Marina Lesnikova

α-1-Antitrypsin (AAT)-modified donor cells suppress GVHD but enhance the GVL effect: a role for mitochondrial bioenergetics.

Hematopoietic cell transplantation is curative in many patients. However, graft-versus-host disease (GVHD), triggered by alloreactive donor cells, has remained a major complication. Here, we show an inverse correlation between plasma α-1-antitrypsin (AAT) levels in human donors and the development of acute GVHD in the recipients (n = 111; P = .0006). In murine models, treatment of transplant donors with human AAT resulted in an increase in interleukin-10 messenger RNA and CD8(+)CD11c(+)CD205(+) major histocompatibility complex class II(+) dendritic cells (DCs), and the prevention or attenuation of acute GVHD in the recipients. Ablation of DCs (in AAT-treated CD11c-DTR donors) decreased CD4(+)CD25(+)FoxP3(+) regulatory T cells to one-third and abrogated the anti-GVHD effect. The graft-versus-leukemia (GVL) effect of donor cells (against A20 tumor cells) was maintained or even enhanced with AAT treatment of the donor, mediated by an expanded population of NK1.1(+), CD49B(+), CD122(+), CD335(+) NKG2D-expressing natural killer (NK) cells. Blockade of NKG2D significantly suppressed the GVL effect. Metabolic analysis showed a high glycolysis-high oxidative phosphorylation profile for NK1.1(+) cells, CD4(+)CD25(+)FoxP3(+) T cells, and CD11c(+) DCs but not for effector T cells, suggesting a cell type-specific effect of AAT. Thus, via altered metabolism, AAT exerts effective GVHD protection while enhancing GVL effects.

A novel monoclonal antibody specific for canine CD25 (P4A10): selection and evaluation of canine Tregs.

A monoclonal antibody (mAb), P4A10, was made to the canine interleukin-2 receptor alpha chain (IL-2Ralpha; p55; Tac antigen; CD25) to facilitate studies of canine regulatory T-cells (Treg). By non-reduced Western blot, P4A10 bound to a 55kDa protein, the size of human IL-2Ralpha. In flow cytometry assays, it reacted with a minor population of circulating dog CD3(+)CD4(+) T-cells and the majority (>60%) of in vitro PMA-Ionomycin (PMA-IO)-activated canine CD3(+) T-cells. P4A10 recognized a hematopoietic cell population enriched for FoxP3+ cells as measured by flow cytometry. The P4A10-selected fractions of T-cells had significantly increased copy numbers of CD25, FoxP3, IL-10, and TGFbeta as detected by RT-PCR (reverse transcriptase-PCR) compared to the negative fractions. The P4A10-selected cells inhibited (3)H (tritiated) thymidine incorporation in a mixed leukocyte reaction (MLR) containing responders of the same origin. P4A10-selected T-cells from fresh peripheral blood mononuclear cells had less FoxP3 (p=0.07) by qRT-PCR (quantitative RT-PCR) and were less suppressive (p=0.01) than in vitro alloantigen-activated Treg. The mAb P4A10 is specific for canine CD25 and can be used to facilitate studies of CD25+FoxP3+ Treg in this clinically relevant large animal model.

Lentiviral vector conferring resistance to mycophenolate mofetil and sensitivity to ganciclovir for in vivo T-cell selection

Several clinical studies of gene-modified T cells have shown limited in vivo function of the cells, immunogenicity of the transgene, and lack of a selective advantage for gene-modified T cells. To address these problems, we developed a lentiviral vector (LV) that provides a selectable, proliferative advantage and potentially decreases immunogenicity for transduced T cells. The bicistronic vector expressed two genes linked with an internal ribosomal entry site. One gene is a variant of the inosine monophosphate dehydrogenase 2, inosine monophosphate dehydrogenase (IMPDHIY), conferring resistance to the immunosuppressive drug mycophenolate mofetil (MMF). The other is a suicide gene, herpes simplex virus thymidine kinase (HSV-TK), rendering proliferating cells sensitive to ablation with ganciclovir, fused to the selectable transmembrane marker ΔCD34 (ΔCD34/TK). Cells transduced with LV-ΔCD34/TK.IMPDHIY were efficiently enriched by immunomagnetic selection for CD34, proliferated in 0.5–5 μM MMF, and were killed by 0.5–25 μg ml−1 ganciclovir. We demonstrate efficient selection and killing of gene-modified cells and suggest LV-ΔCD34/TK.IMPDHIY-transduced T cells could be used to facilitate allogeneic hematopoietic cell engraftment. The expression of IMPDHIY would allow in vivo selection with MMF, and ΔCD34/TK expression would allow rapid and safe elimination of transduced T cells if graft-versus-host disease developed.

