Haploidentical Setting Research Articles

At the Bench: Preclinical rationale for exploiting NK cells and γδ T lymphocytes for the treatment of high-risk leukemias

NK cells and γδ T lymphocytes display potent cytolytic activity against leukemias and CMV-infected cells and are thus, promising immune effector cells in the context of allo-HSCT. NK cells express HLA class I-specific inhibitory receptors and preferentially kill HLA class I(low) tumors or virus-infected cells. Killing occurs upon engagement of activating NKRs with ligands that are up-regulated on tumors and infected cells. A similar activating receptor/ligand interaction strategy is used by γδ T cells, which in addition, use their TCRs for recognition of phosphorylated antigens and still largely undefined ligands on tumor cells. In the haploidentical allo-HSCT setting, alloreactive NK cells, derived from donor HSCs, can exert potent antileukemia activity and kill residual patient DCs and T cells, thus preventing GvHD and graft rejection. However, generation of KIR(+) alloreactive NK cells from HSCs requires many weeks, during which leukemia relapses, and life-threatening infections may occur. Importantly, mature NK cells and γδ T cells can control certain infectious agents efficiently, in particular, limit CMV reactivation, and infusion of such donor cells at the time of HSCT has been implemented. Development of novel, cell-based immunotherapies, allowing improved trafficking and better targeting, will endow NK cells and γδ T lymphocytes with enhanced anti-tumor activity, also making them key reagents for therapies against solid tumors. The clinical aspects of using NK cells and γδ T lymphocytes against hematological malignancies, including the allo-HSCT context, are reviewed in the related side-by-side paper by Locatelli and colleagues [1].

Adoptive Immunotherapy with Polyclonal T Cells and Natural Killer Cells for Hematological Malignancies: Current Status and Future Prospects

SUMMARY Adoptive cellular therapy with polyclonal T cells and natural killer cells are immunotherapeutic modalities being studied in solid tumors and hematological malignancies to treat disease and prevent relapse. These include unexpanded polyclonal T cells, short-term activation by cytokine into lymphokine-activated killer cells, longer term expansion by cytokine stimulation giving rise to cytokine-induced killer cells or expansion under costimulation with beads expressing anti-CD3 and anti-CD28. Similarly natural killer cells can be given with or without activation and expansion. Here we review the published work and clinical trials involving each cell type in the autologous, matched allogeneic, haploidentical and nontransplant settings, comparing and contrasting each cell type and discussing their potential applications.

Long-term safety and survival outcomes after TK-expressing donor lymphocyte infusion (TK-DLI) in allogeneic hematopoietic stem cell transplantation (HSCT).

7007 Background: Suicide gene therapy (SGT) was firstly applied to allogeneic HSCT, addressing the need for modulation of graft vs host disease (GvHD) reactions while preserving graft vs leukemia (GvL) effect of alloreactive T cells. HSV-TK gene insertion in donor T-cells modulates alloreactivity by selectively destroying dividing alloreactive cells involved in GvHD. Methods: Long-term safety and survival was assessed in 128 pts entering worldwide 10 phase I-II trials that used TK-DLI to improve GvL, immune reconstitution (IR) and GvHD control. In all, 57 pts received TK DLI at our Institution: 23 to treat relapse after HLA-identical HSCT (Ciceri, 2007) and 34 to improve IR after haploidentical HSCT (Ciceri, Bonini, 2009). Results: SGT was feasible, safe and effective in promoting a dynamic and specific modulation of alloreactivity. TK-DLI clinical benefit, defined by chimerism, tumor response and/or IR, was achieved by 65 pts (51%). Grade 2 to 4 GvHD (n=28, 22%) was fully controlled by SGT. TK-DLI engrafted in 51 pts (90%) and, being detectable at low frequency up to 14 yrs, no SGT-related adverse events occurred. In HLA-identical setting (n=23; median follow-up, 15 yrs), 11 pts (48%) had disease response and 2 pts (9%) were alive in complete response (CR). In haploidentical setting (n=34; median follow-up, 7 yrs), 25 pts (73%) had IR and 9 pts (26%) were alive in CR. All pts were monitored according to guidelines on long-term survivors (Majhail, 2012). There were no major infections, while 3 pts had a second tumor. Immunity against TK-DLI was reported exclusively after HLA-identical allo-HSCT indicating that TK-DLI is not limited by SGT-specific immunity after haploidentical HSCT. Conclusions: Long-term follow-up confirms the high benefit to risk ratio of TK-DLI. A phase III trial is ongoing in haploidentical HSCT (NCT00914628). Clinical trial information: NCT00423124.

