Signs Of GVHD Research Articles

Enhanced Allogeneic Engraftment Achieved Via Prenatal Tolerance Induction and Postnatal Anti-CD45 Immunotoxin Conditioned Hematopoietic Cell Transplantation

INTRODUCTION: In utero hematopoietic cell transplantation (IUHCT) is a non-myeloablative, non-immunosuppressive transplant approach that takes advantage of the immunologic immaturity of the fetus to engraft donor cells across immune barriers and induce donor specific immune tolerance (DST). Though it has the potential to treat many congenital hematologic disorders, IUHCT is limited by levels of engraftment below those anticipated to be therapeutic for most target diseases, including sickle cell anemia. Thus, a likely initial clinical application of IUHCT is to induce DST to allow for non-myeloablative, non-immunosuppressive postnatal "booster" transplants to increase engraftment. We have previously validated this approach in murine sickle cell, beta-thalassemia, and non-diseased models using low dose total body irradiation and busulfan prior to postnatal bone marrow transplantation (BMT). Although encouraging, less toxic approaches that specifically target the hematopoietic system would make IUHCT + postnatal BMT more applicable. CD45 is a membrane glycoprotein expressed by all leukocytes and hematopoietic stem cells. Recent studies have demonstrated enhanced engraftment in a congenic postnatal transplant mouse model following pre-transplant conditioning with an anti-CD45-saporin (SAP) immunotoxin (Palchaudhuri et al., Nat Biotech 2016; 34:738-45). We hypothesized that mixed allogeneic chimerism achieved by IUHCT can be enhanced after birth by conditioning with an anti-CD45-SAP immunotoxin followed by a same-donor allogeneic BMT.METHODS: 5x106 BM mononuclear cells from C57Bl/6 mice (H2Kb, CD45.2+) were injected intravenously into gestational day 14 (E14) Balb/c fetuses (H2Kd, CD45.2+). Donor cell peripheral blood (PB) chimerism was confirmed at 4 weeks of age, at which time chimeric mice were injected via tail vein with PBS (control) or CD45.2-SAP. Six days following treatment, chimeric animals were intravenously injected with 30x106 B6Pep3b (H2Kb, CD45.1+) BM cells. Additional control groups consisted of recipients of IUHCT alone without a postnatal BMT and naïve 5 week-old Balb/c mice which did not undergo IUHCT and only received CD45.2-SAP + postnatal BMT. Following postnatal BMT, PB was assessed bi-weekly by flow cytometry for total donor chimerism and the contribution of prenatal and postnatal donor cells to engraftment. Mice were weighed before each bleeding and evaluated for signs of GVHD. Statistical comparison was performed using the Student’s t-test for 2 samples at each time point assuming unequal variances.RESULTS: There was no significant difference in initial donor cell chimerism levels at 4 weeks of age between treatment (IUHCT+CD45.2-SAP+BMT, 12.4±2.2%, n=5) and control (IUHCT alone, 9.3±2.7%, n=18; IUHCT+BMT, 10.0±2.6%, n=7) groups prior to therapy. In contrast to naïve non-chimeric Balb/c mice undergoing a postnatal BMT (n=10) which demonstrated no donor cell engraftment, recipients of IUHCT and a postnatal BMT (both PBS and CD45.2-SAP treated groups) demonstrated postnatal donor cell engraftment, supporting DST achieved by IUHCT. While postnatal BMT after IUHCT failed to demonstrate a significant enhancement of engraftment compared to IUHCT alone (4.4±1.3% vs. 7.1±4.2% donor cell engraftment at 22 weeks, p = 0.7), low-level allogeneic chimerism following IUHCT was significantly enhanced to high-level chimerism following conditioning with CD45.2-SAP+BMT (55.7±9.8% vs. 7.1±4.2% donor cell engraftment at 22 weeks, p=0.0001) (Fig. 1). The majority of donor cell chimerism resulted from the postnatal donor population in recipients of CD45.2-SAP, supporting a mechanism of enhanced engraftment via a competitive advantage following recipient CD45.2-SAP treatment (Fig. 2). Chimerism was stable and multilineage at 22 weeks indicating enhanced stem cell/early progenitor engraftment. All mice demonstrated appropriate weight gain without GVHD.CONCLUSIONS: Prenatal tolerance induction achieved by IUHCT followed by postnatal CD45-SAP conditioning and same-donor BMT results in enhanced stable multilineage allogeneic engraftment at therapeutic levels for many target diseases. This work supports a potentially less toxic approach for the strategy of prenatal tolerance induction to facilitate non-myeloablative, non-immunosuppressive postnatal BMT and has the potential to significantly expand the applicability of IUHCT. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Transplantation of T Cell Depleted Haploidentical HSCT Following Non-Myeloablative (NMA) Conditioning: Combining the Power of Megadose Transplants with Post Transplant Cyclophosphamide (PTCY)

The use of PTCY is associated with reduced risk for GVHD in T cell replete NMA haplo-human stem cell transplants (HSCT); however, this intervention is still not sufficiently safe to justify treatment of non-malignant diseases or as a platform for organ transplantation. Furthermore, use of immune suppression to ameliorate GVHD likely adversely impacts anti-tumor immunity. Conversely, risk of rejection after NMA conditioning presents a major challenge for graft survival in T cell depleted (TCD) haplo-HSCT.We now show in a total of 66 mice (9 experiments) that combining the power of megadose TCD HSCT with high dose PTCY (Fig.1A), enables marked and durable chimerism following NMA conditioning, while each modality alone was ineffective (Fig1B). Chimerism included all myeloid and lymphoid lineages, and LDA analysis of alloreactive T cells revealed specific immune tolerance towards donor stimulators (Fig.1C), also associated with acceptance of donor but not 3rd party skin. A similar protocol (Fig.2) was therefore developed for clinical use in patients with high risk hematologic malignancies.The first patient, a 54 yr old male with high-risk multiple myeloma (unfavorable cytogenetics, micromolecular disease and compromised renal function at diagnosis) was treated with 4 courses of Velcade-Thalidomide-Dexametasone and autologous HSCT, attaining complete remission with good performance status and normal renal function. Six months later, he received megadose (15.4 x106 CD34+ cells/kg) CD3/CD19 depleted (1.17 x105 CD3+T cells/kg) haloidentical PBPCs. Hematopoietic engraftment was achieved at day +15 with over 96% donor type chimerism during the first 6 months in the myeloid and B cell lineages. T cells during this period were predominantly of host type (10-23% donor type), gradually increasing to 63-72% at 9-12 months post transplant. EliSpot analysis of anti-donor CTLp revealed specific non-responsiveness. The patient overcame CMV (Fig.3A) and subsequently EBV reactivation without any treatment. Dextramer FACS analysis revealed that CMV specific CD8 T cells were exclusively of host origin (Fig3B-C). At 12 months, complete hematological remission and normal Free Light Chain ratio were confirmed.The second patient, a 50 yr old male with MM achieved a partial remission after 4 courses of Lenalidomide-Dexametasone. He subsequently underwent autologous HSCT, and was put on Lenalidomide maintenance until relapse after 3 yrs. Following 8 courses of Bortezomib-Dexametasone he achieved CR. The patient received a megadose (10.8 x106 CD34+ cells/kg) of CD3/CD19 depleted (1.2x105 CD3+T cells/kg) haploidentcal PBPCs. Despite transient engraftment (50% donor cell on day +17), graft failure with autologous recovery (0.04% donor-type chimerism) was documented on day +30. This may be due to the extended treatment (38 months) with lenalidomide, but rejection cannot be excluded. After 5 months, this patient tolerated a second haploidentical HSCT (different donor) after standard myeloablative conditioning (ATG, treosulfan, thiotepa and fludarabine) and alfa/beta TCR/CD19-depleted PBPCs. Two months since the second HSCT, he shows no sign of GvHD, very good immunological reconstitution, excellent quality of life, and remains in complete remission. Collectively, our murine proof of concept data supported by clinical experience in the first high risk MM patient, suggests that combining megadose TCD HSCT with PTCY can enable prompt and durable engraftment with almost complete chimerism in the myeloid and B cell lineages. The marked level of host T cells persisting during the first 6 months after HSCT can provide anti-viral immune protection until thymus-derived donor T cells are generated. Furthermore, our ability to avoid post transplant immune suppression with this GVHD-free protocol, thereby attaining robust anti-viral immunity, could also potentially offer enhanced GVL effect, valuable for high risk MM patients.The rejection experienced by the 2nd patient, although corrected by a 2nd myeloablative TCD HSCT, indicates that the conditioning must be fine-tuned to optimize engraftment in every patient.We are therefore testing, increasing TBI from 2Gy to 3Gy. Further studies will determine the efficacy of this approach in elderly MM patients, in non-malignant hematopoietic diseases, or as a prelude for organ transplantation and cell therapy.*F.A and E.B.L contributed equally [Display omitted] [Display omitted] [Display omitted] DisclosuresBacharLustig:Yeda LTD: Patents & Royalties. Or Geva:Yeda LTD: Patents & Royalties. Reisner:Cell Source LTD: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Prospective Phase II Study of Prophylactic Azacitidine and Donor Lymphocyte Infusions Following Allogeneic Hematopoietic Stem Cell Transplantation for High Risk Acute Myeloid Leukemia and Myelodysplastic Syndrome