Flt3 ligand promotes engraftment of allogeneic hematopoietic stem cells without significant graft-versus-host disease

Background. Graft-versus-host (GVH) reactions contribute to stable engraftment of allogeneic hematopoietic stem cell transplants. It was hypothesized that the in vivo expansion of recipient dendritic cells (DC) with the administration of ligand for Flt3 (FL) could promote allogeneic engraftment after reduced-intensity conditioning by enhancing the GVH effect. Methods. FL was first administered to three nonirradiated healthy dogs for 13 days at a dosage of 100 &mgr;g/kg/day. Next, nine dogs received 4.5 Gy total-body irradiation (TBI) and unmodified marrow grafts from dog leukocyte antigen (DLA)-identical littermates without posttransplant immunosuppression. FL was administered to the recipients at a dosage of 100 &mgr;g/kg/day from day −7 until day +5. Results. In normal dogs, FL produced significant increases in monocytes (CD14+) and neutrophils in the peripheral blood, a marked increase in CD1c+ cells with DC-type morphology in lymph nodes, and increased alloreactivity of third-party responders to peripheral blood mononuclear cells in mixed lymphocyte reactions (P <0.001). Sustained engraftment was observed in eight of nine (89%) FL-treated dogs compared with 14 of 37 (38%) controls (P =0.02, logistic regression). All engrafted FL-treated dogs became stable complete (n=2) or mixed (n=6) hematopoietic chimeras without significant graft-versus-host disease (GVHD). Recipient chimeric dogs (n=4) were tolerant to skin transplants from their marrow donors but rejected skin grafts from unrelated dogs within 7 to 9 days (median, 8 days). Conclusions. In this study, the authors showed that FL administered to recipients promotes stable engraftment of allogeneic marrow from DLA-identical littermates after 4.5 Gy TBI without significant GVHD.

Prevention of Fas-mediated hepatic failure by transferrin

Recent studies in lymphohemopoietic cells show that transferrin (Tf), a pivotal component of iron transport and metabolism, also exerts cytoprotective functions. We show here in a murine model that Tf interferes with Fas-mediated hepatocyte death and liver failure. The mechanism involves the downregulation of apoptosis via BID, cytochrome c, caspase-3 and caspase-9, and upregulation of antiapoptotic signals via Bcl-xL. The results obtained with iron-saturated Tf, Apo-Tf and the iron-chelator salicylaldehyde isonicotinoyl hydrazone indicate that the observed antiapoptotic effect of Tf was not mediated by iron alone. In conclusion, the data suggest that Tf has broader functions than previously recognized and may serve as a cytoprotective agent.

Antagonistic and agonistic anti-canine CD28 monoclonal antibodies: tools for allogeneic transplantation

Background. It has been presumed that antibody-mediated selective costimulatory molecule blockade of CD28 is superior to cytotoxic T lymphocyte antigen 4 (CTLA4)-Ig. This is based on the premise that specifically blocking CD28 allows inhibitory signals through CTLA-4 to proceed, which furthermore suppresses T-cell function. Methods. The extracelluar domain of canine (ca)CD28 was cloned from dog peripheral blood mononuclear cells. Mice were immunized with a caCD28/murine IgG2a fusion protein. Hybridomas were produced by fusing splenocytes with mouse NSO cells and screened for caCD28 binding by ELISA. Agonistic and antagonistic activities of the monoclonal antibodies (mAb) were tested in mixed leukocyte reactions. Canine regulatory T cells were expanded using plate-bound anti-CD3 and an anti-CD28 agonist mAb. Results. One agonistic and seven antagonistic mAbs to canine (ca)CD28 were cloned. Binding studies indicated that an agonistic (5B8) and an antagonistic (1C6) mAb bound equally well to a caCD28/caIgG1 fusion protein and to CD28 expressed on CD4+ and CD8+ peripheral blood T cells. Antagonistic antibody blocked mixed lymphocyte reactions (MLR) in a dose-dependent manner similar to CTLA4-Ig, whereas the agonistic antibody to caCD28 enhanced MLR. The 5B8 was superior to 1C6 when either was combined with anti-caCD3 to stimulate lymphocyte proliferation. Furthermore, the agonistic mAb, 5B8, together with anti-CD3 mAb induced 100-fold proliferation of canine regulatory T cells. Relative to untreated control cells, anti-caCD28 (1C6) and CTLA4-Ig equivalently inhibited cytotoxic T lymphocyte-mediated killing of alloreactive target cells. Conclusion. These studies demonstrated that mouse anti-caCD28 mAbs can be generated with agonistic or antagonistic function.

Immunomodulatory effects of mixed hematopoietic chimerism: immune tolerance in canine model of lung transplantation.

Long‐term survival after lung transplantation is limited by acute and chronic graft rejection. Induction of immune tolerance by first establishing mixed hematopoietic chimerism (MC) is a promising strategy to improve outcomes. In a preclinical canine model, stable MC was established in recipients after reduced‐intensity conditioning and hematopoietic cell transplantation from a DLA‐identical donor. Delayed lung transplantation was performed from the stem cell donor without pharmacological immunosuppression. Lung graft survival without loss of function was prolonged in chimeric (n = 5) vs. nonchimeric (n = 7) recipients (p ≤ 0.05, Fishers test). There were histological changes consistent with low‐grade rejection in 3/5 of the lung grafts in chimeric recipients at ≥1 year. Chimeric recipients after lung transplantation had a normal immune response to a T‐dependent antigen. Compared to normal dogs, there were significant increases of CD4+INFγ+, CD4+IL‐4+ and CD8+ INFγ+ T‐cell subsets in the blood (p < 0.0001 for each of the three T‐cell subsets). Markers for regulatory T‐cell subsets including foxP3, IL10 and TGFβ were also increased in CD3+ T cells from the blood and peripheral tissues of chimeric recipients after lung transplantation. Establishing MC is immunomodulatory and observed changes were consistent with activation of both the effector and regulatory immune response.

