Response of Steroid-Refractory Acute GVHD to α1-Antitrypsin.

α1-Antitrypsin (AAT) is a serine protease inhibitor with anti-inflammatory, antiapoptotic, and immunomodulatory properties. It has therapeutic efficacy in animal models of autoimmune diseases, inflammatory disorders, and transplantation. In a phase I/II open-label single-center study, we administered AAT (Glassia; Baxalta/Kamada, New Ziona, Israel) as salvage therapy to 12 patients with steroid-refractory acute graft-versus-host disease (GVHD). AAT was given i.v. at 2 dose levels over a 15-day course. All patients had grades III or IV GVHD with stage 4 gut involvement. After treatment, plasma AAT levels increased in both cohorts and remained within 2 to 4 mg/mL for the duration of treatment. No clinically relevant toxicities attributable to AAT were observed. GVHD manifestations improved in 8 of 12 patients, and 4 responses were complete. Six patients (50%) were alive at last follow-up (>104 to >820 days). These findings show that AAT is well tolerated and has efficacy in the treatment of steroid-refractory severe acute GVHD. Further studies are warranted.

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.

Neuroimmunomodulation and aging: a role for transferrin and the hypothalamus/thymus axis.

Advanced age is associated with an increased incidence of immune and degenerative disorders, mediated by metabolic changes, dysregulation of proinflammatory signals, and apoptosis. Concurrently, there is a progressive decline in self-recognition. Investigations on biologic functions of transferrin (Tf) other than iron transport showed that Tf has a profound cytoprotective (anti-apoptotic) effect on lympho-hematopoietic cells and the thymus, and interferes with stress-induced signals. Tf protects hepatocytes against Fas-induced cell death by reducing BID cleavage, inhibiting caspase-3 and -9 activation and up-regulating survival signals such as Bcl-xL. The involvement in the regulation of alloreactivity and apoptosis suggests that Tf participates in the maintenance of "self-identity" mechanisms, which are tightly linked to the capacity of the immune system to recognize and react against any noxious agent. Some of the disorders associated with aging are thought to be related to thymic involution, reflecting alterations in the interplay of neural, endocrine and immune factors. We established a murine model of thymic involution induced by stereotactically placed electrolytic lesions in the anterior hypothalamic area. The events observed in this model mimic those observed during senescence including thymus involution, i.e. enhanced glucocorticoid reaction to distress, and obesity. The described properties of Tf can be exploited to modify immune responses and provide cytoprotection against pro-apoptotic and cytotoxic signals when neuroimmunomodulatory mechanisms are impaired, as is the case with aging.

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.

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.

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

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.

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.

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.

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 microg/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 microg/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.

Minor histocompatibility antigen-specific cytotoxic T lymphocytes generated with dendritic cells from DLA-identical littermates.

Donor cytotoxic T lymphocytes (CTL) specific for minor histocompatibility antigens (mHA) mediate the graft-versus-host effect whereas host mHA-specific CTL mediate graft rejection in the setting of major histocompatibility complex identical allogeneic hematopoietic stem cell transplantation. Development of a large animal model from which mHA-specific CTL can be isolated would accelerate translation in clinical studies to improve control of the graft-versus-host effect as well as prevention of graft rejection in sensitized hosts. The aims of the current study were to isolate mHA-specific CTL from dog leukocyte antigen-identical littermate nonsensitized recipients before transplantation, from stable mixed hematopoietic chimeras, and from dogs sensitized to mHA after graft rejection. Donor dendritic cells (DCs) were cultured from bone marrow-derived CD34(+) cells and were used to stimulate recipient T lymphocytes on days 1, 10, and 20 of CTL culture. We reliably generated and expanded mHA-specific CTL ex vivo from sensitized dogs that were given a donor-specific blood transfusion to boost immune recall after graft rejection after a nonmyeloablative transplantation. The mHA-specific cytotoxicity measured by (51)Cr release assay was enriched from less than 5% in the starting population of sensitized peripheral blood mononuclear cells to a median of 63% after 4 weeks in CTL culture. The expanded mHA-specific CTLs were not tissue-specific: hematopoietic cells, fibroblast, and stromal cell lines were lysed in an mHA-specific manner. Allogeneic DCs, but not peripheral blood mononuclear cells, were necessary for stimulating ex vivo expansion of mHA-specific CTL. We were unable to generate mHA-specific CTL from nonsensitized dogs before transplantation, from previously sensitized dogs but without recent recall immunization, or from stable mixed hematopoietic chimeras. We conclude that after recent in vivo sensitization, large-scale ex vivo expansion of mHA-specific CTL was feasible using allogeneic DCs.