Essential requirements for setting up a stem cell processing laboratory.

The Graft Processing subcommittee of the Worldwide Network for Blood and Marrow Transplantation wrote this guideline to assist physicians and laboratory technologists with the setting up of a cell processing laboratory (CPL) to support a hematopoietic stem cell transplant program, thereby facilitating the start-up of a transplant program in a new location and improving patient access to transplantation worldwide. This guideline describes the minimal essential features of designing such a laboratory and provides a list of equipment and supply needs and staffing recommendations. It describes the typical scope of services that a CPL is expected to perform, including product testing services, and discusses the basic principles behind the most frequent procedures. Quality management (QM) principles specific to a CPL are also discussed. References to additional guidance documents that are available worldwide to assist with QM and regulatory compliance are also provided.

Severe autologous GVHD after hematopoietic progenitor cell transplantation for multiple myeloma.

GVHD is a recognized complication of autologous hematopoietic progenitor cell transplantation (HPCT), but has typically been reported to respond well to primary therapy with corticosteroids. In this study, we report the development of severe autologous GVHD in five patients who underwent HPCT for multiple myeloma. In all cases, response to corticosteroids was unsatisfactory and three of these patients ultimately died from complications that ensued from prolonged immunosuppressive therapy. Severe autologous GVHD occurred only in patients transplanted for multiple myeloma and was observed at a much higher frequency in patients undergoing their second HPCT. The severity of this syndrome primarily in patients undergoing second HPCTs suggests that repetitive exposure to high-dose therapy may compromise endogenous peripheral regulatory mechanisms and predispose these patients to autoimmunity. Given the evolving role of second autologous transplantations in the therapeutic armamentarium for multiple myeloma, consideration of this potential toxicity may be appropriate when considering treatment options for these patients.

Stimulation by means of dendritic cells followed by Epstein-Barr virus-transformed B cells as antigen-presenting cells is more efficient than dendritic cells alone in inducing Aspergillus f16-specific cytotoxic T cell responses.

Adoptive immunotherapy with in vitro expanded antigen-specific cytotoxic T lymphocytes (CTLs) may be an effective approach to prevent, or even treat, Aspergillus (Asp) infections. Such lines can be generated using monocyte-derived dendritic cells (DC) as antigen-presenting cells (APC) but requires a relatively high volume of starting blood. Here we describe a method that generates Asp-specific CTL responses more efficiently using a protocol of antigen presented on DC followed by Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (BLCL) as APC. Peripheral blood mononuclear cells were stimulated weekly (2-5x) with a complete pool of pentadecapeptides (PPC) spanning the coding region of Asp f16 pulsed onto autologous mature DC. Cultures were split and stimulated subsequently with either PPC-DC or autologous PPC-pulsed BLCL (PPC-BLCL). Lines from the DC/BLCL arm demonstrated Asp f16-specific cytotoxicity earlier and to a higher degree than lines generated with PPC-DC alone. The DC/BLCL-primed lines showed a higher frequency of Asp f16-specific interferon (IFN)-gamma producing cells but an identical effector cell phenotype and peptide specificity compared to PPC-DC-only-primed lines. Tumour necrosis factor (TNF)-alpha, but not IL-10, appeared to play a role in the effectiveness of BLCL as APC. These results demonstrate that BLCL serve as highly effective APC for the stimulation of Asp f16-specific T cell responses and that a culture approach using initial priming with PPC-DC followed by PPC-BLCL may be a more effective method to generate Asp f16-specific T cell lines and requires less starting blood than priming with PPC-DC alone.

Rapamycin enriches for CD4(+) CD25(+) CD27(+) Foxp3(+) regulatory T cells in ex vivo-expanded CD25-enriched products from healthy donors and patients with multiple sclerosis.

