Robust Immune Reconstitution Research Articles

Immune Reconstitution and Infection Patterns after Early Alemtuzumab and Reduced Intensity Transplantation for Nonmalignant Disorders in Pediatric Patients

Hematopoietic stem cell transplantation (HSCT) is a therapeutic option for many nonmalignant disorders (NMD) and is curative or prevents disease progression. Reduced-intensity conditioning (RIC) in HSCT for NMD may reduce regimen-related acute toxicities and late complications. Myeloablation is often replaced by immune suppression in RIC regimens to support donor engraftment. The pace of immune reconstitution after immune suppression by RIC regimens is influenced by agents used, donor source, and graft-versus-host disease prophylaxis/treatment. In a multicenter trial (NCT 00920972) of HSCT for NMD, a RIC regimen consisting of alemtuzumab, fludarabine, and melphalan was substituted for myeloablation. Alemtuzumab was administered early (days -21 to -19) to mitigate major lymphodepletion of the incoming graft and the risk of graft rejection. Immune reconstitution and infectious complications were prospectively monitored for 1-year post-HSCT. Seventy-one patients met inclusion criteria for this report and received marrow or peripheral blood stem cell transplants. Immune reconstitution and infections are reported for related donor (RD) and unrelated donor (URD) transplants at 3 time-points (100days, 6 months, and 1 year post-HSCT). Natural killer cell recovery was rapid, and numbers normalized in both cohorts by day +100. Mean CD3, CD4, and CD8 T-lymphocyte numbers normalized by 6 months after RD HSCT and by 1 year in the URD group. CD4 and CD8 T-lymphocyte counts were significantly higher in patients who received RD HSCT at 6 months and at 1 year, respectively, post-HSCT compared with patients who received URD HSCT. The pace of CD19 B-cell recovery was markedly different between RD and URD cohorts. Mean B-cell numbers were normal by day 100 after RD HSCT but took 1 year post-HSCT to normalize in the URD cohort. Despite these differences in immune reconstitution, the timing and nature of infections did not differ between the groups, presumably because of comparable T-lymphocyte recovery. Immune reconstitution occurred at a faster pace than in prior reports using RIC with T-cell depletion. The incidence of infections was similar for both cohorts and occurred most frequently in the first 100days post-HSCT. Viral and fungal infections occurred at a lower incidence in this cohort, with "early" alemtuzumab compared with regimens administering serotherapy in the peritransplantation period. Patients were susceptible to bacterial infections primarily in the first 100days irrespective of donor source and had no increase in mortality from the same. The overall mortality rate from infections was 1.4% at 1 year. Close monitoring and prophylaxis against bacterial infections in the first 100days post-HSCT is necessary but is followed by robust immune reconstitution, especially in the T-cell compartment.

Coinfection with Human Cytomegalovirus Genetic Variants in Transplant Recipients and Its Impact on Antiviral T Cell Immune Reconstitution.

Reconstitution of T cell immunity is absolutely critical for the effective control of virus-associated infectious complications in hematopoietic stem cell transplant (HSCT) recipients. Coinfection with genetic variants of human cytomegalovirus (CMV) in transplant recipients has been linked to clinical disease manifestation; however, how these genetic variants impact T cell immune reconstitution remains poorly understood. In this study, we have evaluated dynamic changes in the emergence of genetic variants of CMV in HSCT recipients and correlated these changes with reconstitution of antiviral T cell responses. In an analysis of single nucleotide polymorphisms within sequences encoding HLA class I-restricted CMV epitopes from the immediate early 1 gene of CMV, coinfection with genetically distinct variants of CMV was detected in 52% of patients. However, in spite of exposure to multiple viral variants, the T cell responses in these patients were preferentially directed to a limited repertoire of HLA class I-restricted CMV epitopes, either conserved, variant, or cross-reactive. More importantly, we also demonstrate that long-term control of CMV infection after HSCT is primarily mediated through the efficient induction of stable antiviral T cell immunity irrespective of the nature of the antigenic target. These observations provide important insights for the future design of antiviral T cell-based immunotherapeutic strategies for transplant recipients, emphasizing the critical impact of robust immune reconstitution on efficient control of viral infection. Infection and disease caused by human cytomegalovirus (CMV) remain a significant burden in patients undergoing hematopoietic stem cell transplantation (HSCT). The establishment of efficient immunological control, primarily mediated by cytotoxic T cells, plays a critical role in preventing CMV-associated disease in transplant recipients. Recent studies have also begun to investigate the impact genetic variation in CMV has upon disease outcome in transplant recipients. In this study, we sought to investigate the role T cell immunity plays in recognizing and controlling genetic variants of CMV. We demonstrate that while a significant proportion of HSCT recipients may be exposed to multiple genetic variants of CMV, this does not necessarily lead to immune control mediated via recognition of this genetic variation. Rather, immune control is associated with the efficient establishment of a stable immune response predominantly directed against immunodominant conserved T cell epitopes.

476. No GVHD, but Human Inflammatory “Cytokine Storm” and Mouse Macrophage Activation Upon Accelerated Development of Human CD4+ Effector Memory T Cells in Long-Term Humanized Mice

