conjugated, irrelevant, isotype-matched mAbs were used as controls. PBMCs obtained from normal males and females served as positive and negative controls, respectively, for PCR analysis. SSC, side scatter channel; FSC, forward scatter channel; PGE, percentage gated events.