Influence of Hematopoietic Cell Lysate Vaccinations and Dendritic Cell Enriched Marrow Grafts on Engraftment after Nonmyeloablative Hematopoietic Stem Cell Transplantation.
Authors: Lange Sandra , Lange Bettina , Vogel Heike , Weirich Volker , Hilgendorf Inken , Wolff Daniel , Hofmeister Nicole , Freund Mathias , Junghanss Christian 
Published: April 2020

Background: Stable mixed hematopoietic chimerism can be established in a canine marrow transplantation model using nonmyeloablative conditioning consisting of total body irradiation (TBI, 2Gy) and postgrafting immunosuppression with mycophenolate mofetil (MMF) and cyclosporine A (CSA). Reduction of TBI to 1Gy resulted in rejection of marrow grafts in this model even if CD3 depleted donor PBMC were added to the graft. Here, we investigated whether addition of immature dendritic cells or postgrafting in vivo stimulation of donor T cells against recipient derived hematopoietic antigens are capable of enhancing targeted GVH reactions and allow engraftment.

Methods: Dog leukocyte antigen-identical littermates received 2Gy TBI (group 1, control, n=7) and 1Gy TBI (group 2, n=7; group 3, n=6) before allogeneic stem cell transplantation (SCT). All recipients were given postgrafting immunosuppression consisting of 15mg/kg CSA BID PO (days -1 to +35) and 10mg/kg MMF BID PO (days 0 to +27). In addition, group 2 was vaccinated ID and SQ with recipient blood cell lysates (3x/week w1-w5; 1x/week w6-w9) together with locally applied granulocyte macrophage-colony stimulating factor (125μg/vaccination). The vaccine consisted of PBMC and granulocytes at a ratio of 1:1 (105 cells/kg before lysis). Dogs of group 3 received ex-vivo generated immature monocyte-derived dendritic cells (MoDC) of donor origin in addition to marrow at day 0. MoDC doses ranged from 6.4–10.9x105 cells/kg (median 7.9x105). Littermate donor marrow was infused IV at a median of 3.4x108 (1.6–11.4x108) nucleated cells/kg.

Results: The median platelet and leukocyte nadirs in group 1 were 30x109/l (median at day 11) and 2.4x109/l (median at day 8), respectively. In groups 2 and 3 the median platelet and leukocyte nadirs were 110x109/l (median at day 12) and 4.8x109/l (median at day 7). All dogs in group 1 engrafted and showed mixed hematopoietic chimerism among PBMC and granulocytes (after 4 weeks (w4): median 32% (5–44%) and 68% (10–74%), respectively). 4/7 animals remained stable mixed chimeras &gt;25 weeks, 3/7 rejected their graft (w10, w14, w16). All dogs in group 2 showed initial engraftment and chimerism of donor PBMC and granulocytes (w4: median 12% (10–19%) and 19% (11–24%), respectively), but rejected their grafts a median of 10.3 weeks (w8-w11) after SCT. 4/6 dogs in group 3 had a median w4-chimerism in PBMC and granulocytes of 14% (11–15%) and 20% (16–27%), respectively and 2/6 dogs are too early after SCT to be evaluated. Currently 3/4 evaluable dogs in group 3 remain mixed chimeric. In comparison to group 1 engraftment kinetics of groups 2 and 3 indicate a delayed engraftment combined with significantly lower donor PBMC and granulocytes chimerisms and significantly reduced engraftment duration (group 2). No differences in w4-chimerism patterns between groups 2 and 3 could be observed.

Conclusion: Our data show that vaccinations with recipient hematopoietic cell lysates failed to expedite long term bone marrow engraftment following a 1Gy TBI conditioning. Addition of donor-MoDC to the graft might favour sustained engraftment but a longer follow up is needed. Furthermore, equal dosed MMF applied orally seems to be inferior to SQ applications in postgrafting immunosuppression since compared to historical controls less mixed chimeras could be generated following 2Gy TBI.