ALBERT

Description: The standard cytoreduction for bone marrow transplantation (BMT) of pts with HGBpathies has been the combination of Busulfan (BU) and Cyclophosphamide However, for pts with advanced disease, high rates of graft rejection and toxicity were reported. For such high risk pts, a BU and Fludarabine (FLU) cytoreductive combination was used at low dose in the context of non-myeloablative BMT and was associated with a poor outcome with high rates of graft rejection. In view of the low toxicity associated with the BU-FLU combination, we used both agents at higher, potentially myeloablative doses for the cytoreduction of six pts with high risk HGBpathies. Between 12/00 and 04/05, 4 pts with thalassemia (Thal) and 2 pts with sickle cell disease (SCD), including 2 males and 4 females aged 4.6–15.3 years were transplanted using this regimen. All 4 pts with Thal had advanced Lucarelli Class 2 disease, while the 2 pts with SCD had stroke, recurrent vaso-occlusive crises (VOC), sickle lung disease and alloimmunization (n=1) and recurrent VOC, acute chest syndrome and osteomyelitis (n=1). Cytoreduction included intravenous BU (0.8–1 mg/Kg/dose x 14), FLU (30 mg/m2/day x 5) and Rabbit ATG (2.5 mg/Kg x 2). GvHD prophylaxis consisted of Tacrolimus (n=3) or cyclosporine (n=3) and methotrexate (n=5) or Steroids (n=1). Pts received an unmodified BMT from their HLA-identical sibling with total nucleated cell doses of 0.7–5.7 x 108 cells/Kg. The regimen was well tolerated with minimal toxicity. With a median follow-up of 21.5 mo (range 3–55 mo), all 6 pts are disease- and transfusion- free. There was no graft rejection and no GvHD. Chimerism status for pts with Thal was 98–100% donor, 5 mo to 2 years post BMT, while for the 2 pts with SCD, it was mixed with 50 and 80% donor cells at 3 mo and 2 years post BMT. The pt with 50% donor cells received a low graft cell dose (0.7 x 108 nucleated cells/Kg). One additional pt with SCD and a history of 2 strokes and moya-moya disease received BU FLU + melphalan (70 mg/m2 x 2). This pt had minimal post BMT toxicity, and is engrafted with 100% donor cells at 1 year post BMT. In summary, the combination of high dose BU and FLU for pts with high risk HGBpathies was well tolerated and induced full engraftment for pts with Thal, but mixed chimerism for pts with SCD. Nevertheless, all pts so treated survive with normal hematologic function. The addition of melphalan to BU FLU was also well tolerated and could be beneficial in attaining complete myeloablation and full chimerism in pts with SCD.

Description: Natural killer (NK) cells are capable of cytotoxic targeting of virally infected cells and some tumor cells. It has been well-demonstrated that NK cells recognize target cells that have down-regulated MHC class I antigen expression (i.e. “missing self recognition”), and that it is the lack of class I engagement of inhibitory receptors such as the killer Ig-like receptors (KIR) that thereby allows NK activation and effector function. How these same inhibitory receptors achieve self-tolerance and simultaneously avoid autoimmunity in humans has not been clear, as more than 60% of individuals have inhibitory KIR for which they lack the HLA ligand. We demonstrate that mature NK cells achieve self-tolerance by preferentially endowing functional competence to the inhibitory KIRs for which they exhibit the cognate HLA ligands. To allow evaluation of inhibitory KIR and avoid interference from potentially class-I recognizing activating KIR, we analyzed NK cells from 10 individuals with various HLA backgrounds, but who were all homozygous for KIR haplotype-A. KIR haplotype-A contains the inhibitory KIR receptors 2DL3, 2DL1, and 3DL1, specific for HLA-Cw3, -Cw4, and -Bw4 ligands respectively, in addition to at most one other activating KIR whose ligand is unknown. Using 6-color staining and flow cytometric analysis of intracellular IFN-γ production, we evaluated the responsiveness of 30 inhibitory KIR-expressing NK subsets following activation with 721.221, a target cell line deficient in class I expression, with 721.221 transfectants expressing HLA-Cw3, -Cw4, or -Bw4 ligands, and with B-lymphocyte cell lines with diverse HLA phenotypes. NK cells exclusively expressing an inhibitory KIR for self-HLA demonstrated increased IFN-γ when coincubated with target cells lacking the cognate HLA ligand, whereas NK cells exclusively expressing an inhibitory KIR for non-self HLA were hyporesponsive to all targets. In all individuals, NK cells expressing inhibitory KIR specific for self-HLA were significantly more responsive than NK cells expressing inhibitory KIR for non-self HLA (p

