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Background Aims: Traditional weight-based dosing of rabbit anti-thymocyte globulin (rATG) used in allogeneic hematopoietic cell transplantation (HCT) to prevent graft-versus-host disease (GVHD) and graft rejection leads to variable exposures. High exposures induce delayed CD4+immune reconstitution (CD4+IR) and greater mortality. We sought to determine the impact of rATG exposure in children and young adults receiving various types of EX-VIVO T-cell-depleted (EX-VIVO-TCD) HCT., Methods: Patients receiving their first EX-VIVO-TCD HCT (CliniMACS CD34+, Isolex or soybean lectin agglutination), with removal of residual T cells by E-rosette depletion (E-) between 2008 and 2018 at Memorial Sloan Kettering Cancer Center were retrospectively analyzed. rATG exposure post-HCT was estimated (AU*d/L) using a validated population pharmacokinetic model. Previously defined rATG-exposures, <30, 30-55, ≥55 AU*d/L, were related with outcomes of interest. Cox proportional hazard and cause-specific models were used for analyses., Results: In total, 180 patients (median age 11 years; range 0.1-44 years) were included, malignant 124 (69%) and nonmalignant 56 (31%). Median post-HCT rATG exposure was 32 (0-104) AU*d/L. Exposure <30 AU*d/L was associated with a 3-fold greater probability of CD4+IR (P < 0.001); 2- to 4-fold lower risk of death (P = 0.002); and 3- to 4-fold lower risk of non-relapse mortality (NRM) (P = 0.02). Cumulative incidence of NRM was 8-fold lower in patients who attained CD4+IR compared with those who did not (P < 0.0001). There was no relation between rATG exposure and aGVHD (P = 0.33) or relapse (P = 0.23). Effect of rATG exposure on outcomes was similar in three EX-VIVO-TCD methods., Conclusions: Individualizing rATG dosing to target a low rATG exposure post-HCT while maintaining total cumulative exposure may better predict CD4+IR, reduce NRM and increase overall survival, independent of the EX-VIVO-TCD method., Competing Interests: Declaration of competing interest KJC: research support: Novartis, Celgene, Cellectis, Atara Bio; consultant: Novartis, Atara Bio. SEP: support for the conduct of clinical trials: AlloVir, Jasper Therapeutics, Atara Biotherapeutics; consultation (last 24 months) CellEvolve, ADMA, Regeneron; intellectual property related to the use of third-party VSTs licensed to Atara Biotherapeutics with all rights assigned to Memorial Sloan Kettering Cancer Center. RJO: royalties following licensure of EBV-specific T-cell bank by Atara Biotherapeutics, research support and consultant fees from Atara Biotherapeutics. JJB: consulting Sobi, Bluebird Bio, Avrobio, BlueRock, SmartImmune, Sanofi, Omeros, Advanced Clinical (DMC Chair). All other authors have no commercial, proprietary or financial interest in the products or companies described in this article., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Background Aims: Cytomegalovirus (CMV) reactivation is a significant complication following allogeneic hematopoietic stem cell transplant (HSCT) and affects upwards of 40% of pediatric HSCT patients. Pre-emptive therapy remains the only effective treatment strategy available for pediatric patients following CMV reactivation. Little is known about how the timing of induction treatment following CMV reactivation impacts outcomes in pediatric patients, especially following ex vivo T-cell-depleted (TCD) HSCT., Methods: The authors evaluated how the timing of induction treatment after CMV reactivation impacts overall survival (OS) and CMV disease in pediatric patients undergoing TCD HSCT at a single institution. The authors retrospectively analyzed patients treated on the pediatric service who received an initial ex vivo TCD HSCT at Memorial Sloan Kettering Cancer Center (MSKCC) from January 2010 to June 2018. CMV reactivation was defined as ≥1 CMV polymerase chain reaction >500 copies/mL in whole blood or >137 IU/mL in plasma within the first 180 days after allogeneic HSCT. To analyze the impact of the timing of induction treatment, the authors' primary study outcome was OS and secondary outcome was CMV disease., Results: A total of 169 patients who underwent an initial allogeneic TCD HSCT on the pediatric service at MSKCC from January 2010 to June 2018 were included in the analysis. Thirty-seven (22%) patients reactivated CMV during the first 180 days following HSCT. Of those patients who reactivated CMV, CMV donor/recipient (D/R) serostatus was as follows: D+/R+ n = 28 (76%) and D-/R+ n = 9 (24%). There was no CMV reactivation observed among recipients who were CMV-seronegative irrespective of donor serostatus. In those patients who reactivated CMV, the median time from HSCT to CMV reactivation was 24 days (interquartile range, 20-31). Eleven patients ultimately developed CMV disease in addition to CMV viremia, whereas the remaining patients had only CMV viremia. The cumulative incidence of CMV reactivation at 60 days was 45.2% (95% confidence interval [CI], 32.8-57.5) in the D+/R+ subgroup and 31% (95% CI, 14.2-47.9) in the D-/R+ subgroup. For those patients who reactivated CMV, 30 (81%) received induction treatment with ganciclovir or foscarnet. To analyze the impact of the timing of induction treatment on clinical outcomes, the authors restricted the analysis to those patients who reactivated CMV and received induction treatment (n = 30). The timing of induction treatment was significantly associated with OS, with optimal timing of initiation within a week of CMV reactivation (P = 0.02). There was no significant impact on the timing of induction treatment and risk of CMV disease (P = 0.30)., Conclusions: In ex vivo TCD HSCT in pediatric patients, early initiation of induction treatment after CMV reactivation is associated with improved OS., (Copyright © 2021 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Background: An association between early CD4+ T cell immune reconstitution (CD4+ IR) and survival after T-replete allogeneic hematopoietic cell transplantation (HCT) has been previously reported. Here we report validation of this relationship in a separate cohort that included recipients of ex vivo T-cell-depleted (TCD) HCT. We studied the relationship between CD4+ IR and clinical outcomes., Methods: A retrospective analysis of children/young adults receiving their first allogeneic HCT for any indication between January 2008 and December 2017 was performed. We related early CD4+ IR (defined as achieving >50 CD4+ T cells/µL on two consecutive measures within 100 days of HCT) to overall survival (OS), relapse, non-relapse mortality (NRM), event-free survival (EFS) and acute graft-versus-host disease (aGVHD). Fine and Gray competing risk models and Cox proportional hazard models were used., Results: In this analysis, 315 patients with a median age of 10.4 years (interquartile range 5.0-16.5 years) were included. The cumulative incidence of CD4+ IR at 100 days was 66.7% in the entire cohort, 54.7% in TCD (N = 208, hazard ratio [HR] 0.47, P < 0.001), 90.0% in uCB (N = 40) and 89.6% in T-replete (N = 47) HCT recipients. In multi-variate analyses, not achieving early CD4+ IR was a predictor of inferior OS (HR 2.35, 95% confidence interval [CI] 1.46-3.79, P < 0.001) and EFS (HR 1.80, 95% CI 1.20-2.69, P = 0.004) and increased NRM (HR 6.58, 95% CI 2.82-15.38, P < 0.001). No impact of CD4+ IR on relapse or aGVHD was found. Within the TCD group, similar associations were observed., Conclusion: In this HCT cohort, including recipients of TCD HCT, we confirmed that early CD4+ IR was an excellent predictor of outcomes. Finding strategies to predict or improve CD4+ IR may influence outcomes., (Copyright © 2020 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Chimeric antigen receptor (CAR) T cells have demonstrated clinical benefit in patients with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). We undertook a multicenter clinical trial to determine toxicity, feasibility, and response for this therapy. A total of 25 pediatric/young adult patients (age, 1-22.5 years) with R/R B-ALL were treated with 19-28z CAR T cells. Conditioning chemotherapy included high-dose (3 g/m2) cyclophosphamide (HD-Cy) for 17 patients and low-dose (≤1.5 g/m2) cyclophosphamide (LD-Cy) for 8 patients. Fifteen patients had pretreatment minimal residual disease (MRD; <5% blasts in bone marrow), and 10 patients had pretreatment morphologic evidence of disease (≥5% blasts in bone marrow). All toxicities were reversible, including severe cytokine release syndrome in 16% (4 of 25) and severe neurotoxicity in 28% (7 of 25) of patients. Treated patients were assessed for response, and, among the evaluable patients (n = 24), response and peak CAR T-cell expansion were superior in the HD-Cy/MRD cohorts, as compared with the LD-Cy/morphologic cohorts without an increase in toxicity. Our data support the safety of CD19-specific CAR T-cell therapy for R/R B-ALL. Our data also suggest that dose intensity of conditioning chemotherapy and minimal pretreatment disease burden have a positive impact on response without a negative effect on toxicity. This trial was registered at www.clinicaltrials.gov as #NCT01860937., (© 2019 by The American Society of Hematology.)

