Cell-depleted Grafts Research Articles

Innate Lymphoid Cells Groups 1 and 3 in the Epithelial Compartment of Functional Human Intestinal Allografts

We examined intraepithelial lymphocytes (IELs) in 213 ileal biopsies from 16 bowel grafts and compared them with 32 biopsies from native intestines. During the first year posttransplantation, grafts exhibited low levels of IELs (percentage of CD103(+) cells) principally due to reduced CD3(+) CD8(+) cells, while CD103(+) CD3(-) cell numbers became significantly higher. Changes in IEL subsets did not correlate with histology results, isolated intestine, or multivisceral transplants, but CD3(-) IELs were significantly higher in patients receiving corticosteroids. Compared with controls, more CD3(-) IELs of the grafts expressed CD56, NKp44, interleukin (IL)-23 receptor, retinoid-related orphan receptor gamma t (RORγt), and CCR6. No difference was observed in granzyme B, and CD3(-) CD127(+) cells were more abundant in native intestines. Ex vivo, and after in vitro activation, CD3(-) IELs in grafts produced significantly more interferon (IFN)-γ and IL-22, and a double IFNγ(+) IL-22(+) population was observed. Epithelial cell-depleted grafts IELs were cytotoxic, whereas this was not observed in controls. In conclusion, different from native intestines, a CD3(-) IEL subset predominates in grafts, showing features of natural killer cells and intraepithelial ILC1 (CD56(+) , NKp44(+) , CCR6(+) , CD127(-) , cytotoxicity, and IFNγ secretion), ILC3 (CD56(+) , NKp44(+) , IL-23R(+) , CCR6(+) , RORγt(+) , and IL-22 secretion), and intermediate ILC1-ILC3 phenotypes (IFNγ(+) IL-22(+) ). Viability of intestinal grafts may depend on the balance among proinflammatory and homeostatic roles of ILC subsets.

Brief Report: Autologous Stem Cell Transplantation Restores Immune Tolerance in Experimental Arthritis by Renewal and Modulation of the Teff Cell Compartment

Autologous stem cell transplantation (ASCT) induces long-term drug-free disease remission in patients with juvenile idiopathic arthritis. This study was undertaken to further unravel the immunologic mechanisms underlying ASCT by using a mouse model of proteoglycan-induced arthritis (PGIA). For initiation of PGIA, BALB/c mice received 2 intraperitoneal injections of human PG in a synthetic adjuvant on days 0 and 21. Five weeks after the first immunization, the mice were exposed to total body irradiation (7.5 Gy) and received (un)manipulated bone marrow (BM) grafts from mice with PGIA. Clinical scores, T cell reconstitution, (antigen-specific) T cell cytokine production, and intracellular cytokine expression were determined following autologous BM transplantation (ABMT). ABMT resulted in amelioration and stabilization of arthritis scores. BM grafts containing T cells and T cell-depleted grafts provided the same clinical benefit, with similar reductions in PG-induced T cell proliferation and the number of PG-specific autoantibodies. In vivo reexposure to PG did not exacerbate disease. Following ABMT, basal levels of disease-associated proinflammatory cytokines (interferon-γ [IFNγ], interleukin-17 [IL-17], and tumor necrosis factor α [TNFα]) were reduced. In addition, restimulation of T cells with PG induced a strong reduction in disease-associated proinflammatory cytokine production. Finally, although the remaining host T cells displayed a proinflammatory phenotype following ABMT, IFNγ, IL-17, and TNFα production by the newly reconstituted donor-derived T cells was significantly lower. Taken together, our data suggest that ABMT restores immune tolerance by renewal and modulation of the Teff cell compartment, leading to a strong reduction in proinflammatory (self antigen-specific) T cell cytokine production.

CMV reactivation is associated with a lower incidence of relapse after allo-SCT for CML

Preemptive therapy at CMV reactivation has diminished post-transplant CMV mortality. Furthermore, recent studies suggest a favorable 'virus-versus-leukemia' effect from reactivating CMV, reducing relapse of AML after SCT. We studied the relationship of CMV reactivation with leukemic relapse in 110 patients with CML receiving HLA-identical sibling SCT between 1993 and 2008. Of these, 79 (72%) were in chronic phase, 5 in second chronic phase, 17 in accelerated phase and 9 in blast phase. A total of 97 patients (88%) received a myeloablative conditioning regimen, 97 received 4-log ex vivo T cell-depleted grafts and 13 received T-replete grafts. CMV reactivation before day 100 was observed in 72 patients (65.5%). At a median follow-up of 6.2 years, CMV reactivation < day 100 as a time-dependent covariate was an independent factor associated with decreased relapse. We conclude that CMV reactivation may contribute to a beneficial GVL effect in CML transplant recipients.