Description: The Th1 and Th2 lineages of CD4+ T helper cells are essential for control of host infection. Both lineages respond to antigenic stimulation with distinct effector functions and cytokine profiles. Differential homing patterns permit localization within specific tissue sites where these cells interact with other immune cells to promote the immune response. Variability in T helper lineage homing is due, in part, to differing chemokine receptor expression patterns. This laboratory and others recently described another CD4+ T helper lineage, Th17. Following stimulation, Th17 cells also produce a unique cytokine profile, including interleukin (IL)-17, IL-21, and IL-22. The Th17 lineage has now been implicated in the pathogenesis of several human autoimmune diseases, including psoriasis and inflammatory bowel disease, and appears to be critical for the inflammation of both the skin and gastrointestinal tract, respectively, seen in these diseases. It is not well understood whether Th17 cells arise within the inflammatory milieu in these tissues, or whether these cells possess a distinct homing pattern. We have performed studies using in vitro polarized Th17 cells for the study of tissue homing patterns in vivo. Experiments were performed using the well-described HLA Class II-disparate C57BL/6 (B6) to B6.C-H-2bm12 (bm12) model. Previous studies have established CD4+ T cell-dependent inflammation in this model. Naïve CD4+ T cells from B6 mice were polarized to the Th17 lineage in vitro using standard techniques, including IL-6 and TGF-β. FACS analysis of the Th17 cells prior to adoptive transfer revealed IL-17-positive staining in 〉60% cells and IFN-γ-positivity in

Description: Human graft-versus-host disease (GVHD) biology beyond 3 months after hematopoietic stem cell transplantation (HSCT) is complex. The Applied Biomarker in Late Effects of Childhood Cancer study (ABLE/PBMTC1202, NCT02067832) evaluated the immune profiles in chronic GVHD (cGVHD) and late acute GVHD (L-aGVHD). Peripheral blood immune cell and plasma markers were analyzed at day 100 post-HSCT and correlated with GVHD diagnosed according to the National Institutes of Health consensus criteria (NIH-CC) for cGVHD. Of 302 children enrolled, 241 were evaluable as L-aGVHD, cGVHD, active L-aGVHD or cGVHD, and no cGVHD/L-aGVHD. Significant marker differences, adjusted for major clinical factors, were defined as meeting all 3 criteria: receiver-operating characteristic area under the curve ≥0.60, P ≤ .05, and effect ratio ≥1.3 or ≤0.75. Patients with only distinctive features but determined as cGVHD by the adjudication committee (non-NIH-CC) had immune profiles similar to NIH-CC. Both cGVHD and L-aGVHD had decreased transitional B cells and increased cytolytic natural killer (NK) cells. cGVHD had additional abnormalities, with increased activated T cells, naive helper T (Th) and cytotoxic T cells, loss of CD56bright regulatory NK cells, and increased ST2 and soluble CD13. Active L-aGVHD before day 114 had additional abnormalities in naive Th, naive regulatory T (Treg) cell populations, and cytokines, and active cGVHD had an increase in PD-1− and a decrease in PD-1+ memory Treg cells. Unsupervised analysis appeared to show a progression of immune abnormalities from no cGVHD/L-aGVHD to L-aGVHD, with the most complex pattern in cGVHD. Comprehensive immune profiling will allow us to better understand how to minimize L-aGVHD and cGVHD. Further confirmation in adult and pediatric cohorts is needed.