Fanconi anemia (FA) is an inherited bone marrow failure syndrome characterized by chromosomal fragility, progressive marrow failure, and cancer predisposition. Hematopoietic cell transplantation (HCT) is curative for FA-related marrow failure or leukemia, but both radiation exposure during transplant and graft-versus-host disease (GVHD) may increase risk of later malignancies of the head and neck and anogenital area. In this study, we tested a radiation-free conditioning regimen with a T-cell-depleted graft to eliminate radiation exposure and minimize early and late toxicities of transplant. Forty-five patients (median age, 8.2 years; range 4.3-44) with FA underwent HCT between June 2009 and May 2014. The preparative regimen included busulfan, cyclophosphamide, fludarabine, and rabbit anti-thymocyte globulin. Busulfan levels were monitored to avoid excess toxicity. All grafts were CD34-selected/T-cell-depleted using the CliniMacs CD34 columns (Miltenyi). Thirty-four patients (75.6%) with marrow failure and 11 (24.4%) with myelodysplastic syndrome underwent HCT using matched unrelated (n = 25, 55.5%), mismatched unrelated (n = 14, 31.1%), or mismatched related donors (n = 6, 13.4%). One year probabilities of overall and disease-free survival for the entire cohort, including patients with myeloid malignancy and those receiving mismatched related/haploidentical grafts, were 80% (±6%) and 77.7% (±6.2%), respectively (median follow-up 41 months). All young children (<10 years of age) undergoing HCT for marrow failure using low-dose busulfan-containing regimen survived. No patients developed acute grade 3-4 GVHD. Sequential reduction of busulfan dose was successfully achieved per study design. Our results show excellent outcomes in patients with FA undergoing alternative donor HCT without radiation exposure. The study is registered to www.clinicaltrials.gov as #NCT01082133., (© 2017 by The American Society of Hematology.)

Hepatic veno-occlusive disease (VOD), also called sinusoidal obstruction syndrome (SOS), is a potentially life-threatening complication of hematopoietic stem cell transplantation (HSCT). Untreated hepatic VOD/SOS with multi-organ failure (MOF) is associated with >80% mortality. Defibrotide has shown promising efficacy treating hepatic VOD/SOS with MOF in phase 2 studies. This phase 3 study investigated safety and efficacy of defibrotide in patients with established hepatic VOD/SOS and advanced MOF. Patients (n = 102) given defibrotide 25 mg/kg per day were compared with 32 historical controls identified out of 6867 medical charts of HSCT patients by blinded independent reviewers. Baseline characteristics between groups were well balanced. The primary endpoint was survival at day +100 post-HSCT; observed rates equaled 38.2% in the defibrotide group and 25% in the controls (23% estimated difference; 95.1% confidence interval [CI], 5.2-40.8;P= .0109, using a propensity-adjusted analysis). Observed day +100 complete response (CR) rates equaled 25.5% for defibrotide and 12.5% for controls (19% difference using similar methodology; 95.1% CI, 3.5-34.6;P= .0160). Defibrotide was generally well tolerated with manageable toxicity. Related adverse events (AEs) included hemorrhage or hypotension; incidence of common hemorrhagic AEs (including pulmonary alveolar [11.8% and 15.6%] and gastrointestinal bleeding [7.8% and 9.4%]) was similar between the defibrotide and control groups, respectively. Defibrotide was associated with significant improvement in day +100 survival and CR rate. The historical-control methodology offers a novel, meaningful approach for phase 3 evaluation of orphan diseases associated with high mortality. This trial was registered at www.clinicaltrials.gov as #., (© 2016 by The American Society of Hematology.)

While cord blood transplantation (CBT) is an effective therapy for hematologic malignancies, acute graft-versus-host disease (aGVHD) is a leading cause of transplant-related mortality (TRM). We investigated if biomarkers could predict aGVHD and TRM after day 28 in CBT recipients. Day 28 samples from 113 CBT patients were analyzed. Suppressor of tumorigenicity 2 (ST2) was the only biomarker associated with grades II-IV and III-IV aGVHD and TRM. Day 180 grade III-IV aGVHD in patients with high ST2 levels was 30% (95% confidence interval [CI], 18-43) vs 13% (95% CI, 5-23) in patients with low levels (P = .024). The adverse effect of elevated ST2 was independent of HLA match. Moreover, high day 28 ST2 levels were associated with increased TRM with day 180 estimates of 23% (95% CI, 13-35) vs 5% (95% CI, 1-13) if levels were low (P = .001). GVHD was the most common cause of death in high ST2 patients. High concentrations of tumor necrosis factor receptor-1, interleukin-8, and regenerating islet-derived protein 3-α were also associated with TRM. Our results are consistent with those of adult donor allografts and warrant further prospective evaluation to facilitate future therapeutic intervention to ameliorate severe aGVHD and further improve survival after CBT., (© 2015 by The American Society of Hematology.)

We investigated the unit characteristics associated with engraftment after double-unit cord blood (CB) transplantation (dCBT) and whether these could be reliably identified during unit selection. Cumulative incidence of neutrophil engraftment in 129 myeloablative dCBT recipients was 95% (95% confidence interval: 90-98%). When precryopreservation characteristics were analyzed, the dominant unit CD34(+) cell dose was the only characteristic independently associated with engraftment (hazard ratio, 1.43; P = .002). When postthaw characteristics were also included, only dominant unit infused viable CD34(+) cell dose independently predicted engraftment (hazard ratio, 1.95; P < .001). We then examined the determinants of infused viable CD34(+) cell dose (precryopreservation count, postthaw recovery, and postthaw viability) in 402 units thawed at our center. This revealed close correlation between precryopreservation and postthaw CD34(+) cell counts (r(2) = 0.73). Median CD34(+) cell recovery was 101%, although it ranged from 12% to 1480%. Notably, units from non-Netcord Foundation for the Accreditation of Cellular Therapy (Netcord-FACT)-accredited banks were more likely to have low recovery (P < .001). Furthermore, although median postthaw CD34(+) cell viability was 92%, 33 (8%) units had <75% viable CD34(+) cells. Units from non-Netcord-FACT-accredited banks and units with cryovolumes other than 24.5 to 26.0 mL were more likely to have poor postthaw viability. Precryopreservation CD34(+) cell dose and banking practices should be incorporated into CB unit selection., (© 2014 by The American Society of Hematology.)