Quantitative assessment of T cell repertoire recovery after hematopoietic stem cell transplantation

Delayed T-cell recovery and restricted T-cell receptor (TCR) diversity after allogeneic hematopoietic stem cell transplantation (allo-HSCT) are associated with increased risks of infection and cancer relapse. Technical challenges have limited faithful measurement of TCR diversity following allo-HSCT. Here we combined 5′-RACE PCR with deep sequencing, to quantify TCR diversity in 28 allo-HSCT recipients using a single oligonucleotide pair. Analysis of duplicate blood samples confirmed that the frequency of individual TCRs was accurately determined. After 6 months, cord blood graft recipients approximated the TCR diversity of healthy individuals, whereas recipients of T-cell-depleted peripheral blood stem cell grafts had a 28-fold and 14-fold lower CD4+ and CD8+ T-cell diversity, respectively. After 12 months, these deficiencies had improved for the CD4+, but not the CD8+ T-cell compartment. Overall, this method provides unprecedented views of T-cell repertoire recovery after allo-HSCT and may identify patients at high risk of infection or relapse.

In Vivo DPP-4 Inhibition to Enhance Engraftment of Single-Unit Cord Blood Transplants in Adults with Hematological Malignancies

Delayed engraftment is a significant limitation of umbilical cord blood (UCB) transplantation due to low stem cell numbers. Inhibition of dipeptidyl peptidase (DPP)-4 enhanced engraftment in murine transplants. We evaluated the feasibility of systemic DPP-4 inhibition using sitagliptin to enhance engraftment of single-unit UCB grafts in adults with hematological malignancies. Twenty-four patients (21-58 years) received myeloablative conditioning, followed by sitagliptin 600 mg orally days -1 to +2, and single UCB grafts day 0. Seventeen receiving red cell-depleted (RCD) grafts, matched at 4 (n=10) or 5 (n=7) of 6 human leucocyte antigen (HLA) loci with median nucleated cell dose 3.6 (2.5-5.2)×10(7)/kg, engrafted at median of 21 (range, 13-50) days with cumulative incidence of 94% (95% confidence interval, 84%-100%) at 50 days. Plasma DDP-4 activity was reduced to 23%±7% within 2 h. Area under DPP-4 activity-time curve (AUCA) correlated with engraftment; 9 of 11 with AUCA <6,000 activity·h engrafted within ≤21 days, while all 6 with higher AUCA engrafted later (P=0.002). Seven patients receiving red cell replete grafts had 10-fold lower colony forming units after thawing compared with RCD grafts, with poor engraftment. Systemic DPP-4 inhibition was well tolerated and may enhance engraftment. Optimizing sitagliptin dosing to achieve more sustained DPP-4 inhibition may further improve outcome.

Negative Depletion of B Cells and T Cells Expressing the αβ Chain of the T-Cell Receptor (TCR) for Haploidentical Stem Cell Transplantation