Sirolimus and post transplant Cy synergistically maintain mixed chimerism in a mismatched murine model

Because of the toxicity associated with myeloablative conditioning, nonmyeloablative regimens are increasingly being used in vulnerable patient populations. For patients with sickle cell disease, stable mixed chimerism has proven sufficient to reverse the phenotype. Because the vast majority of patients do not have an HLA-matched sibling, a safe nonmyeloablative regimen that could be applied to the haploidentical setting would be ideal. We employed a mismatched mouse model using BALB/c donors and C57BL/6 recipients. Recipient mice were conditioned with 200 cGy TBI and sirolimus or CSA with or without post transplant Cy (PT-Cy). Our data show that when sirolimus or PT-Cy alone is given to C57BL/6 recipients, donor cells are not detected. However, when sirolimus is administered for 15 or 31 days starting 1 day before or up to 6 days after transplant with PT-Cy, all mice maintain stable mixed chimerism. In contrast, conventional therapy employing CSA with or without PT-Cy does not result in stable mixed chimerism. Lastly, mice with stable mixed chimerism after sirolimus display decreased reactivity to donor Ag both in vitro and in vivo. These data identify a novel strategy for inducing mixed chimerism for the treatment of nonmalignant hematologic diseases.

Haploidentical Bone Marrow Transplantation Without T-Cell Depletion

Approaches for haploidentical bone marrow transplantation (BMT) without T-cell depletion have been designed using new transplant strategies, including anti-thymocyte globulin (ATG) preparative regimens, granulocyte colony-stimulating factor-primed grafts, post-transplantation rapamycin, or high-dose cyclophosphamide (Cy) in combination with other immunosuppressive agents for graft-versus-host disease (GVHD) prophylaxis. These strategies ensured fast hematologic engraftment across the human leukocyte antigen (HLA) barrier with an acceptable incidence of GVHD. Long-term follow-up results from different transplant centers suggest that unmanipulated transplantation may provide an alternative strategy in the haploidentical setting without requiring the technical expertise and cost of ex vivo T-cell depletion. This review discusses immune reconstitution and factors associated with clinical outcomes following unmanipulated haploidentical hematopoietic stem cell transplantation (HSCT), and compares outcomes between unmanipulated haploidentical transplant versus HLA-matched sibling donor (MSD) transplantation, HLA-matched unrelated donor (MUD) transplantation, or unrelated double umbilical cord blood (dUCB) transplantation. Advantages and disadvantages of unmanipulated haploidentical HSCT and strategies to improve outcome after haploidentical BMT without ex vivo T-cell depletion are discussed.

TK-Cells Safety and Efficacy in Allogeneic Hematopoietic Stem Cell Transplantation: Long-Term Clinical and Molecular Assessment in 128 Treated Patients.

Abstract 3061 Background and methods.Suicide gene therapy (SGT) applied to allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been one of the first clinical applications of gene therapy. In allo-HSCT the antileukemic potential mediated by alloreactive lymphocytes towards patient-specific antigens, such as minor and major histocompatibility antigens, is counterbalanced by the Graft-versus-Host-Disease (GvHD). The risk of GvHD increases with the level of HLA disparity between host and donor, and leads to impaired quality of life and reduced survival, particularly in patients (pts) transplanted from HLA-mismatched donors. In this contest SGT has been applied to modulate alloreactivity, by inserting the HSV-tk gene in donor lymphocytes. This suicide gene/prodrug system requires cell cycle for optimal killing. In the context of allo-HSCT, this characteristic ensures a further level of specificity in GvHD control, by allowing to selectively kill highly proliferating alloreactive cells during GvHD, while sparing resting T cells. We assessed long-term safety in TK cells treated pts. Results.Overall 128 pts have been treated worldwide in 10 phase I-II clinical trials with donor lymphocytes expressing the HSV-tk suicide gene, with the purpose of enforcing the graft-versus-tumor (GvT) effect and/or promoting a functional post-transplant immune reconstitution (IR) while allowing control of GvHD. This approach proved highly feasible, safe and effective in promoting a dynamic and patient-specific modulation of alloreactivity. TK cells engrafted in the majority of pts and a clinical benefit, measured as improvement of hematopoietic chimerism, malignant regression and/or IR, was reported for 65 pts (51%). GvHD grade II-IV was observed in 28 pts (22%) and was always rapidly and completely controlled by the activation of suicide machinery.A selected population of 57 pts, treated at San Raffaele Institution, was studied in more detail for long-term analysis: 23 pts received cells to treat relapse occurring after an HLA-identical allo-HSCT (Ciceri F et al, Blood. 2007: 109; 4698–470) and 34 pts to improve IR after haploidentical HSCT (Bonini C et al, Blood. 2002; 100: 115a; Ciceri F, Bonini C et al, Lancet Oncology. 2009; 10: 489–500). No adverse event correlated to the gene transfer procedure was ever reported.Genetically modified cells engrafted in 90% of treated pts and could be detected in vivo, at low frequencies for up to 14 years (y).In the HLA-identical setting 11 pts obtained clinical response of the malignant disease and 3/11 are alive with a median follow-up of 15y. Two pts are in complete remission (CR), while one pt affected by chronic myelomonocytic leukemia relapsed 15y post transplant. This pt was subsequently treated with a second transplant from an unrelated match donor and is now in CR 1 year after transplant.In the haploidentical setting, 25 pts/34 reached the target of IR and 9 are alive and in CR with a median follow-up of 7y. Four out of 9 experienced GVHD in the early phase post IR, none presented signs, symptoms, complications related to GvHD and none needed pharmacological treatment in long-term.According to international guideline for long-term follow-up (Majhail NS et al, BBMT. 2012; 18: 348–371) all pts underwent regular screening and clinical evaluation. No major infections occurred in the late phase, no ocular, oral, respiratory, hepatic, renal, genitourinary, muscle-skeletal or neurological distress. Noteworthy 3 pts developed second cancer (2 skin cancer non-melanoma, 1 endometrial cancer), 2 pt hypothyroidism, 1 pt coronary artery disease and 3 pts metabolic syndrome. No evidence of psychosocial symptoms was reported.Immunity against the transgene was reported in pts who received TK cells late after an HLA-identical allo-SCT. The quality of IR at the time of TK cells administration was the most predictive variable. On the contrary, no immunity against HSV-tk was ever detected in pts who received TK cells after transplantation of haploidentical CD34-selected cells, indicating that the infusion of TK cells to highly immunosuppressed pts is not limited by the development of transgene-specific immune responses. Conclusions.Long-term assessment confirmed the overall high benefit/risk ratio of the TK-cell approach in allo-HSCT.A phase III multicentric, randomized clinical trial sponsored by MolMed, is currently undergoing in the context of haploidentical HSCT. Disclosures:Bordignon:MolMed SpA: Employment. Bonini:MolMed S.p.A.: Consultancy.