We evaluated HLA-compatible donor leukocyte infusions (DLIs) and HLA-compatible or HLA-disparate EBV-specific T cells (EBV-CTLs) in 49 hematopoietic cell transplantation recipients with biopsy-proven EBV-lymphoproliferative disease (EBV-LPD). DLIs and EBV-CTLs each induced durable complete or partial remissions in 73% and 68% of treated patients including 74% and 72% of patients surviving ≥ 8 days after infusion, respectively. Reversible acute GVHD occurred in recipients of DLIs (17%) but not EBV-CTLs. The probability of complete response was significantly lower among patients with multiorgan involvement. In responders, DLIs and EBV-CTLs regularly induced exponential increases in EBV-specific CTL precursor (EBV-CTLp) frequencies within 7-14 days, with subsequent clearance of EBV viremia and resolution of disease. In nonresponders, EBV-CTLps did not increase and EBV viremia persisted. Treatment failures were correlated with impaired T-cell recognition of tumor targets. Either donor-derived EBV-CTLs that had been sensitized with autologous BLCLs transformed by EBV strain B95.8 could not lyse spontaneous donor-derived EBV-transformed BLCLs expanded from the patient's blood or biopsied tumor or they failed to lyse their targets because they were selectively restricted by HLA alleles not shared by the EBV-LPD. Therefore, either unselected DLIs or EBV-specific CTLs can eradicate both untreated and Rituxan-resistant lymphomatous EBV-LPD, with failures ascribable to impaired T-cell recognition of tumor-associated viral antigens or their presenting HLA alleles.

The influence of cell dose and human leukocyte antigen (HLA) match on double-unit cord blood (CB) engraftment is not established. Therefore, we analyzed the impact of cell dose and high-resolution HLA match on neutrophil engraftment in 84 double-unit CB transplant recipients. The 94% sustained engraftment rate was accounted for by 1 unit in nearly all patients. Higher CD3(+) cell doses (P = .04) and percentage of CD34(+) cell viability (P = .008) were associated with unit dominance. After myeloablative conditioning, higher dominant unit total nucleated cell (TNC), CD34(+) cell, and colony-forming unit doses were associated with higher sustained engraftment and faster neutrophil recovery (P = .07, P = .0008, and P < .0001, respectively). Total infused TNC (P = .0007) and CD3(+) cell doses (P = .001) also significantly influenced engraftment. At high-resolution extensive donor-recipient HLA disparity was frequent, but had no influence on engraftment (P = .66), or unit dominance (P = .13). Although the unit-unit HLA match also did not affect sustained engraftment (P = 1.0), recipients of units closely (7-10 to 10-10) HLA-matched to each other were more likely to demonstrate initial engraftment of both units (P < .0001). Our findings have important implications for unit selection and provide further insight into double-unit biology.

Outcomes of unrelated donor cord blood transplantation in 191 hematologic malignancy children (median age, 7.7 years; median weight, 25.9 kg) enrolled between 1999 and 2003 were studied (median follow-up, 27.4 months) in a prospective phase 2 multicenter trial. Human leukocyte antigen (HLA) matching at enrollment was 6/6 (n = 17), 5/6 (n = 58), 4/6 (n = 111), or 3/6 (n = 5) by low-resolution HLA-A, -B, and high-resolution (HR) DRB1. Retrospectively, 179 pairs were HLA typed by HR. The median precryopreservation total nucleated cell (TNC) dose was 5.1 x 10(7) TNC/kg (range, 1.5-23.7) with 3.9 x 10(7) TNC/kg (range, 0.8-22.8) infused. The median time to engraftment (absolute neutrophil count > 500/mm(3) and platelets 50 000/muL) was 27 and 174 days. The cumulative incidence of neutrophil engraftment by day 42 was 79.9% (95% confidence interval [CI], 75.1%-85.2%); acute grades III/IV GVHD by day 100 was 19.5% (95% CI, 13.9%-25.5%); and chronic GVHD at 2 years was 20.8% (95% CI, 14.8%-27.7%). HR matching decreased the probability of severe acute GVHD. The cumulative incidence of relapse at 2 years was 19.9% (95% CI, 14.8%-25.7%). The probabilities of 6-month and 2-year survivals were 67.4% and 49.5%. Unrelated donor cord blood transplantation from partially HLA-mismatched units can cure many children with leukemias. The study was registered at www.clinicaltrials.gov as #NCT00000603.

Antithymocyte globulin (ATG) has been used in allogeneic stem-cell transplantation to prevent graft rejection and graft-versus-host disease (GvHD). Its use, however, has been associated with delayed T-cell reconstitution and prolonged susceptibility to opportunistic infections (OIs) especially in patients undergoing T cell-depleted (TCD) transplantation. Recently, a prospective trial was conducted in 52 adult patients (median age, 47 years) with various hematologic malignancies undergoing TCD transplantation from HLA-matched related donors without the use of ATG. The cytoreductive regimen consisted of hyperfractionated total body irradiation (HFTBI), thiotepa, and fludarabine. The preferred source of the graft was peripheral blood stem cells (PBSCs). No additional graft rejection or GvHD prophylaxis was given. All evaluable patients engrafted without any immune-mediated graft rejections. Disease-free survival (DFS) at 3 years was 61% in all patients, and 70% in patients with standard-risk disease. Acute GvHD was limited to grade 2 in 8% and chronic GvHD in 9% of patients. Life-threatening OIs occurred in 3 of 52 patients and was fatal in 1. This study demonstrates durable engraftment with a low incidence of GvHD despite the lack of ATG, as well as the curative potential of this regimen.

Current European and US guidelines for recombinant hepatitis B vaccine (rHBV) after hematopoietic-cell transplantation (HCT) vary. The European Group for Blood and Marrow Transplantation (EBMT) recommends rHBV starting 6 to 12 months after HCT. Immunization is optional in the Centers for Disease Control and Prevention (CDC) guidelines. Nevertheless, rHBV is required for re-entry to school and certain workplaces. To determine the immunogenicity of rHBV following HCT, the prevaccine and postvaccine titers of 292 allogeneic transplant recipients who were immunized with rHBV were analyzed. Immunization was initiated in patients off immunosuppression who achieved specific minimal milestones of immune competence. Overall, 64% of patients seroconverted. In multivariate analyses, response was adversely affected by age older than 18 years (P < .01) and history of prior chronic graft-versus-host disease (GVHD; P < .001) but not by donor type or by use of T-cell depletion, adoptive immunotherapy, or rituximab. By comparison, 89% of rHBV nonresponders mounted a 3-fold or greater rise in polio titers following 3 doses of inactivated poliovirus. These data demonstrate that the rate of seroconversion following rHBV is lower in allogeneic HC transplant recipients compared with age-matched healthy controls. The data emphasize the need to document prevaccine and postvaccine titers to ensure response and suggest that immunization guidelines based on time interval from HCT, irrespective of immune competence, may not ensure adequate protection against certain vaccine-preventable diseases.

Donor-derived T cells have been proposed to play a role in pathogenesis of chronic graft-versus-host disease (cGVHD). The impact of ex vivo T-cell depletion (TCD) on cGVHD was analyzed in a randomized multicenter trial involving unrelated donor marrow transplants. A total of 404 patients diagnosed with hematologic malignancies received a total body irradiation-based myeloablative conditioning regimen. GVHD prophylaxis included TCD plus cyclosporine (CSA) or unmodified grafts with CSA plus methotrexate (M/C). Median recipient age was 31.2 years (range, 0.5-55.6 years); median follow-up time since randomization was 4.2 years. The mean number of T cells infused was 1 log lower on the TCD arm. The incidence of cGVHD at 2 years was similar between the TCD and M/C arms, 29% versus 34% (P = .27), respectively. Survival at 3 years from diagnosis of cGVHD was also similar, (TCD 51% versus M/C 58%; P = .29). The proportion of patients with cGVHD who discontinued immunosuppression at 5 years was not different (TCD 72% versus M/C 63%; P = .27), and incidence of serious infections and leukemia relapse were similar on both treatment arms. In spite of a significant reduction of acute GVHD, TCD did not reduce the incidence of cGVHD or improve survival in patients who developed cGVHD.