Although allogeneic hematopoietic stem cell transplantation is presently the only curative option for many patients with AML or MDS, relapse remains the main cause of morbidity and mortality. Strategies are therefore developed to prevent relapse. Post-transplant immune intervention with administration of prophylactic or pre-emptive donor lymphocyte infusion (DLI) and/or chemotherapy maintenance using DNA-demethylating agents such as azacitidine (aza) is being investigated. The exact mechanism of action of aza remains obscure and might be due in part to tumor antigen upregulation or other gene induction by tumor cells causing an immune response. We enrolled, in a clinical trial (ClinicalTrials.gov Identifier:NCT01541280), patients (pts) with high risk AML or MDS and candidates for allo-HSCT transplantation to receive azacitidine (aza) and DLI post-transplant as prophylactic treatment with the primary objective to reduce the relapse rate at 2 years following allo-HSCT and secondary objectives to increase disease free survival at 2 years post-transplant, increase overall survival at 2 years, investigate feasibility and safety of maintenance strategy combining chemotherapy and immunotherapy and follow the incidence and severity of acute and chronic GVHD. High risk AML was defined as AML in CR1 with unfavorable cytogenetics, AML in CR2 or greater remission, refractory AML or in relapse prior allogeneic transplantation. High risk MDS was defined as MDS with intermediate-2 group and higher risk group according to IPSS criteria. Aza was scheduled to begin between d+56 and d+112 post-transplant at the doses of 32 mg/m²/d sc for 5 days every 28 days for up to a total of 12 cycles if the pt had not acute GVHD >1 or severe infection. The first DLI was started following 3 cycles of aza and discontinuation of immunosuppressive prophylaxis, and if the pt had no clinical signs of GVHD, uncontrolled infection or a recent history of gr>2 acute GVHD. Two other DLI were scheduled every 8 weeks after the 5th and 7th cycle of aza. The doses of DLI 1, 2 and 3 were respectively 5x106, 1x107, 5x107 CD3+cells/kg for related donor, and 1x106, 5x106, 1x107 CD3+cells/kg for unrelated donor. Sixty-four patients were pre-included prior transplantation, 30 pts were subsequently included, 20 pts with AML and 10 pts with MDS, median age 58 y (22-70). The status at transplantation was: CR1 = 16 pts (53%), CR2 = 6 pts (20%), refractory = 5 pts (16%), upfront transplantation for MDS = 3 pts (10%). Cytogenetics was normal or intermediate for 15 pts and unfavorable for 15 pts (namely 8 pts with complex caryotype). Conditioning was myeloablative for 11 pts, reduced for 19 pts (including 2 sequential). Donors were unrelated volunteers in 18 pts (60%).The time between allografting and first aza cycle was 66 days (38-93). The median number of cycles of aza administered was 5 (1-12) with 10 pts (33%) completing the 12 cycles. Forty one DLI were injected in 17 pts: 5 pts received one DLI, 2 pts received 2 DLI, 8 pts received 3 DLI. Two additional pts received 4 and 5 DLI because of a mixed chimerism. The first DLI was given at a median of 142 d (129-221) post transplantation. Aza was well tolerated, but was discontinued in 20 pts: because of GVHD (n=11), relapse (n=5), GVHD/infection (1pt), sudden death due to heart failure (n=1), withdrawal of consent (n=2, one after 1 cycle and another after 5 cycles). Four months following transplantation, 24 (80%) demonstrated full donor chimerism (>95%) in CD3+ cells. Nine patients developed grade 1 to 3 acute GVHD (CI 29.8±9%), 6 who did not receive DLI and 3 following DLI (grade 1 n=2, grade 2 n=3, grade 3 n=4). No grade 4 acute GVHD was observed. Nine pts developed chronic GVHD (2 limited, 7 extensive), 3 who did not receive DLI, 6 following DLI. Twenty patients are alive. With a median follow-up from the allotransplant for those alive of 36 months (range 12 - 46 months), the survival at three years is 66%. Causes of death were infection (n=1), relapse (n=8), sudden death due to heart failure (n=1). The median time to relapse was 5 months (2.5-9) and the cumulative incidence of relapse at 3 years 28.1±8.5%. These results confirm that aza is well tolerated as a prophylactic treatment to reduce the risk of post-transplantation relapse. The incidence of GVHD following aza + DLI was not overwhelming. Analysis of T cell population and immune response as well as comparison to matched-pair control are currently performed and will be presented. DisclosuresMoreau:Amgen: Honoraria; Novartis: Honoraria; Janssen: Honoraria, Speakers Bureau; Takeda: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria.

Preemptive administration of human αβ T cell receptor-targeting monoclonal antibody GZ-αβTCR potently abrogates aggressive graft-versus-host disease in vivo.

GVHD, both acute and chronic, remains the major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Thus, there is still a great need for therapeutic tools for the prevention and treatment of GVHD. Several biologics have shown promising results in salvage therapies but are attendant on an increased risk for opportunistic infections, lymphoproliferative disorders, and relapse. This is partly due to efficient T cell elimination that neither dissects alloreactive from non-alloreactive T cells nor considers functional and structural distinctiveness of pathogen- and malignancy-reactive γδ and iNKT T cells. A novel, humanized monoclonal antibody, GZ-αβTCR, specific for the human αβ T cell receptor, was evaluated in a xenogeneic GVHD model for its potential to prevent or ameliorate GVHD and prolong survival. We could show that GZ-αβTCR significantly attenuated clinical signs of GVHD and prolonged survival by preferential depletion of CD4 cells and the naïve T cell compartment, the trigger and driver of GVHD. In a regimen that included a preemptive dose, GZ-αβTCR treatment sufficiently abrogated GVHD. Importantly, GZ-αβTCR's specificity spared host cell-mediated immune competence of cell types other than αβT cells: namely γδT cells. GZ-αβTCR's outstanding capacity to prevent GVHD and ameliorate an ongoing GVHD while sparing immune cells other than αβT cells strongly recommends GZ-αβTCR for the prevention and treatment of acute GVHD in clinical settings.