Non clinical assessment of immunotoxicity of human cellular products in vivo is limited by the lack of availability of pharm-tox models enabling a full range of human innate and adaptive responses. We previously demonstrated improved development of human adaptive T and B cell responses in mice receiving human hematopoietic stem cell transplantation (HSCT) in combination with prime/boost immunization with induced dendritic cells (iDCs), i.e. monocytes derived the stem cell donor reprogramed with a lentiviral vector for co-expression of GM-CSF, IFN-alpha and the CMV antigen pp65 that self differentiate into DCs in vivo (Salguero et al, 2014 J. Immunology; Daenthanasanmak et al, 2015 Mol. Ther. Meth.). For these previous studies, mice were kept for 6-10 weeks after iDC administration, and we did not observe signs of tumorigenicity or graft-versus-host disease (GVHD). After consultation with the German regulatory authorities, we were requested to perform pharm-tox analyses 26 weeks after iDC immunization. We report here a pilot-feasibility study with humanized mice showing long-term (more than 33-36 weeks after HSCT) robust immune reconstitution with human T cells (control mice, n=2 Vs. iDC-immunized mice, n=6). Longitudinal analyses of human immune reconstitution in blood samples confirmed accelerated development of CD4+ effector memory T cells 5-10 weeks after iDC immunization. No weight loss, GVHD or malignancies were observed after iDC immunization. 3/6 immunized mice developed skin erythema and inflammation around 23 weeks after iDC immunization and were sacrificed earlier for analyses. Analyses of all mice were performed at autopsy by macroscopic observations, H&E histopathology, immunohistochemistry and flow cytometry of target organs. Positivity for anti-human nuclei antibody, anti-human HLA-DR and anti-human CD3 leucocytes were detected in several tissues (spleen, bone marrow, brain, lungs, skin, eyelids, liver, and kidneys), confirming the long-term persistency of human T cells in the humanized mice. In inflamed skin, abundant human CD4+ T cells were localized in the basal layers of the epidermis and hair follicles in the dermis. Human macrophages (CD68+) could be detected in several tissues, but were rare in inflamed skin. However, in the inflamed dermis areas of ulceration, mouse macrophages (F4/80+) were observed frequently with strongly positive cytoplasm. Mice with ulcerations had significantly higher levels of several human inflammatory cytokines in plasma (IFN-gamma, IL-6, GM-CSF and IL-8) than immunized mice with no skin inflammation or control mice. In summary, a long-term pilot study exploring a hybrid human-mouse model system was feasible and iDCs accelerated mature human T cell reconstitution with no signs of tumorigenicity or GVHD. However, accumulation of human CD4+ memory T cells and activated mouse macrophages in inflamed skin was associated with elevated levels of human inflammatory cytokines resembling a “cytokine storm”.

IPEX syndrome caused by a novel mutation in FOXP3 gene can be cured by bone marrow transplantation from an unrelated donor after myeloablative conditioning

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a rare primary immunodeficiency caused by inherited defects in the FOXP3 gene that impair regulatory T cells. IPEX syndrome can be cured by hematopoietic stem cell transplantation (HSCT) from HLA-matched unrelated donors (MUD); however, the best conditioning prior to HSCT for IPEX syndrome is not known. Here we report on a patient suffering from IPEX syndrome, including immune-mediated colitis and membranous nephropathy, without polyendocrinopathy, caused by a novel mutation in the Forkhead domain of the FOXP3 gene. The patient's symptoms resolved following MUD HSCT after myeloablative conditioning performed at 16 months of age. The patient is clinically well, 3 years after HSCT, with robust immune reconstitution and fully engrafted. The lack of extensive autoimmune damage might have contributed to the patient's favourable outcome following MUD HSCT with myeloablative conditioning. Statement of novelty: We describe a novel mutation in the FOXP3 gene causing IPEX syndrome and the correction of IPEX syndrome with bone marrow transplant from a HLA-matched unrelated donor following myeloablative conditioning.

Robust Immune Reconstitution in Children with Severe Primary Immunodeficiency after Reduced-Intensity Conditioning Hematopoietic Stem Cell Transplantation

Hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for severe primary immunodeficiency diseases (PID), but it is still unclear what is the optimal transplant approach. To help answer this question, the stem cell transplant team at Ann & Robert H. Lurie Children’s Hospital of Chicago (formerly Children’s Memorial Hospital) evaluated the risks and benefits of a reduced-intensity conditioning (RIC) transplant approach for treatment of severe PID. Between 2000 and 2013, 42 children with severe PID were treated with allogeneic HSCT following a RIC regimen consisting of fludarabine (30mg/m2/day x 6), rabbit anti-thymocyte globulin (once daily x 4), and intravenous busulfan (once daily x 2). Total busulfan exposure, as measured by the plasma concentration-time area-under-the-curve (AUC), was targeted individually for each patient at 4000 µM*min/day in an earlier cohort (n=14), and 5000 in a later cohort (n=28). Patients were treated in an ambulatory setting and were hospitalized only for specific medical or social reasons. There were 31 male and 11 female patients. The median age at the time of transplant was 8.2 months (range 1 month-17.4 years). The median weight was 7.6 kg (range 2.5-81.5 kg). Patients were diagnosed with severe combined immunodeficiency (n=17), Wiscott-Aldrich syndrome (n=7), hyper-IgM syndrome (n=5), major histocompatibility complex II deficiency (n=3), X-linked lymphoproliferative disease (n=3), NEMO syndrome (n=2), Omenn syndrome, reticular dysgenesis, Chediak-Higashi syndrome, IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome, and IPEX-like syndrome (n=1 each). Fourteen patients had related donors and 28 had unrelated donors. Twenty-eight patients received peripheral blood HSC, 12 received cord blood, and 2 received bone marrow as the source of HSC. All patients tolerated the conditioning regimen with minimal nausea, vomiting, or pain. No severe mucositis, sinusoidal occlusion syndrome, seizure, grade III/IV acute GVHD or extensive chronic GVHD were seen. In most patients, the engraftment was rapid, with ANC ≥ 500 at a median of +15 days post-transplant, following a nadir at day +10 with a median ANC nadir of 60. In the AUC 4000 cohort, 2 patients (14%) developed primary graft failure and 2 patients (14%) developed immune-mediated secondary graft failure; in the AUC 5000 cohort, 2 patient (7%) developed primary graft failure and 1 patient developed secondary graft failure after primary EBV infection. Two patients died from complications associated with primary graft failure, 2 from complications caused by EBV infections, and 2 from complications related to their underlying diseases in the early post-transplant period. Kaplan–Meier analysis shows overall and event-free survival rates of 81.9% and 73.6%, respectively, at a median follow-up of 4.3 years. Most engrafted patients have stable donor chimerism over time. At a mean of 3.5 years post-transplant, the median donor chimerism in total white blood cells was 97% and the first and third quartiles were 77% and 99%, respectively. Engrafted patients demonstrated robust immune reconstitution with B and T cell counts normalized by 3 months and 9 months post-transplant, and CD45RA+ naive T cells by 1 year. Between 1 to 4 years post-transplant, the median absolute CD4+, CD8+ and CD19+ cell counts were 1388, 976 and 720 per mm3, respectively, percentage of CD45RA+ naive CD4 and CD8 cells were 68% and 81%, and endogenous IgG and IgM levels were 840 and 97 mg/dL. T cells showed regular responses to antigen and mitogen stimulation and exhibited normal TCRVβ repertoires. Endogenous IgG levels returned to normal by day +100, and no intravenous immunoglobulin infusion was required after day +100 in all but 1 patients. Positive vaccine-specific antibody responses were seen in patients after reimmunization at 12-24 months post-transplant. Clinically, patients have normal growth and development and do not exhibit increased susceptibility to infection. Our data showed that HSCT after RIC offers an optimal treatment for severe PID by providing robust post-transplant immune reconstitution while minimizing side effects. To reduce the risk of graft failure, we recommend a targeted busulfan AUC of 5000 µM*min/day for 2 days. DisclosuresNo relevant conflicts of interest to declare.