Donor Lymphocyte Infusions (DLI) Following Haplo Bone Marrow Transplantation (hBMT) with High-Dose of Cyclophosphamide Post-Transplant As Gvhd Prophylaxis

AbstractAbstract 1950 Background:In the haploidentical setting, the use of donor lymphocyte infusions (DLI) is associated with a significant risk of acute and chronic graft vs host disease (GvHD): in one report, a median T-cell dose of 2.4 × 10⋀8/kg was administered to recipients of unmanipulated haploidentical stem cell transplants, and a cumulative incidence of chronic GVHD of 50% was reported (Huang X, BBMT 2009). It is possible that patients receiving high dose Cyclophosphamide post-transplant (PT-CY) may be able to receive DLI with a lower risk of GvHD Aim of the study:We tested the feasibility and occurrence of GVHD, following 32 DLI administered in sixteen patients, who relapsed after an unmanipulated haploidentical T-cell replete BMT, and PT-CY. Patients and Methods:All sixteen patients were transplanted from haploidentical related donor, after a myeloablative conditioning (n=10) or a non myeloablative conditioning (n=6) with PT-CY as GVHD prophylaxis, in association with CyA and micophenolate. The diagnosis was Hodgkin’s lymphoma (n 6), acute lymphoblastic B-cell leukemia (n=3), acute myeloid leukemia (n=5), pro-lymphocytic T-cell leukemia (n=1), chronic myeloid leukemia blast crisis (n=1). The median time of relapse was 211 days from transplant (range 45–697 days); the median time at DLI was 48 days from relapse (range 29–540 days) and 252 days from transplant (range 109–690). Ten patients had hematologic relapse, while six had molecular relapse.At time of first DLI all patients were off GVHD prophylaxis.The minimum dose of lymphocytes was 1 × 103′/kg (n=2); higher doses were as follows: 1 × 104′/kg (n=13), 1 × 105′/kg (n=11), 5 × 105′/kg (n=4), 1 × 106′/kg (n=2). The median number of DLI/patient, was 1 (range 1–5). In eleven cases DLI followed chemotherapy: gemcitabine or bendamustine in Hodgkin’s lymphoma and pro-lymphocytic leukemia; fludarabine, ara-C and antracycline containing regimens in acute leukemia. Median time of DLI was 12 days from chemotherapy (range 10–14 days), during the chemotherapy-induced nadir. Five patients received DLI alone. Results:The infusions were well tolerated and no major adverse effect was observed.The cumulative incidence of acute GvHD grade II-III, was 6%, and 0% for chronic GVHD. No aplasia related to haplo-DLI occurred.Five patients died with progressive disease; 11 patients are surviving (69%), 4 of them are disease-free, seven have active disease; two patients relapsed after 313 and 135 days from DLI, respectively. The median follow-up is 221 days (range 46–559 days). Conclusions:This study suggests that patients grafted with haplo-mismatched BMT and high dose post-transplant cyclophosphamide, can be treated with DLI at doses up to 1× 106′/kg, with a very low risk of developing acute or chronic GVHD. Disclosures:No relevant conflicts of interest to declare.

Evaluation of NIH-Defined Chronic Graft-Versus-Host-Disease in a Rapamycin-Based Haploidentical Stem Cell Transplantation: Analysis of 113 Consecutive Patients with High Risk Haematological Malignancies