Abstract 343 Introduction.Allogeneic hematopoietic stem cell transplantation (HSCT) from a HLA-haploidentical relative is a suitable option for patients (pts) lacking a compatible donor, either related or unrelated. The two main approaches for overcoming the obstacles of HLA barriers are based either on the infusion of large numbers of T-cell-depleted HSC or on intensive pharmacological prevention of graft-versus-host disease (GVHD). While for many years T-cell depletion (TCD) of the graft has been based on either immunomagnetic positive selection of CD34+ cells or on physical removal of all subsets of T cells by virtue of mAb, we and other groups have recently developed a novel method of ex vivo TCD based on the selective elimination of αβ+ T cells through labeling with a biotinylated anti-TCRαβ Ab, followed by incubation with an anti-biotin Ab conjugated to paramagnetic beads (Miltenyi Biotec, Germany). This approach also allows the removal of B cells to prevent post-transplant EBV-associated lymphoproliferative disease (PTLD). Here, we report the results of graft manipulation using this approach. Methods.Twenty-two children entered the study, 15 with hematological malignancies and 7 with non-malignant disorders. No post-transplant GvHD prophylaxis was employed. HLA-haploidentical family donors received G-CSF (12–16 μg/kg of body weight) to mobilize HSC prior to large-volume leukapheresis, which was commenced when circulating CD34+ HSC were >20 cells/μl. Cell therapy products containing up to 60×109 white blood cells (WBC) were processed according to the manufacturer’s protocol. In some cases, leukapheresis bags were stored overnight at 4°C in appropriate media at a WBC concentration <200×106/ml. Aliquots of the graft were used to enumerate residual αβ T cells and B cells, as well as other immune effector cells, including type 1 and 2 dendritic cell (DC) precursors, invariant NKT cells, classical CD14+CD16− and non-classical CD14+CD16+ monocytes. All TCR αβ+/CD19+cell-depleted grafts were re-infused within 2 hours from the end of processing. Results.Median recovery of CD34+ HSC and median number of infused CD34+ HSC were 99.3% (range 55.4–100) and 14.7×106/kg (range 7.9–37), respectively. The graft contained 3.9×106 CD3+ T cells/kg (range 0.9–30.9) and 0.08×106 B cells/kg (range 0.002–0.32). The log-depletion of αβ+ T cells was 4.5 (range 3.2–6.1), with a median number of transplanted αβ+ T cells equal to 36×103/kg (range 1–139.9). Patients received 36×106 CD56+ NK cells/kg (range 1.6–80.3) and 4.1×106 γδ+ T cells/kg (range 0.9–30.8). A median of 40.8% NK cells (range 30.3–60.2) co-expressed Tim-3, an activating co-receptor promoting IFN-γ production and being scarcely expressed on NK cells that reconstitute after allogeneic HSCT. The αβ/CD19-depleted grafts also contained DC1 and DC2 precursors, which were identified based on BDCA-1 (0.23% of all nucleated cells, range 0.02–2.4), BDCA-3 (0.93%, range 0.57–9.4) and BDCA-2 (0.66%, range 0.49–1.28) or BDCA-4 expression (0.69%, range 0.02–1.5), respectively. Invariant NKT cells were enumerated using mAb reacting against the TCR Vα24-Jα18 chain and were detected in minute percentages (<0.05% of all nucleated cells) within the graft. The αβ/CD19-depleted grafts were also enriched in non-classical CD14+CD16+ monocytes (13.7% of all CD14+cells, range 9.3–29.4), which have been reported to predict a reduced incidence of GVHD after allogeneic HSCT. All patients rapidly engrafted, the median time to reach 500 neutrophils and 50,000 platelets per μl of blood being 12 days (range 10–16) and 13 days (range 12–18), respectively. Only 2 pts developed skin grade I/II acute GVHD, while no pt had visceral acute GVHD. With a median follow-up of 6 months (range 2–8), no pt experienced EBV-related PTLD or died of transplant-related complications. Conclusions.The immunomagnetic removal of αβ+ T cells and B cells was effective and gave reproducible results. In all cases, a remarkable depletion of unwanted T and B cells was attained. The depleted graft contained high numbers of CD34+ HSC, as well as of immune effector cells implicated in the control of leukemia growth and GVHD, such as γδ+ T cells, NK cells, DC1, DC2 and non-classical CD14+CD16+ monocytes. From a clinical standpoint, the infusion of αβ/CD19-depleted grafts from HLA-haploidentical family donors was safe, resulting into sustained donor engraftment without life-threatening complications. Disclosures:No relevant conflicts of interest to declare.

Long-Term Survival and Late Deaths after Hematopoietic Cell Transplantation for Primary Immunodeficiency Diseases and Inborn Errors of Metabolism

It is uncertain whether late mortality rates after hematopoietic cell transplantation for severe combined immunodeficiency (SCID), non-SCID primary immunodeficiency diseases (non-SCID PIDD), and inborn errors of metabolism (IEM) return to rates observed in the general population, matched for age, sex, and nationality. We studied patients with SCID (n = 201), non-SCID PIDD (n = 405), and IEM (n = 348) who survived for at least 2 years after transplantation with normal T cell function (SCID) or >95% donor chimerism (non-SCID PIDD and IEM). Importantly, mortality rate was significantly higher in these patients compared with the general population for several years after transplantation. The rate decreased toward the normal rate in patients with SCID and non-SCID PIDD beyond 6 years after transplantation, but not in patients with IEM. Active chronic graft-versus-host disease at 2 years was associated with increased risk of late mortality for all diseases (hazard ratio [HR], 1.87; P = .05). In addition, late mortality was higher in patients with non-SCID PIDD who received T cell-depleted grafts (HR 4.16; P = .007) and in patients with IEM who received unrelated donor grafts (HR, 2.72; P = .03) or mismatched related donor grafts (HR, 3.76; P = .01). The finding of higher mortality rates in these long-term survivors for many years after transplantation confirms the need for long-term surveillance.

Haploidentical stem cell transplantation for children with high‐risk leukemia

The Chilean population is ethnically diverse, and more than 50% of children referred for hematopoietic stem cell transplantation (HSCT) lack a suitable donor. To expand the donor pool, we assessed the feasibility, tolerance, and efficacy of using a haploidentical (HI) donor and a reduced-intensity conditioning regimen for high-risk pediatric leukemia. This study was facilitated by technology transfer from St. Jude Children's Research Hospital over the 2 preceding years. Between March 2006 and April 2009, 10 patients (median age, 9.8 years) received T cell-depleted grafts at Calvo Mackenna Hospital in Santiago. Median cell doses were CD34+: 7.45 × 10(6)/kg (range, 4.00-20.20 × 10(6)/kg); CD3+: 0.88 × 10(5)/kg (0.11-1.35 × 10(5)/kg); and CD56+: 71.30 × 10(6)/kg (31.50-131.80 × 10(6)/kg). Nine patients experienced complete engraftment; six of the nine remain alive and clinically well 13-50 months post-HSCT. Three patients died after bone marrow relapse, while only one died of transplant-related causes. Virus reactivation was the main post-transplant complication: 5/10 had positive CMV PCR but none had CMV disease. One patient developed acute GvHD > grade II and only one had chronic GvHD. HI-HSCT is feasible in our setting, offers a rational treatment option, and expands the donor pool significantly for children with high-risk leukemia in a developing country. This information is especially relevant to other ethnically diverse populations that are poorly represented in international donor registries.