BACKGROUND: CD4(+) CD25(bright+) regulatory T cells (Treg) can be expanded to clinical doses using CD3/CD28 Ab-coated beads plus IL-2. However, this method requires high purity of the starting population to prevent overgrowth by non-regulatory T cells. Rapamycin, an agent that inhibits T-cell proliferation but selectively spares Treg, may be a means to expand Treg from less pure CD25-enriched cells. METHODS: CD25-enriched cells were prepared by a single-step immunomagnetic-selection using anti-CD25 microbeads. The cells were activated with a single addition of anti-CD3/CD28 beads and expanded in ex vivo 15-5% HS and autologous CD4(+) CD25(-) feeder cells,+/-rapamycin (0.01-20 ng/mL). IL-2 was added on day 3. Cells were rested for 2 days in ex vivo 15-5% HS and tested for phenotype, intracellular Foxp3 protein and suppressor activity. RESULTS: In the absence of rapamycin, CD25-enriched fractions expanded >17 000-fold by 21 days. Although suppressor activity was detected to day 14, it declined significantly by 21 days as non-regulatory cells expanded. The addition of rapamycin inhibited expansion of non-regulatory T cells at doses > or =1 ng/mL while increasing suppressor activity and the percentage of CD4(+) CD25(+) CD27(+) Foxp3(+) cells. Rapamycin did not enrich for Foxp3(+) cells in expanded cultures of CD4(+) CD25(-) cells. Treg were also readily expanded in cultures of CD25-enriched cells obtained from patients with multiple sclerosis in the presence of rapamycin. DISCUSSION: The addition of 1-20 ng/mL rapamycin to CD25-enriched cultures increased the purity of cells with the phenotype and function of Treg. This approach may alleviate the need for rigorous enrichment of Treg prior to activation and expansion for potential clinical use.

Alternative donor bone marrow transplant for children with Philadelphia chromosome ALL.

Children with Philadelphia chromosome positive (Ph+) acute lymphocytic leukemia (ALL) have only a 20% event-free survival when treated with chemotherapy alone. Bone marrow transplant (BMT) for patients with matched siblings has been associated with significantly better long-term survival. We asked whether children who lack a matched sibling donor would do as well if an alternative donor was utilized. Between 1987 and 2002, we transplanted 29 children and adolescents using either an unrelated donor (23) or a mismatched family member (six). The conditioning regimen included cytosine-arabinoside, cyclophosphamide and total body irradiation. Graft-versus-host disease (GVHD) prophylaxis consisted of T-cell depletion (antibody T10B9 or OKT3 and complement) with post transplant cyclosporine (CSA). All patients engrafted. Four developed grades III-IV acute GVHD. Three of 24 evaluable patients developed extensive chronic GVHD. Two patients died of relapse (7%). Two long-term survivors (>6 years) died of malignant glioblastoma multiforme. Event-free survival at 3, 5, and 10 years is 56, 51, and 46%, respectively. Five of six patients in >CR2 or relapse at the time of transplant died. Our data should encourage the use of alternative donor transplants early in the course of disease for children with Ph+ ALL.

The implication of follicular lymphoma patients receiving allogeneic stem cell transplantation from donors carrying t(14;18)-positive cells.

We performed real-time quantitative polymerase chain reaction (RQ-PCR) in peripheral blood (PB) and/or bone marrow (BM) samples collected pre- and post transplant from 23 recipient-donor pairs receiving allogeneic stem cell transplantation (allo-SCT) for follicular lymphoma (FL). Of 23 donors, 11 had a PB and/or BM sample positive for t(14;18) (BCL2/IGH fusion) at low levels (

Generation of cytotoxic T cell responses directed to human leucocyte antigen Class I restricted epitopes from the Aspergillus f16 allergen.

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)-gamma 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.

Generation of Th1 T cell responses directed to a HLA Class II restricted epitope from the Aspergillus f16 allergen.

