Regulatory T cells (T reg ) are CD4+ T lymphocytes with constitutive expression of CD25 and FOXP3, as well as the ability to modulate cellular immune responses. In this study, the phenotypic characteristics, function and feasibility of enrichment and expansion of canine T reg were examined. Canine peripheral blood mononuclear cells were isolated and enriched by labelling of CD25, and expansion of T reg was achieved by adding interleukin (IL)-2 for 1 week. Phenotypic and functional analyses of T reg were performed prior to and after expansion. Canine T reg could be phenotypically characterized by CD4, CD25, and FOXP3 expression. Isolation and enrichment of canine T reg is possible, but high purities are difficult to achieve without significant cell loss. Expansion of canine T reg was possible by adding IL-2 without other growth factors. Higher initial cell numbers seeded allow more substantial T reg expansion in vitro. Canine T reg have the potential to suppress proliferation of effector T cells (T eff ). By adding expanded T reg , a higher capability for suppressing T eff could be shown in comparison with freshly isolated T reg . Enrichment and expansion of canine T reg is feasible, and canine T reg had similar characteristics to T reg from other species.

The mammalian target of rapamycin inhibitor everolimus (RAD001) is a successfully used immunosuppressant in solid-organ transplantation. Several studies have already used RAD001 in combination with calcineurin inhibitors after hematopoietic stem cell transplantation (HSCT). We investigated calcineurin inhibitor-free pre- and post-transplantation immunosuppression of RAD001 combined with mycophenolate mofetil (MMF) in a nonmyeloablative HSCT setting. After nonmyeloablative conditioning with 2 Gy total body irradiation, 8 dogs received HSCT from dog leukocyte antigen-identical siblings. Immunosuppressives were given at doses of 1.5 mg RAD001 twice daily from day -1 to +49, then tapered until day +56, and 20 mg/kg MMF from day 0 to +28, then tapered until day +42. An historical cyclosporin A (CsA)/MMF regimen was used in the control group. All dogs engrafted. Median platelet nadir amounted in all dogs to 0 × 10(9)/L (median, day +10; duration <50 × 10(9)/L, 22 days) and median leukocyte nadir was 1.0 × 10(9)/L (range, .1 to 2.5 × 10(9)/L; median, day +13). Eventually, 5 of 8 (63%) animals rejected their grafts. Two dogs died of infections on day +19 and +25. Pharmacokinetics of RAD001 and MMF showed median trough levels of 19.1 (range, 10.5 to 43.2) μg/L and .3 (.1 to 1.3) mg/L, respectively. The median area under the curve was 325 (range, 178 to 593) μg/L × hour for RAD001 and 29.6 (range, 7.9 to 40.5) ng/L × hour for MMF. All dogs developed clinically mucosal viral infections during the clinical course. Compared with the control group, the level of toxicities for RAD001/MMF increased in all qualities. Combined immunosuppression of RAD001 and MMF after nonmyeloablative HSCT is associated with significant toxicities, including a prolonged platelet recovery time as well as increased infections compared to the CsA/MMF regimen.

BackgroundLangerhans cells (LC) are bone marrow-derived cells in the skin. The LC donor/recipient chimerism is assumed to influence the incidence and severity of graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HSCT). In nonmyeloablative (NM) HSCT the appearance of acute GVHD is delayed when compared with myeloablative conditioning. Therefore, we examined the development of LC chimerism in a NM canine HSCT model.Methods2 Gy conditioned dogs received bone marrow from dog leukocyte antigen identical littermates. Skin biopsies were obtained pre- and post-transplant. LC isolation was performed by immunomagnetic separation and chimerism analysis by PCR analyzing variable-number-of-tandem-repeat markers with subsequent capillary electrophoresis.ResultsAll dogs engrafted. Compared to peripheral blood chimerism the development of LC chimerism was delayed (earliest at day +56). None of the dogs achieved complete donor LC chimerism, although two dogs manifested a 100 % donor chimerism in peripheral blood at days +91 and +77. Of interest, one dog remained LC chimeric despite loss of donor chimerism in the peripheral blood cells.ConclusionOur study indicates that LC donor chimerism correlates with chimerism development in the peripheral blood but occurs delayed following NM-HSCT.

