•The study cohort comprised children with leukemia who underwent partially CD3+-depleted peripheral stem cell transplantation (PSCT).•Three-year overall survival was 61.8% (95% confidence interval ...CI, 50.2% to 71.4%) and event-free survival was 52.0% (95% CI, 40.3% to 62.4%).•Age ≥15 years and second complete remission were associated with worse outcomes.•The incidence of severe chronic graft-versus-host disease was lower in CD3+-depleted PSCTthan in T cell-replete PSCT.
Most children who may benefit from stem cell transplantation lack a matched related donor. Alternative donor transplantations with an unrelated donor (URD) or a partially matched related donor (PMRD) carry an increased risk of graft-versus-host-disease (GVHD) and mortality compared with matched related donor transplantations. We hypothesized that a strategy of partial CD3+/CD19+ depletion for URD or PMRD peripheral stem cell transplantation (PSCT) would attenuate the risks of GVHD and mortality. We enrolled 84 pediatric patients with hematologic malignancies at the Children's Hospital of Philadelphia and the Children's Hospital of Wisconsin between April 2005 and February 2015. Two patients (2.4%) experienced primary graft failure. Relapse occurred in 23 patients (27.4%; cumulative incidence 26.3%), and 17 patients (20.2%) experienced nonrelapse mortality (NRM). Grade III-IV acute GVHD was observed in 18 patients (21.4%), and chronic GVHD was observed and graded as limited in 24 patients (35.3%) and extensive in 8 (11.7%). Three-year overall survival (OS) was 61.8% (95% confidence interval CI, 50.2% to 71.4%) and event-free survival (EFS) was 52.0% (95% CI, 40.3% to 62.4%). Age ≥15 years was associated with decreased OS (P= .05) and EFS (P= .05). Relapse was more common in children in second complete remission (P = .03). Partially CD3+-depleted alternative donor PSCT NRM, OS, and EFS compare favorably with previously published studies of T cell-replete PSCT. Historically, T cell-replete PSCT has been associated with a higher incidence of extensive chronic GVHD compared with limited chronic GVHD, which may explain the comparatively low relapse and NRM rates in our study cohort despite similar overall rates of chronic GVHD. Partial T cell depletion may expand donor options for children with malignant transplantation indications lacking a matched related donor by mitigating, but not eliminating, chronic GVHD.
Hematopoietic progenitor cells (HPC) collected by apheresis HPC(A) have become the most common source of HPC used for transplantation. HPC(A) products have a more variable content of CD34+ progenitor ...cells than marrow or umbilical cord blood, especially when collected from autologous donors. Most often the CD34+ cell content of previous collections is used to decide whether or not additional collections are needed to reach the target CD34+ cell infusion dose. Accurate assessment of CD34+ cell counts are imperitive in order to properly determine the need for additional apheresis procedures. CD34 content is typically measured by flow cytometry, either using a dual platform method that requires determination of the percentage of CD34+ cells within the total nucleated cells or a single platform method that uses counting beads to directly determine the number of CD34+ cells in a given volume of product. Both methods depend on accurate determination of the volume of the product in order to calculate the total number of CD34+ cells that have been collected. Volume estimates of HPC source products are most often performed using the product weight in grams (g) after subtracting the weight of the bag in which the product is collected. The weight is then divided by the product’s specific gravity to obtain the volume. The specific gravity of a substance is the ratio of the density of the substance to the density of a reference substance, such as water (1 g = 1 mL). The specific gravity of whole blood has been defined as approximately 1.058, whereas the specific gravity of platelet products is approximately 1.030. The difference between the two products is a function of the cellular content. Blood has a higher specific gravity since it contains more high-density cells (red blood cells (RBCs) and granulocytes) than platelet concentrates (largely platelets and lymphocytes). The composition of marrow is similar to blood, so a specific gravity of 1.058 is used. However, there are no published data that estimates the specific gravity of HPC(A) products and since these products are enriched for cells that are more dense than platelet products but less dense than whole blood products neither of the two established specific gravities is optimal to use. We sought to determine what the specific gravity of an apheresis product truly is so as to determine the optimal factor to estimate volume from weight for HPC(A) products. To this end our laboratory sampled 54 well-mixed HPC(A) products at receipt and used a reverse pipetting method and a sensitive analytical balance to weigh 1 mL aliquots of product. A hematology analyzer was used to measure the total number of white blood cells (WBC) and RBCs per mL of product. The 54 sampled products contained on average 2.7 ± 1.1 x 10E8 WBCs per mL and 5.5 ± 2.0E8 RBCs per mL, which is similar to the average seen for 823 products collected since 2010 of 2.81 ± 1.33 x 10E8 WBCs per mL. The weight in grams was significantly associated with the total number of RBCs + WBCs in a given product (p=0.026) but not with total WBC alone, indicating that RBC content was a contributing factor. The average weight (i.e. specific gravity) of 1.0 mL HPC(A) aliquots was 1.047 ± 0.009 g, a value between that of whole blood and platelet products. Based on these data we recommend that laboratories use a factor of 1.047 to convert the weight of HPC(A) products in grams to volume to more accurately determine the number of CD34+ cells contained in a given product.