The Aspergillus allergen Asp f16 has been shown to confer protective Th1 T cell-mediated immunity against infection with Aspergillus conidia in murine models. Here, we use overlapping (11-aa overlap with preceding peptide) pentadecapeptides spanning the entire 427-aa coding region of Asp f16 presented on autologous dendritic cells (DC) to evaluate the ability of this antigen to induce Th1 responses in humans. Proliferative responses were induced in five out of five donors, and one line with a high frequency of interferon (IFN)-gamma-producing CD4(+) T cells in response to the complete peptide pool was characterized. This line was cytotoxic to autologous pool-pulsed and Aspergillus culture extract-pulsed targets. Limitation of cytotoxicity to the CD4(+) T cell subset was demonstrated by co-expression of the degranulation marker CD107a in response to peptide pool-pulsed targets. Cytotoxic T lymphocytes (CTL) killed Aspergillus hyphae and CTL culture supernatant killed Aspergillus conidia. By screening 21 smaller pools and individual peptides shared by positive pools we identified a single candidate sequence of TWSIDGAVVRT that elicited responses equal to the complete pool. The defined epitope was presented by human leucocyte antigen (HLA)-DRB1-0301. These data identify the first known Aspergillus-specific T cell epitope and support the use of Asp f16 in clinical immunotherapy protocols to prime protective immune responses to prevent or treat Aspergillus infection in immunocompromised patients.

Generation of Aspergillus- and CMV- specific T-cell responses using autologous fast DC.

BACKGROUND: Recent reports have described a new strategy for differentiation and maturation of monocyte (Mo)-derived DCs within only 48 h of in vitro culture (fast-DC). Here we assess the efficacy of the fast-DC to process and present different Aspergillus fumigatus and CMV Ag preparations to autologous T cells, compared with DCs generated in standard 7-day cultures (standard-DC). METHODS: Adherent blood Mo were treated with GM-CSF and IL-4 (1 day for fast-DC, 5 days in the standard-DC) to generate immature DCs, and then were matured for either 1-2 days (fast-DC) or 2 days (standard-DC) with inflammatory cytokines. DCs were pulsed with A. fumigatus or CMV Ag preparation immediately prior to maturation, or infected after maturation with adeno-pp65. Mature DCs were then used to prime Ag-specific proliferative and cytotoxic T lymphocytes (CTL) responses. RESULTS: Fast-DC were CD14- and expressed mature DC surface markers to the same degree as standard-DC, and maintained this phenotype after withdrawing cytokine from the cultures. Fast-DC and standard-DC were equally capable of inducing A. fumigatus and CMV-specific T-cell proliferation, as well as priming Ag-specific CTL activity. The Aspergillus- and CMV-specific CTL were of mixed CD3+/CD4+ and CD3+/CD8+ phenotype, and specifically killed autologous DC pulsed with A. fumigatus Ag and autologous CMV infected fibroblasts, respectively. DISCUSSION: Fast-DC are as effective as standard-DC in the generation of Ag-specific T-cell responses. Moreover, use of fast-DC not only reduces labor and supply cost, as well as workload and time, but also increases the number of DCs derived from adherent Mo, which may facilitate the use of DCs in clinical trials of cellular immunotherapy.

Suppression of EBV release from irradiated B lymphoblastoid cell-lines: superior activity of ganciclovir compared with acyclovir.