Transplantation of Maternal Haploidentical TcRαβ-Depleted Stem Cells for Malignant Infantile Osteopetrosis – Optimal Timing and Rapid Hematological Recovery

BACKGROUND: Osteopetrosis (OP), characterized by improper bone resorption is in most cases caused by dysfunctional, hematopoietic stem cell (HSC) derived osteoclasts. A number of genetic mutations are described to underlie OP and disease phenotypes varies from benign to very severe, rapidly progressive forms of OP. In most cases hematopoietic stem cell transplantation (HSCT) is the only curative treatment. Especially in infantile malignant OP early HSCT is critical in means to recover allogeneic osteoclast function to inhibit and prevent otherwise mutilating disease progression. The use of an alternative donor is required in the absence of an HLA-identical healthy sibling. While donor search is time consuming, umbilical cord blood transplantation is usually related to higher transplant related morbidity/mortality. We present a case of infantile malignant autosomal recessive OP that illustrates the advantage of the use of haploidentical maternal TcRαβ+ cells depleted graft as an attractive curative approach.PATIENT: The boy, carrying a common mutation involving the TCIRG1 gene was diagnosed with OP at 3 months of age while showing signs of progressing anemia, hypogammaglobulemia, esotropia/exophthalmia with suspicion of blindness, impaired hearing and multiple skeletal malformations. Three weeks later conditioning regimen was initiated and consisted of ATG-F (30mg/kg), i.v. busulfan (TDM; AUC 90mg +/- 5mg/L*h) fludarabin (160mg/m2) and thiotepa (15mg/mg). Maternal G-CSF mobilized mononuclear cells were harvested from peripheral blood and TcRαβ+ cell were depleted from the graft (CliniMACS). The patient was transplanted with a single graft including 44.8x106 CD34+ cells/kg, 48.6x106 TcRγδ+ cells/kg and 0.0011x106 of residual TcRαβ+ cells/kg, followed by post-transplantation in vivo CD20-depletion (Rituximab 375mg/m2). Platelets (>50x109/L) and neutrophils (>0.5x109/L) recovery was reached day +14, respectively day +15 post-HSCT. Immunosuppression consisted of Cyclosporine A and Methylprednisolon and was discontinued 4, respectively 6 weeks post-HSCT without any signs of GvHD. There was no significant transplant related morbidity. OP characteristics did not show obvious progression following HSCT and at one year post-transplant the patient is in a stable general condition with good quality of life. He has decreased but stable hearing performance, deeply impaired vision (already before HSCT), normalization of the phenotypic facial and skeletal characteristics, normal bone marrow function as well as normal physical and mental development. T-cell chimerism showed convertion from initially mixed chimerism (up to 40% of autologous cell) to 100% donor chimersim at 1 year post transplant.CONCLUSION: We conclude that haploidentical HSCT with TcRαβ+ depleted graft is an encouraging approach in infantile malignant OP that enables 1) prompt HSCT with immediate available parental donor and 2) rapid and sustained hematological recovery. DisclosuresNo relevant conflicts of interest to declare.

Establishment and identification of a H-2 completely mismatched microtransplantation model of leukemia mouse

This study was purposed to establish and identify a H-2 completely mismatched microtransplantation model of leukemia mouse. The recipients were female BALB/c mice, while donors were male C57BL/6J mice. Recipients were inoculated intravenously with 1×10(6) of WEHI-3 cells, a cell line of myelomonocytic leukemia. Donors received 100 µg/kg G-CSF mobilization through hypodermic injection, every 12 hours, and it last 5 days. Chemotherapy regimens was MA (mitoxantrone+cytarabine), and it last 4 days. Recipients were given chemotherapy conditioning without GVHD prophylaxis after inoculation of leukemic cells for 2 days, and within 8 hours after last chemotherapy received donor mobilized spleen mononuclear cells (sMNC). The number of sMNC was (3, 6, 12) ×10(7), respectively. The early death rate, recovery level of WBC in peripheral blood and leukemia load were compared between chemotherapy and microtransplantation groups. The donor chimerism was detected by RT-PCR. From the clinical manifestation and pathological features, the GVHD in recipients was evaluated. The results showed that the early mortality in chemotherapy group was 25%, meanwhile those in the (3, 6, 12)×10(7) groups were 16.67%, 8.33%, 8.33%, respectively. The(3, 6)×10(7) groups has a stronger hematopoietic recovery capability than that in chemotherapy and 12×10(7) groups (P < 0.05) . There were more leukemic cells in chemotherapy mice than that in microtransplantation mice (P < 0.01) , and (12, 6)×10(7) groups had lower leukemia load than that in 3×10(7) group (P < 0.05) . No signs of GVHD were observed in microtransplantation mice. The donor microchimerism could be discovered at eraly 2 weeks after donor cell transfusion. It is concluded that a H-2 completely mismatched microtransplantation model of leukemia mouse has been successfully established, and it will provide a experimental base for studying microtransplantation in clinic.

The Use Of a Novel Nonmyeloablative Conditioning Regimen and Sirolimus For GvHD Prophylaxis To Transplant Patients With Chronic Granulomatous Disease

Chronic Granulomatous Disease (CGD) results from a mutation in the NADPH oxidase complex. As a result, patients are prone to recurrent infections and an increased risk of autoimmune disorders such as colitis. Currently, the only available cure is hematopoietic stem cell transplantation using a related or unrelated donor. In 2002, the NIH published their results using a nonmyeloablative regimen and sibling matched donors. Although the results were overall promising, there was still significant GvHD in older patients and a number of graft rejections in the younger patients. Further accrual was limited due to donor unavailability.In 2007, after establishing an agreement with the National Marrow Donor Program, we were able to initiate a protocol for patients with primary immunodeficiencies using an HLA matched unrelated donor. The goal of this protocol was to achieve engraftment in patients with CGD, including high risk patients due to the presence of an ongoing infection or inflammation, without increasing the rate of graft versus host disease. We therefore devised a novel conditioning regimen of Busulfan, Campath, and TBI along with sirolimus as the sole GVHD prophylaxis.To date we have transplanted 21 evaluable patients. Results are summarized below:AgeInfectionInflammationaGvHDcGvHDadditional cells?A&Wcause of death32XX1Nrefused dialysis25XGrade 1Y21XXGrade 1Y19X (colostomy)Grade 4XNinfection, GvHD of skin17XX2NEvan's/TRALI/GvHD17XNpulmonary hemorrhage17XGrade 2Y12XlimitedXY7XY8XY8X (colostomy)Grade 1Y8XXY6XX3NGvHD after 3rd transplant5XY4XY4XXGrade 2Y17Y11Grade 1Y10Y10Y8Y1. Received peripheral blood stem cells from same donor after receiving bone marrow2. Received cells after additional conditioning in the setting of Evan's syndrome3. Received additional cells with initial graft failure. Went on to a second then third transplant with a different conditioning regimen and different donor.Overall survival was 76%; however all deaths occurred in high risk patients and 2 of the 5 were unrelated to the initial transplant. Further, all surviving patients transplanted with high risk disease (11 of the 16) continue to have stable engraftment and had complete resolution of their inflammation and/or infection. This includes a patient with P40phox deficiency whose primary manifestation of CGD was colitis as well as a patient with an invasive fungal infection requiring emergency laminectomy 3 weeks prior to transplant.We have had limited GvHD to date and this occurred primarily in the high risk patients (6 of the 7). The most severe GvHD occurred in a patient given additional cells due possible poor engraftment and persistent thrombocytopenia. In retrospect, this may have been a sign of GvHD and not graft failure; however the result was severe GvHD of the skin and ultimately death from sepsis. Further, the only chronic GvHD (transient, now resolved) was also in a patient given additional cells for concerns of possible graft failure. Subsequently, the protocol was modified to no longer give additional unmanipulated cells and no graft failures or any severe GvHD has occurred in any of the subsequent patients.In general, patients tolerated the transplant well, although we did see a higher than expected rate of engraftment syndrome, again in the high risk patients only (5 of 21). Many patients needed only 1 or 2 transfusions of either platelets or red blood cells and 3 did not require any transfusions at all. We also transplanted two patients with CGD/McLeod's, banking autologous blood prior to the transplant, and only one patient required any blood (1 autologous unit). Three patients did require multiple infusions due to prolonged time to engraftment or slow platelet recovery including the one patient to receive bone marrow as their initial donor product. For the one patient with late graft failure, there was autologous recovery.Thus in this single centre study we have transplanted 21 patients to date including 16 of those considered high risk using a novel non-myeloablative transplant regimen and an unrelated donor. We have had significantly lower rates of GvHD (33%) of which <10% was greater than Grade 2 and only one patient with graft failure. Overall, the combination of Campath and Sirolimus, along with Busulfan and low dose radiation is well tolerated and has a low risk of graft versus host disease while still allowing engraftment in patients, even those with high risk disease. Disclosures:No relevant conflicts of interest to declare.