No relevant conflicts of interest to declare.
Background
SCD is a chronic debilitating disease secondary to frequent veno-occlusive events resulting in chronic organ damage, including cerebral vasculopathy, acute chest syndrome (ACS), pulmonary ...hypertension and a significantly shortened life-span (Bunn et al, NEJM, 1997; Lanzkron et al, PHR, 2013; Platt et al, NEJM, 1994). Importantly, SCD survivors also develop significant defects in neurocognition, especially processing speed and have a poor HRQL (Stotesbury et al, Neurology, 2018); Vichinsky et al, JAMA, 2010; Panepinto et al, BJH, 2005). To date the only cure for SCD patients has been HLA matched sibling AlloSCT following either myeloablative (MAC) or reduced toxicity conditioning (RTC), (Walters et al, NEJM, 1996; Bhatia/Cairo et al, BMT, 2014; Talano/Cairo et al, EJH, 2015; Gluckman et al, Blood, 2017). Unfortunately, only approximately 15% of patients have an unaffected HLA matched sibling donor (Mentzer et al, AJPHO, 1994). We previously demonstrated the safety and efficacy of CD34 enrichment and mononuclear cell (MNC) addback (2 x 105 CD3/kg) in pediatric matched unrelated donor recipients (Geyer/Cairo et al, BJH, 2012). We now report the long term results of MAC and familial HISCT utilizing CD34 enrichment and MNC (CD3) addback in high risk SCD recipients.
Methods
SCD patients with one or more high risk features (cerebral vasculopathy, repetitive ACS, repetitive VOC, abnormal TCD requiring RBC TX) underwent MAC and parental HISCT consisting of hydroxyurea, azathioprine, fludarabine, busulfan, cyclophosphamide, thiotepa, r-ATG and TLI and PBSCT utilizing CD34 enrichment (10x106 CD34/kg) (Miltenyi®) and MNC addback (2x105 CD3/kg) and tacrolimus AGVHD prophylaxis x 100 days as we have previously described (Talano/Cairo et al, ASH, 2017). Donor chimerism in WBC and RBC (CD71) enriched fractions was determined centrally as we have previously described (Geyer/Cairo et al, BJH, 2012). AGVHD & CGVHD were determined utilizing the Glucksberg and NIH consensus criteria, respectively. Transthoracic echocardiogram, TRJ velocity, PFTs, MRI/MRA were performed at baseline and 2 years. Neurocognitive testing utilizing DIVERGT screening was performed at baseline and 2 years as we have previously described (Vichinsky et al, JAMA, 2010; Krull et al, JCO, 2008). HRQL testing utilized the CHRIS-General and CHRIs-HSCT (age appropriate) at baseline and Days +45, 100, 180, 365 and 730 post HISCT as we have previously described (Kelly/Parsons et al, PBC, 2012).