Addition of interleukin-6 inhibition with tocilizumab to standard graft-versus-host disease prophylaxis after allogeneic stem-cell transplantation: a phase 1/2 trial

Interleukin 6 mediates graft-versus-host disease (GVHD) in experimental allogeneic stem-cell transplantation (allogeneic SCT) and represents an attractive therapeutic target. We aimed to assess whether the humanised anti-interleukin-6 receptor monoclonal antibody, tocilizumab, could attenuate the incidence of acute GVHD. We undertook a single-group, single-institution phase 1/2 study at the Royal Brisbane and Women's Hospital Bone Marrow Transplantation unit, QLD, Australia. Eligible patients were 18-65 years old and underwent T-replete HLA-matched allogeneic SCT with either total body irradiation-based myeloablative or reduced-intensity conditioning from unrelated or sibling donors. One intravenous dose of tocilizumab (8 mg/kg, capped at 800 mg, over 60 mins' infusion) was given the day before allogeneic SCT along with standard GVHD prophylaxis (cyclosporin [5 mg/kg per day on days -1 to +1, then 3 mg/kg per day to maintain therapeutic levels (trough levels of 140-300 ng/mL) for 100 days plus methotrexate [15 mg/m(2) on day 1, then 10 mg/m(2) on days 3, 6, and 11]). The primary endpoint was incidence of grade 2-4 acute GVHD at day 100, assessed and graded as per the Seattle criteria. Immunological profiles were compared with a non-randomised group of patients receiving allogeneic SCT, but not treated with tocilizumab. This trial is registered with the Australian and New Zealand Clinical Trials Registry, number ACTRN12612000726853. Between Jan 19, 2012, and Aug 27, 2013, 48 eligible patients receiving cyclosporin and methotrexate as GVHD prophylaxis were enrolled into the study. The incidence of grade 2-4 acute GVHD in patients treated with tocilizumab at day 100 was 12% (95% CI 5-24), and the incidence of grade 3-4 acute GVHD was 4% (1-13). Grade 2-4 acute GVHD involving the skin developed in five (10%) patients of 48 treated with tocilizumab, involving the gastrointestinal tract in four (8%) patients; there were no reported cases involving the liver. Low incidences of grade 2-4 acute GVHD were noted in patients receiving both myeloablative total body irradiation-based conditioning (12% [95% CI 2-34) and fludarabine and melphalan reduced-intensity conditioning (12% [4-27]). Immune reconstitution was preserved in recipients of interleukin-6 receptor inhibition, but qualitatively modified with suppression of known pathogenic STAT3-dependent pathways. Interleukin 6 is the main detectable and dysregulated cytokine secreted after allogeneic SCT and its inhibition is a potential new and simple strategy to protect from acute GVHD despite robust immune reconstitution; a randomised, controlled trial assessing tocilizumab in addition to standard GVHD prophylaxis in these patients is warranted. National Health and Medical Research Council and Queensland Health.

The Addition Of Interleukin-6 Inhibition To Standard Gvhd Prophylaxis Prevents Acute Gvhd: Interim Results Of a Phase I/II Clinical Study