A Bi-Specific T Cell Engaging Monoclonal Antibody (mAb) Derived From a TCR-Like Mab Specific For WT1/HLA-A0201 (ESK-BiTE) Shows a Potent Activity Against Human AML and Ph+ ALL In Mouse Models

The Wilms' tumor oncogene protein (WT1) is an intracellular, oncogenic transcription factor that is over-expressed in a wide range of leukemias and solid cancers. RMFPNAPYL (RMF), a WT1-derived CD8 T cell HLA-A0201epitope, is a validated target for T cell-based immunotherapy. We generated a high affinity, fully human IgG1 mAb specific for the RMF/HLA-A0201 complex. The mAb shows potent anti-leukemia activity both in vitro and in vivo in mouse models. Bi-specific T cell engaging antibodies (BiTE) have been used effectively to target cell surface proteins and kill cancers. We have developed a new, potent form of the ESK mAb that is a bi-specific T cell engaging antibody (BiTE) specific for tumor cells coexpressing the intracellular oncoprotein, WT-1 and HLA A0201. ESK-BiTE and an irrelevant control BiTE were constructed with ESK1 scFv or irrelevant ScFv on one arm, and anti-CD3 ScFv fragment as the other arm. The BiTE constructs were expressed in CHO cells. Both the ESK-BiTE and the control BiTE bind human CD3 T cells. The ESK-BiTE selectively bind to leukemia cells that express both WT1 and HLA-A0201. The ESK-BiTE activated human resting T cells and EBV-specific T cells retargeting potent cytotoxicity against WT-1+ HLA A0201+ human leukemia cells in vitro. In an NSG mouse xenograft model, injection of ESK-BiTE (20 ug/ml) twice a week, following I.V. administration of 2x107 human EBV-specific T cells, once a week, significantly inhibited the growth of a previously established disseminated HLA A0201+, WT-1+ human Ph+ ALL, BV173 expressing luciferase, as measured by bioluminescence imaging. In a second NSG mouse model mice injected I.V. with an aggressive human AML, SET-2, on day 0, were treated on day 4 with ESK-Bite for 6 days consecutively at 20 ug/day together with EBV-specific T cells given twice a week. In this setting, the ESK-BiTE and T cells resulted in undetectable leukemic growth for 14 days post-leukemia inoculation, with a minimal tumor burden detected by day 18, while all control groups showed massive increases in leukemia burden by day 14. Mice bearing SET-2 leukemia, that received ESK-BiTE and T cells also showed longer survival and delayed limb paralysis. As expected, the human T cells, which were EBV-specific, did not induce signs of GVHD in mice. Our data provide evidence that ESK-BiTE is a potent and specific therapeutic agent against aggressive human leukemias expressing WT1 and HLA-A0201. This is the first study showing efficacy of a TCR-like BiTE antibody targeting an intracellular tumor antigen expressed at low density.Supported by the Leukemia and Lymphoma Society, NIH R01CA55349 and P01 23766. Disclosures:No relevant conflicts of interest to declare.

Phenotypic and Functional Characterization Of a Novel Immature Hematopoietic Myeloid Suppressive Progenitor Cells Mobilized By G-CSF. Application To Gvhd Treatment

Background Myeloid derived suppressive cells (MDSCs) represent a heterogeneous population of cells endowed with immunosuppressive properties. They have been first described in the tumor microenvironment. Some mature MDSCs either induced by GM-CSF and IL-13 (Highfill et al., Blood 2010) or mobilized by G-CSF (Joo et al., Immunology 2009) have been reported to control experimental GVHD by inhibiting alloreactive T cell proliferation. We describe here the existence in mice and humans of a not yet characterized population of GCSF-mobilized hematopoïetic cells with phenotypic characteristics of immature MDSCs (called therefore pro-MDSCs) that can inhibit GVHD by a distinct mechanism than those described with classical mature MDSCs. Methods In the C57BL6 mouse and human, G-CSF mobilized MDSCs were collected and analyzed in the spleen and PBSC using several antibodies directed against various markers of maturity, lineage specific antigens and chemokine receptors. Depending on the expression of maturity antigens various population were sorted. In vitro, functions of sorted MDSC were analyzed by co-cultures with T cells activated either by anti-CD3 and CD28 mAbs or allogeneic dendritic cells. In vivo, the effect of various population of MDSCs on GVHD was assessed either by the transfer of murine C57BL6 (H-2b) cells (2x106 splenic T cells + 5x106 T depleted bone marrow cells +/- 0.5x106 MDSC subtypes) into lethally irradiated BALB/c (H-2d) recipients or by injecting 2x105 human pro-MDSCs with 2.5x106 human PBMC into 2 Gy irradiated Nod/SCID/gammac-/- mice. In 19 allografted patients, proportions of MDSC subpopulations contained in the peripheral stem cell graft were correlated to the occurrence of acute GVHD and to the post-transplant peripheral blood levels of conventional proliferating T cells and CD4+ CD25+ CD127low reguatory T cells (T regs). Results In the G-CSF mobilized cells, immature Lin- Sca1high cKithigh CD34+ CX3CR1+ CD16/32+ CD11b+ Ly6C+ and Lin- CD34+ HLA-DR- CD33high CD11blow CD14+ cell populations were identified in mice spleen and human PBSC, respectively. Because the pattern of maturity antigen expression, these populations were named pro-MDSCs. The mature MDSC counterparts shared the same differentiation phenotype without the markers of maturity. In vitro, both murine and human pro-MDSCs, but not the corresponding mature MDSCs, could inhibit the proliferation and induced the apoptosis of activated T cells (p&lt;0,001). The inhibition of T cell activation by pro-MDSCs required IFN-gamma produced by activated T-cells and the production of NO by pro-MDSCs in response to IFN-gamma. NO suppressed T-cell functions through impaired responses to IL2 and induction of apoptosis. In vivo, in the C57BL6 to BALB/c GVHD model, the administration of murine pro-MDSCs significantly reduced the development of clinical and histological GVHD signs as compared to allografted mice without pro-MDSCs or with GCSF-mobilized mature MDSCs (p=0,03). Murine pro-MDSCs could migrate to site of allo-priming and induced the apoptosis of allogeneic T cells when compared to mice allografted without pro-MDSCs (p&lt;0,01). In mice that had received pro-MDSCs, we observed that apoptotic T cells could be engulfed by phagocytes and that those phagocytes produced high levels of cytokines (IL-10, TGF-beta), which was associated with increased induced CD4+CD25+Foxp3+ T regs leading to the induction of tolerance. These observations were not seen in mice allografted without pro-MDSCs (p&lt;0,05). Human pro-MDSCs could protect all xeno-grafted Nod/SCID/gamma c-/- mice from GVHD mortality as compared to 100% GVHD lethality in controlled xeno-grafted mice without pro-MDSCs (p&lt;0,001). Allografted patients having received a stem cell graft containing levels of Pro-MDSCs &gt;10% of the CD34+ fraction had a significantly reduced risk of developing grade II-IV acute GVHD (p= 0,04) and reduced numbers of proliferating conventional T cells but higher numbers of T regs in the peripheral blood on days 15 and 30 post-HSCT (p&lt;0.05). No correlation between the occurrence of acute GVHD and the proportions of mature MDSCs contained in the graft was observed. Conclusion We have characterized a new homogeneous population of G-CSF mobilized immature MDSCs, which has been named pro-MDSC that can regulate alloreactive T cell activation in vitro and in vivo by inducing tolerance with potential therapeutic application in allogeneic HSCT. Disclosures: No relevant conflicts of interest to declare.