Results
Among the 19 HISCT recipients, the mean±SEM age was 13.1±1.2 years (3.3-20.0) years, gender 12/7 (M/F). There were 18 parental haploidentical donors with mean±SEM age of 41.3±1.8 (30-55) years, gender 15/3 (F/M). The mean±SEM CD34 enriched PBSC infused was 10.94±0.4x106/kg and MNC addback (2 x 105 CD3/kg). The median time to neutrophil and platelet engraftment was day +9 and +19, respectively. The 1yr mean±SEM whole WBC and RBC (CD71) mixed donor chimerism was 97.1±1.4 and 96.4±2.0%, respectively. The cumulative incidence of grade II-IV AGVHD and CGVHD was 6.2% and 6.7%, respectively (Figure 1A). The probability of 1yr EFS was 90% (CI95 64-97%). In comparison from baseline to 2yr post HISCT, PFTs were stable to improved with a significant improvement in conductance (sGAW) (p<0.026), cardiac SF and TRJ velocity were stable to improved, MRI/MRA showed no new overt and/or silent strokes and no new cerebral vasculopathy. Intellectual functioning, memory, language, and executive function were also stable to improved. Most importantly, processing speed was significantly improved (p<0.026) (Figure 1B), emotional functioning (HRQL) (p<0.03) (Figure 1C) and physical functioning (HRQL) (p<0.01) (Figure 1D) were significantly improved at 2 years vs. pre-HISCT.
Conclusion
MAC and familial HISCT utilizing parental donors and CD34 enrichment and MNC addback (2 x 105 CD3/kg) in high risk patients with SCD was well tolerated, resulted in rapid hematological reconstitution, long term stable WBC and RBC (CD71) donor chimerism, low cumulative incidence of A+CGVHD and stable to improved pulmonary and cardiac function. There was also a significant improvement in processing speed and emotional and physical HRQL 2yrs post HISCT. This study was registered at clinicaltrials.gov (NCT02675959) and conducted under IND (14359) (MSC) and supported in large part by FDA R01FD004090.
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Cairo:Janssen: Research Funding. Parsons:Seattle Genetics: Research Funding. Friedman:MedImmune/AstraZeneca: Consultancy, Speakers Bureau. Vichinsky:Global Blood Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; bluebird bio: Membership on an entity's Board of Directors or advisory committees; Protagonist: Research Funding. Walters:Sangamo Therapeutics: Consultancy; bluebird bio: Research Funding; ViaCord Processing Lab: Other: Medical Director; AllCells Inc.: Other: Medical Director. Shenoy:Novartis, Vertex, Bluebird Bio: Honoraria, Membership on an entity's Board of Directors or advisory committees.
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Background: SCD is characterized by chronic vaso-occlusive crises and multiorgan failure resulting in poor quality of life and early mortality (Bhatia/Cairo et al, BMT 2014). There is presently no ...curative therapy for patients with high risk SCD other than HLA-identical sibling AlloSCT. (Freed/Cairo et al BMT 2012). However, less than 15% of eligible SCD patients have an unaffected MSD with a 10-15% increase of graft failure and TRM (Talano/Cairo et al, EJH, 2015). Similarly, most patients lack a matched related donor and UCB is an inferior source in SCD recipients (Radhakrishman/Cairo et al, BBMT 2013). Haploidentical familial donors with SCD trait offers an opportunity for a new donor source for children with high risk SCD. To overcome HLA barriers, Geyer/Cairo et al (BJH, 2012) demonstrated that T cell depletion using CD34 enriched HPC products with PB MNC addback transplanted in pediatric recipients utilizing MUD was associated with sustained engraftment, low risk of aGVHD but limited by delayed immune reconstitution. Efforts to use FHI donors and T replete AlloSCT in patients with SCD were associated with high rates of graft failure (Bolanes-Meade J et al Blood 2012; Ruggieri et al BBMT 2011). We previously reported FHI CD34 enriched/PB MNC addback AlloSCT is feasible and well tolerated in patients with high risk SCD (Abikoff/Cairo, ASBMT 2015).
Objective: To characterize immunological reconstitution following FHI AlloSCT with CD34 enriched grafts with PB MNC addback in children and adolescents with high risk SCD.
Methods: 15 patients were evaluatedpretransplant at D+30, 60, 100 and 180 following FHI AlloSCT. GCSF mobilized HPC were collected by apheresis (Spectra OPTIA, Terumo BCT) and products underwent CD34 enrichment using the CliniMACS cell separation system (materials generously supplied by Miltenyi Biotec, Cambridge , MA) with a PB MNC addback dose of 2x10*5 CD3/kg. Immune cell and subset reconstitution was assessed by flow cytometry. NK function was determined by cytotoxic activity against K562 tumor targets at 10:1 E:T ratio by europium release assay and intracellular LAMP-1 (CD107a) and granzyme B expression by flow cytometry. Whole blood, T cell and RBC chimerism (CD71) determined by flow cytometry and by STR.