We and others have demonstrated the dysregulation of interleukin-6 (IL-6) early after experimental bone marrow transplantation (BMT) and protection from acute GVHD following the administration of an anti-IL-6 receptor (IL-6R) antibody. In these models, where GVHD prophylaxis is not administered, systemic IL-6, IFNγ and TNF levels peak 7 days after BMT before returning to baseline by the third week. We have determined cytokine dysregulation in a large clinical cohort of allogeneic stem cell transplant (SCT) recipients conditioned with myeloablative Cy/TBI (12 Gy, n = 25) or reduced intensity Flu/Mel (120mg/m2, n = 25) receiving standard GVHD prophylaxis with cyclosporine and MTX (d 1 at 15mg/m2, d 3, 6, 11 at 10mg/m2). IL-6 levels rose from pre-transplant levels of 6.4 ± 0.7 pg/ml to a peak of 58.8 ± 8.8 pg/ml at day 7 (P < 0.0001) with a fall at day 14 to 39.0 ± 12.5 pg/ml (P < 0.0001) and return to baseline by day 30 (6.2 ± 0.9 pg/ml), consistent with the preclinical data. IL-6 dysregulation was not different in recipients of matched sibling or unrelated donor grafts but was proportional to the intensity of conditioning (day 7 levels after Cy/TBI vs. Flu/Mel: 83.3 ± 12.2 pg/ml vs. 31.0 ± 10.1 pg/ml, P< 0.0001). In contrast to preclinical mouse data, no systemic increases were seen in any other cytokine including IFNγ, TNF, IL-17, IL-4, IL-13 and IL-10.We thus initiated a phase I/II study whereby a human neutralizing monoclonal antibody (mAb) against the IL-6R was administered on day -1 to patients receiving Cy/TBI or Flu/Mel conditioned allogeneic SCT from HLA (10/10)–matched sibling or unrelated donors with standard cyclosporine/MTX GVHD prophylaxis. There was no T cell depletion. The primary endpoint was the incidence of grade II-IV acute GVHD and the study has achieved its planned enrollment (n = 48). There was no toxicity attributable to IL-6R antibody administration. Pharmacokinetic analysis confirmed high levels of IL-6R Ab at day 3 (mean 64.7 ug/ml) which persisted in all patients 3 weeks after BMT (mean = 9.8 ug/ml) and remained above the level of detection (0.1ug/ml) in 75% of patients at day 30 (mean = 1.9 ug/ml). IL-6 levels were dramatically increased (relative to baseline) in patients receiving antibody due to the inability to excrete the inactive IL-6 – soluble IL-6R antibody complex (peak IL-6 levels at day 7 = 773.6 ± 207.9 pg/ml; P < 0.0001) and remained increased at day 30 (60.9 ± 24.4 pg/ml; P < 0.0001), returning to baseline by day 60 (9.5 ± 1.7 pg/ml), consistent with antibody clearance. Soluble IL-6R levels also rose over the first month of SCT and levels at day 30 correlated with residual antibody levels (r2 = 0.72, P = 0.02). Neutrophil (> 0.5x109/L) and platelet (> 20x109/L) recovery was normal relative to a matched untreated control cohort at a median of 16 and 18 days respectively. Donor chimerism and immune reconstitution (conventional T, regulatory T and B cells) was equivalent at day 30 in recipients of IL-6R inhibition versus the control cohort. In contrast, changes in innate immunity were seen in patients receiving IL-6R inhibition with increases in plasmacytoid DC (P = 0.002), CD1c+ conventional DC (P = 0.04) NKT cells (P =0.03) and marked reductions in inflammatory (CD14+CD16+) monocytes (P < 0.0001). Transcriptional profiling of T cell subsets is underway. With 36 patients evaluable (beyond day 100, median follow up of 297 days), the incidence of grade II-IV GVHD is 11.1% in recipients of IL-6R inhibition versus 39.6% in the matched (n = 53) control cohort (P = 0.004). The incidence of grade III/IV acute GVHD is 5.6% in recipients of IL-6R inhibition versus 20.8% in the control cohort (P = 0.045). Protection from grade II-IV acute GVHD was noted in patients receiving both Cy/TBI (7.7% vs. 40.7%, P=0.045) and Flu/Mel conditioning (13.0% vs. 38.5%, P = 0.044). Rates of CMV reactivation were very low in the IL-6R neutralized patients (16.7% vs. 35.8% in controls, P = 0.04), likely due to the prevention of acute GVHD and its' consequent therapy. At one year, the relapse incidence and disease free survival in patients receiving IL-6R inhibition versus the control cohort is 21.2% vs. 30.0% (P = 0.28) and 73.1% vs. 62.4% (P = 0.14) respectively. IL-6 is thus the principal inflammatory cytokine dysregulated after clinical allogeneic SCT and its inhibition appears to offer profound protection from acute GVHD despite robust immune reconstitution, without compromise of the GVL effect. Disclosures:Off Label Use: The use of Tocilizumab to prevent GVHD is experimental and an off label use.

A 2 Step Approach To Myeloablative Haploidentical Hematopoietic Stem Cell Transplantation (HSCT): Low Non-Relapse Related Mortality (NRM) and High Overall Survival (OS) Rates Confirmed In Second Generation Trial

Over 7 years ago, we developed a 2 step myeloablative approach to haploidentical HSCT in which patients, after conditioning with 12 Gy of total body irradiation (TBI days -9 to -6), receive a fixed dose of 2 x 108/kg of donor T cells (DLI-step 1 of HSCT) immediately after the last fraction of TBI. This large dose of haploidentical lymphocytes results in high fever, and in some cases, diarrhea and rash, developing on days-5 and -4. Cyclophosphamide (CY) 60 mg/kg, given on days -3 and -2 for T cell tolerization, results in complete resolution of this “alloreaction.” Tacrolimus and MMF are begun on day-1, followed by a CD 34 selected donor product infused on day 0 (Step 2 of HSCT). The separation of the lymphoid and myeloid portions of the graft (1) avoids the exposure of allogeneic stem cells to CY, (2) avoids the polarization of T cells to a TH2 phenotype because the donor T cells are collected prior to the initiation of G-CSF, (3) allows the administration of a fixed dose of T cells establishing a consistent platform from which to compare outcomes, and (4) provides a method to deliver higher doses of T cells. Recent data suggests that T cell doses > 1.1 x108/kg are associated with superior GVT effects as compared to lower T cell doses (Guo et al. JCO, 2012;30:4084 and Colvin et al. BBMT, 2009;15:421). To date, over 180 patients have undergone 2 step haploidentical myeloablative and reduced intensity HSCT at our institution. In the initial myeloablative haploidentical trial (2006-2009), twenty-seven patients underwent treatment using this approach. Immune reconstitution was brisk with median CD3/4 and CD3/8 counts at day +28 of 33.6 and 28.7 cells/ul respectively Cumulative incidences of grades III-IV graft versus host disease (GVHD), NRM, and relapse-related mortality were 7.4%, 22.2%, and 29.8% respectively. OS for the 12 patients without disease at HSCT was 75% and 48% for the whole cohort with 51-79 (median 63) months of follow-up.A 2nd generation myeloablative 2 step trial specific to patients without morphologic evidence of disease at the time of their haploidentical HSCT completed accrual in 2013. Twenty-eight patients with AML (15), ph+ ALL (4), B cell ALL (4), T cell ALL (2), MDS (1), mantle cell NHL (1), and hepatosplenic NHL (1), were treated and are 3-32 (median 14) months post HSCT. All patients engrafted and no patients died of GVHD or infection. Median CD3/4 and CD3/8 counts at 28 days were 74 and 47 cells/ul respectively. The only death related to treatment occurred in a patient who suffered a subdural hematoma after an LP performed for fever. Four patients have relapsed (14%), 3 of whom have died of their disease. The probability of OS of the patients treated on the current trial is 85% at 15 months. The combination of the most recently treated 28 patients with the 12 patients treated on the initial trial who were in morphologic CR at the time of HSCT, has resulted in a probable OS rate of 78% with 3 to 79 (median 18 months of follow-up).For patients without morphologic evidence of their disease at the time of HSCT, the 2 step myeloablative approach to haploidentical HSCT has been associated with robust immune reconstitution resulting in low rates of infectious death. There have been no deaths from GVHD, and very low rates of treatment-related mortality. OS rates of the good risk patients treated on this approach are very high due to this low degree of NRM. The results of this 2nd generation trial have confirmed that the 2 step myeloablative approach to haploidentical HSCT is safe and efficacious and produces outcomes that are comparable to those reported by CIBMTR (2000-2010) for recipients of matched related or other alternative donor grafts. This approach has become our alternative HSCT strategy of choice allowing virtually every patient to be treated with HSCT rapidly at our institution. Disclosures:Kasner: Roche: Research Funding.