Tissue Distribution of a Minor Antigen, but Not PD-1 Expression, Predicts the Antileukemia Efficacy of Adoptively Transferred, Antigen-Specific T-Cells in a Preclinical Model of Allogeneic Transplant

Abstract 456The clinical success of T-cell directed allogeneic immunotherapy for hematologic malignancy relies on the selection of antigens that elicit effective antitumor responses. While it has been demonstrated that vaccination against a single minor histocompability antigen (miHA) can cure solid and hematologic tumors, we have observed that alloreactivity produced by broad expression of miHA can reduce antigen-specific, solid tumor vaccine responses. In this study, we hypothesized that shared expression of a miHA would diminish the antileukemia potency of adoptively transferred, miHA-specific CD8 T-cells.We developed a murine allotransplant model that allowed us to identify and analyze HY-specific alloreactive CD8 T-cells after adoptive transfer using congenic markers. Following lethal irradiation, T-cell depleted female marrow was transplanted into allogeneic male [F B6(CD45.1+) > M B6(CD45.1+)= F>M] and syngeneic female [F B6(CD45.1+) > F B6(CD45.1+) = F>F] recipients. CD8 T cells (CD45.2+) specific for the Class I immunodominant HY peptide (HY-CD8) were then adoptively transferred. Using this method, we have generated clinically significant, nonlethal GVHD in male recipients and have demonstrated that, despite robust expansion of HY-CD8, these T cells are unable to mediate effective antitumor responses to an HY-expressing epithelial tumor (MB49). This phenomenon cannot be overcome with dendritic cell vaccination and is associated with high levels of PD-1 expression on HY-CD8.In the present study, we first determined whether tissue restriction of HY expression could improve HY-CD8 responses. [M>F] bone marrow chimeras were created to restrict HY expression to hematopoietic tissues, followed by adoptive transfer of HY-CD8 and MB49 subcutaneous challenge. In this setting, immunity to MB49 was preserved (55% +/− 7.4% tumor-free survival) without clinical signs of GVHD (p<0.0001) and maintenance of male marrow engraftment. As expected, HY-CD8 recovered from the bone marrow of tumor-free survivors maintained the ability to proliferate to HY peptide ex vivo by CFSE dilution. Unexpectedly, nearly 100% of HY-CD8 recovered from secondary lymphoid tissues of all [M>F] recipients expressed PD-1, regardless of whether they controlled MB49 in vivo. Syngeneic [F>F] recipients controlled MB49 equally well, (62 +/− 5.3% tumor-free survival), but neither tumor-bearing nor tumor-free survivors expressed PD-1 on recovered HY-CD8.Next, we determined whether hematopoietic restriction of HY could also preserve antileukemia responses. An immunogenic, HY-expressing, GFP+ murine pre-B cell leukemia was intravenously injected at the time of transplant and followed by HY-CD8 adoptive transfer. Unlike the solid tumor model, hematopoietic restriction of HY resulted in 100% leukemia-associated mortality (GFP+ marrow leukemia burden 64.6 +/− 9.7%) at a rate similar to [M>F] recipients that did not receive HY-CD8. HY-CD8 recovered from bone marrow of leukemia-bearing mice did not express PD-1. In contrast, [F>M] allogeneic recipients had 82.1 +/− 5.8% leukemia-free survival with 100% of HY-CD8 expressing PD-1. Similar to the solid tumor model, syngeneic [F>F] recipients had 62 +/− 5.2% survival, and no expression of PD-1 on HY-CD8.In summary, while adoptive transfer of CD8 T cells specific for a hematopoietically restricted minor antigen improved solid tumor immunity, it did not preserve antileukemia responses. Effective antileukemia responses were generated when the antigen was redistributed to nonhematopoietic tissues. These data suggest that the tissue location of an endogenous minor antigen relative to the tumor can directly impact the function of adoptive T-cells targeting that antigen. Further, PD-1 expression on antigen specific CD8 does not correlate with antileukemia efficacy, suggesting that PD-1 biology may be uniquely modulated by the alloimmune environment. Disclosures:No relevant conflicts of interest to declare.

Vascularized Bone Marrow in CTA Provides the Unique Biological Opportunity to Promote Stable Mixed Chimerism and Tolerance

Introduction: The induction of mixed chimerism (MC), as a result of bone marrow cell (BMC) transplantation, is an effective approach to achieve transplantation tolerance. However, host conditioning with irradiation or cytotoxic drugs is required in many protocols. Our previous study demonstrated that composite tissue allograft (CTA) as a complex unit, is more pro-tolerogenic than its component: the skin. We hypothesized that vascularized bone marrow (VBM) in CTA not only provides (BMC), but also provides bone marrow stromal microenvironment, may provides a unique biological opportunity to induce stable MC and tolerance without harsh host conditioning. Methods: A same immunosuppressive regimen, two doses of antilymphocyte serum (ALS) plus 7-day cyclosporine A followed by 14-day rapamycin, were used to compare the MC and allograft survival between the recipients received Vascularized skin/muscle/bone (VSMB) allografts with that received vascularized skin/muscle transplantation (VSM) plus 50 million of bone marrow cells transfusion (VBM), equivalent to that from VSMB. GFP-transgenic SD rats served as donors and MHC mismatched LEW rats as recipients. Results: 1) GFP+ multilineage MC (2.9-5.5%) persisted > 150 days in the peripheral blood and VSMB were accepted indefinitely in all recipients without sign of GVHD. 2) Transient low-level GFP+ cells (1-1.5%) were detected in VSM+BMC recipients and disappeared 4-6 weeks thereafter and VSM graft were rejected soon after the cease of immunosuppression and lost of MC. 3) In VSMB chimeras, donor GFP+ cells homed to recipient bone marrow, and migrated to recipient thymus where they underwent T-cell development. 4) The bone components of VSMB maintained normal bone structure and contained 40% of GFP+ cells with typical bone marrow cell phenotype > 150 days post transplant. 5) The long-term VSMB recipients accepted same donor strain secondary skin allografts without any immunosuppression, while acutely rejected skin allografts from third party strain donor. Conclusion: Vascularized bone marrow in CTA, carrying not only bone marrow cells but also bone marrow stromal microenvironment, provides the unique biological opportunity to promote stable MC and tolerance.