Results: Patients achieved neutrophil and platelet engraftment in a median time of 10 and 16 days, respectively. By D+30, median whole blood donor chimerism was ≥93% and ≥95% at most recent followup (D+30-730). Median donor chimerism in the erythroid lineage was 95% by D+60, with 7 of 13 patients ≥99% at D+30. This was maintained at most recent followup (D+30-730). Median T cell chimerism was 90% (D+60-550) and median NK cell chimerism was 90% by D+30 and maintained at ≥95% through D+730. NK (CD3-/56+) and NKT (CD3+/56+) cell reconstitution following FHI AlloSCT was rapid and peaked at D+30 (35.5±8.6%, 271x10*3/ul; 14.2±4%, 179x10*3/ul, respectively). Moreover, there was robust NK cell receptor expression reconstitution with high levels of activating receptors, NKp46, NKG2D and KIR2DS and inhibitory receptors NKG2A, CD94 and KIR2DL2/3 at D+30 Fig 1. NK cytotoxicity against K562 at E;T 10:1 peaked at D+30 (26±3%) and D+180 (28±3%) compared to pretransplant (16±2%, p<0.01). NK activation marker, CD107a, peaked at D+30 (37±9%) and D+180 (41±6%) and there was robust granzyme B degranulation at D+30. CD3+, CD4+, CD8+ and CD19+ immune reconstitution occurred between D+180 and D+270. One year absolute (mean±SEM) cells/ul of CD3+, CD4+, CD8+, CD19+ and CD56+ was 795±168, 408±102, 375±90, 815±352 and 204±37, respectively. Fig 2
Conclusion: Immune reconstitution and donor chimerism was relatively rapid after FHI AlloSCT with CD34 enriched grafts with PB MNC addback in high risk SCD patients. The donor MNC addback after CD34 selection may in part contribute to rapid engraftment and immune reconstitution along with sustained donor chimerism.
This research was supported by FDA grant 5R01FD004090.
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Cairo:Celgene: Research Funding.
Background:
There are a limited number of studies that have evaluated the proteome of MDS patients (pts). Two chemokines (CXCL-4 and CXCL-7) were found to be differentially depressed in patients with ...MDS compared to patients with other hematologic disorders (Aviado et al, Proc Nat Acad Sci 2007). One study found that normal levels of these proteins are essential for stem cell quiescence, and with their depletion stem cell exhaustion ensues (Bruns I et al, Nat Med). In the current study, we sought to confirm the MDS-specific reduction of these chemokines, and in an exploratory fashion examine the association of CXCL4/CXCL7 pre-HCT plasma levels with MDS disease status pre-HCT and subsequent post HCT outcomes.
Methods:
We analyzed three cohorts of pts >18 years of age reported to CIBMTR from 2008-2011: MDS cohort (N= 80); Non-MDS cohort undergoing allogeneic HCT for either aplastic anemia or non-Hodgkin lymphoma (N= 70); and Normal donors (ND) (N= 79). The primary objectives were to compare the pre-HCT plasma levels of the two chemokines (CXCL-4 and CXCL-7) among the three cohorts, and to explore whether these chemokines correlate with post-HCT disease free survival (DFS), overall survival (OS), relapse and non-relapse mortality (NRM) in the MDS cohort. Abcam’s Human ELISA kits were used to measure pre-HCT serum CXCL4 and CXCL7 concentrations. Optimal cut-off points to classify patients as "high" vs. "low" were identified using maximum likelihood method. We also examined whether the use of hypomethylating agents (HMA) pre-HCT correlated with chemokine levels pre-HCT in the MDS cohort.
Results:
Median ages were 58 yrs, 47 yrs, and 30 yrs for MDS, non-MDS, and ND, respectively. Forty-five pts (56%) in the MDS cohort had advanced stage disease at transplant and 46% had poor or very poor risk cytogenetics based on the International Prognostic Scoring System-Revised (IPSS-R) criteria. Fifty pts (63%) were male and 65% had Karnofsky performance score of 90-100 in MDS cohort. Myeloablative regimen was used in 55% of MDS pts compared to 23% in non-MDS cases. Low CXCL4 levels were seen in 57 (71%) MDS vs. 38 (48%) ND vs. 69 (99%) non-MDS pts (P<0.001) (Fig A). Low CXCL7 levels were seen in 63 (79%) MDS, 47 (59%) ND and 65 (93%) non-MDS pts (p<0.001) (Fig B). In MDS pts with low CXCL4 levels, 39% had high or very high IPSS-R score compared to only 11% among those with high CXCL4 levels (p=0.07) (Table 2). Similarly, among MDS pts with low CXCL7, 36% had high or very high IPSS-R score compared to only 7% among those with high CXCL7 levels (p=0.18) (Table 2). There was no correlation between the use of HMA pre-HCT and the pre-HCT chemokine levels in MDS pts. For MDS pts >50 yrs, there was no correlation between age and CXCL4 and CXCL7 levels. In univariate analysis, neither chemokine was associated with post-HCT OS, DFS, NRM or relapse in MDS pts.