Abstract 4198 Background and Methods.Chronic Graft-versus-Host-Disease (GvHD) remains a serious complication after allogeneic stem cell transplantation. In 2005 the National Institutes of Health (NIH) established new criteria for chronic GvHD based on retrospective data and expert recommendations.Here we are reporting the prospectively evaluation of the incidence of NIH-defined chronic GvHD and its prognostic impact in 113 consecutive patients who underwent unmanipulated peripheral blood graft from an HLA-haploidentical relative after a treosulfan based conditioning at our Institution.The peculiarity of this setting relies on the exploitation of a rapamycin-based, calcineurin-inhibitor-free regimen for prophylaxis of GvHD to induce in-vivo expansion of T-regulatory cells (Tregs). Results.Chronic GvHD was evaluable in 81 patients: 41 patients were diagnosed with chronic GvHD according to the revised Seattle criteria, 28 were classified as having extensive chronic GvHD and 13 were classified as having limited. The 1-year cumulative incidence was 43 +/− 9%.According to the NIH Consensus Criteria, 1 patient was reclassified as having late-acute GvHD, 20 patients as having overlap syndrome and 20 patients as having classic chronic GvHD. The late-acute consisted of late onset subtype.The 40 patients with classic chronic GvHD and overlap syndrome were graded as moderate (26 patients) and severe (14 patients) by NIH consensus criteria. The onset was classified as progressive in 4 cases, de novo in 27 cases, quiescent in 9 cases.The median number of organs involved was 3 (range 1–7), the majority of patients presented with skin and oral involvement, but also eyes, liver, joint, genitalia and gastro-intestinal damages were present – maximum severity of chronic GvHD according to organ manifestation is presented in figure 1.Twenty-seven out of 40 patients started a specific immunosuppressive treatment for chronic GvHD, noteworthy 21 patients restarted rapamycin (in combination with prednisone, or alone) with clinical benefit.In univariable analysis patients with overlap GvHD tended to present a lower overall survival (OS) compared to those with classic chronic GvHD (36% versus 63%, p 0.07). Patients with severe chronic GvHD at onset had a significantly lower OS compared to those with moderate chronic GvHD (19% versus 69%, p0.017). OS was significantly worse for patients with platelets counts below 100 g/l at onset of chronic GvHD (29% vs 67%, p0.003)– figure 2. Conclusion.This analysis confirms in haploidentical setting thrombocytopenia, overlap category and severe presentation as prognostic items for overall survival after allogeneic stem cell transplantation. Further investigations on implementation of immunosuppressive treatment strategy for high-risk GvHD patients (namely patients with overlap GvHD diagnosis and concomitant thrombocytopenia and/or progressive onset) are actually ongoing to enhance response and improve outcome. [Display omitted] [Display omitted] Disclosures:Bordignon:MolMed SpA: Employment. Bonini:MolMed S.p.A.: Consultancy.

Haploidentical hematopoietic stem cell transplantation with unmanipulated granulocyte colony stimulating factor mobilized marrow and blood grafts

This article summarizes recent improvements and progress with unmanipulated haploidentical blood and marrow transplantation (HBMT) and discusses the difference in outcomes between patients receiving HBMT and those receiving unmanipulated granulocyte colony stimulating factor (G-CSF) mobilized haploidentical peripheral blood (G-PB) grafts as allografts. Long-term follow-up confirmed that unmanipulated HBMT is a promising protocol that can be successfully extended to treat severe aplastic anemia. Recent observations regarding immune recovery, infections, and strategy for modified donor lymphocyte infusions have provided insight into the prevention of infections and the decrease in relapse after HBMT. Extensive chronic graft-versus-host disease (GVHD) strongly and negatively impacts patient health-related quality of life, suggesting that it should be successfully controlled. A prospective study suggested the inclusion of HBMT in treatment algorithms as a viable option for adults with acute myeloid leukemia with unfavorable cytogenetics who lack a matched donor. Randomized clinical trials are warranted to investigate whether mixture grafts of G-CSF-mobilized blood and marrow or G-PB alone should be chosen as allografts in haploidentical settings. Unmanipulated HBMT is a reliable protocol. New strategies should be investigated to decrease the incidences of GVHD and relapse. Novel mobilization regimens such as AMD3100 alone or G-CSF+AMD3100 for allograft engineering may improve transplant outcomes following HBMT.

Overview of T-cell depletion in haploidentical stem cell transplantation.

Overview of T-cell depletion in haploidentical stem cell transplantation.

Immune Reconstitution in Recipients of Photodepleted HLA-Identical Sibling Donor Stem Cell Transplantations: T Cell Subset Frequencies Predict Outcome

We evaluated an exvivo photodepletion (PD) technique to selectively deplete graft-versus-host disease (GVHD) alloreacting T cells given to 24 human leukocyte antigen (HLA)-identical sibling stem cell transplantation (SCT) recipients. Donor lymphocytes were activated by 72-hour exposure to irradiated invitro expanded recipient T lymphocytes and pulsed with a TH9402 photosensitizer. Alloactivated T cells preferentially retaining the photosensitizer were eliminated by light exposure. The PD product showed an inverted CD4(+)/CD8(+) ratio with greatest depletion occurring in the CD4(+) naive and central memory populations. In contrast, the CD8(+) naive and effector cells were relatively conserved, reflecting the differential extrusion of TH9402 by T cell subsets. Cytomegalovirus reactive T cells were reduced in the PD product and in recipient blood 100 days after SCT when compared with contemporaneous HLA-identical sibling donor T cell-depleted SCT recipients. Although PD SCT recipients experienced similar absolute lymphocyte counts during the first 100 days after SCT, they achieved 100% donor T cell chimerism more rapidly and had higher CD8(+) naive T cell counts early after SCT. SCT recipients of PD products with the lowest CD4 central memory content had the highest risk of developing chronic GVHD (cGVHD) (P = .04) and a poorer survival (P = .03). Although the persistence of CD8(+) naive T cells may have contributed to important antileukemia responses resulting in a relatively low relapse rate, our findings emphasize the role of donor memory T cells and CD4 cells in establishing immune competence post-SCT. Although PD is associated with excellent outcomes in the haploidentical setting, the low frequency of alloactivations in HLA-matched pairs makes the PD approach used by our group for allodepletion in HLA-matched sibling transplantations an inefficient technique.