Allogeneic Hematopoietic Stem Cell Transplantation for Pediatric Patients with Treatment-Related Myelodysplastic Syndrome or Acute Myelogenous Leukemia

The development of treatment-related myelodysplastic syndrome (tMDS) or treatment-related acute myelogenous leukemia (tAML) is a complication that can occur after chemotherapy or radiation therapy. Eighteen patients with a previous malignancy treated at our institution and three patients with a nonmalignant primary tumor received an allogeneic hematopoietic stem cell transplant (HSCT) on the pediatric bone marrow (BM) transplantation service for the treatment of tMDS/tAML over a 15-year period. Five patients proceeded to HSCT without induction chemotherapy. Fourteen patients received high-dose cytarabine according to the Capizzi II regimen as first-line induction therapy with 13 of them achieving complete remission (CR) or refractory anemia (RA) with persistent cytogenetic abnormalities after this treatment. Two patients received an anthracycline-based induction therapy. Conditioning regimens were selected according to previous therapies: 11 patients received busulfan-melphalan-fludarabine (BU-MEL-FLU), which consisted of busulfan (0.8 mg/kg/dose every 6 hours ×10 doses), melphalan (70 mg/m(2)/dose × two doses), and fludarabine (25 mg/m(2)/dose × five doses) for cytoreduction; three patients received a total body irradiation (TBI)-containing regimen; seven patients received myeloablative regimens containing busulfan and/or melphalan and/or thiotepa with doses modified for organ toxicity. Sixteen patients received T cell-depleted (TCD) grafts; four patients received unmodified grafts; one patient received a double-unit cord blood transplantation (DUCBT). Donors included HLA-matched (n = 9), or mismatched (n = 3) related donors, or HLA-matched (n = 4), or mismatched (n = 4) unrelated donors, or DUCBT (n = 1). Disease status at the time of HSCT was: morphologic and cytogenetic CR (n = 12); RA with positive cytogenetics (n = 6); and refractory disease (n = 3). With a median follow-up of 5.9 years (2.2-15.7 years), the 5-year overall survival (OS) and disease-free survival (DFS) rates for the entire group were 61.1% with 12 patients alive without evidence of either primary disease or tMDS/tAML. The OS and DFS rate for the 11 patients who received the BU-MEL-FLU cytoreduction with TCD grafts was 54.5%. DFS was 65.7% for patients in RA or CR at HSCT compared with 0% for patients with >5% residual marrow blasts (P = .015). Nine patients died; the cause of death was relapse of MDS/AML (n = 4) or primary disease (n = 2), graft-versus-host disease (GVHD; n = 2), and infection (n = 1). Four patients developed grade II to IV acute GVHD. One patient developed localized chronic GVHD. Our results suggest that the strategy of induction with high-dose cytarabine therapy followed by allogeneic stem cell transplantation improves the overall outcome for patients with tMDS/tAML. In addition, the use of a TCD transplantation with BU-MEL-FLU as cytoreduction may decrease the toxicity of transplantation in heavily pretreated patients without an increase in relapse rate.

Characteristics of CliniMACS® System CD34-Enriched T Cell-Depleted Grafts in a Multicenter Trial for Acute Myeloid Leukemia-Blood and Marrow Transplant Clinical Trials Network (BMT CTN) Protocol 0303

Eight centers participated in the Blood and Marrow Transplant Clinical Trials Network (BMT CTN) protocol 0303 to determine the effect of extensive T cell depletion (TCD) on the outcome of HLA matched sibling donor transplantation for acute myeloid leukemia. One goal of the study was to determine if TCD could be performed uniformly among study sites. TCD was achieved using the CliniMACS(®) CD34 Reagent System for CD34 enrichment. Processed grafts needed to contain ≥ 2.0 × 10(6) CD34(+)cells/kg with a target of 5.0 × 10(6) CD34(+) cells/kg and <10(5) CD3(+) T cells/kg. Up to 3 collections were allowed to achieve the minimum CD34(+) cell dose. In total, 86 products were processed for 44 patients. Differences in the starting cell products between centers were seen in regard to total nucleated cells, CD34(+) cells, and CD3(+) T cells, which could in part be ascribed to a higher dose of granulocyte-colony stimulating factor used for mobilization early in the trial. Differences between centers in processing outcomes were minimal and could be ascribed to starting cell parameters or to differences in graft analysis methods. Multivariate analysis showed that CD34(+) cell recovery (66.1% ± 20.3%) was inversely associated with the starting number of CD34(+) cells (P = .02). Median purity of the CD34 enriched fraction was 96.7% (61.5%-99.8%) with monocytes and B cells the most common impurity. All patients received the minimum CD34(+) cell dose, and 39 patients (89%) came within 10% or exceeded the target CD34(+) cell dose without exceeding the maximum T cell dose. All patients proceeded to transplantation and all achieved initial engraftment. Products processed at multiple centers using the CliniMACS System for CD34 enrichment were comparably and uniformly highly enriched for CD34(+) cells, with good CD34(+) cell recovery and very low CD3(+) T cell content.