In Vivo T Cell Costimulation Blockade with Abatacept for Acute Graft-versus-Host Disease Prevention: A First-in-Disease Trial

We performed a first-in-disease trial of in vivo CD28:CD80/86 costimulation blockade with abatacept for acute graft-versus-host disease (aGVHD) prevention during unrelated-donor hematopoietic cell transplantation (HCT). All patients received cyclosporine/methotrexate plus 4 doses of abatacept (10 mg/kg/dose) on days −1, +5, +14, +28 post-HCT. The feasibility of adding abatacept, its pharmacokinetics, pharmacodynamics, and its impact on aGVHD, infection, relapse, and transplantation-related mortality (TRM) were assessed. All patients received the planned abatacept doses, and no infusion reactions were noted. Compared with a cohort of patients not receiving abatacept (the StdRx cohort), patients enrolled in the study (the ABA cohort) demonstrated significant inhibition of early CD4+ T cell proliferation and activation, affecting predominantly the effector memory (Tem) subpopulation, with 7- and 10-fold fewer proliferating and activated CD4+ Tem cells, respectively, at day+28 in the ABA cohort compared with the StdRx cohort (P < .01). The ABA patients demonstrated a low rate of aGVHD, despite robust immune reconstitution, with 2 of 10 patients diagnosed with grade II-IV aGVHD before day +100, no deaths from infection, no day +100 TRM, and with 7 of 10 evaluable patients surviving (median follow-up, 16 months). These results suggest that costimulation blockade with abatacept can significantly affect CD4+ T cell proliferation and activation post-transplantation, and may be an important adjunct to standard immunoprophylaxis for aGVHD in patients undergoing unrelated-donor HCT.

A Two-Step Approach to Allogeneic Haploidentical Hematopoietic Stem Cell Transplantation

Strategies that exploit natural killer (NK) cell alloreactivity or attenuate rather than deplete T cells have resulted in improved outcomes after haploidentical hematopoietic stem cell transplantation (HSCT). However, no approach has consistently produced the triad of optimal immune reconstitution, avoidance of significant graft-versus-host disease (GVHD), and durable control of malignancy. We developed a two-step approach to haploidentical HSCT in which the lymphoid and myeloid portions of the graft are given in two separate steps in order to control and optimize T-cell dosing. The initial results from these trials have included robust immune reconstitution, low rates of toxicity and significant GVHD, and durable disease control in good-risk patients, as well as insights regarding a threshold for T-cell dosing above which graft-versus-tumor (GVT) effects might be expected. Patients who were not in remission at the time of HSCT had higher rates of relapse requiring efforts to further strengthen GVT effects. Second-generation trials are underway to further exploit changes in the dosing and timing of administration of T cells and to optimize donor selection in an effort to decrease relapse rates in high-risk patients.

A Novel Chemotherapy-Based Allogeneic Hematopoietic Stem Cell Transplant Regimen for Very Young Children with Leukemia.