Effective prevention and treatment of CD8 T cell-mediated graft-versus-host-like disease (GvHD) using a JAK inhibitor. (51.3)

Abstract A Janus kinase (JAK) inhibitor, tofacitinib (tofa), inhibits JAK 1, 2 and 3 of some cytokine receptor pathways, affects CD4+ T cell respnses, and is being tested in more than 20 clinical trials in autoimmune diseases.To determine if tofa affects CD8+ T cells, we analyzed its effect on a CD8+ T-cell-dependent murine model of GvHD (OT-I T cell transfer into Tg mice expressing ovalbumin (OVA) in their skin and mucosae). Tofa at 50 mg/kg/day (50) p.o. completely prevented GvHD development in our model vs. vehicle-treated mice with final weights: -0.6%±5.6 and -15.4%±4.1 (p&amp;lt;0.01; 2-way ANOVA) and clinical scores: 0 and 7.3±0.52 (p&amp;lt;0.01; U test) 13 days post OT-I cell transfer. Mice treated with tofa 50 starting at day 5 were completely rescued, with no clinical signs of GVHD. FACS analysis demonstrated reduced expression of OVA-specific TCR and reduced activation of OT-I cells in skin-draining lymph nodes from mice transferred with GFP/OT-I cells and treated with tofa 50 vs. vehicle alone. Tofa inhibited proliferation of OT-I cells stimulated by SIINFEKL (OVA peptide) in vitro. qRT-PCR showed increased chemokine mRNA (CXCL9; 39-fold and CXCL10; 22-fold) in the epidermis of vehicle-treated mice vs. tofa 50-treated mice 4 days post OT-I cell transfer. The effect of tofa could be explained by its inhibition of JAK signaling in IL-2 and IL-15 receptor-pathways of OT-I cells and in IFN-γ receptor-pathways of keratinocytes and tofa may thereby be effective in autoimmune diseases.

Lenalidomide plus donor-lymphocytes infusion after allogeneic stem-cell transplantation with reduced-intensity conditioning in patients with high-risk multiple myeloma

Myeloma relapse is the main cause of death after allogeneic stem cell transplantation. The aim of our observational study was to evaluate the anti-myeloma effect of lenalidomide followed by donor-lymphocyte infusion (DLI) as post-transplantation adoptive immunotherapy. Twelve patients with refractory myeloma were analyzed. The median age at transplantation was 56 years (range, 46-64 years). All patients received reduced-intensity conditioning. Patients were included if progressive or residual disease was observed at day +100 and if no signs of graft-vs-host disease were evident. DLIs were administered after two cycles of lenalidomide. Median dose of lenalidomide was 15 mg (range, 10-25 mg). Patients received a median of six cycles (range, 1-10 cycles). Nine patients (60%) received an escalating dose of DLI. The 1 and 2-year probability of progression-free survival was 75% and 50%, and overall survival was 83% and 69%, respectively. Median overall survival was not reached and median progression-free survival was 23 months. Lenalidomide is well tolerated after allogeneic stem cell transplantation; the combination with DLI did not cause a higher risk of graft-vs-host disease; an immunological synergistic effect was probably present with this strategy. This combination should be evaluated further in a larger cohort of patients.

Endothelial Stress in Steroid-Refractory Gvhd: A Burden Statins Cannot Lift

Abstract 1012Graft-versus-host disease (GvHD) is the major complication of allogeneic stem cell transplantation (alloSCT) and its therapy-resistant form causes significant morbidity and mortality. The pathomechanism of steroid resistance is currently not completely understood; however, we have recently suggested that endothelial dysfunction seems to play an important role (Luft et al., Blood 2011). The aim of this study was to validate in a large cohort of patients that steroid refractory acute GvHD is associated with endothelial stress. Secondly we assessed if this endothelial stress can be overcome by statins, which are known to have endothelial protective effects.We have therefore performed a retrospective study of 393 patients who underwent alloSCT in our institution between 2002–2010. Endothelial stress serum markers were correlated with the course of GvHD, non relapse mortality (NRM) and statins intake. Overall, 169 of 393 patients (43%) developed GVHD. Of these, 101 patients (60%) had steroid-sensitive GvHD grade 1–2, 26 patients (15%) steroid-sensitive GvHD grade 3–4 and 42 patients (25%) had steroid-refractory GvHD which was always grade 3–4 at its maximum severity. 224 patients had no signs of GvHD. In order to elucidate the pathomechanisms that contribute to steroid resistance, serum markers of endothelial stress and vascular growth (Angiopoietin-2 (ANG2), IL-8, soluble thrombomodulin (sTM), and vascular endothelial growth factor (VEGF)) were measured by ELISA and multiplex assays before and on days +50 and +100 after alloSCT. RESULTS:Landmark analyses at days +50 and +100 after alloSCT showed that NRM was dramatically high in the steroid refractory group but was equivalently low in the no GvHD-, sensitive grade 1–2 - and grade 3–4 - groups (p<0.001, Figure 1). ANG2, a mediator of endothelial cell vulnerability, measured before transplant was predictive for a steroid refractory course of GvHD (p=0.05) and higher NRM (p=0.06) under the prerequisite that patients developed grade 3–4 GvHD. Moreover, patients who developed an acute steroid refractory GvHD displayed significantly higher levels of the endothelial stress markers IL-8 (day +50: p<0.001; day +100: p<0.001) and sTM (day +50: p<0.009; day +100: p=0.003) compared to steroid-sensitive GvHD patients and patients with no GvHD. High levels of sTM, IL8 and ANG2 were significantly associated with increased NRM rates (day +50: IL8 p=0.02, sTM p=0.0008, ANG2 p=0.0001; day +100: IL8 p=0.0007, sTM p<0.0001, ANG2 p=0.05); even after multivariate adjustment for donor, conditioning intensity, disease status at alloSCT and sex mismatch. In contrast, no influence of VEGF levels on steroid-refractory GvHD. [Display omitted] When IL8, sTM, ANG2 and VEGF kinetics were compared in patients with and without concomitant statin therapy (n=81), no differences were observed in the whole patient cohort nor in the subset with steroid-refractory GVHD. Consequently, patients with or without statins had similar NRM, relapse rates and overall survival in univariate as well as multivariate analysis. CONCLUSION:This supports the hypothesis that steroid-refractory GVHD is associated with progressive microangiopathy. Statins, although reported to have protective effects on endothelial cells, were inefficient to alleviate endothelial stress in this context and, accordingly, did not change the outcome of acute GVHD patients. Disclosures:Luft:Celgene: Research Funding.

Allogeneic Stem Cell Transplantation for Sickle Cell Disease in Brazil: The Time Is Now!

Abstract 1064▪▪This icon denotes a clinically relevant abstract Introduction:Sickle Cell Diseases (SCD) are the most frequent inherited monogenic diseases worldwide. Data from the neonatal screening program showed that about 4% of the brazilian population harbors the sickle cell trait, reaching until 10% in the afro-brazilians. In the northern states of Brazil this frequency is much higher, as an example in Bahia 1/17 births are of children with sickle cell trait. The neonatal screening program doesn't cover the whole country so the estimate of about 3500 newborns with SCD per year in Brazil is certainly underestimated. Nevertheless the first official document regarding the treatment of these patients was published by the brazilian ministry of health only in 2004. Hydroxiurea (HU) is already used in more severe forms of SCD by the public health system since several years but is not available for all patients yet. In a recent publication Machado et al (ASH 2010 #843) demonstrated for the first time that HU can prolong survival in SCD, although the mortality rate for this disease is still high in Brazil. Stem Cell Transplanation (SCT) is the only available curative treatment for the inherited hemoglobinopathies, in spite of this there is no such program for SCD in Brazil. Here we describe the first brazilian cases transplanted in 5 brazilian centers of severely affected patients with no other treatment options not responding to HU. Patients and Methods:Sixtheen patients (15 HbSS, 1 case Sbeta thalassemia), median age 16 years (3–39), all except for one with HLA identical sibling donors (3 donors with sickle cell trait)received a alo SCT. Indications consisted mainly of severe acute chest syndrome, priapism, alloimunization and silent cerebral infarctions and overt previous stroke. One patient transplanted because of advanced Hodgkin’s disease. The conditioning consisted of BuCy or FluBu with ATG in all but 3 patients who received FluCy and ATG. The decision was based on the age of one of the patients (39 years old and grade IV liver fibrosis) and 2 patients with previous stroke. Results:Primary graft failure occurred only with the unrelated donor. All other patients engrafted, but one patient conditioned with FluCy lost the graft about 100 days after transplant. She received a second transplant 3 years after with BuCy from the same donor, had a 100% engraftment without any sign of GVHD but unfortunately died 3 years later in remission from a SNC bleeding caused by her acquired vascular abnormality. Median follow up for all surviving patients is 2 years (152 days – 12,2 years). Overall survival is 13/16. Death occurred in 3 cases (1 primary graft failure and sepsis, 1 Hodgkin disease, 1 hemorrhagic stroke in a patient with severe cerebral vasculopathy with MoyaMoya). No veno-occlusive disease was observed. Acute GVHD grade II were observed in 4 patients (gut and skin) easily treated with short time prednisone. All surviving patients but one are full chimeras and the 39 years old patient remain a stable mixed chimera 6 years after transplant. Quality of life has improved in all patients considerably referring extremely grateful to be free of pain. Conclusion:Different from thalassemia SCT can be offered safely for older patients with SCD in our opinion. Our data confirm the curative potential of SCT in SCD and reinforce us to offer this curative approach to patients not responding to HU. A prospective trial is planned in Brazil who should address also long term toxicities and quality of life. Disclosures:No relevant conflicts of interest to declare.