In Vitro or In Vivo Expansion Before Adoptive T-Cell Therapy?

carried out in a GMP environment for a prolonged period. In addition, the cytotoxic T‐lymphocyte products may not be fully HLA compatible, thus increasing the risk of complications such as graft‐ versus‐host disease where, instead of attacking the virus target specifically, the adopted T cells attack other tissues of the recipient. Fortunately, with the new techniques and tools of today and a broader knowledge of T‐cell epitopes, faster and more cost–effective approaches have emerged. The invention of combining several recombinant HLA/peptide molecules with streptavidin and a fluorochrome created a revolutionary tool to both study and separate specific T cells of interest [9]. Cobbold et al. successfully used this labeling technique, combined with magnetic separation, to infuse CMV‐specific T cells from the donor following transplantation [10]. In this case, the numbers of cells infused are obviously much smaller and trust must be put in the capacity of an in vivo antigen‐driven expansion. This separation has also been modified by our group for EBV in a haploidentical setting, to rapidly treat life‐ threatening PTLD [11]. The EBV‐specific T cells from the patient’s mother apparently expanded in vivo and were detected over a prolonged period of time following infusion. The clinical effect was dramatic, despite widespread tumors, and the patient is presently alive, 3 years after treatment. In the last few years, the clinically adapted IFN‐g capture assay (Miltenyi Biotec, Germany) has been tested in several Phase I and II studies with promising results [12]. This approach requires a 6‐h step for activation of the blood product with relevant antigens before separation of specific T cells based on the production of IFN. So what is preferable – a time‐consuming in vitro expansion creating a massive amount of cells, or direct separation and infusion of only a few cells? Over the past decade, there has been a consider‐ able evolution in the field of immune therapy, including the use of specific T cells as a treat‐ ment modality for various infectious agents and cancers. As early as two decades ago, Riddell et al. successfully documented the first use of donor‐derived T cells to treat life‐threatening cytomegalo virus (CMV) disease following allo‐ geneic stem cell transplantation (ASCT) [1]. This approach was also quickly adopted to treat Epstein–Barr virus (EBV)‐associated post‐ transplantation lymphoproliferative disease (PTLD) [2]. It was common practice in those days to use a laborious protocol for the expansion of T cells over several weeks, including restimu‐ lations with either EBV‐immortalized cell lines or donor‐derived cells pulsed with the relevant antigens [3,4]. This approach, with modifications, is still used successfully today by several groups to counteract both cancer and virus diseases [5,6].

Use of G-CSF-stimulated marrow in allogeneic hematopoietic stem cell transplantation settings: a comprehensive review

In recent years, several researchers have unraveled the previously unrecognized effects of granulocyte colony-stimulating factor (G-CSF) on hematopoiesis and the immune cell functions of bone marrow in healthy donors. In human leukocyte antigen-matched or haploidentical transplant settings, available data have established the safety of using G-CSF-stimulated bone marrow grafts, as well as the ability of this source to produce rapid and sustained engraftment. Interestingly, G-CSF-primed bone marrow transplants could capture the advantages of blood stem cell transplants, without the increased risk of chronic graft-versus-host disease that is associated with blood stem cell transplants. This review summarizes the growing body of evidence that supports the use of G-CSF-stimulated bone marrow grafts as an alternative stem cell source in allogeneic hematopoietic stem cell transplantation.

A Potent Thymic Function Is Associated with a Low Risk of Relapse In Leukemia Patients Treated with Haploidentical Stem Cell Transplantation