Safety and Immunogenicity of the Tetravalent Protein-Conjugated Meningococcal Vaccine (MCV4) in Recipients of Related and Unrelated Allogeneic Hematopoietic Stem Cell Transplantation

Given the high morbidity and mortality associated with meningococcal disease, in 2007 the Advisory Committee of Immunization Practices recommended immunization of all children ages 11-18 with a protein-conjugated meningococcal vaccine. There are limited data on the immunogenicity of this vaccine after allogeneic hematopoietic stem cell transplantation (allo-HCT). Since 2007, we have immunized 48 patients with the MCV4 vaccine. Two vaccinated patients who lacked follow-up titers were excluded from this analysis. Stem cells were derived from an HLA-identical sibling (n = 17) or an alternative donor (n = 29). The median time to vaccination was 2.34 years after allo-HCT. Only 7 patients responded to all 4 serogroups, and 16 patients responded to none of the serogroups. The response to serogroups A, C, Y, and W-135 was 52%, 30%, 46%, and 33%, respectively. The ability to respond to 2 or more serogroups was not affected by age, diagnosis, time to vaccination, or history of graft-versus-host disease. Receipt of a T cell-depleted graft was associated with a poorer response (P = .044). Eight of 16 patients who received a second MCV4 vaccination responded to all 4 serogroups. This retrospective study suggests that response to a single MCV4 vaccination is poor after allo-HCT. Administration of a 2-dose series, as currently recommended for patients with asplenia, complement deficiency, and HIV infection, should be evaluated in this patient population.

Safety and Immunogenicity of the Live Attenuated Varicella Vaccine Following T Replete or T Cell-Depleted Related and Unrelated Allogeneic Hematopoietic Cell Transplantation (alloHCT)

There are limited studies assessing the live attenuated varicella vaccine following allogeneic hematopoietic cell transplantation (alloHCT). Because of the morbidity of varicella acquired after childhood, we immunized and retrospectively analyzed the safety and immunogenicity of this vaccine in 46 varicella zoster virus (VZV) seronegative patients <20 years old at HCT who achieved a CD4 cell count ≥200/μL, were off immunosuppression, and responded to ≥1 post-HCT vaccines. Two vaccinated patients lacking follow-up titers were excluded from analysis. Stem cells were derived from an HLA-matched sibling (n = 18) or an alternative (HLA mismatched related or unrelated) donor (n = 26). Median time to vaccination was 4 years. Sixty-four percent of patients seroconverted following 1 immunization. There was no significant difference in response between recipients of a matched related or alternative donor graft (P = .2) or between those given a T cell-depleted or T-replete alternative donor graft (P = .27). Three of 44 patients developed a self-limited varicella-like rash within 2.5 weeks of immunization. With a median follow-up of 29.1 (range: 6.9-167.1) months, there were no subsequent cases of varicella-like rashes. No patient developed shingles. This study suggests that this vaccine is safe and immunogenic when given according to preset clinical and immunologic milestones, warranting larger prospective studies in patients ≥24 months following HCT as outlined in current post-HCT vaccine guidelines.

Haploidentical Stem Cell Transplantation for Children with High-Risk Leukemia In Chile: Report of the First 15 Cases.

AbstractAbstract 4581The Chilean population is ethnically diverse, and more than 50% of the children referred for hematopoietic stem cell transplantation lack a suitable donor. To expand the donor pool we assessed the feasibility, tolerance, and efficacy of using a parental haploidentical (HI) donor and a reduced-intensity conditioning regimen for patients with high-risk leukemia; this study was preceded by 2 years of technology transfer from St. Jude Children's Research Hospital. Between March 2006 and November 2009 fifteen patients (median age, 9.6 years) received T cell-depleted grafts at Calvo Mackenna Hospital, from peripheral stem cells mobilized with G-CSF from parental donors. N° aphaeresis/patient = one; 6/15 products required a second depletion, final Log of depletion= 3, 11 (5, 05-1, 64). Median cell doses were CD34+: 9.5 × 106 cells/kg (range, 2.13 – 20.0), CD3+: 1.0 ×105cells/kg (0.11 – 1.59), and CD56+: 67, 9 × 106cells/kg (7, 6–131, 8). 14/15 patients engrafted one1ry primary engraftment failure and in 2 cases a 2ry engraftment failure (CMV and Toxoplasmosis treatment). Eight remain alive and clinically well at a median follow up of 12.4 months post-transplant (7.2 – 52.2). Four patients died after bone marrow relapse, while only one died of transplant-related causes. Virus reactivation was the main post-transplant complication: 9/15 had positive CMV PCR but none had CMV disease. One patient developed acute GvHD > grade II and one had chronic GvHD. In this setting, HI transplantation offers a rational option for children who lack suitable stem cell donors. This information is especially relevant for populations that are poorly represented in international donor registries. On the other hand HISCT with RIC with ATG and TNI permits a good OS and EFS in this especially high risk population low TRM. This approach is associated with low incidence of GvHD and high viral reactivation incidence but no lethal infection even without CD3+ lymphocyte recovery due probably to pre-emptive treatment and fast NK cell recovery. Disclosures:No relevant conflicts of interest to declare.