Lenalidomide Followed with Donor Lymphocytes Infusion (DLI) After Allogeneic Stem-Cell Transplantation (Allo-SCT) with Reduced-Intensity Conditioning in Patients with High Risk Multiple Myeloma

Abstract 4310 Purpose:Relapse remains the main issue after allogeneic stem cell transplantation (Allo-SCT) in high risk Multiple Myeloma (MM) patients. The aim of this study is to assess the anti-myeloma effect of lenalidomide followed by donor lymphocyte infusion (DLI) as adoptive immunotherapy after transplantation. Patients and methods:Twelve patients with refractory and high risk myeloma were analyzed. Median age at transplantation was 56 years (46–64); 6 patients (50%) received lenalidomide before Allo-SCT. All patients received a RIC including Fludarabine 30 mg/m2 5 days, ATG 2,5 mg/kg for 2 days and Busilvex 3.2 mg/kg/day (3 days in 6 patients and 2 days in 6 patients). All but one received peripheral blood stem cells (PBSC). Donor was HLA id in 6 patients and matched unrelated in 6 patients. It is our standard long term practice to consider post-transplant DLI in patients with progressive or persistent disease after day 100 if no GVHD signs were evident. In 2010, we introduced the use of lenalidomide after day 100 in patients with MM presenting the same characteristics. Doses ranged from 10 to 25 mg/day. Lenalodomide treatment could be completed with DLI, administered afterward, at least after 2 cycles. Results:The median time between Allo-SCT and lenalidomide was 10 months (3–38). The median initial dose of lenalidomide was 15 mg (10–25). Patients received a median of 6 cycles (1–10). Nine patients (60%) received an escalating dose of DLI; 1 × 107/Kg of CD3+cells for the first DLI and 1 × 108/Kg of CD3+cells for the second DLI. One patient with GVHD (after tapering of the cyclosporine A and only after 10 days of lenalidomide) and two patients with progressive disease after lenalidomide did not receive DLI.The toxicity related to lenalidomide was mainly haematological (grade II in 4 patients (33%) and grade I in 3 patients (25%); 7 patients (58%) had moderate asthenia. One patient developed a reversible renal insufficiency after 10 cycles of lenalidomide, none of our patients developed thrombo-embolism under treatment.At the last follow up, 9 patients are alive and all of them are under ongoing treatment. Four patients achieved complete remission (CR) and five patients partial remission at last evaluation. The 1 and 2 years probability of the progression-free survival (PFS) was 75% and 50% and overall survival (OS) was 83 % and 69% respectively. The median OS was not reached and the median PFS was 23 months. Conclusions:These data show that lenalidomide has an acceptable toxicity. Combination with DLI should be further evaluated in a larger cohort of patients. Disclosures:No relevant conflicts of interest to declare.

Hematopoietic Multipotent Progenitor Cells Mobilized by G-CSF Can Inhibit Gvhd

Abstract 2969 Backgound.Acute graft-versus-host-disease (aGVHD) is a frequent life threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT). Despite the infusion of higher doses of T cells with the use of G-CSF-mobilized HSC grafts, the incidence of aGVHD is not increased. The mechanisms by which G-CSF-mobilized HSC can control GVHD are imperfectly elucidated. We previously described the mobilization of murin hematopoïetic progenitor cells (HPCs) by G-CSF and FLT3 ligand capable of inducing tolerance against autoimmune diabetes in the nude mice (Kared, Immunity 2006). We now show that G-CSF can mobilize murin HPCs with immunoregulatory functions in the allogeneic immune response and describe their mechanisms of action. Methods.Mobilization of HPCs is performed by subcutaneous administration of human recombinant G-CSF at 200μg/kg per day, for 4 consecutive days in the C57BL6 (H-2b) mouse. HPCs are collected in the spleen by FACS sorting according to their phenotype: Lin- Sca1high cKithigh FLT3low CD34+ CD106+ CD127−. In vitro, functions and mechanisms of action were analyzed by co-cultures with i) T cells (from C57BL6) activated by anti-CD28 and -CD3 mAbs or activated by BALB/c (H-2d) allogeneic splenic LPS matured dendritic cells, ii) C57BL6 splenic selected CD4+CD25high T regulatory T cells activated by anti-CD28 and -CD3 mAbs iii) activated antitumor specific CD8 T cells (C57BL6 ovalbumin specific TCR transgenic T cells). These different cultures were performed in the presence or absence of inhibitors of selective cytokines or other regulatory molecules. In vivo, we assessed the effect of donor HPCs on GVHD development by injecting C57BL6 derived HPCs (0.5×106/mouse), splenic T cells (1×106/mouse) and T depleted bone marrow cells (5×106/mouse) into lethally irradiated (8 Gy) Balb/c recipients. Results.In vitro, as compared to controls without HPCs, after 3 days of culture, HPCs: 1) promote the proliferation of natural T regs activated by anti-CD3 and anti-CD28 (>80% at 3 days of culture compared to control <50%), 2) inhibit the proliferation of activated T cells (>80% T cells blocked before 4 divisions as compared to control-T cells alone >80% after 4 divisions- p<0, 001) and 3) induce the apoptosis of activated T cells (30% increased, p=0, 01). The proliferation of T regs was cell contact dependant and required the presence of TGF-b. The inhibition of T cell activation required IFN γ produced by activated T-cells and some contact-dependent stimuli. In such pro-inflammatory conditions, HPCs differentiate after 4 days in myeloid derived suppressor cells (MDSC). These cells could then produce NO in response to IFN γ and suppress the proliferation of activated T cell. However, T cell suppression was not dependant on L-arginine depletion. Induction of apoptosis of T cells was Fas/Fas-L dependant. Although in the presence of HPCs the proliferation of CD8+ T TCR transgenic against the dominant ovalbumin epitope SIINFEKL was reduced, the cytotoxic response against the SIINFEKL-pulsed EL4 cell line was enhanced (cytotoxicity >90% with HPCs versus <90% w/o HPCs, p<0, 001). In addition, HPCs express CCR7 and CD62L, which should allow their migration to the sites of allopriming. In vivo, none of the mice that had received allogeneic HSCT with HPCs developed clinical or histological GVHD signs as compared to 50% of the control allografted mice without HPCs. Conclusion.Hematopoietic progenitor cells acquire an immunosuppressive potential after G-CSF mobilization. These cells can be isolated from mobilized peripheral stem cells and suppress GVHD while possibly preserving the GVL effect. Work is underway in humans to identify and amplify this population ex vivo for potential therapeutic application in allogeneic HSCT. Disclosures:No relevant conflicts of interest to declare.