Background: Graft-versus-host disease (GVHD) reduces the efficacy of unrelated donor bone marrow transplantation in patients with lymphohaemopoietic malignancy. A multi-centre, randomised trial was undertaken to determine the effects of ex-vivo T-cell depletion versus methotrexate and cyclosporine immunosuppression on 3-year disease-free survival., Methods: Between Mar 1, 1995, and Oct 31, 2000, 405 patients with lymphohaemopoietic malignancy, from 15 participating centres, were randomly assigned to undergo transplantation with either T-cell depleted marrow and cyclosporine A (TCD arm; n=201) or methotrexate and cyclosporine A after transplantation of T-replete marrow (M/C arm; n=204). The primary outcome was 3-year disease-free survival and was analysed by intention to treat., Findings: Five patients died before transplantation. Seven in the TCD arm received T-replete grafts. Disease-free survival at 3 years was 27% (95% CI 21-33) and 34% (27-40) in recipients of TCD and M/C, respectively (p=0.16). TCD was associated with significantly more rapid neutrophil recovery (15 days vs 20 days, p<0.0001), less grade III-IV acute GVHD (18%vs 37%, p<0.0001), reduced grade III-IV toxicities (19%vs 29%, p=0.017), reduced duration of initial hospitalisation, but higher risk of chronic myelogenous leukaemia relapse (20%vs 7%, p=0.009) and cytomegalovirus infection (28%vs 17%, p=0.023) than was M/C., Interpretation: Disease-free survival at 3 years did not differ between TCD and M/C groups. Relapse and opportunistic infection are important obstacles to successful unrelated donor bone marrow transplantation, irrespective of the method of GVHD prophylaxis used.

Successful allogeneic hematopoietic stem cell transplantation (HSCT) requires reconstitution of normal T-cell immunity. Recipient thymic activity, biologic features of the allograft, and preparative regimens all contribute to immune reconstitution. We evaluated circulating T-cell phenotypes and T-cell receptor rearrangement excision circles (TRECs) in 331 blood samples from 158 patients who had undergone allogeneic HSCTs. All patients had received myeloablative conditioning regimens and were full donor chimeras in remission. Younger patients exhibited more rapid recovery and higher TRECs (P =.02). Recipients of T-cell-depleted allografts initially had lower TRECs than unmodified allograft recipients (P <.01), but the difference abated beyond 9 months. TREC level disparities did not achieve significance among adults with respect to type of allograft. Measurable, albeit low, TREC values correlated strongly with severe opportunistic infections (P <.01). This finding was most notable during the first 6 months after transplantation, when patients are at greatest risk but before cytofluorography can detect circulating CD45RA(+) T cells. Low TRECs also correlated strongly with extensive chronic graft-versus-host disease (P <.01). Recipients of all ages of either unmodified or T-cell-depleted allografts therefore actively generate new T cells. This generation is most notable among adult recipients of T-cell-depleted allografts, most of whom had also received antithymocyte globulin for rejection prophylaxis. Low TREC values are significantly associated with morbidity and mortality after transplantation. T-cell neogenesis, appropriate to age but delayed in adult recipients of T-cell-depleted allografts, justifies interventions to hasten this process and to stimulate desirable cellular immune responses.

Allogeneic bone marrow transplantation (BMT) offers the only curative therapy for chronic myelogenous leukemia. We compared prospectively collected results of 2464 unrelated donor (URD) transplantations with 450 HLA-identical, matched sibling donor (MSD) transplantations performed at collaborating National Marrow Donor Program institutions. A total of 63% of URDs were matched at HLA-A, -B, and at -DRB1 alleles; all MSDs were genotypically identical at major histocompatibility loci. URD recipients were younger (median 36 vs 39, P =.001) than MSDs and underwent BMT later after diagnosis (median 17 [0-325 months] vs 7 [1-118 months], P =.001) and less often in chronic phase (CP) (67% vs 82%, P =.001). Multivariate analysis demonstrated a significantly increased risk of graft failure and acute graft versus host disease after URD BMT. The risk of hematologic relapse was low after either matched URD or MSD transplantations. We observed significantly though modestly poorer survival and disease-free survival (DFS) after URD transplantations. However, for those undergoing transplantation during CP within 1 year from diagnosis, 5-year DFS was similar or only slightly inferior after matched URD versus MSD transplantation (age < 30: URD 61% +/- 8% vs MSD 68% +/- 15%, P =.18; 30-40: URD 57% +/- 9% vs MSD 67% +/- 10%, P =.05; > 40: URD 46% +/- 9% vs MSD 57% +/- 9%, P =.02). Delay from diagnosis to BMT in CP patients led to substantially poorer 5-year DFS after matched URD than MSD BMT (CP 1-2 years: URD 39% +/- 6% vs MSD 63% +/- 12%; beyond 2 years: URD 33% +/- 7% vs MSD 50% +/- 20%). Outcome of matched URD BMT for early CP chronic myelogenous leukemia yields survival and DFS approaching that of MSD transplantation. However, delay may compromise URD outcomes to a greater extent. Improvements in URD and MSD transplantation are still needed, and results of newer, nontransplantation therapies should be evaluated against the established curative potential of URD and MSD marrow transplantation.

The National Marrow Donor Program (NMDP) maintains a registry of approximately 4 million volunteer unrelated donors for patients in need of a stem cell transplant. When several comparably HLA-matched volunteers are identified for a patient, various criteria are used to select a donor. A retrospective analysis of 6978 bone marrow transplantations facilitated by the NMDP from 1987 to 1999 was conducted to study the effects of various donor characteristics on recipient outcome. The evaluation addressed possible effects of donor age, cytomegalovirus serologic status, ABO compatibility, race, sex, and parity on overall and disease-free survival, acute and chronic graft-versus-host disease (GVHD), engraftment, and relapse. Age was the only donor trait significantly associated with overall and disease-free survival. Five-year overall survival rates for recipients were 33%, 29%, and 25%, respectively, with donors aged 18 to 30 years, 31 to 45 years, and more than 45 years (P =.0002). A similar effect was observed among HLA-mismatched cases (28%, 22%, and 19%, respectively). A race mismatch between recipient and donor did not affect outcome. The cumulative incidences of grade III or IV acute GVHD were 30%, 34%, and 34%, respectively, with donors aged 18 to 30 years, 31 to 45 years, and more than 45 years (P =.005). The corresponding incidences of chronic GVHD at 2 years were 44%, 48%, and 49% (P = 0.02). Recipients with female donors who had undergone multiple pregnancies had a higher rate of chronic GVHD than recipients with male donors (54% versus 44%; P <.0001). The use of younger donors may lower the incidence of GVHD and improve survival after bone marrow transplantation. Age should be considered when selecting among comparably HLA-matched volunteer donors.