Abstract 3149▪▪This icon denotes a clinically relevant abstractYoung children in need of allogeneic hematopoietic stem cell transplant (HSCT) are at increased risk of unacceptable side effects from total body irradiation (TBI) and have historically been considered candidates for non-TBI containing regimens. However, disease free survival (DFS) has been poorer in cohorts of very young patients transplanted without TBI and novel chemotherapy based regimens are needed. We report results in a cohort of 14 children all under three years of age at the time of transplant (6 – 32 months; median 19.8 months) using a clofarabine-based ablative regimen. Fourteen patients in this age group have undergone transplant with a regimen consisting of clofarabine 20 mg/m2/day × 5, thiotepa 10 mg/Kg/day × 1 and melphalan 70 mg/m2/day × 2. All patients had high risk disease. Seven (7) pts were transplanted for ALL, 6 for AML and 1 for JMML. Patients with ALL or AML in first remission (CR1) or CR2, were categorized as patients with good risk disease while all other pts were considered as poor risk irrespective of all other factors. Transplant risk was good for 6/7 with ALL, and 3/6 with AML. The patient with JMML had stable disease. Stem cell grafts consisted of unmodified bone marrow (BMT) (N=6), double cord blood (dCBT) (N=7) and T cell depleted PBSCT (N=1). Donors were matched unrelated (N=5) or mismatched unrelated (N=9) including 7 double umbilical cord blood grafts, and one T cell depleted graft. Graft versus host disease (GvHD) prophylaxis was with tacrolimus and methotrexate for unmodified BMT, tacrolimus and mycophenolate for dCBT or T cell depleted HSCT. Two patients died early post transplant of infection (1) and acute GvHD (1). Neutrophil engraftment for the 13 evaluable patients was at a median of 13 days (10 –29 days) for PBSC and BM grafts and 17.5 days (12 –23 days) for recipients of CB grafts. Platelet engraftment for the 12 evaluable patients was at a median of 23 days (16 – 36 days) for recipients of PBSC and BM grafts and 43.5 days (36 –66 days) for recipients of CB grafts. In all five patients developed grade II-IV GvHD, and two patients chronic GvHD. Seven patients developed transaminitis which resolved in all cases. No patients developed Grade IV mucositis. One patient (AML) died after relapsing 5.5 months post transplant. Two patients are alive after relapsing at 1.3 months (AML) and 10.8 months (JMML) post-transplant. Nine of the 14 patients are alive in continuous complete remission seven of whom are greater than 36 months from transplant (40.2 – 71 months). The seven patients without chronic GvHD have had robust immune reconstitution, have responded to vaccination, and continue to meet growth and developmental milestones. Only one patient (transplanted at 14 months of age) has mild neurocognitive deficits. This novel chemotherapy based regimen is associated with durable engraftment of unmodified and cord blood HSCT grafts and promising disease free survival in very young children with leukemia. Based on the low toxicity profile in this cohort of patients higher dosing of clofarabine will be explored as a possible way to improve leukemia remission in the highest risk patients. Disclosures:Off Label Use: Clofarabine.

Tregs combined with mature donor T cells hasten immune reconstitution without triggering GvHD in HLA haploidentical transplantation

Haploidentical transplantation, with extensive T cell depletion to prevent GvHD, is associated with a high incidence of infection-related deaths. The key challenge is to improve immune recovery with allogeneic donor T cells without triggering GvHD. As T regulatory cells (Tregs) controlled GvHD in preclinical studies, the present phase I/II clinical trial evaluated the impact of early infusion of donor CD4/CD25+ Tregs, followed by an inoculum of donor mature T cells (Tcons) and positively immunoselected CD34+ cells. Twenty-eight patients (median age 41, range 21-60) were enrolled from September 2008 onwards; 22 had AML (10 in CR1 at high risk, 10 in ≥CR2 and 2 in relapse), 5 had ALL (4 in CR1; 1 in relapse) and 1 had high grade NHL in relapse. Conditioning was: 8 Gy single fraction TBI, thiotepa (4 mg/kg × 2), fludarabine (40 mg/m2 × 5), cyclophosphamide (35 mg/kg × 2). All patients received immunoselected Tregs (CliniMACS, Miltenyi Biotec) (23/28 2 × 106/kg bw; 5/28 4 × 106/kg bw) and 4 days later positively immunoselected CD34+ cells (median 8.2 × 106/kg bw, range 5.0-19.1) together with Tcons (4/28 0.5 × 106/kg bw; 17/28 1 × 106/kg bw; 5/28 2 × 106/kg bw; 2/28 did not receive Tcons). CD4/CD25+ Tregs (purity 92.7 ± 2.1) consisted of 33.6% ± 13.1 CD25high; 58.1% ± 6.6 CD25int; 5.8% ± 2.5 CD25low; 65.7% ± 11.8 FoxP3; 17.4% ± 7.2 CD127 (mean ± SD). No GvHD prophylaxis was administered. 26/28 patients engrafted. No GvHD developed in 24/26 patients, 2 developed ≥ grade II GvHD. Ten patients died (3 VOD, 2 fungal pneumonia, 1 bacterial sepsis, 1 CNS aspergillosis, 1 systemic toxoplasmosis, 1 adenoviral infection, 1 MOF). CD4 and CD8 counts reached, respectively, 50/μL medianly on days 34 (range 19-63 days) and 24 (range 15-87); 100/μL medianly on days 47 (range 28-100 days) and 34 (range 19-95); 200/μL on days 70 (range 41-146 days) and 61 (range 21-95). A wide T-cell repertoire developed rapidly with high frequencies of specific CD4+ and CD8+ for opportunistic pathogens. Episodes of CMV reactivation were significantly fewer than after our standard haplo transplants. In KIR ligand-mismatched transplants, speed of NK cell reconstitution/maturation and size of donor vs recipient alloreactive NK cell repertoires were preserved. In conclusion, in the setting of haploidentical transplantation infusion of Tregs makes administration of a high dose of T cells feasible for the first time. This strategy provides a long-term protection from GvHD and robust immune reconstitution.

Higher Infused CD34+ Cell Dose Improves Survival In Patients Undergoing In Vivo T-Cell Depleted, but Not T-Cell Replete Peripheral Blood Hematopoietic Cell Transplant