Description: Background: The Wiskott-Aldrich syndrome (WAS) including X-linked thrombocytopenia (XLT) is a complex disorder with a wide range of disease severity and unique hematological and immunological manifestations. Based on this complexity, several approaches are available to these patients, including observation, symptomatic treatment, splenectomy, gene therapy (GT), or allogeneic hematopoietic stem cell transplantation (HSCT). In many instances more than one of these therapeutic options may seem appropriate for any given patient. A prospective, randomized study comparing the pros and cons of these therapeutic options in WAS/XLT would be desirable, but is not feasible due to the rarity and variable severity of the disease, as well as the need for long-term follow-up. Methods and Definitions: We retrospectively assessed via an international, anonymized, file-based survey the consequences of different therapies based on the severity of the disease phenotype and how these therapies affected patients' quality of life as perceived by their treating physician. The frequency of disease- and therapy-related complications with respect to the specific treatment was recorded. "Severe events" were defined as: all fatal events or sepsis, meningitis, pneumonia requiring respiratory support, systemic viral/fungal infections or serious bleeding episodes (intracranial and gastrointestinal) requiring transfusion support. Allogeneic HSCT, splenectomy and GT were defined as "procedures", HSCT and GT as "definitive". Results: A total of 575 patients with a documented WAS gene mutation from 51 centers in 27 countries with a median follow-up of 7.4 years (range: 0.2-75.6), resulting in 5632 patient years, were included in the study. Of these, 240 (42%) carried missense, 67 (12%) nonsense, 90 (16%) splice-site mutations, 77 (13%) deletions, 40 (7%) insertions and 61 (11%) had incomplete or inconclusive mutation information. An allogeneic HSCT was performed in 252 (44%), splenectomy in 78 (14%), GT in 14 (2%) patients, while 264 (46%) patients never had a procedure. At the time of last follow-up or before the first procedure the WAS disease severity score was 1 in 55 (10%), 2 in 144 (25%), 3 in 161 (28%), 4 in 109 (19%) and 5 in 86 (15%) patients. Overall survival of the entire cohort (censored at the time of first definitive procedure, thereby representing the "natural" disease outcome) was 82% (95% confidence interval 78-87) at 15 years and 70% (61-80) at 30 years of age. Ten year overall survival after HSCT was 80 % (74-85). The cumulative incidence (CI) of severe bleeding, severe infection, autoimmunity or malignancy in patients without a procedure at last follow-up or censored before the first procedure was 45% (39-50), 61% (55-66), 46% (40-52) and 31% (25-37) respectively at 15 years of age and 61% (51-69), 70% (62-76), 62% (52-70) and 45% (35-53) at 30 years. The frequency of definitive procedures (HSCT or GT) increased in patients with higher WAS scores, while better natural disease outcomes were associated with lower WAS scores. Overall quality of life (QoL) as perceived by the treating physician was very good, good, limited or unacceptable in 85/457 (19%), 172/457 (38%), 176/457 (39%) and 24/457 (5%) of patients without or before a procedure respectively. QoL was also strongly correlated with the WAS score. At last follow-up after successful HSCT QoL improved to very good in 123/184 (67%), good in 47/184 (26%), limited in 12/184 (7%) and unacceptable in 2/184 (1%). Splenectomy also had a favorable effect on QoL with 16/52 (31%) very good, 24/52 (46%) good, 9/52 (17%) limited and 3/52 (6%) unacceptable. Platelet counts improved from a baseline mean of 36G/l to 91G/l after GT, 159G/l after splenectomy and 204G/l after HSCT. Conclusion: This study presents outcome data of the largest cohort of patients with a WAS gene mutation studied so far and confirms the anticipated spectrum of disease severity and the curative effect of HSCT. The data show that untreated patients with WAS suffer from increasing rates of disease-associated complications over time which correlates well with a significant reduction of QoL. Both HSCT and splenectomy have a positive effect on physician-perceived QoL. Due to the large cohort size this study's data will allow us to assess the influence of specific genotypes on outcome in WAS (analysis ongoing), possibly allowing for more individualized treatment recommendations in the future. Disclosures Albert: GSK: Research Funding.