We analyzed engraftment of unrelated-donor (URD) bone marrow in 5246 patients who received transplants facilitated by the National Marrow Donor Program between August 1991 and June 1999. Among patients surviving at least 28 days, 4% had primary graft failure (failure to achieve an absolute neutrophil count > 5 x 10(8)/L before death or second stem-cell infusion). Multivariate logistic regression analysis showed that engraftment was associated with marrow matched at HLA-A, HLA-B, and DRB1; higher cell dose; younger recipient; male recipient; and recipient from a non-African American ethnic group. More rapid myeloid engraftment was associated with marrow serologically matched at HLA-A and HLA-B, DRB1 match, higher cell dose (in non-T-cell-depleted cases), younger recipient, recipient seronegativity for cytomegalovirus (CMV), male donor, no methotrexate for graft-versus-host disease prophylaxis, and transplantation done in more recent years. A platelet count higher than 50 x 10(9)/L was achieved by 47% of patients by day 100. Conditional on survival to day 100, survival at 3 years was 61% in those with platelet engraftment at day 30, 58% in those with engraftment between day 30 and day 100, and 33% in those without engraftment at day 100 (P <.0001). Factors favoring platelet engraftment were higher cell dose, DRB1 allele match, recipient seronegativity for CMV, HLA-A and HLA-B serologically matched donor, and male donor. Secondary graft failure occurred in 10% of patients achieving initial engraftment, and 18% of those patients are alive. These data demonstrate that quality of engraftment is an important predictor of survival after URD bone marrow transplantation.

Over a period of 8.5 years (February 1988 to October 1996), 1423 patients with chronic myelogenous leukemia (CML) underwent unrelated donor (URD) bone marrow transplants (BMTs) facilitated by the National Marrow Donor Program (NMDP) at 85 transplant centers. One hundred thirty-seven evaluable (9.9%) patients failed to engraft, and an additional 83 (6.6%) evaluable patients experienced late graft failure. Grade III/IV acute graft-versus-host disease (GVHD) developed in 33% of patients (95% confidence interval [CI], 30%-36%). The incidence of extensive chronic GVHD was 60% (95% CI, 56%-63%) at 2 years. Only 5.7% of patients (95% CI, 3.6%-7.8%) transplanted in chronic phase developed hematologic relapse at 3 years. Several factors were independently associated with improved disease-free survival (DFS), including transplant in chronic phase, transplant within 1 year of diagnosis, younger recipient age, a cytomegalovirus seronegative recipient, and development of no or mild acute GVHD. The combined effect of these factors on outcome is manifest in a subset (n = 157) of young (less than 35 years), chronic phase patients transplanted within 1 year of diagnosis using HLA-matched donors who had 63% (95% CI, 53%-73%) DFS at 3 years. URD BMT therapy for CML is both feasible and effective with more frequent and more rapid identification of suitable donors. Early URD transplant during chronic phase yields good results and should be considered in CML patients otherwise eligible for transplant but without a suitable related donor.

The efficacy and toxicity of donor leukocyte infusions (DLI) after unrelated donor bone marrow transplantation (BMT) is largely unknown. We identified 58 recipients of unrelated DLI (UDLI) for the treatment of relapsed disease from the National Marrow Donor Program database. A retrospective analysis was performed to determine response, toxicity, and survival after UDLI and to identify factors associated with successful therapy. UDLI was administered for relapsed chronic myelogenous leukemia (CML) (n = 25), acute myelogenous leukemia (AML) (n = 23), acute lymphoblastic leukemia (ALL) (n = 7), and other diseases (n = 3). Eight patients were in complete remission (CR) before UDLI, and 50 were evaluable for response. Forty-two percent (95% confidence interval [CI], 28%-56%) achieved CR, including 11 of 24 (46%; 95% CI, 26%-66%) with CML, 8 of 19 (42%; 95% CI, 20%-64%) with AML, and 2 of 4 (50%; 95% CI, 1%-99%) with ALL. The estimated probability of disease-free survival (DFS) at 1 year after CR was 65% (95% CI, 50%-79%) for CML, 23% (95% CI, 9%-38%) for AML, and 30% (95% CI, 6%-54%) for ALL. Acute graft-versus-host disease (GVHD) complicated UDLI in 37% of patients (grade II-IV, 25%). A total of 13 of 32 evaluable patients (41%) developed chronic GVHD. There was no association between cell dose administered and either response or toxicity. In a multivariable analysis, only a longer interval from BMT to relapse and BMT to UDLI was associated with improved survival and DFS, respectively. UDLI is an acceptable alternative to other treatment options for relapse after unrelated donor BMT. (Blood. 2000;95:1214-1221)

Daclizumab, a humanized monoclonal IgG1 directed against the alpha chain of the interleukin-2 receptor (IL-2R), is a competitive inhibitor of IL-2 on activated lymphocytes. To test the hypothesis that specific inhibition of activated lymphocytes in patients with ongoing acute graft-versus-host disease (GVHD) might ameliorate the process, we treated 43 patients with advanced or steroid-refractory GVHD with daclizumab. The first cohort of 24 patients was treated with daclizumab 1 mg/kg on days 1, 8, 15, 22, and 29. On day 43, the complete response (CR) rate was 29% (95% confidence interval [CI], 13%-51%). Survival on day 120 was 29% (95% CI, 13%-51%). A second cohort of 19 patients was treated with daclizumab 1 mg/kg on days 1, 4, 8, 15, and 22. For these patients, the CR rate on day 43 was 47% (95% CI, 24%-71%), and survival on day 120 was 53% (95% CI, 29%-76%). There were no infusion-related reactions and no serious side effects related to daclizumab. Following treatment, there was a reduction in serum concentrations of soluble IL-2R and peripheral blood CD3( + )25(+) lymphocytes, but these changes were not predictive of response. Daclizumab has substantial activity for the treatment of acute GVHD, and the second regimen evaluated is recommended for a controlled study. (Blood, 2000; 95:83-89)

High incidences of graft failure and graft-versus-host disease in the recipients of bone marrow transplantations (BMT) from unrelated donors (URD) may reflect the existence of allelic disparities between the patient and the URD despite apparent HLA identity at HLA-A, HLA-B, and HLA-DRB1 loci. To identify the extent and pattern of allelic disparities at HLA-A and HLA-B loci, 128 patients and 484 potential URD were evaluated by DNA typing. DNA typing for HLA-A, HLA-B, and HLA-DRB1 was performed at Memorial Sloan Kettering Cancer Center. HLA-A and HLA-B serotyping on URD was provided by the registries. By original typing (serology for HLA-A and HLA-B; DNA typing for DRB1) 187, 164, and 133 URD were 6/6, 5/6, and 4/6 matches, respectively. Following DNA typing, however, only 52.9% of the originally 6/6 matched URD remained 6/6, while 38.5%, 7.5%, and 1.1% were found to be 5/6, 4/6, and 3/6 matches. The level of disparity was higher in the originally 5/6 (P <.01) and 4/6 (P <.01) matched URD. A higher level of disparity was seen for HLA-B as compared to HLA-A. In addition, a serotype related variation was also noticed. For example, 24.1% of HLA-A2 and 60.1% of HLA-B35 seromatched URD were genotypically disparate, but no disparities were seen for HLA-A1 and HLA-B8. A higher percentage of HLA-A (67. 4%) compared with HLA-B (35.4%) serologic homozygous URD remained genotypically homozygous (P =.01). The level of allelic disparity was lower (P <.01 for 6/6; P =.02 for 5/6) if the patient had one of the 15 most common haplotypes (A1B8DR3, A2B7DR15, A3B7DR15, etc) in comparison to the rest of the group. Outcome studies will answer the question whether these disparities are associated with a higher rate of immunological complications seen with URD-BMT.