AbstractAbstract 1258Hematopoietic Stem Cell Transplantation (HSCT) is an effective treatment for many malignant and non malignant diseases in children. Since less than 30% of the patients have an HLA identical related donor, alternative donor/sources of Hematopoietic Stem Cells need to be considered, such as unrelated bone marrow (BM) and Cord Blood (CB) donors or HLA-haploidentical related donors. However, these types of HSCT are associated with a significant delay in immune reconstitution that favors both a high incidence of opportunistic infection and, in the absence of an alloreactive, natural killer cell-mediated effect, disease relapse. Indeed, T-cell depletion of the allograft in the haploidentical setting or T-cell naivety in CB, as well as HLA-disparity and use of serotherapy before HSCT, contribute to this impaired immune reconstitution. In view of the role played by the thymus in immune reconstitution post-allogeneic HSCT, it would be informative to compare thymic-dependent immune reconstitution after these types of HSCT.We have studied immune reconstitution and thymic function through signal joint (sj) and beta T cell Receptor Excision Circles (TREC) quantification in a group of 33 haplo-HSCT pediatric patients and in a group of 24 unrelated CB-HSCT. Patients were transplanted mainly for hematological malignancies (n=46, including 31 Acute Lymphocytic Leukemia and 9 Acute Myeloid Leukemia). All children received a myeloablative conditioning regimen, including the combination of total body irradiation and chemotherapy (n=33) or chemotherapy alone (n=24). In haplo-HSCT, no pharmacological immune-suppression was given after the allograft, while patients given CB-HSCT received cyclosporine-A and steroids as Graft vs. Host Disease (GvHD) prophylaxis. In haplo-HSCT patients the median number of CD34+ cells infused/kg was 20.7 × 106 (range 8.7–41), while in patients given CB-HSCT the median number of nucleated cells infused/kg was 7.1) × 107 (range 1.4–12.5). The only significant difference between the 2 groups was that haplo-HSCT patients were older (p=.0008) than CB-HSCT patients (median age being 7.7 and 3.4 yrs, range 3–17 and 0.75–16 yrs, respectively). Patients treated for an hematological malignancy had a significant lower pre-transplant TREC value (p = .0002 and .004 for sj and beta TREC respectively) than those affected by non-malignant diseases in both groups. Number of sj and betaTREC per 150 000 PBMC or absolute counts per μl of blood, showed a very similar thymic function in both groups despite the older age of haplo-HSCT patients. TREC levels were comparable to those found before transplantation starting from 6 months after HSCT. Cumulative incidence of acute or chronic GvHD was low in both groups, with no significant impact on thymic function.In haplo-HSCT, but not in CB transplantation, patients treated for hematological malignancies (n=27) who relapsed (n=8) had a significantly lower level of thymic function (sjTREC) than those who did not relapse (n=19), before (p=0.03), after 3 (p=0.014) and 6 months (p=0.015) from the allograft. In this group of patients, relapse was not associated with age, conditioning regimen or number of CD34+ cells infused. In conclusion, we demonstrate the crucial importance of thymic function in the Graft-vs. Leukemia effect in the haplo-HSCT setting. Monitoring thymic function could be of predictive value in these patients. Disclosures:No relevant conflicts of interest to declare.

Chronic Myelomonocytic Leukemia and Related Disorders Provide Particular Disease-Specific Challenges to Effective Bone Marrow Transplant: a Multicenter Experience.

AbstractAbstract 4561Chronic myelomonocytic leukemia (CMML) typically has a dire prognosis with limited treatment options. We retrospectively reviewed the outcomes of 41 patients diagnosed with CMML (n= 35) or MDS/MPD overlap (n=6) who underwent allogeneic stem cell transplantation at three centers (University of Minnesota (n= 19), Johns Hopkins University (n=11), and the Cleveland Clinic (n=11)) between1990-2009. The majority of patients were male (59%) with a mean age of 51. At diagnosis nearly half of the patients had normal cytogenetics (n=20, 49%) with abnormalities of chromosome 7 the most commonly identified clonal finding (n=5, 12%). The majority had <5% blasts (n=22, 54%), and the majority had an MD Anderson Prognostic Score (MDAPS) of 2 (n=16, 39%) or 3 (n=13, 32%) at diagnosis. Fifteen patients (37%) received no pre-transplant therapy while 12 (29%) received hydroxyurea, 11 (27%) induction-type chemotherapy, and 3 (7%) miscellaneous other therapies. Nine patients (22%) had progressed to AML prior to transplant. At the time of transplant, blast percentage was <5% in the majority (n=23, 56%) of patients and more than half of patients now had a MDAPS of 1 (n=13, 32%) or 2 (n=12, 32%). Comorbidity index (HCT-CI) at transplant was retrospectively calculated on 32 patients and was 0 (n=5, 12%), 1–2 (n=13, 32%), or 3+ (n= 15, 37%). Myeloablative conditioning was used in 23 (56%) and stem cell source was bone marrow in 14 (34%), PBSC in 16 (39%), and UCB in 9 (22%). The majority of donors were matched siblings (n=22, 54%) while unrelated donors (URD) (n=16, 39%) and haploidentical donors (n=3, 7%) comprised the remaining. Graft versus host disease (GVHD) prophylaxis consisted of a calcineurin inhibitor (cyclosporine or tacrolimus) + methotrexate or mycophenolate mofetil (MMF) in the majority of patients (n=29, 71%) with the remainder receiving post-transplant cyclophosphamide (n=6, 15%), elutriation (n=2, 5%), or calcineurin inhibitor alone (n=4, 10%). While the median time to follow-up was only 5.8 months (range 0.5–140), 24 patients had died by one year and there was extensive follow-up available in the surviving patients. Overall survival (OS) for the entire cohort at 1 and 3 years was 41% (95% CI, 26–56%) and 16% (95% CI, 6–30%), respectively. No disease or transplant factors significantly impacted survival. Progression free survival (PFS) at 1 and 3 years was 29% (95% CI, 16–43%) and 16% (95% CI, 7–29%), respectively. Sibling donor showed improved PFS at both 1 (45%, 95% CI 24–64%), (p=0.027) and 3 (27%, 95% CI 10–46%), (p=0.04) years. Transplant related mortality (TRM) at Day +100 and 1 year was 27% (95% CI, 13–40%) and 41% (95% CI, 26–57%) respectively. Age at transplant, year transplanted, HCT-CI at transplant, conditioning intensity, donor source, transplant site, or presence of acute graft versus host disease (GVHD) did not impact TRM. Relapse at 1 and 3 years was 29% (95% CI, 15–44%) and 40% (95% CI, 23–56%), respectively. High MDAPS at diagnosis and sibling donor source were the only significant factors impacting relapse. At 1 year, those with an MDAPS of 3–4 had a 53% chance of relapse compared with 29% with a score of 0–1 (p=0.05) and those with a sibling donor had a 9% relapse incidence compared with 50% in the URDs and 67% in the haploidentical setting (p=0.003). Interestingly the presence of acute GVHD at Day +100 was not protective against relapse (40% incidence at 1 year in those with aGVHD versus only 19% for those without). Our data suggest that high MD Anderson Prognostic score at diagnosis predicts for high incidence of relapse post allogeneic stem cell transplantation while a sibling donor source improves rates of relapse and progression free survival. Our data highlight the need for improved CMML treatment paradigms. Augmentation of pre and post transplant therapy including maintenance therapy post transplant are possible approaches to improve outcomes and could be considered for prospective trials. Disclosures:No relevant conflicts of interest to declare.

