CpG-induced myeloid CD11b+Gr-1+ cells efficiently suppress T cell-mediated immunoreactivity and graft-versus-host disease in a murine model of allogeneic cell therapy.

Transplantation of mismatched allografts in irradiated recipients results in lethal graft- versus-host disease (GVHD). In our study, pretransplantation donor treatment with CpG, administered either alone or emulsified in incomplete Freund's adjuvant, efficiently prevented GVHD in sublethally irradiated recipients of haploidentical (H-2(b) into H-2(b/d)) and fully mismatched (H-2(b) into H-2(d)) allografts. CpG treatment of donor mice caused an accumulation of double-positive CD11bGr-1 cells in their blood and spleens, whereas treatment with CpG+IFA resulted in an even greater accumulation of these cells. Isolated CD11b(+) cells from the spleens of CpG+IFA-treated mice efficiently suppressed alloreactivity in vitro (> 92%), as determined by co-culturing these cells in mixed lymphocyte reactions. After CpG+IFA treatment, a T cell-depleted fraction enriched with CD11b(+)Gr-1(+) cells, acting as myeloid suppressor cells, was able to efficiently prevent GVHD induced by naïve T cells in the sublethally irradiated recipients: 20/21 mice remained GVHD-free survivors for more than 200 days. Splenocytes from CpG+IFA-treated mice displayed enhanced interleukin (IL)-6, IL-10, and interferon-gamma production, reduced T cell allogeneic and mitogenic responses, as well as failure of T cells to induce GVHD. In summary, CpG treatment led to impaired T cell function, enriched myeloid suppressor cells and regulatory cytokine production, which together appear to suppress alloreactivity and protect against the development of GVHD. We hypothesize that similar immunoregulatory effects could be applied experimentally in a clinical setting when inhibition of alloreactivity is required in recipients of stem cell allografts.

Mesenchymal stromal cells lose their immunosuppressive potential after allotransplantation.

OBJECTIVE: The stem cell fraction of mesenchymal stromal cells (MSCs) is capable of self-renewal and under inductive conditions differentiates into bone, cartilage, hematopoietic stroma, and other mesenchymal tissues. Therefore, MSCs represent a promising source for hard tissue repair therapies. MSCs are also immunosuppressive and prevent activation of allogeneic lymphocytes in vitro. Thus it has been suggested that they might be able to engraft in allogeneic recipients and downregulate recipients' immunity. In this study we examined whether MSCs retain their immunomodulating properties in vivo after allotransplantation. MATERIALS AND METHODS: MSCs were propagated from bone marrow (BM), placenta, or umbilical cord tissues. Using a murine parental-into-F1 model of graft-vs-host disease (GVHD) we tried to control GVHD by intravenous transplanting parental or recipient MSCs together with parental lymphocytes (day 0) and on days +7 and +14. MSCs' immunosuppressive potential in vivo was also examined by comparing their ability to construct ectopic bone after local transplantation with osteogenic inductor (demineralized bone matrix) under the kidney capsule of syngeneic and allogeneic recipients. RESULTS: Repeated IV MSC injections failed to reduce GVHD-related recipient mortality. Local implantation of MSCs propagated from BM, placenta or umbilical cord resulted in ectopic bone formation in syngeneic recipients and in transplant rejection by allogeneic mice. CONCLUSION: MSCs lose their immunosuppressive potential in mismatched setting.

Pretransplant treatment of donors with immunomodulators to control graft-versus-host disease (GVHD) in transplant recipients.

OBJECTIVE: Prevention of graft-versus-host disease (GVHD) by pretransplant donor treatment with known immunomodulators like complete Freund's adjuvant (CFA) and synthetic oligo-deoxynucleotides expressing CpG motifs (CpG). METHODS: Induction of GVHD by inoculation of C57BL/6 (C57) splenocytes into sublethally irradiated (BALB/c x C57BL/6) F1 (F1) mice. Splenocytes were derived from either naive C57 mice or from C57 mice that were treated previously with the immunomodulators. RESULTS: Inoculation of CFA or CpG into C57 mice led to an increase in the total number of spleen cells and resulted in activation of immunoregulatory cells that significantly suppressed mixed allogeneic lymphocyte reaction in vitro. CFA-treated C57 splenocytes led to GVHD-related death in only 14 out of 61 F1 recipients while the remaining 47 mice survived without disease for more than 200 days. Pretransplant treatment of donor C57 mice with GpG emulsified in incomplete Freund's adjuvant resulted in 19/20 GVHD-free survivors of sublethally irradiated F1 mice for more than 200 days. In contrast, naive C57 splenocytes injected into sublethally irradiated F1 recipients induced severe GVHD, which resulted in the death of 77/78 recipient mice (median of survival was 16 days). CONCLUSION: Our results suggest that adjuvant-induced immunoregulation of donor cells prior to allogeneic cell therapy may augur a new strategy that will bring the benefits of safe cellular immunotherapy aiming to eradicate malignant and nonmalignant pathological cells while avoiding or minimizing the risk of GVHD.

Effect of KRN7000 on induced graft-vs-host disease.

OBJECTIVE: Graft-vs-host disease (GVHD) is a serious complication of allogeneic stem cell transplantation (SCT) and donor lymphocyte infusion (DLI), for which no effective therapy exists. In our study, KRN7000, a synthetic analog of alpha-galactosylceramide, known for its ability to activate natural killer T cells, was tested for its ability to prevent onset of GVHD in a murine model of haploidentical major histocompatible (MHC) mismatched hematopoietic cells. METHODS: Irradiated (BALB/cXC57BL/6)F1 mice were inoculated with parental C57BL/6 splenocytes with or without SCT. KRN7000 was given intraperitoneally as single or multiple doses at 100 microg/kg/dose and mice were followed up for GVHD clinical symptoms and for survival. The effect of KRN7000 treatment was also tested in vitro for the induction of suppression of alloreactivity in mixed lymphocyte reaction (MLR). RESULTS: KRN7000 prevented development of GVHD symptoms in almost all mice and 52/53 mice maintained a healthy profile for more than 235 days. Most vehicle-treated mice or untreated controls died of GVHD within a median of 3 weeks. KRN7000 treatment did not prevent engraftment of donor cells following sublethal total-body irradiation (TBI) and allowed durable persistence of donor cells following lethal TBI and SCT. Splenocytes derived from KRN7000-treated mice suppressed efficiently mixed lymphocyte reaction (MLR) in vitro. CONCLUSION: GVHD induced by alloreactive lymphocytes can be prevented by KRN7000. GVHD prevention may be accomplished by regulation of T cell function and might thus provide a new modality for safer SCT and DLI.