Conclusions:
Our study confirms that levels of chemokines CXCL-4 and CXCL-7 in MDS pts were significantly lower compared to ND. However, in contrast to previous findings by Aivado et al.2007, we did not observe MDS specific reduction as these chemokines were significantly lower among those with NHL and AA compared to MDS patients at HCT. Consistent with previous findings by Aivado et al. we observed a trend towards further reduction of these chemokines among MDS patients with more advanced disease (defined by higher IPSS-R scores) compared to MDS pts with less advanced disease. In our exploratory analysis, we did not observe an association between pre-HCT chemokines levels and post-HCT outcomes among MDS patients. Similarly, the use of HMA among MDS pts was not associated with pre-HCT chemokines levels. An important caveat to this conclusion is that we did not have data on the duration of HMA use. In MDS pts who were >50 yrs, there was no correlation between age and chemokine levels. Large-scale prospective studies are needed to understand more fully the prognostic role of the MDS proteome as well as the complex interplay between proteomic perturbations and acquired somatic mutations these patients often display in determining outcomes.
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No relevant conflicts of interest to declare.
AlloSCT from HLA-matched MSD has been successful for high-risk SCD, and is the only known curative therapy (Talano/Cairo et al EJH, 2014). We have recently demonstrated 100% EFS and absence of sickle ...cell symptoms following reduced toxicity conditioning in HLA matched sibling donor (MSD) or cord blood AlloSCT (Bhatia/Cairo et al BMT, 2014). However, 5 out of 6 children lack an HLA MSD without SCD. Only 19% of recipients of African descent will identify a well-matched unrelated donor (MUD) and results after unrelated UCBT are poor (Radhakrishnan/Cairo et al BBMT, 2013). Recent results utilizing matched unrelated donors in a multi-center trial showed unacceptable rates of cGVHD at 62% (95% CI 41-77) (Shenoy et al Blood, 2016). We demonstrated CD34+ selection followed by T-cell addback from MUD in pediatric recipients led to 100% engraftment with minimal aGVHD (Geyer/Cairo et al BJH, 2011). Limitations in the past of various T-cell depletion methods have included a higher incidence of graft failure, delayed immune reconstitution, opportunistic fungal infection and higher incidence of cGVHD. FHI TCD AlloSCT utilizing the approach of CD34+ enrichment and T-cell addback could expand the donor pool and improve outcomes for patients with high risk SCD and have similar outcomes to AlloSCT from HLA MSD.
This SCD consortium trial (www.sicklecelltransplantconsortium.org) (NCT01461837) is investigating the safety, feasibility, EFS, donor chimerism, graft failure, aGVHD and cGVHD after FHI TCD AlloSCT in high-risk SCD patients.
High risk included: ≥1 CVA, ≥2 ACS, ≥3 VOC in past 2 years, or 2 abnormal TCDs. Patients (2-<21 yrs) who have ≥1 high-risk SCD features were eligible. Patients received hydroxyurea and azathioprine, day -59 - day -11, fludarabine (150mg/m2), busulfan (12.8mg/kg) (targeted trough Css of 600-900), thiotepa (10 mg/kg), cyclophosphamide (200mg/kg), R-ATG (8mg/kg), and TLI (500cGy) followed by FHI T-cell depleted AlloSCT. AGVHD prophylaxis: tacrolimus. We utilized the CliniMACS to enrich for peripheral blood HPC's; target dose of 10 x 106 CD34+ cells/kg with a fixed dose of 2 x 105 CD3+ T cells/kg. The Child Health Rating Inventories (CHRIs) was used to serially collect parent proxy-rated health-related quality of life (HRQL).