Description: Background: The use of post-transplant cyclophosphamide (PT-Cy) in combination with other immunosuppressive agents for graft versus host disease (GVHD) prophylaxis in matched unrelated donor (MUD) allogeneic hematopoietic stem cell transplantation (HSCT) is not well defined. PT-Cy mitigates GVHD after T-cell replete HLA haploidentical (Haplo) bone marrow transplant. Extrapolating from the success of PT-Cy in haplo transplants, we investigated the benefit of PT-Cy (at a lower dose than that used in haplo HSCT) in preventing GVHD following myeloablative peripheral blood stem cell (PBSC) MUD HSCT. Methods: We conducted a phase II clinical trial between September 2013 and June 2018 of PT-Cy for GVHD prophylaxis following myeloablative MUD HSCT. GVHD prophylaxis consisted of 1 dose of PT-Cy 50mg/kg on day +3, mycophenolate mofetil starting day +5 till day +35 and tacrolimus starting on day +5 with taper starting at day +100. The primary endpoint of the study was to determine the incidence of grade II-IV acute GVHD. Secondary endpoints included overall survival (OS), disease free survival (DFS), non-relapse mortlity (NRM) and time to engraftment. Approval for the study was obtained from the Institutional Review Board. Results: There were 39 patients enrolled in the study (Table 1). The mean age of the study population was 47.36 years (SD 13.41). There were 23 females (59%) and majority of the patients were white (85%). Thirty six donors were 8/8 HLA MUD and 3 were 7/8 matched unrelated. The stem cells were collected from peripheral blood in all cases. Indications for HSCT included AML/MDS (62%), ALL (15%), myelofibrosis (10%), NHL/HL (10%) and CML (3%). Based on disease risk index (DRI) assessment, 6 (15%) patients were DRI high, 32 (82%) were intermediate and 1 (3%) was low. The most commonly used myeloablative conditioning regimen was Busulfan and Fludarabine (74%). All 6 patients with ALL received TBI (12 Gy) based conditioning. Three patients with NHL received Fludarabine and Melphalan. There was 1 death within the first 30 days before engraftment. The remaining 38 patients (97%) successfully engrafted. The median time to ANC engraftment was 12 days (range 9-14 days). The incidence of day 100 acute GVHD maximum grade was 36% for grade I/II and 5% for grade III/IV (Table 2). The overall incidence of limited and extensive chronic GVHD was 10% and 36% respectively. There were 8 (21%) confirmed relapses within the first year after HSCT. Twenty four patients were alive at the 1-year mark after transplant and 17 of them were in complete remission. The 1-year and 2-year OS rates were 61.5% and 51.2% respectively. The median OS for the entire cohort was 21.2 months with a median follow up of 50 months (Figure 1). The day 100, 1-year and overall NRM rates were 10%, 28% and 33% respectively with infectious complications being the most common cause of death. Conclusion: We report a low incidence of acute severe GVHD with the combination of one dose of PT-Cy in combination with MMF and tacrolimus following myeloablative PBSC MUD HSCT. The single dose of PT-Cy may explain the modest control over chronic GVHD with this regimen. We also report favorable survival outcomes along with acceptable levels of NRM for the entire cohort. The use of PT-Cy in combination with other immunosuppressant agents for GVHD prevention appears to be a promising strategy in MUD HSCT and may play a vital role in mismatched unrelated donor transplants as well. Disclosures Costa: Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy; Fujimoto Pharmaceutical Corporation Japan: Other: Advisor; GSK: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Speakers Bureau; Abbvie: Consultancy. Saad:Actinium Pharma Inc: Consultancy; Amgen: Other: Research Support; Kadmon: Other: Research Support; OrcaBio: Other: Research Support.