CMV-Reactive γ/δ T Cells Are Equivalent to Their α/β Counterparts In the Lysis of CMV-Infected Targets – Potential for Post Transplant Adoptive Immunotherapy.

AbstractAbstract 1277Reactivation of cytomegalovirus (CMV) post allogeneic stem cell transplantation (SCT) can be prevented and treated by adoptive immunotherapy with donor-derived alpha/beta CMV-reactive T cells (CMV-T). We have reported that VD1+ gamma/delta T cells arising post allogeneic SCT are also CMV-reactive and that these cells can be identified in and isolated from CMV-seronegative donors (Knight et al 2010). The relative ability of VD1 gamma/delta cells to lyse CMV-infected targets compared to CMV-reactive alpha/beta T cells has not been determined.Here we have isolated gamma/delta T cells (VDelta 1) from normal, healthy HLA-A2+, CMV-seropositive donors and tested them freshly isolated and after expansion on allogeneic feeder cells (as described in Knight et al 2010) for their ability to lyse MRC5 fibroblasts infected with CMV strain VHLE in vitro. In parallel we isolated HLA-A2 restricted CMV-reactive alpha/beta T cells using a CMV-specific pentameric HLA reagent (ProImmune Ltd), rested overnight to ensure reagent release and tested for lysis of the same CMV-infected targets.MRC5 fibroblasts were cultured in MEM/10%FCS and infected with VHLE (kind gift from Dr Sinzger, Tubingen, Germany) at an MOI of 1 for three days. Infected cells were recovered by trypsinisation and mixed with effector cells at 5:1 and 10:1 E:T ratios in duplicate in a four hour flow cytometric killing assay. Dead fibroblasts were identified by failure to exclude ToPro Iodide. Control cultures were established with uninfected MRC5 and “percent specific lysis” calculated by subtraction of the degree of lysis of uninfected cells from that of the infected targets at each E:T ratio.Lysis of non-infected MRC5 by both alpha/beta CMV-T cells and VD1 gamma/delta T cells was low (0.7%-13.2%) and was not significantly different between the two cell subsets. In contrast alpha/beta CMV T were highly lytic of CMV-infected targets (mean 33.85, sd 1.48) as were VD1 gamma/delta T cells (mean 26.3% sd 3.53) at 5:1 E:T. There was no significant difference between the specific lysis mediated by alpha/beta CMV-T compared to matched-pair VD1 gamma/delta T cells (p>0.05); nor was there any significant difference in CMV-specific lysis by freshly isolated versus ex-vivo cultured VD1 gamma/delta T cells from these donors. The specific lysis mediated by the VD1 gamma/delta T cells from these CMV seropositive donors was comparable to that we previously reported from CMV seronegative donors although slightly higher.Since clinical trials of alpha/beta CMV-T have shown their protective effect as adoptive immunotherapy post transplant, the fact that we show comparable lytic function by VD1 gamma/delta T cells suggests that these cells may also be clinically useful since they can be readily isolated without functional selection and are known not to mediate GvHD even in the haploidentical transplant setting. More importantly, since the lytic function of VD1 gamma/delta T cells from CMV seropositive donors is comparable to that of VD1 gamma/delta T cells from seronegative individuals we are encouraged that these cells could be used for successful adoptive immunotherapy of CMV in the “CMV+ve receipient:CMV-ve donor” transplant setting. Disclosures:No relevant conflicts of interest to declare.

An Improved Bicistronic CD20/tCD34 Vector for Efficient Purification and In Vivo Depletion of Gene-Modified T Cells for Adoptive Immunotherapy

T-cell-based adoptive immunotherapy is widely used to treat graft rejection and relapse after stem cell transplantation (SCT). However, this approach is hampered by a high risk of life-threatening graft-versus-host-disease (GvHD). Clinical trials have demonstrated the value of suicide genes to modify T cells for the effective control of GvHD. Herewith, we show that the combination of a codon-optimized B-cell antigen (CD20op) with a selection marker based on a cytoplasmic truncated version of the human stem cell antigen CD34 (tCD34) allows the generation of highly enriched gene-modified T cells. We demonstrate coordinate co-expression of both transgenes and high expression of CD20op resulting in an increased susceptibility to Rituximab (RTX)-induced cell death. In addition, T cells partially retained their alloreactive potential and their CD4/CD8 ratio after transduction and expansion. Long-lasting transgene expression was sustained in vivo after adoptive transfer into Rag-1(-/-) mice. Moreover, gene-modified T cells were quickly and efficiently depleted from peripheral blood (PB) and secondary lymphoid organs of transplanted animals after RTX treatment. These results warrant further steps toward a clinical application of CD20op as a suicide gene for adoptive immunotherapy.