Early expression of plasma CCL8 closely correlates with survival rate of acute graft-vs.-host disease in mice

To elucidate the significance of early expression of CC-chemokine ligand motif 8(CCL8) in mice with graft-vs.-host disease (GVHD), we investigated its induction mechanisms and correlation with overall survival rate in GVHD mice. Plasma CCL8 increases on day 5 of allogeneic transplantation, when signs of GVHD are barely detectable. Increase of allogeneic splenocytes in grafts exacerbates GVHD and leads to upregulation of plasma CCL8 on day 5. Overall survival is the gold standard in determining the severity of acute GVHD in mice, but the absence of clinical and/or pathological manifestations in the early phase make it difficult to estimate vital outcomes at this stage of allogeneic marrow transplantation. After lethal irradiation, BALB/c mice received bone marrow transplantation from C57BL/6 mice. Survival rate was monitored and clinical and pathological scores of GVHD were examined. Coculture of BALB/c-derived dendritic cells and C57BL/6-derived splenocytes was performed. CCL8 was measured by immunoassay. The plasma CCL8 level at day 5 of transplantation was closely correlated with survival rate and clinical/pathological scores on day 14. Invitro study revealed that the BALB/c-derived dendritic cells expressed CCL8 upon stimulation of C57BL/6 CD4(+) T cells by cell interactions through major histocompatibility complex class II molecules. These investigations indicate that early and preclinical expression of CCL8 in plasma predicts overall survival of GVHD mice. Together with an involvement of allo-recognition in CCL8 expression, it suggests that CCL8 plays an important role in GVHD pathology.

Contribution of Human T Cell Subpopulations to Xenogeneic Graft-Versus-Host Disease In RAG2−/−gc−/− Mice

AbstractAbstract 2542Graft-versus-host disease (GVHD) and opportunistic infections represent two major causes of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Both of these conditions are respectively mediated or controlled to a large extend by donor-derived T cells. Moreover T cells play a critical role in promoting stem cell engraftment and decreasing the probability of disease relapse. Therefore the separation of the effects of GVHD from the beneficial effects mediated by T cells has been a long-standing challenge of transplantation immunology. The contribution of different subsets of donor T cells to the pathogenesis of GVHD has been studied in different mouse models: both purified naïve CD4+ and CD8+ T cells were able to induce acute GVHD. On the contrary memory T cells failed to induce GVHD. In order to dissect the contribution of different human T cells, we sought to define a xenogeneic model for GVHD in RAG2−/−gc−/− mice. Newborn RAG2−/−gc−/− mice were intraperitoneally (i.p.) injected with either peripheral blood mononucleated cells (PBMC), immunomagnetically selected CD4+ or CD8+ cells, or FACS sorted CD45RA+CD4+ or CD45RO+CD4+ cells. Mice were weaned 3 weeks after birth and were scored weekly for signs of GVHD and bled for human engraftment evaluation. Both PBMC and CD4+ injected groups developed xenogeneic GVHD (x-GVHD) and the survival at 12 weeks was 42% and 53% respectively. The peak of peripheral blood (PB) engraftment was reached between week 6 and 7 and it was about 40% in both groups. Human cells were present in high percentage (>60%) in the spleen and liver of mice that displayed signs of x-GVHD. Moreover in these mice developed detectable thymi and mesenteric lymph nodes (MLN) which are not normally present in the RAG2−/−gc−/− mice and which were populated by human cells. In PBMC injected mice, CD4+ and CD8+ T cells were the only populations observed with no survival of myeloid, B or NK cells. PBMC or CD4+ groups developed GVHD in 55%, while mice adoptively transferred with CD45RA+CD4+ cells manifested a positive score for x-GVHD (30%) but displayed milder symptoms compared with the PBMC or CD4+ groups. The lesser morbidity and mortality correlated with a lower human engraftment in PB (25% at week 7) and in spleen (about 40%). Only 10% of mice injected with CD45RO+CD4+ cells showed x-GVHD symptoms and the human engraftment level in PB remained below 20%. Finally mice injected with CD8+ cells did not develop any x-GVHD symptoms. The human PB engraftment was low (<15%) in these mice and it decreased over time. In summary we established an easy and reliable xenogeneic mouse model suitable for dissecting the contribution of different human T cell populations to x-GVHD. Our results suggest that depletion of CD4+CD45RA+ naïve T cells and/or adoptive transfer of CD4+CD45RO+ memory T cells may find use in the allo-HSCT setting, allowing for rapid reconstitution of T cell-mediated immunity while minimizing GVHD. Disclosures:No relevant conflicts of interest to declare.

Role of Il-17 Varies at Different Periods After Hematopoietic Stem Cell Transplantation: Protection From Acute Graft-Versus-Host Disease and Exacerbation of Chronic Graft-Versus-Host Disease.

AbstractAbstract 3741Th17 is a newly identified T cell lineage that secretes the proinflammatory cytokine IL-17. Th17 cells have been shown to play a crucial role in mediating autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE), arthritis, and colitis. Anti-IL-17 therapy for some autoimmune diseases in clinical settings has been started and promising results have been reported. However the role of IL-17 on developing acute and chronic GVHD in hematopoietic stem cell transplantation (HSCT) is not yet fully understood. Interaction between IL-17 and IL-17 receptor is complicated because IL-17 is produced in various kinds of immune cells other than CD4+ T-cells, and IL-17 receptors express on not only immune cells but also various epithelial cells, including lung and intestine, both of which are target organs of GVHD. To explore the role of host derived or donor derived IL-17 separately in acute GVHD, lethally irradiated wild type (WT) or IL-17 knockout (KO) Balb/c (H-2d) were given WT or IL-17 KO C57BL/6 (H-2b) bone marrow (BM) cells with WT splenocytes to induce acute GVHD. Infused cell number of WT splenocytes in this study induced acute GVHD, but not lethal in IL-17 WT host mice. In contrast, IL-17 KO host mice receiving WT BM plus WT splenocytes developed severe acute gut GVHD and finally half of them died (p<0.05). To exclude the possibility that alloreactivity of host IL-17 KO derived dentritic cells (DCs) could be much more than that of WT DCs, mixed leukocyte reaction (MLR) was performed using stimulators from WT or IL-17 KO DCs and responders from WT CD4+ T-cells. No significant differences were observed between WT DCs and IL-17 KO DCs in thymidine uptake and percentage of responder cells producing IFN-g or TNF-a. Taken together, host-derived IL-17 has a protective effect against acute GVHD. Moreover similar results were observed when IL-17 KO Balb/c mice were given BM cells from another strain B10.D2 plus splenocytes shown in the figure below (p<0.05). Next, we compared the development of chronic GVHD between the lethally irradiated WT Balb/c mice given IL-17 KO C57BL/6 BM cells or WT BM cells with low dose WT splenocytes to induce sublethal acute GVHD and chronic GVHD subsequently. After day 60 the mice receiving WT BM cells plus WT splenocytes experienced weight loss accompanied by skin histological changes (p<0.05, shown in the figure below), while mice receiving IL-17 KO BM plus WT splenocytes showed minimal signs of GVHD as well as mice receiving IL-17 KO BM or WT BM alone. Increased number of donor-BM derived IL-17 producing cells was observed in the mice showing chronic GVHD compared to BM control (p<0.05). Moreover, a significant increase of T-cell proliferation was observed by adding rIL-17 into MLR culture (p<0.05). These results suggest that donor BM derived IL-17 producing cells involved in the pathogenesis of chronic GVHD by exacerbating the alloimmune response in part. In conclusion IL-17, especially from host-derived, has a protective effect against acute GVHD. On the contrary, donor BM derived IL-17 exacerbates chronic GVHD. Neutralizing IL-17 would be a potent strategy only for preventing chronic GVHD, not for acute GVHD. [Display omitted] Disclosures:No relevant conflicts of interest to declare.