AbstractAbstract 1272 Introduction:Allogeneic hematopoietic cell transplant (HCT) remains a potentially curative modality for various hematological disorders. The cellular composition of the infused allograft has important ramifications for transplantation outcomes, for example higher infused CD34+ cell doses have previously been shown to be is associated with early engraftment, improved survival and possibly increased acute graft-versus-host disease (GVHD) following HCT. The influence of cellular composition of infused allograft on transplant outcomes has been the subject of many previous studies. There is paucity of data on the impact of cellular composition of allograft on transplant outcomes of patients undergoing HCT with in vivo T-cell depletion (TCD) compared to patients receiving T-cell replete allografts. We report here a comparative analysis of the impact of CD34+, CD3+, CD4+ and CD8+ cell doses and survival outcomes of allogeneic, peripheral blood HCT patients receiving in vivo T-cell depletion with alemtuzumab or anti thymocyte globulin (TCD group) versus patients who underwent T-cell replete HCT (non-TCD group). Methods:The study cohort includes 150 consecutive patients who underwent allogeneic HCT between January 2003 through December 2009. All patients received peripheral blood allografts from matched sibling or unrelated donors (URD). In vivo T-cell depletion consisted of alemtuzumab 40mg in two divided doses on days -4 and -1 (n=39) or Thymoglobulin at a total dose of 6 mg/kg for ablative and reduced intensity conditioning (RIC) transplants and 7.5 mg/kg total dose for non myeloablative allografts (n=51).4 patients received Atgam at 30mg/kg on days -5, -4 and -3. Impact of CD34+, CD3+, CD4+ and CD8+ cell doses divided into two groups; >/= 50th and < 50th percentile on overall survival (OS), progression free survival (PFS) and non relapse mortality (NRM) was initially measured by univariate analysis. Multivariate logistic regression analysis was constructed for variables showing significance on univariate analysis (p<0.1). Cellular components of allografts was done by standard flow cytometric techniques. Results:Of the 150 patients, 94 (62.7%) were males. Median age was 49 (range 17–69). Baseline diagnosis included acute leukemia and myelodysplastic syndrome (n=88; 58.6%), chronic myeloid leukemia (n=19; 12.7%), non-Hodgkin lymphoma (n=27; 18%) and others (10.7%). There were 95 patients (63.3%) in the TCD group and 55 (36.7%) in the non-TCD group. The baseline characteristics of the TCD group and non-TCD group were well matched except that significantly more patients in the TCD group had high risk disease (86.3% vs. 61.8%, p = 0.0005) and received allografts from unrelated donors (62.1% vs. 29.1%, p < 0.001). Median doses of the infused cellular components in the allograft were; CD 34+ = 5.8 × 106/Kg (range 1.2 – 16), CD3+ = 30.8 × 107 (4.5 – 100.8), CD4+ = 18.6 × 107 (1.9 – 63) and CD8+ = 11.3 × 107 (0.8 – 52.4). Median follow-up time for surviving patients was 3 years.In the TCD group, multivariate analysis showed that CD34+ cell doses >/= 5.8 × 106 was associated with improved OS (p=0.0085; CI 0.28–0.83), PFS (p=0.03; CI 0.31–0.93) and NRM (p=0.02; CI 0.21–0.89). Multivariate analysis also showed that CD3+ cell dose >/= 30.8×107 improved OS (p=0.03; CI 0.25–0.92), but not PFS (p=0.14; CI 0.16–1.31) and NRM (p=0.15; CI 0.23–1.26). No association was noted between CD4+ and CD8+ cell doses and OS, PFS and NRM (p>0.05), in the TCD group.In the non-TCD group, univariate analysis of CD34+, CD3+, CD4+ and CD8+ cell doses failed to show any statistical significance for NRM, OS and PFS (p>0.1). Conclusion:Our limited, retrospective analysis of 150 peripheral blood allogeneic HCT shows improved OS, PFS and NRM in patients receiving CD34+ cell dose >/= 5.8×106/Kg and improved OS with CD3+ dose >/= 30.8×107/Kg, limited only to the TCD group. No such association was seen in the non-TCD group. We hypothesize that higher CD34+ in TCD transplants probably improved survival by rapid engraftment and by robust immune reconstitution thereby reducing infectious complication otherwise associated with TCD. Disclosures:Abraham:Genentech: Membership on an entity's Board of Directors or advisory committees. Hamadani:Celgene: Honoraria, Speakers Bureau; Otsuka: Research Funding, Speakers Bureau.

Adoptive Immunotherapy with Tregs Prevents GvHD and Favours Immune Reconstitution After HLA Haploidentical Transplants for Hematological Malignancies.