Transplant Outcomes in Acute Leukemia (II)

Currently, it is possible to find a hematopoietic stem cell (HSC) donor for virtually all patients with acute leukemia who have an indication to receive an allogeneic hematopoietic stem cell transplant (HSCT) and lack a human leukocyte antigen (HLA)-identical sibling or a well-matched HLA unrelated donor (URD). According to the ethnicity of the patients and the donor registry, approximately 25% to 60% of patients will not find an 8/8 HLA-matched unrelated donor. Other alternative donors, such as HLA-mismatched related donor or unrelated donor umbilical cord blood (UCB), have emerged to solve the lack of a sibling or well-matched URD. In the haploidentical HSCT setting, new techniques of T-cell depletion, new approaches using combinations of immunosuppressive drugs or different conditioning regimens, and developments on immunotherapy have focused attention on this option. Therefore, any physician has to carefully evaluate, for each patient in need of an allograft, all of the possible alternatives in order to choose the best HSC donor, taking into account type of disease to be transplanted, urgency of transplantation, donor characteristics, and center experience. This review evaluates the current status of haploidentical HSCT in acute leukemia, its advantages and remaining limitations compared to other stem cell sources, and how these data may be used in the development of donor selection algorithms.

Post-Transplant Cyclophosphamide and Sirolimus Are Synergistic in Preventing Rejection and Inducing Stable Mixed Chimerism Independently of Regulatory T Cells.

Abstract 3540Poster Board III-477A strategy that can induce stable mixed chimerism across human leukocyte antigen (HLA) barriers would be beneficial in extending the application of hematopoietic stem cell transplantation (HSCT) to patients with severe sickle cell disease (SCD) who are in need of this potentially curative procedure. Indeed, we have recently demonstrated the feasibility of an HLA-matched sibling protocol employing low dose total body irradiation (TBI, 300cGy), the lymphocyte depleting agent alemtuzumab, and sirolimus to reverse the phenotype with minimal side effects. Due to the lack of HLA-matched siblings in the majority of patients, our goal is to develop a safe haploidentical regimen. In this work, we focused on determining optimal postgrafting immunosuppression and examined sirolimus and post-transplant cyclophosphamide (PT-cy), agents known to induce transplantation tolerance. To determine the optimal sequence for combining these drugs and whether this combination is synergistic in promoting stable donor chimerism despite the antiproliferative effects of sirolimus, we used a mismatched murine model with BalbC donors and C57Bl6 recipients. Twenty-five to 40 recipient mice received 200cGy TBI and PT-cy (200mg/kg intraperitoneally (IP) 2 days post transplant) with or without sirolimus (3mg/kg IP) for 14 to 30 days starting 1 day before or 4, 6, or 10 days post transplant. We found that in contrast to sirolimus or PT-cy alone, the combination of PT-cy and a limited course of sirolimus resulted in stable mixed chimerism: all mice that received PT-cy and sirolimus starting between 1 day before and 6 days after transplant attained donor chimerism levels ranging from 15-35%. Further, a 14 day course of sirolimus was sufficient to maintain stable mixed chimerism in our model (See Figures 1 and 2). To examine whether this synergistic effect is mediated by regulatory T cells, we administered anti-CD25 monoclonal antibody (CD25 mAb), an agent known to transiently deplete these cells in vivo. Fifteen mice received 200cGy TBI, sirolimus, PT-Cy, and either no CD25 mab, CD25 mab (1mg IP) on 7 and 3 days before and 1 day after transplant, or CD25 mab starting 14 days after transplant. CD25 mab was given biweekly for 5 weeks to mice in both groups. Donor engraftment levels did not differ in the three groups, with donor chimerism levels ranging from 30-40%. Our data show that the anti-proliferative effects of sirolimus do not inhibit the efficacy of the cytotoxic agent cyclophosphamide. Rather, our data demonstrate that the combination of PT-cy and a limited course of sirolimus synergistically promote mixed bone marrow chimerism in a complete mismatched setting. Further, the synergistic effect of this drug combination appears to be mediated independently from CD25+ regulatory T cell expression. In light of our previous success using sirolimus in an HLA-matched HSCT protocol, these findings lay the groundwork for developing PT-cy and sirolimus as a novel, safe, and effective means of promoting stable mixed chimerism in the haploidentical setting and thus greatly enhancing our ability to successfully apply this approach to patients with severe SCD. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Alloreactive natural killer cells in targeting high-risk leukaemias

Natural killer (NK) cells express surface receptors that regulate potent effector functions, such as cytolytic activity and release of cytokines, which play a central role in inflammatory response and immunoregulation....