Thirty-one consecutive patients with acute myelogenous leukemia (AML) in first complete remission and 8 with AML in second complete remission received T cell-depleted allogeneic bone marrow transplants from HLA-identical sibling donors. Patients received myeloablative cytoreduction consisting of hyperfractionated total body irradiation, thiotepa, and cyclophosphamide. Those patients at risk for immune-mediated graft rejection received additional immune suppression with antithymocyte globulin and methylprednisolone in the early peritransplant period. Patients with AML who underwent allogeneic T-cell-depleted bone marrow transplantations (BMT) in first or second remission have achieved respective disease-free survival (DFS) probabilities of 77% (median follow-up at approximately 56 months) and 50% (median follow-up at approximately 48 months). Ten of 31 patients transplanted in first remission were > or = 40 years old and have attained a DFS at 4 years of 70%. For patients with AML transplanted in first or second remission, the respective cause-specific probabilities of relapse were 3.2% or 12.5%, and those of nonleukemic mortality were 19.4% or 37.5%. There were no cases of immune-mediated graft rejection and no cases of grade II to IV acute graft-versus-host disease (GVHD). All survivors enjoy Karnofsky performance scores (KPS) of 100%, except 2 patients with KPS of 80% to 90%. T-cell-depleted allogeneic BMT can provide durable DFS together with an excellent performance status in the majority of patients with de novo AML. In addition, GVHD is not an obligatory correlate of the graft-versus-leukemia benefit or freedom from relapse afforded by allogeneic BMT administered as postremission therapy for AML. This study provides a basis for prospective comparison with other postremission therapies considered standard in the management of patients with this disease.

Infusions of large numbers (> 10(8)/kg) of donor leukocytes can induce remissions in patients with chronic myeloid leukemia (CML) who relapse after marrow transplantation. We wanted to determine if substantially lower numbers of donor leukocytes could induce remissions and, if so, whether this would reduce the 90% incidence of graft-versus-host disease (GVHD) associated with this therapy. Twenty-two patients with relapsed CML were studied: 2 in molecular relapse, 6 in cytogenetic relapse, 10 in chronic phase, and 4 in accelerated phase. Each patient received escalating doses of donor leukocytes at 4- to 33-week intervals. Leukocyte doses were calculated as T cells per kilogram of recipient weight. There were 8 dose levels between 1 x 10(5) and 5 x 10(8). Lineage-specific chimerism and residual leukemia detection were assessed using sensitive polymerase chain reaction (PCR) methodologies. Nineteen of the 22 patients achieved remission. Remissions were achieved at the following T-cell doses: 1 x 10(7) (n = 8), 5 x 10(7) (n = 4), 1 x 10(8) (n = 3), and 5 x 10(8) (n = 4). To date, 15 of the 17 evaluable patients have become BCR-ABL negative by PCR. The incidence of GVHD was correlated with the dose of T cells administered. Only 1 of the 8 patients who achieved remission at a T-cell dose of 1 x 10(7)/kg developed GVHD, whereas this complication developed in 8 of the 11 responders who received a T-cell dose of > or = 5 x 10(7)/kg. Three patients died in remission, 1 secondary to marrow aplasia, 1 of respiratory failure and 1 of complications of chronic GVHD. Sixteen patients who were mixed T-cell chimeras before treatment became full donor T-cell chimeras at the time of remission. Donor leukocytes with a T-cell content as low as 1 x 10(7)/kg can result in complete donor chimerism together with a potent graft-versus-leukemia (GVL) effect. The dose of donor leukocytes or T cells used may be important in determining both the GVL response and the incidence of GVHD. In many patients, this potent GVL effect can occur in the absence of clinical GVHD.

Allogeneic bone marrow transplantation is limited by the availability of suitable marrow donors and risk of graft-versus-host disease (GVHD) and opportunistic infection. In an attempt to ameliorate these limitations, umbilical cord blood has been postulated as an alternative source of allogeneic haemopoietic stem cells for transplantation. From September, 1994, umbilical cord blood from sibling donors has been used to reconstitute haemapoiesis in 44 children with acquired or congenital lympho-haemapoietic disorders, neuroblastoma, or metabolic diseases. Patients who had HLA-identical and HLA-1 antigen disparate grafts, had a probability of engraftment at 50 days after transplantation of 85%. No patient had late graft failure. The probability of grade II-IV GVHD at 100 days was 3% and the probability of chronic GVHD at one year was 6%. With a median follow-up of 1.6 years, the probability of survival for recipients of HLA-identical or HLA-1 antigen disparate grafts is 72%. We conclude that umbilical cord blood is a sufficient source of transplantable haemopoietic stem cells for children with HLA-identical or HLA-1 antigen disparate sibling donors with very low risk of acute or extensive chronic GVHD. The feasibility of umbilical-cord-blood transplantation with HLA-2 and HLA-3 antigen disparate sibling donors remains to be determined.

To help elucidate the mechanism responsible for graft failure (GF) following a T-cell depleted bone marrow transplant (BMT) from an unrelated donor, five patients (2 chronic myelogenous leukemia, 1 acute undifferentiated leukemia, 2 myelodysplastic syndrome) who experienced this complication were studied. All patients were HLA class I identical with their donors as determined by serology and one-dimensional isoelectric focusing (IEF); two were serologically matched with their donors for HLA class II antigens, whereas three donor-recipient pairs were serologically mismatched for one HLA-DR antigen. All patients received total body irradiation (fractionated, 1,500 rads), VP-16 (750 mg/m2), and cyclophosphamide (120 mg/kg) pre-BMT and antithymocyte globulin (15 mg/kg every other day) and methylprednisolone (2 mg/kg) post-BMT. Three patients experienced primary nonengraftment and two experienced secondary GF. Peripheral blood mononuclear cells obtained from the patients at the time of GF were studied to examine their functional and phenotypic characteristics. Emerging cells were of host origin and were found to be specifically cytotoxic to donor target cells and suppressive to the in vitro growth of donor BM, especially in the cases of primary nonengraftment. Peripheral blood mononuclear cells from these patients were expanded to form T-cell lines (TcLs). The cytotoxic activities of TcLs were tested in the presence of blocking MoAbs directed against various HLA determinants in an attempt to determine if HLA antigens expressed on donor cells were the target for cytotoxicity. The observed cytotoxic activity was blocked by antibodies to HLA-B, -C (1 patient), HLA-DR (1 patient), and HLA-DQ (1 patient). In two cases, antidonor cytotoxicity could not be blocked by MoAb directed against HLA-A, -B, -C, or -DR. Phenotypic characterization of four successfully maintained TcLs showed 100% CD3+ cells with 100% CD4+ (3 patients) or 50% CD4+/50% CD8+ (1 patient). In two of the three patients with 100% CD4+ cells, antidonor cytotoxicity was blocked by an anti-HLA class II MoAb. In contrast to our previous findings in cases of GF following T-cell-depleted HLA nonidentical family member BMT in which host T cells were CD8+ and cytotoxicity was directed against HLA class I antigens, our present study indicates host T cells emerging at the time of GF following BMT from an HLA class I IEF-identical unrelated donor can be of the CD4+ subset and seem to be capable of recognizing antigenic disparities in the HLA class II region.