Safety and Feasibility of Posaconazole as Oral Antifungal Prophylaxis In Pediatric Patients Under 12 Years of Age Following Allogeneic Stem Cell Transplantation.

AbstractAbstract 1308Pediatric patients undergoing hematopoietic stem cell transplantation (HSCT) are at high risk of acquiring fungal infections. Post-transplant immune deficiency, immunosuppressive medication, viral infections, and acute graft-versus-host disease (GvHD) are known risk factors for fungal infections especially with Aspergillus spp. and Candida spp. Thus, antifungal prophylaxis early after transplantation is indicated, but data for pediatric patients under twelve years of age is scarce. Oral antifungal prophylaxis with extended spectra azoles, e. g. voriconazole or itraconazole, is preferentially used in pediatric patients after allogeneic HSCT, while only few studies have been published. Under either one prophylaxis, break-through invasive fungal infections were still observed in our center. In adults, another broad-spectrum triazole, posaconazole, showed activity against Candida spp., Aspergillus spp., Cryptococcus spp., Zygomycetes, Fusarium spp. Based on these data pediatric BMT patients received oral prophylaxis using posaconazole in our clinic, and we retrospectively assessed the safety, feasibility, and initial data on efficacy. The patient group consisted of sixty pediatric patients (median of age 6.0 years) early after high dose chemotherapy and allogeneic HSCT for hemato-oncological malignancies and inborn errors of metabolism. 31 patients (51.7%) received a T cell-depleted graft. Posaconazole was commenced after discharge from the BMT unit. The observation period was defined as the time from treatment start of posaconazole till the end of oral antimycotic prophylaxis with posaconazole with a maximum of 200 days after transplant. The dosage of posaconazole given on the basis of adult dosage of 200 mg three times per day (tid) and was adapted according to the weight of the pediatric patients accordingly. Twenty-eight of the sixty pediatric patients received 5 mg per kg body weight twice a day (bid) (5 mg/kg BW bid) in an oral suspension, and thirty-two pediatric patients received 4 mg/kg BW tid. Pediatric patients, who received posaconazole 4 mg/kg BW tid had more stable trough levels in the morning (median 377 μg/L, mean 390±137.1 μg/L) in comparison to patients, who received posaconazole 5 mg/kg BW bid (median 134 μg/L, mean 217±187.9 μg/L). Both regimens were well tolerated without severe side effects. There was no decline in white blood cell counts, granulocytes, platelets, the number of CD3 positive T cells, CD4 positive T cells and CD16/56 positive NK cells during treatment with posaconazole. There were no adverse effects on the organ functions of any of the 60 pediatric patients due to the administration of posaconozole. After start of posaconazole we observed an increase in the liver parameters AST (baseline value smaller than 39 U/L) in 3 (5%) out of sixty pediatric patients amount equivalent to 2 × baseline values, in 14 (23.3%) patients amount equivalent 3 × baseline and ALT in 9 (15%) pediatric patients amount equivalent to 2 × baseline, in 14 patients amount equivalent to 3 × baseline of 39 U/L. The increase of liver parameters occurred in 83% of cases within the first fourteen days after start of posaconazole and normalized by day twenty-six after discontinuation of the drug. The analysis of cyclosporine A (CsA) whole blood concentrations was performed in a total of 28 pediatric patients, who were treated with CsA during the observation period with posaconazole. There was a statistically significant, but moderate increase of CsA blood concentrations by 28% on days 2 to 6 (p<0.01) after start of posaconazole in a total of 16 (57.14%) pediatric patients. Adverse events potentially related to posaconazole were observed in six (10.0%) out of the sixty patients and included itching in one case (1.7%), nausea in two cases (3.3%), vomiting in one case (1.7%), and reduced appetite in two cases (3.3%).No incidence of proven or probable invasive mycosis was recorded during the observation period. Taken together, posaconazole prophylaxis was safe and well tolerated in a cohort of sixty children under the age of 12 years following HSCT. Disclosures:Off Label Use: Posaconazole for prophylaxis in pediatric patients after BMT.