Transient proteasome inhibition as a strategy to enhance lentiviral transduction of hematopoietic CD34(+) cells and T lymphocytes: implications for the use of low viral doses and large-size vectors.

The proteasome system restricts lentiviral transduction of stem cells. We exploited proteasome inhibition as a strategy to enhance transduction of both hematopoietic stem cells (HSC) and T lymphocytes with low dose or large-size lentiviral vectors (LV). HSC showed higher transduction efficiency if transiently exposed to proteasome inhibitor MG132 (41.8% vs 10.7%, p<0.0001). Treatment with MG132 (0.5 μM) retained its beneficial effect with 3 different LV of increasing size up to 10.9 Kb (p<0.01). We extended, for the first time, the application of proteasome inhibition to the transduction of T lymphocytes. A transient exposure to MG132 significantly improved lentiviral T-cell transduction. The mean percentage of transduced T cells progressively increased from 13.5% of untreated cells, to 21% (p=0.3), 30% (p=0.03) and 37% (p=0.01) of T lymphocytes that were pre-treated with MG132 at 0.1, 0.5 and 1 μM, respectively. MG132 did not affect viability or functionality of HSC or T cells, nor significantly increased the number of integrated vector copies. Transient proteasome inhibition appears as a new procedure to safely enhance lentiviral transduction of HSC and T lymphocytes with low viral doses. This approach could be useful in settings where the use of large size vectors may impair optimal viral production.

A Preclinical Model of Double- versus Single-Unit Unrelated Cord Blood Transplantation

Cord blood transplantation (CBT) with units containing total nucleated cell (TNC) dose >2.5 x 10(7)/kg is associated with improved engraftment and decreased transplant-related mortality. For many adults no single cord blood units are available that meet the cell dose requirements. We developed a dog model of CBT to evaluate approaches to overcome the problem of low cell dose cord blood units. This study primarily compared double- versus single-unit CBT. Unrelated dogs were bred and cord blood units were harvested. We identified unrelated recipients that were dog leukocyte antigen (DLA)-88 (class I) and DLA-DRB1 (class II) allele-matched with cryopreserved units. Each unit contained <or=1.7 x 10(7) TNC/kg. Recipients were given 9.2 Gy total-body irradiation (TBI) and DLA-matched unrelated cord blood with postgrafting cyclosporine and mycophenolate mofetil. After double-unit CBT, 5 dogs engrafted and 4 survived long term with 1 dominant engrafting unit and prompt immune reconstitution. In contrast, 0 of 5 dogs given single-unit CBT survived beyond 105 days (P = .03, log-rank test); neutrophil and platelet recovery was delayed (both P = .005) and recipients developed fatal infections. This new large animal model showed that outcomes were improved after double-unit compared to single-unit CBT. After double-unit CBT, the nonengrafted unit facilitates engraftment of the dominant unit.

Partial donor-specific tolerance to delayed skin grafts after rejection of hematopoietic cell graft.

Background. Donor-specific tolerance (DST) is induced after allogeneic hematopoietic cell transplantation (HCT) and is a potential strategy for prolonging survival of solid organ grafts. DST may persist in recipients with transient mixed hematopoietic chimerism (MC) when solid organ transplantation and HCT are done concomitantly. Methods. In a canine model of allogeneic HCT after nonmyeloablative conditioning, DST to skin grafts was evaluated in dog leukocyte antigen (DLA)-identical recipients with stable MC (n=11), or after rejection of the hematopoietic cell (HC) graft (n=19). Results. There was significant improvement in the survival of DLA-identical HC donor-derived skin grafts in recipients with MC compared to normal recipients (n=7; P<0.0001). However, HC donor-derived skin grafts in four recipients with MC developed an inflammatory reaction without skin graft loss. This may represent partial DST. Survival of DLA-identical HC donor-derived skin grafts was also significantly prolonged compared to normal recipients even when skin grafting was delayed until after rejection of the HC graft (P=0.002). An inflammatory reaction developed in all nine of the surviving HC donor-derived skin grafts in this group, but there was no graft loss at last follow-up (median, 30 [range, 9–84] weeks). An increased time to rejection of the hematopoietic graft was associated with prolonged survival of the subsequent skin graft (P=0.02). Conclusion. In a model of stable MC, DST to skin grafts may be complete or partial. Partial DST can persist after HC graft rejection even if solid organ transplantation is delayed. Further investigations are required to understand the mechanisms responsible for DST after allogeneic HCT.