Engraftment of marrow following autologous or allogeneic bone marrow transplantation (BMT) may be influenced by quantity and function of stem cells. T lymphocytes, supporting microenvironmental cells, and hematopoietic growth factors (HGF). To elucidate the physiologic role of interleukin-3 (IL-3) in the engraftment process, serum IL-3 levels were measured in over 400 samples from 77 transplant recipients before and for up to 3 weeks following transplantation using a novel enzyme-linked immunoabsorbent assay (ELISA) with a sensitivity of > or = 78 pg/mL. Thirty-seven patients received two to three log T-cell-depleted allografts. In the remaining 40 patients (18 autologous marrow, 12 allogeneic marrow, and 10 autologous peripheral blood [PB] stem cell), T cells were not depleted (non-TCD) from the grafts. A burst of IL-3 (peak levels, 1,500 to 6,000 pg/mL) was detected in the immediate posttransplant period between day 0 and day 14 in all non-TCD recipients and in 21 of 37 (57%) of TCD recipients. A strong inverse relationship between IL-3 levels and absolute neutrophil count (ANC) was observed in both non-TCD recipients (r = -.796) and in TCD recipients (r = -.897). However, both peak IL-3 levels and mean IL-3 levels from day 0 through 14 were significantly lower in TCD recipients compared with either autologous or unmodified allogeneic marrow recipients (P < .01). The lowest peak or mean day 0 through 14 IL-3 levels were observed in matched related recipients undergoing the most aggressive (2.5 to 3.0 log) T-cell-depleted BMT. Autografted patients receiving blood stem cell transplants alone or posttransplant granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony stimulating factor (GM-CSF) also had significantly lower peak IL-3 levels (P < .01). In patients receiving TCD grafts, administration of antithymocyte globulin (ATG) posttransplant significantly increased peak IL-3 levels compared with patients not treated with ATG (P < .04). This study shows that endogenous release of IL-3 is strongly associated with myeloid engraftment and inversely related to ANC. Removal of T lymphocytes from donor marrow or acceleration of engraftment by use of stem cells or growth factors appears to blunt the endogenous release of IL-3 whereas use of ATG posttransplant increases IL-3 release.

In the interval from December 1987 to November 1990, 196 consecutive patients with chronic myelogenous leukemia (CML) received unrelated donor marrow transplantation using marrow procured by the National Marrow Donor Program (NMDP) at 21 NMDP-affiliated marrow transplant centers. Baseline donor and recipient data as well as follow-up data were obtained systematically in all cases by the NMDP. The median interval from the initiation of a search for an unrelated donor to bone marrow transplantation was 8.4 months (range, 1.7 to 34.6 months). Median age of the recipients was 33.3 years (4.5 to 54.5 years). Seventy-five recipients were female and 121 were male. At time of transplant, 115 patients were in chronic phase, 51 in accelerated phase, 14 in blast crisis, and 16 in a second or subsequent chronic phase. In 133 cases, donors and recipients were identical at the HLA A, B, and DR loci using standard serologic typing, and in 63 cases, there was nonidentity at one HLA locus. Patients were prepared for transplantation with a combination of high-dose chemotherapy and total body irradiation (N = 169) or with high-dose chemotherapy only (N = 27). Thirty-five patients received marrow depleted ex vivo of T lymphocytes, whereas 161 patients received non-T-depleted marrow. One hundred seventy-four of 196 patients engrafted (absolute neutrophil count > or = 500/mm3 for 3 consecutive days). The median time to engraftment was 22 days (6 to 69 days). Twenty-two patients failed to engraft, and an additional 10 patients experienced late graft failure. The incidence of grades III or IV acute graft-versus-host disease (GVHD) was 0.54 +/- 0.10, and that of extensive chronic GVHD was 0.52 +/- 0.12. A lower incidence of both grades III and IV acute GVHD (P = .0003) and of extensive chronic GVHD (P = .01) were independently associated with use of T-depleted marrow. The actuarial incidence of hematologic relapse at 2 years is 0.11 +/- 0.06. The 2-year actuarial incidence of disease-free survival for patients transplanted in first chronic phase within 1 year of diagnosis is 0.45 +/- 0.21, in chronic phase more than 1 year from diagnosis is 0.36 +/- 0.11, in accelerated phase is 0.27 +/- 0.12, in second or subsequent chronic phase is 0.22 +/- 0.21, and in blast crisis is 0. Fifteen of 55 patients transplanted at 40 to 50 years of age survive.(ABSTRACT TRUNCATED AT 400 WORDS)

Conflicting results have been published on whether or not myelodysplastic syndromes (MDS) affect all cell lineages. Involvement of myeloid and erythroid cell lineages has been regularly observed, but it remains controversial whether the different lymphoid cell lineages are involved. In this study of eight patients with MDS associated with monosomy 7, fluorescent in situ hybridization (FISH) was used to enumerate the chromosomes 7 in interphase cells. With the probe D7Z1, the rate of false-positive detection of monosomy 7 was 3% +/- 2% in normal cells. T- and B-cell lines were established from eight patients with MDS and monosomy 7. As determined by FISH in interphase cells, 1.9% (0% to 3%) of the cells in the B-cell lines showed one fluorescent spot and 1.1% (0% to 2.9%) of the cells in the T-cell lines. These values do not differ from normal values. However, the possibility that normal cells were selected when the T- and B-cell lines were established could not be excluded. Therefore, peripheral blood cells were obtained, separated according to surface markers specific for lymphoid and myeloid cell lineage with a cell sorter, and analyzed for the expression of monosomy 7 by FISH. Antibodies recognizing T cells (CD3), B cells (CD20), natural killer (NK) cells (CD57), monocytes and granulocytes (low and high expression of CD11b antigen), and myeloid progenitors (CD33) were used to separate cells. The expression of monosomy 7 in the T cells, NK cells, and B cells did not differ from control values. These results in the lymphoid subpopulations are in stark contrast with the observations in the myeloid populations; the percentage of cells with monosomy 7 ranged from 9% to 78% (controls: 6% +/- 2%) in cells with low CD11b expression, 20% to 89% in cells with a high expression of the CD11b antigen (controls: 7% +/- 3%), and 23% to 91% in the CD33 positive cells (controls: 5% +/- 3%). The results of this study suggest that monosomy 7 does not usually affect lymphoid subpopulations but is restricted to committed progenitor cells with the capacity to differentiate into mature myeloid cells.

Acute steroid-resistant graft-versus-host disease (AGVHD) after allogeneic bone marrow transplantation is frequently fatal. A new treatment for this T-lymphocyte-mediated condition uses an immunotoxin, H65-RTA, comprised of a monoclonal antibody that recognizes the CD5 lymphocyte differentiation antigen coupled to ricin A chain, a cytotoxic enzyme that inhibits protein synthesis. The safety and efficacy of this lymphocyte-targeted immunotoxin was evaluated in patients with severe AGVHD in a phase I-II dose escalation study with group expansion at the two middle doses. Thirty-four patients received up to 14 daily intravenous infusions of the immunotoxin. The principal side effects were constitutional symptoms such as fatigue and myalgias, and hypoalbuminemia with weight gain was seen at all doses. Thirty-two patients were evaluated for improvement or resolution of disease. Durable complete or partial responses were not dose-related and were seen in 16 patients. Skin GVHD had the highest incidence of response (73%), although improvement or resolution in gastrointestinal tract (45%) and liver (28%) GVHD was also noted. Survival in responding patients was significantly prolonged at all times as compared with those with no response (P = .03). Treatment was associated with a rapid decrease in peripheral blood T lymphocytes, which persisted for greater than 1 month after therapy. Anti-immunotoxin antibodies were seen in 6 of the 23 patients tested; these were of low titer and did not block immunotoxin binding to T cells. Results of this study indicate that anti-T-lymphocyte immunotoxins may form a new class of immunosuppressive agents useful in T-lymphocyte-mediated diseases.