Leukemia Lineage-Specific Chimerism Analysis and Molecular Monitoring Improve Outcome of Donor Lymphocyte Infusions

A retrospective analysis was performed of 118 patients with hematologic malignancies who received donor lymphocyte infusions (DLIs) after allogeneic stem cell transplantation (ASCT). Treatment was either given because of hematologic relapse (n = 44), molecular/cytogenetic relapse (n = 52), or other causes (n = 22). Molecular relapse was in most cases based on leukemia lineage-specific chimerism analysis. Patients with acute leukemia and myelodysplastic syndrome showed a 3-year survival of 42% if DLI treatment was given because ofmolecular relapse, compared to 16% in hematologic relapse (P < .006). In multivariate analysis, there was a correlation between response to DLI and nonhematologic relapse (risk ratio [RR] 3.36, P = .001), chronic graft-versus-host disease (cGVHD) (RR = 1.51, P = .005), and late relapse (RR = 2.06, P = .017). The overall incidences of acute GVHD (aGVHD) grades I-II and grades III-IV aGVHD were 33% and 8.5%, respectively. Probability of cGVHD was 33%. The development of aGVHD or cGVHD did not significantly influence the response of DLI in patients with molecular/cytogenetic relapse. However, the development of cGVHD was significantly associated with a better response in patients with hematologic relapse because only 4 of 29 patients without cGVHD responded compared to 7 of 12 with cGVHD (P = .007). The development of cGVHD increased significantly if DLI was given >12 months after ASCT (46% versus 27%, P = .04). In contrast, time between ASCT and start of DLI treatment had no significant influence on the risk of developing aGVHD. To conclude, monitoring of leukemia lineage-specific chimerism is of utmost importance for DLI response after ASCT.

Generation of cytotoxic T-cell lines using overlapping pentadecapeptides derived from conserved regions of the adenovirus hexon protein

Many of the 51 serotypes of adenovirus have been associated with clinically relevant infection. Adenovirus can disseminate rapidly in patients with a compromised immune system, such as that which occurs secondary to haematopoietic progenitor-cell transplantation. The higher rate of infection in recipients of T cell-depleted grafts and in those undergoing T cell-targeted treatment during graft versus host disease demonstrates the importance of a T-cell response in preventing disseminated infection. Studies have shown that the memory response to adenovirus is directed primarily to the hexon protein and is dominated by CD4+ T cells, probably due to the ability of the virus to block its presentation on HLA class I antigens. We have developed an approach to expand adenovirus-specific T cells using a pool of overlapping pentadecapeptides derived from selected conserved regions of hexon. We characterized responses to identify the peptides that are recognized, the responding T-cell subsets and their HLA restriction. Of eight lines that were characterized extensively, seven included both CD4+ and CD8+ T cells and each recognized between two and eight unique peptide sequences. By focusing the response on the conserved sequences of hexon, the cell lines are likely to recognize most of the serotypes responsible for clinically relevant disease. The 15 aa peptides used to prime the responses are more likely than whole virus or longer peptides to expand the less frequent CD8+ memory subset. Lines prepared by using our method may be more effective in adoptive immunotherapy protocols designed to prevent or treat disseminated adenovirus infections in high-risk patients.

Partial T Cell-Depleted Allogeneic Stem Cell Transplantation following Reduced-Intensity Conditioning Creates a Platform for Immunotherapy with Donor Lymphocyte Infusion and Recipient Dendritic Cell Vaccination in Multiple Myeloma

Allogeneic stem cell transplantation (SCT) in multiple myeloma (MM) may induce a curative graft-versus-myeloma (GVM) effect. Major drawback in unmanipulated reduced-intensity conditioning (RIC) SCT is the risk of severe and longstanding graft-versus-host-disease (GVHD). This study demonstrates that transplantation with a partial T cell-depleted graft creates a platform for boosting GVM immunity by preemptive donor lymphocyte infusion (DLI) and recipient dendritic cell (DC) vaccination, with limited GVHD. All 20MM patients engrafted successfully. Chimerism analysis in 19 patients evaluable at 3 months revealed that 7 patients were complete donor, whereas 12 patients were mixed chimeric. Grade II acute GVHD (aGVHD) occurred in 7 patients (35%) and only 4 patients (21%) developed chronic GVHD (cGVHD). Fourteen patients received posttransplantation immunotherapy, 8 preemptive DLI, 5 patients both DLI and DC vaccination, and 1 patient DC vaccination only. DC vaccination was associated with limited toxicity, and none of these patients developed GVHD. Importantly, overall treatment-related mortality (TRM) at 1 year was low (10%). Moreover, the overall survival (OS) is 84% with median follow-up of 27 months, and none of the patients died from progressive disease. These findings illustrate that this novel approach is associated with limited GVHD and mortality, thus creating an ideal platform for adjuvant immunotherapy.