3711 Introduction: Successful engraftment following transplantation of hematopoietic stem cells (HSCT) depends mainly on pre- and posttransplant immunosuppression, graft type and composition as well as on the HSC numbers infused. Whereas some of the aforementioned parameters can be influenced in the clinical setting, the latter one is more difficult to address. HSCTs of grafts with limited HSC numbers are accompanied by increased graft failure rates, longer cytopenias and increased morbidity. Current concepts to overcome low HSC numbers include the combination of two unrelated grafts, expansion techniques, modification of the graft composition or the site of graft infusion. In preliminary rodent studies intra-bone marrow (IBM) compared to intravenous (IV) HSCT led to faster engraftment which might be explained by closer location of the HSC to the stem cell niches. Aims: To investigate the feasibility and efficiency of IBM-HSCT following a non-myeloablative conditioning regimen in a dog-leukocyte antigen (DLA) identical canine HSCT model. Method: DLA-identical siblings were used as donor/recipient pairs for HSCTs. Recipients received a single dose of 2 Gy total body irradiation before HSCT (day 0). Pre- and postgrafting immunosuppression consisted of CSA (d-1 to d+35) and MMF (d0 to d+27). Two IBM-HSCT cohorts were investigated and data compared to IV controls (CON). BM-grafts of the respective donors were infused unmodified IV (CON, n=9) or IBM after HSC enrichment using a buffy coat followed by ficoll density centrifugation (IBM-I, n=6; 5ml total volume) or IBM after HSC enrichment using buffy coat centrifugation only (IBM-II, n=6; 25 ml total volume). In the CON group the graft was infused in the cubital vein. In the IBM-groups the grafts were infused through a BM aspiration needle into the BM of the left humerus and femur over a period of 5 minutes. In 4 IBM animals graft migration analyses were performed using technecium99 marking. Chimerism and BM cellularity were determined at injection and opposite sides. Analyses of chimerism were performed via polymorphic nucleotide repeat analyses weekly. BM cellularity was determined biweekly. Complete blood count was performed daily. Result: Infusion of grafts directly into the BM was feasible: both volumes (5ml, 25ml) could be infused without any leakage at the injection sites. Tc99-marked BM cells stayed predominately at the injection site for the first 24 hours. All animals engrafted. Mean TNC numbers infused were 2.6 ×108/kg (range: 1.6–11.4; CON), 1.6 ×108/kg (range: 1–2.4; IBM-I), 3.7 ×108/kg (range: 2.1–5.8; IBM-II) (IBM-I vs CON: p=0.08, IBM-II vs CON: p=0.9, IBM-I vs II: p<0.02,). Mean CD34+ numbers infused were 0.6 ×106/kg (range: 0.3–2.2; CON), 2.5 ×106/kg (range: 0.3–6.4; IBM-I), 4.3 × 106/kg (range: 1.3–6.5; IBM-II) (IBM-I vs CON: p=0.06, IBM-II vs CON: p=0.01, IBM-I vs II: p=0.4). PBMC chimerisms at d+14, d+28 and d+56 were 25% (range: 8–46), 36% (range: 5–63), 28% (range: 4–54) (CON), 12% (range: 6–18), 23% (range: 14–39), 18% (range: 6–64) (IBM-I) and 40% (range: 23–60), 61% (range: 31–84), 45% (range: 25–70) (IBM-II) (IBM-I vs CON: p=0.03, p=0.05, p=0.1, IBM-II vs CON: p=n.s. (all), IBM-I vs II: p=0.06, p=0.04, p=0.1). Recovery of hematopoiesis occurred significantly slower in both IBM-BMT groups compared to CON (p<0.002): mean leukopenia (<1.0 gpt/l) durations were 4 days (range: 0–15; IBM-I), 0.5 days (range: 0–2, IBM-II) versus 0 days (CON); mean thrombocytopenia durations (<50Gpt/l) were 19 days (range: 2–49, IBM-I), 10 days (range: 8–16, IBM-II) versus 4 days (range: 0–6, CON). However, if only grafts with <2.0 ×106 CD34+/kg were analysed differences concerning leukocytes recovery times diminished between IBM and the IV groups, whereas they persisted in regards to thrombocytopenia. Analyses of cellularity and chimerism within a HSCT recipient (injection vs opposite site) revealed higher initial cellularities and significantly higher BM donor chimerisms up to day +56, both in favour of the injection site (p<0.03). Conclusion: Infusion of HSC grafts up to volumes of 25ml directly into the BM is feasible and allows successful donor engraftment following non-myeloablative conditioning. Duration of cytopenias following IBM-HSCT is still significant, perhaps due to the loss of precursor cells during graft preparation. Further studies are warranted to determine optimal graft preparation and IBM application techniques. Disclosures: No relevant conflicts of interest to declare.

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