Serial cytogenetic studies were performed on 64 patients with chronic myelogenous leukemia (CML) after T cell-depleted allogeneic bone marrow transplantation (BMT). Forty patients with CML in chronic phase (CP) received cytoreduction followed by BMT with HLA-matched T cell-depleted allogeneic marrow. The remaining 24 patients were transplanted in second chronic, accelerated, or blastic phase, or received T cell-depleted grafts with a dose of T cells added back. The Y chromosome and autosomal heteromorphisms were used to distinguish between donor and host cells. Mixed hematopoietic chimerism (presence of donor and host cells) was identified in 90% of patients in first CP. The Philadelphia (Ph) chromosome reappeared in 16 of the 40 first CP CML patients. As expected, patients who had detectable Ph chromosome positive cells at any time during the posttransplant period had a high likelihood of subsequent clinical relapse. Transient disappearance of the Ph positive clone was rarely observed, and was followed by reappearance of the Ph chromosome or clinical relapse. A subset of engrafted patients with greater than 25% host cells within 3 months post-BMT had a significantly shorter survival time free of cytogenetic or clinical relapse compared with other patients. In patients who had received donor T cells added to the T cell-depleted graft, there was a higher proportion of complete chimerism. Clonal progression of Ph positive as well as negative cells was observed and may be the result of radiation induced breakage. Serial cytogenetic studies of patients post-BMT can provide useful information regarding the biologic and clinical behavior of CML.

The reconstitution of hematopoietic cells and in vitro assays of immunologic function have been followed in leukemic patients after conventional bone marrow transplantation (BMT) (N = 34) and T-cell depleted BMT (N = 52) from human leukocyte antigen (HLA)-identical sibling donors. No effects of the T-cell depletion could be seen on the recovery of myeloid or lymphoid cells as measured by the day to engraftment or by the absolute number of cells through day 100. Normal numbers of lytically active natural killer cells returned the earliest and were rapidly followed in both groups of patients by the appearance of circulating B cells and normalization of the responses to B-cell mitogens. However, the recovery of normal T-cell proliferative responses were more delayed in recipients of T-cell depleted grafts. Significant quantitative differences were seen only during the first 3 months after transplantation. Neither the number of CD3+ T cells nor the ratio of CD4:CD8 positive cells differed markedly between the two transplant groups. Mitogen-induced immunoglobulin production by peripheral blood lymphocytes (PBL) from patients following T-cell depleted BMT was quantitatively less than that of conventional marrow recipients through the first year, with low normal IgM production reached by 4 to 6 months in both groups. IgG production reached low normal 7 to 9 months after conventional BMT but did not remain at this level until 1 year following either type of transplant. Assessment of the incidence of infections from the day the absolute neutrophil count reached 500 until day 180 after transplant revealed no significant differences between the two groups; indeed, the overall nonleukemic mortality was higher in the recipients of conventional bone marrow. Thus, in our series, the removal of mature cells from the marrow graft did not affect the rate or degree of recovery of myeloid and lymphoid cells but did affect the regeneration of in vitro T-cell dependent functions. We noted early quantitative differences and a delay in the normalization of the T-cell functions measured rather than prolonged absolute deficiencies. The in vitro deficiencies did not result in significant clinically apparent differences between the two groups.

To identify mechanisms potentially contributing to graft failure, 19 leukemic recipients of T-cell-depleted marrow transplants who failed to engraft following a transplant of HLA identical sibling marrow depleted of T cells by soybean agglutinin (SBA) and sheep erythrocytes (E) were evaluated. Peripheral blood mononuclear cells isolated at the time of failure were consistently of host origin, bearing the phenotype of suppressor T cells (CD3+, CD8+, Leu 7+). A direct cytolytic effect on 51Cr-labeled donor-derived target cells was not detected, a finding that contrasts with the donor-specific cytotoxic host T lymphocytes that have been regularly observed in patients rejecting HLA nonidentical SBA -E- BMTs. However, these host T cells did exhibit a strong and specific suppressive activity against the donor marrow CFU-GM in vitro. Furthermore, in contrast to prior findings in durably engrafted recipients of SBA -E- BMTs, the lymphocytes isolated prior to or at the time of graft failure lacked natural killer surface antigen expression and effector function.

Risk factors for graft failure were analyzed in 122 recipients of an allogeneic T-cell-depleted human leukocyte antigen (HLA)-identical sibling marrow transplant as treatment for leukemia. In each case pretransplant immunosuppression included 1,375 to 1,500 cGy hyperfractionated total body irradiation and cyclophosphamide (60 mg/kg/d x 2). No patient received immunosuppression prosttransplant for graft-versus-host disease (GVHD) prophylaxis. Nineteen patients in this group experienced graft failure. The major factors associated with graft failure were transplants from male donors and the age of the patient (or donor). Among male recipients of male donor-derived grafts a low dose per kilogram of nucleated cells, progenitor cells (colony forming unit-GM) and T cells was also associated with graft failure. Additional irradiation to 1,500 cGy, high dose corticosteroids posttransplant, and additional peripheral blood donor T cells did not decrease the incidence of graft failure. In addition, type of leukemia, time from diagnosis to transplant, an intact spleen, or the presence of antidonor leukocyte antibodies did not correlate with graft failure. To ensure engraftment of secondary transplants, further immunosuppression was necessary but was poorly tolerated. However, engraftment and survival could be achieved with an immunosuppressive regimen in which antithymocyte globulin and high dose methylprednisolone were administered both before and after infusions of secondary partially T-cell-depleted marrow grafts.

Early clinical trials using T lymphocyte-depleted human marrow for transplantation have reported that such grafts reduce, to varying degrees, both the incidence and the severity of graft-v-host disease (GVHD). However, to date, no clear estimates have been made as to what degree of T cell depletion is necessary to prevent GVHD in every case. To address this problem, we used a limiting dilution assay (LDA) to quantitate residual clonable T lymphocytes in human T cell-depleted bone marrow in 31 HLA-identical transplants for leukemia. The number of phytohemagglutinin -interleukin 2-responsive T lymphocytes determined by LDA and expressed as T cell per kilogram recipient weight was found to correlate with the subsequent development of GVHD: no patients who received less than 1 X 10(5) T cell per kilogram developed GVHD (N = 24). Of the seven patients who received 1 X 10(5) to 4.4 X 10(5) T cell per kilogram, four patients developed grade I or II skin GVHD. This study thus provides a quantitative estimate of the number of T lymphocytes necessary to initiate clinically detectable GVHD in an HLA-identical host.

NK cells are capable of a variety of effector functions including the unprimed lysis of certain tumor cell targets and the nonspecific suppression of hematopoietic colony formation. In this study we have evaluated in parallel the radiosensitivity of lytic and colony inhibitory activity (CIA) by peripheral blood lymphocytes and large granular lymphocytes before and after activation by 72-96 hr of culture in medium containing interleukin 2 (IL-2). Our results show that (1) cells exhibiting CIA and lytic activities have almost identical patterns of radioresistance both before and following activation by IL-2; (2) the amount of preculture irradiation which completely blocks IL-2-stimulated cell proliferation only minimally affects CIA and lytic activities; (3) lysis of K562 and inhibition of CFU-GM are more resistant to preculture irradiation than is the killing of NK insensitive targets; (4) both the CIA and the lytic activities of PBL, but not of LGL, can be increased by 500-1000 rad of gamma-irradiation; and (5) NK lytic activity and CIA both before and following activation by IL-2 are primarily mediated by cells bearing the NKH1A antigen. Evidence that suggests NK cells mediate both the lysis of K562 and the inhibition of myeloid cells by a common mechanism came from experiments which showed that a monoclonal antibody to the T-200 molecule (13.3) could block both activities.