Twenty-one patients have been enrolled. Nineteen patients have received AlloSCT to date. Fifteen patients utilized maternal donors and 4 patients utilized paternal donors. Two donors encountered thrombocytopenia that resolved spontaneously. The mean ± SD CD34+ enrichment prior to cryopreservation was 12.66 ± 3.3 x 106/kg and log T-cell depletion was 4.8 ± 0.58. Evaluable pts had early neutrophil engraftment (median day +9), and ≥95% whole blood and RBC (CD71) donor chimerism at 1yr. Probability of aGVHD n=17, 7.1% (CI95: 0-70.0); cGVHD n=17, 20% (CI95: 0.7-59.6) (Figure 1A and 1B). Probability of 1-Year Overall Survival (OS) n=17; 88.2% (CI95: 60.6-96.9) (Figure 1C). Immune reconstitution was robust with 1 yr NK, CD3, CD4 and CD8 204±37 cells/μL, 795±168 cells/μL, 408±102 cells/μL and 375±90 cells/μL, respectively. Two-yr PFT results were also robust with % predicted FVC, FEV1, TLC and DLCO of 89%, 84%, 82%, and 68%, respectively. All survivors are free of SCD symptoms and without signs of progression of multi-organ toxicity. One patient developed late hepatic SOS and died at day +59; one patient died at day +390 due to complications of CGVHD, and one patient died at day +141 of steroid refractory aGVHD, the remainder are alive and free of SCD symptoms with a median follow-up of +711 days (range, +231 to +1684 days). Most importantly, mean + SD HRQL score (0-100) reported at 1 yr for emotional, physical functioning, role, and global scores were 82.9±9.7 points, 82.8±15.8 points, 89.6±13.3 points, and 86±8.2 points, respectively.
Early results indicate promising results in 1 yr OS, stable donor chimerism, and low incidence of acute and/or cGVHD after FHI HCT utilizing CD34 enrichment and CD3/MNC addback in high-risk SCD patients who lack a MSD or URD. A longer term follow-up is needed to assess long-term safety and outcomes. This research was supported by RO1FD004090-01A1.
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Walters:AllCells, Inc: Other: Medical Director; bluebird bio: Research Funding; Sangamo Therapeutics: Consultancy; ViaCord Processing Lab: Other: Medical Director. Cairo:Jazz Pharmaceuticals: Speakers Bureau.
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 × 106 CD34+ cells/kg with a target of 5.0 × 106 CD34+ cells/kg and <105 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.
Summary
Invasive aspergillosis (IA) is a major cause of infection‐related mortality in patients with haematological malignancies, especially in recipients of haematopoietic stem cell transplants. We ...have prepared overlapping pentadecapeptides (11‐aa overlap with previous peptide) spanning the entire 427‐aa coding region of the Aspergillus allergen, Asp f16 shown previously in mice to induce Th1‐type cell responses in vivo and in humans to induce proliferative and cytotoxic CD4+ T cell responses. Mature dendritic cells (DC) pulsed with a complete pool of peptides were used to generate T cell lines. Two lines from HLA‐B*3501+ donors were found to be strongly cytotoxic to autologous Asp f16‐peptide pool‐ and Aspergillus culture extract‐pulsed targets after 4–5 weekly primings. Cytotoxic T lymphocyte (CTL) culture supernatant killed Aspergillus conidia, and cells directly killed Aspergillus hyphae. Cytotoxic activity and interferon (IFN)‐γ production were mediated exclusively by CD8+ T cells in response to pool‐pulsed targets. Interleukin (IL)‐4 production was not detected. CTL activity was restricted by HLA‐B*3501 and based on peptide prediction programmes was most probably directed to YFKYTAAAL (YFK), LPLCSAQTW (LPL) and GTRFPQTPM (GTR) in one donor, while only LPL was recognized by CTL from the second donor. Pool‐pulsed B*3503+ BLCL but not B*3502+ or B*3508+ BLCL presented peptide to donor no. 1. B*3503+ BLCL presented YFK and to a lesser extent GTR, but not peptide LPL. Our data show that in addition to our previously identified Class II restricted peptide response, DC pulsed with a pentadecapeptide pool from Asp f16 are capable of inducing polyclonal, HLA‐Class I‐restricted, Aspergillus‐specific T cells that may be capable of conferring immunity to IA.
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