Description: The hematopoietic stem cell transplant (HCT) donor KIR genotype has been correlated with disease-free survival in patients with acute myelogenous leukemia. The Killer Cell Immunoglobulin-like Receptor (KIR) gene family encodes highly homologous pairs of activating and inhibiting receptors, 2DL1–2DS1; 2DL2/3–2DS2; and 3DL1–3DS1. Inhibitory members are known to regulate NK cell function through interactions with HLA Class I antigens. The role of activating KIRs and their ligand specificity is, however, not well defined. The activating receptor, KIR2DS1, is known to bind the HLA-Cw C2 group antigens and we have recently demonstrated a role for this receptor in NK cell allorecognition. In contrast, KIR2DS2 does not bind HLA-Cw C1 group antigens, and a functional role of this receptor even in NK allorecognition has not been established. We now demonstrate, that presence of the activating KIR2DS2 gene in NK donors homozygous for the HLA-KIR ligand group C2 is associated with significant alloreactivity against C1 homozygous target cells (polyclonal NK cells, p=0.006; NK clones, p=0.001). This alloreactivity is mediated by “missing self” on the target and is dominated by “lack of C2 group on target”. The “missing C2” effect was absent, however, in C2 homozygous donors lacking 2DS2 (p=0.99). Only very rare cytotoxic NK clones expressing GL183 (2DL2/3, 2DS2) and with alloreactivity against C1 targets could be generated in vitro from 2DS2-positive, C2 homozygous donors. A majority of these rare GL183-positive clones did not demonstrate inhibitory function against the HLA class I deficient 721.221 transfected with Cw3 (C1-group), and GL183 cross-linking of the clones resulted in increased cytokine production. Thus, KIR2DS2 is an activating receptor in NK clones from C2 homozygous donors, but does not appear to recognize C1 ligand. We next investigated 2DS2 function in donors heterozygous for the C groups (i.e. C1/C2). Analysis of NK cell function in a 2DS2-positive, C1/C2 donor revealed a “missing HLA-KIR ligand” effect for the C2 group. Cytotoxicity by IL2-propagated, polyclonal NK cells and NK clones revealed allocytotoxicity against targets lacking the C2 group (p

Description: The role of Natural Killer (NK) cells in host protection against viral infection and malignant transformation has been well described. NK cells may also lead to a reduction in post-transplant relapse and improved survival in hematopoietic stem cell transplantation (HSCT) for acute myelogenous leukemia (AML). It has been hypothesized that the genotype for the inhibiting killer immunoglobulin-like receptor (KIR) of the hematopoietic stem cell donor in combination with the HLA class I genotype of the recipient could control NK alloreactivity leading to a reduction in post-transplant complications. The KIR gene family encodes however both activating and inhibiting receptors. Here we test the hypothesis that activating KIRs with ligand specificity for HLA class I may contribute to alloreactivity, and potentially could be a genetic factor of significance in allogeneic HSCT. We tested this hypothesis in studies of two pairs of inhibiting and activating KIRs with highly homologous codon sequences in the extracellular domain, namely KIR2DL2/3-KIR2DS2 and KIR2DL1-KIR2DS1. Both the inhibitory 2DL1 and activating 2DS1 have ligand specificity for HLA-Cw group 2, and 2DL2 and 2DL3, have ligand specificity for HLA-Cw group 1, while the activating 2DS2 does not bind in vitro to C1 group. Using an EBV-transformed B-lymphoblastoid cell line (EBV-BLCL) target cell panel homozygous for HLA Class I alleles, we found that NK cells from donors with KIR haplotypes lacking KIR2DS1 or 2DS2 were not cytotoxic to allogeneic EBV-BLCL, independent of the target HLA class I genotype. Polyclonal NK cells obtained from KIR2DS1 positive and C1 group positive donors mediated NK cytotoxicity against C2 positive targets. In contrast, NK cells from KIR2DS1 positive, C2 group homozygous donors displayed minimal cytotoxicity against the C2 group targets (p

Description: Chronic graft-versus-host disease (cGVHD) and late acute graft-versus-host disease (L-aGVHD) are understudied complications of allogeneic hematopoietic stem cell transplantation in children. The National Institutes of Health Consensus Criteria (NIH-CC) were designed to improve the diagnostic accuracy of cGVHD and to better classify graft-versus-host disease (GVHD) syndromes but have not been validated in patients