Investigation of immunological approaches to enhance engraftment in a 1 Gy TBI canine hematopoietic stem cell transplantation model

Abstract

Objective: Stable mixed hematopoietic chimerism can be established in a canine stem cell transplantation model using a conditioning consisting of total body irradiation (TBI; 2 Gy) and postgrafting immunosuppression with mycophenolate mofetil (MMF) and cyclosporin (CSA). Reduction of TBI had resulted previously in graft rejection in this model. We investigated whether postgrafting stimulation of donor T cells against recipient's hematopoietic antigens or graft augmentation with donor monocyte-derived dendritic cells (MoDC) promote engraftment following 1 Gy TBI.

Materials and methods: All dogs received dog leukocyte-antigen-identical bone marrow transplantation. Dogs were conditioned with either 2 Gy TBI (group 1) or 1 Gy TBI, followed by repetitive recipient hematopoietic cell lysate vaccinations (group 2) or graft augmentation with MoDC (group 3). Immunosuppression consisted of CSA and MMF.

Results: In group 1, four animals remained stable chimeras for >110 weeks, and three rejected their grafts (week 10, week 14, week 16). All dogs in groups 2 and 3 rejected their graft (median: week 10 and 11, respectively). Peak chimerism and engraftment duration was shorter in the 1-Gy groups (p < 0.05) compared to group 1.

Conclusion: Neither postgrafting vaccination nor graft augmentation with MoDC were effective in supporting durable engraftment. Additional modifications are necessary to improve potential strategies aimed at establishment of early tissue specific graft-vs-host reactions.

Figure 1

Study design. Three HSCT groups were investigated. Group 1 (control) received unmodified DLA-identical littermate bone marrow (BM) for HSCT following 2 Gy TBI conditioning. Group 2 received unmodified DLA-identical littermate BM for HSCT following 1 Gy TBI conditioning and received recipient haematopoietic cell lysate vaccinations (1 × 10⁵ lysed cells/kg + 125 μg GM-CSF; 3x week [wk 1-5], 1x week [wk 6-9]) for in vivo graft stimulation thereafter. Group 3 received DLA-identical littermate BM enriched with donor monocyte derived dendritic cells (MoDC_Donor) for HSCT following 1 Gy TBI conditioning. All dogs received for immunosuppression CSA (days -1 to +35) and MMF (days 0 to +27).

Figure 2

Development of haematopoietic donor chimerism following HSCT. Dogs received 2 Gy conditioning (group 1; a, b; n = 7), 1 Gy conditioning in combination with vaccination (group 2; c, d; n = 7), and 1 Gy TBI plus enrichment of graft with MoDC (group 3; e, f; n = 6). Analyses were performed within the granulocyte (a, c, e) as well as within the PBMC (b, d, f) compartment.

Figure 3

Recipient T-cell proliferation response at different times after HSCT. T-cells were stimulated with irradiated (25 Gy) PBMC of the same dog collected and croypreserved before transplantation. Proliferation was measured using a [³H]-thymidine incorporation assay and has been illustrated as counts per minute. Data are representative of 5 independent experiments done in triplicates (⁺p < 0.05 compared to pretransplantation [pre Tx]).

Figure 4

Flow cytometric 4 quandrant analyses of the intracellular cytokine production by lymphocytes. Percentages of positive cells are given for each quadrant. The dog received a DLA-identical HSCT but lost its graft eventually on day +98. TNF-α and IFN-γ producing cells seem to increase during the period of graft rejection.