Abstract 4Transplantation of large numbers of highly purified CD34+ cells from haploidentical relatives is a viable strategy for the cure of acute leukaemia at high risk of relapse (Aversa et al., NEJM 1998; JCO 2005). As extensive T cell depletion is required to prevent GvHD, the very narrow T cell repertoire in the inoculum delays recovery of immune response against pathogens, leading to a high incidence of infection-related deaths. Thus the key challenge is to improve immune recovery by administering allogeneic donor T cells without causing GvHD. Preclinical studies demonstrated that freshly isolated or ex vivo expanded T regulatory cells (Tregs) could be used to control GvHD following bone marrow transplantation. The present phase I/II clinical trial evaluated the impact of early infusion of freshly isolated donor CD4/CD25+ Tregs, followed by an inoculum consisting of donor mature T cells (Tcons) and positively immunoselected CD34+ cells, on GvHD prevention and immunological reconstitution. Twenty-two patients (10 male; 12 female; median age 40.5, range, 21 to 60) with AML (n=17; 8 in CR1 at high risk, 7 in ≥CR2 and 2 in relapse), ALL (n=4; 3 in CR1; 1 in relapse) and 1 with high grade NHL in relapse were enrolled from September 2008 onwards. The conditioning regimen consisted of 8Gy single fraction TBI, thiotepa (4 mg/kg×2), fludarabine (40mg/m2×4), and cyclophosphamide (35 mg/kg×2). All patients received CD4/CD25+ GMP immunoselected Tregs (CliniMACS, Miltenyi Biotec) (21/22 2×106/kg bw; 1/22 1×106/kg bw). Three days later they received positively immunoselected CD34+ cells (median 8.2, range 5.0-19.1) together with Tcons (16/22 1×106/kg bw; 4/22 0.5 ×106/kg bw; 2/22 did not receive Tcons). Immunoselected CD4/CD25+ Tregs (purity 91.5±4.5) consisted of CD25high 25.6%±11.2; CD25int 57.4%±5.9; CD25low 8.5%±6; FoxP3 64%±1; CD127 14.9%±13.7 (mean±SD). As suggested by in vitro immunosuppressive assays and by immunophenotypic analysis, the contaminating cells in the Treg fraction were 50% of the CD25int and 100% of the CD25low, so that the infused Tregs:Tcons ratio was established at 1:1.5. No post-transplant prophylaxis against GvHD was used. 20/22 patients engrafted. Neutrophils reached 1×109/L at a median of 15 days (range, 11 to 39 days). Platelets reached 25×109/L and 50×109/L at median of 13 and 15 days, respectively (range, 11 to 48 days, and 13 to 60 days). All engrafted patients showed persistent full donor-type chimerism in peripheral blood and bone marrow. Strikingly, no GvHD was observed in 17/20 valuable patients, 2/20 developed grade I cutaneous self-limited untreated GvHD and 1/20 developed grade III GvHD. This patient had received the fewest Tregs. Six patients died (1 bacterial sepsis, 2 VOD, 1 fungal pneumonia, 1 CNS aspergillosis and 1 GvHD/systemic toxoplasmosis). In contrast with our previous experience, the speed of immune recovery was enhanced. The CD4 and the CD8 counts reached, respectively, 50/μL medianly on days 34 (range, 19 to 63 days) and 24 (range, 15 to 87); 100/μL medianly on days 47 (range, 28 to 100 days) and 34 (range, 19 to 95); 200/μL on days 70 (range, 41 to 146 days) and 61 (range, 21 to 95). We also observed a rapid development of a wide T-cell repertoire and detection of high frequencies of specific CD4+ and CD8+ for opportunistic pathogens such as Aspergillus, Candida, CMV, ADV, HSV, VZV, Toxoplasma. In KIR ligand-mismatched transplants, speed of NK cell reconstitution/maturation and size of donor vs recipient alloreactive NK cell repertoires were preserved (Ruggeri et al., Science 2002). In conclusion, in the setting of haploidentical transplantation infusion of freshly purified Tregs makes administration of high dose of T cells feasible for the first time. This strategy provides a long-term protection from GvHD and robust immune reconstitution. Treg-based cellular therapy may represent an innovative strategy to improve the outcome of haploidentical transplants. Disclosures:No relevant conflicts of interest to declare.

Improvement of Canine XSCID by In Vivo Gene Therapy Using RD114/TR-Pseudotyped SIV Lentiviral Vectors.

X-linked severe combined immunodeficiency (XSCID) is characterized by profound immunodeficiency (dysfunctional B cells; absence of T and NK cells) and early mortality, caused by mutations in the IL2RG gene encoding the common gamma chain (γc) of receptors for interleukins (IL)-2,-4,-7,-9,-15 and -21. The standard therapy for XSCID in infants is a T cell depleted bone marrow transplant with either none or very modest conditioning. While 5-yr survival is >95% with HLA-matched sibling donors, most patients receive a haploidentical graft from a parent, resulting in lower survival and less robust immune reconstitution that may be limited to only the T cell lineage. Ex vivo autologous stem cell gene therapy has emerged as an alternate treatment capable of achieving substantial to complete immune reconstitution in infants without a sibling donor. However, some children have developed lymphocytic leukemia, which appears in part to be related to vector insertional mutagenesis. The gammaretroviruses currently in use have potent enhancers in the LTR and have a predilection to insert at the 5′ end of genes. Self-inactivating (SIN) lentivirus vectors may be advantageous because they do not show this property and can be constructed with internal promotors that have less enhancer activity together with insertion of insulators. We have constructed SIN simian immunodeficiency viral vectors (SIVmac) encoding human γc (hγc), with or without a double copy chicken insulator core element in the 3′ LTR. In addition we have pseudotyped the vector with a chimeric RD114 envelope to enhance targeting of hematopoietic stem cells (HSC) and avoid the cytotoxicity of the VSV-G envelope traditionally used with lentivectors. We have previously shown that it is possible to correct a dog model of XSCID using a RD114-pseudotyped gammaretroviral vector encoding the dog γc for in vivo gene therapy. Unlike the mouse model of XSCID which lacks B cells, the dog model more closely resembles the phenotype in humans. We decided to use our in vivo dog model for preclinical testing of safety and efficacy of our SIV hγc vectors. Viral particles were produced by transient transfection of 293T cells with a 4-plasmid system and concentrated by high-speed centrifugation. 30 mls (average 2.4–3×107 viral particles in total) was injected IV into 2–5 day-old pups. Transgene marking in blood lymphocytes was detected as early as 2 wks after treatment, increased within the first 6–8 wks and became relatively stable thereafter. The absolute lymphocyte count was normalized in one dog (injected on the second day after birth) by wk 6 (4000, 76% hγc+), and improved in two other dogs (422, 29% hγc+ at 18 wks; 498, 27% hγc+, at 16 wks) which had received the same amount of virus over 2 days. Furthermore, up to 5% of the myeloid lineage showed gene marking at 10–18 wks after viral delivery, indicating that early committed progenitors or HSCs had been transduced. It is noteworthy that our finding that the hγc can improve the disease phenotype in XSCID dogs makes it an excellent large animal model for preclinical evaluation of vectors. In summary, we have demonstrated that in vivo delivery of SIV lentiviral vectors expressing hγc efficiently reconstitute the T-lymphoid compartment in the XSCID canine model. We plan to monitor these dogs closely for potential adverse events.

Five-year follow-up of a cohort of profoundly immunosuppressed patients discontinuing therapy for cytomegalovirus retinitis.

Potent antiretroviral therapy stimulates robust and durable immune reconstitution among profoundly immunosuppressed patients that is clinically important. Fifteen patients with cytomegalovirus retinitis (CMVR) were followed after discontinuing anti-cytomegalovirus therapy. No patients had progression or relapse for a median of 51 months. Survival from the diagnosis of CMVR was universal for up to 95 months, with an observation period averaging more than 6 years. The CD4 cell count continued to rise through the fourth year of follow-up.