Hematopoietic Cell Transplantation for Children with Acute Lymphoblastic Leukemia in Second Complete Remission: Similar Outcomes in Recipients of Unrelated Marrow and Umbilical Cord Blood versus Marrow from HLA Matched Sibling Donors

Transplant decisions for children with acute lymphoblastic leukemia (ALL) in second complete remission (CR2) are often based on the type of available donor. In many cases, allogeneic hematopoietic cell transplantation (HCT) is considered only if a human leukocyte antigen (HLA) matched sibling donor (MSD) is available. The role of unrelated donor (URD) HCT in this patient population is not well established. As advances in supportive care and donor selection have improved, the use of URD HCT in such patients should be reevaluated. We analyzed the outcomes of 87 consecutive children with ALL in CR2 who underwent allogeneic HCT at the University of Minnesota between 1990 and 2007. Donor sources included MSD bone marrow (n = 32), well and partially matched (M, n = 18) and mismatched (MM, n = 16) URD bone marrow and URD umbilical cord blood (UCB, n = 21). Although the incidence of neutrophil recovery was similar in all groups, the overall incidence of grades II-IV acute graft-versus-host disease (aGVHD) and chronic GVHD (cGVHD) was 37% and 9%, respectively, with a higher incidence of aGVHD in recipients of URD grafts. Leukemia-free survival (LFS) at 5 years was lower in recipients of MM-URD grafts, but was comparable in all other groups. Although relapse at 5 years was highest in recipients of MSD (50%), results were not significantly different compared to recipients of M-URD (17%), MM-URD (6%), and UCB (33%) (P = .17). The development of grades II-IV aGVHD and a first remission >3 years were associated with a lower risk of relapse (relative risk [RR] 0.2, P = .03; RR 0.2. P = .01 respectively). Together, these results support the continued investigation of URD HCT for ALL in CR2, and suggest the timing of HCT in these children should be based primarily on the risk of relapse with conventional chemotherapy and not on the type of donor available.

Human Cytomegalovirus–Specific T Cell Reconstitution in Young Patients Receiving T Cell–Depleted, Allogeneic Hematopoietic Stem Cell Transplantation

Reconstitution of human cytomegalovirus (HCMV) T cell-specific immunity is of crucial relevance in patients receiving a hematopoietic stem cell transplant (HSCT). Scarce data on this subject are available for children receiving a T cell-depleted HSCT. We investigated HCMV-specific T cell recovery in 48 recipients of a T cell-depleted HSCT from a human leukocyte antigen (HLA)-disparate relative. Autologous HCMV-infected dendritic cells were used to stimulate HCMV-specific CD8+ and CD4+ T cells producing interferon-gamma (IFN). The 1-year cumulative incidence of both HCMV infection and specific T cell reconstitution was 83% among the 23 HCMV-seropositive patients and 4% and 8%, respectively, among the 25 HCMV-seronegative patients (P < .01). HCMV-specific T cell reconstitution was significantly delayed in patients receiving T cell-depleted grafts, compared with patients receiving unmanipulated HSCTs (median time to reconstitution, 75 vs. 47 days, respectively; P < .01). The median time from HCMV infection to immune recovery in recipients of T cell-depleted grafts was 47 days. Detection of HCMV-specific T cells correlated with control of HCMV infection. The number of residual T cells in the graft predicted earlier T cell recovery (P = .02). Latent HCMV in the recipient was the major cause of HCMV reactivation and also promoted specific T cell reconstitution in patients given a T cell-depleted HSCT from an HLA-disparate relative. Routine immunologic monitoring is valuable in identifying patients with early HCMV-specific T cell reconstitution.

Early Immune Response Against Retrovirally Transduced Herpes Simplex Virus Thymidine Kinase-Expressing Gene-Modified T Cells Coinfused with a T Cell-Depleted Marrow Graft: An Altered Immune Response?

Administration of herpes simplex thymidine kinase (HSV-tk)-expressing, gene-modified T cells (GMCs) with T cell-depleted bone marrow transplantation (TCD-BMT) can allow modulation of posttransplantation alloreactivity. Twelve patients received 2 x 10(5) to 2 x 10(6) CD3+ donor GMCs per kilogram with HLA-identical sibling TCD-BMT. Despite extensive T cell depletion of bone marrow, an intensive conditioning regimen, and immunosuppressive graft-versus-host disease (GvHD) prophylaxis, infusion at the time of TCD-BMT of this low number of GMCs sufficed to induce a rapid GMC-specific immune response, as detected by interferon- enzyme- linked immunospot assay in six of eight patients, preferentially targeting HSV-tk. Maximal responses were reached early (median time, 49 [35-68] days post-BMT), with a subsequent rapid and significant decrease in five of six evaluable patients. Immune responses were negatively correlated with the maximal circulating GMC counts. However, such immune response did not result in the elimination of circulating GMCs and was not associated with measurable ex vivo cytotoxic activity against GMCs. Furthermore, alloreactive GMCs still could induce GCV-sensitive GvHD in one patient despite an ongoing immune response. Overall, infusion of HSV-tk-expressing GMCs at the time of BMT results in an early immune response. Such immune response may be altered and may not prevent persistent GCV-sensitive alloreactivity.