BACKGROUND: B lymphoblastoid cell-lines (BLCL), generated by exposure of PBMC to a laboratory strain of EBV, are commonly utilized in the preparation of T cells used for immunotherapy. Although most B cells are latently infected, BLCL contain a subset of cells that harbor infectious virus, which could be released into the infusion product during preparation. To reduce this known risk, laboratories have pretreated BLCL for > or = 14 days with 100 microM acyclovir (ACV), an inhibitor of viral DNA polymerase, prior to use. We tested the effectiveness of ACV in preventing the release of infectious virus from irradiated fresh and previously frozen BLCL, and compared its effects with those of ganciclovir (GCV). METHODS: BLCL were grown for 14 days in medium containing various doses of ACV or GCV, washed, irradiated, and tested for the presence of infectious virus in co-culture assays with cord blood mononuclear cells(CBMC) (21 CBMC to BLCL). B-cell transformation was assessed at 3-4 weeks of culture. RESULTS: Both fresh and previously frozen BLCL released infectious virus, which transformed nearly all (92%) of CBMC co-cultures (n = 52). Transformation was not prevented by treatment with 100 microM ACV (88%, n = 52). Increasing the ACV dose to 200 microM (or 50 microg/mL) still allowed transformation in 4/9 (44%) cultures, while this and higher doses severely reduced the proliferation rate of the BLCL during ACV exposure. Infectious virus release was detectable within 1 day of ACV removal and BLCL irradiation. In contrast, GCV was able to prevent infectious virus release in 12/12 co-cultures at a concentration (15 microM) that only modestly reduced BLCL growth. DISCUSSION: These results indicate that GCV is more effective at preventing release of infectious EBV from irradiated BLCL than ACV at concentrations that do not severely inhibit B-cell growth.

IL-2-based immunotherapy after autologous transplantation for lymphoma and breast cancer induces immune activation and cytokine release: a phase I/II trial.

We determined the safety, immune activating effects, and potential efficacy of i.v. infusion of ex vivo interleukin-2 (IL-2) activated natural killer (NK) cells (part I) or IL-2 boluses (part II) during daily s.c. IL-2 administration following hematopoietic recovery from autologous transplantation. In all, 57 patients with relapsed lymphoma (n=29) or metastatic breast cancer (n=28) were enrolled. In part I of the study, 34 patients were enrolled at three dose levels of ex vivo IL-2-activated NK cells. Lymphaphereses were performed on days 28 and 42 of s.c. IL-2 administration. Following overnight ex vivo IL-2 activation of the pheresis product, the cells were reinfused the following day. In part II, 23 patients were enrolled at three dose levels of supplemental i.v. IL-2 bolus infusions, given on days 28 and 35 during s.c. IL-2 administration. Toxicities were generally mild, and no patient required hospitalization. Lytic function was markedly enhanced for fresh peripheral blood mononuclear cells (PBMNCs) obtained 1 day postinfusion of either IL-2-activated cells or IL-2 boluses. IL-2 boluses transiently increased the levels of IL-6, IFN-gamma, TNF-alpha and IL1-beta, with increases in IL-6 and IFN-gamma being dose dependent. A total of 37 patients (19 patients with lymphoma, 18 with breast cancer) treated with an optimum dose of post-transplant immunotherapy (defined as having received 1.75 x 10(6) IU/m(2)/day of s.c. IL-2 plus at least one of the planned ex vivo IL-2-activated cell infusions/IL-2 boluses) could be matched with controls from the Autologous Blood and Marrow Transplant Registry database. The matched-pairs analysis demonstrated no improvement in disease outcomes of survival and relapse. We conclude that IL-2-activated cells/IL-2 boluses can be safely administered, generate PBMNCs with enhanced cytotoxicity against NK-resistant targets, and increase cytokine levels. With this dose and schedule of administration of IL-2, no improvement in patient disease outcomes was noted. Alternative strategies will be needed to exploit the immunotherapeutic potential of IL-2-activated NK cells.

Tumor load in patients with follicular lymphoma post stem cell transplantation may correlate with clinical course.

The purpose of this study was to evaluate if the tumor load, as determined by a real-time quantitative PCR (RQ-PCR) assay, correlated with the clinical course of follicular lymphoma patients after stem cell transplantation (SCT). Cryopreserved bone marrow and/or peripheral blood samples obtained at different time intervals after SCT from 11 patients (seven allogeneic, T-cell depleted/four autologous) were tested for tumor load, as defined by t(14;18) positive cells/total cells, using RQ-PCR. None of the six patients who remained in remission had samples with a tumor load >0.01% after SCT, although fluctuating tumor loads of 0.01% after SCT (0/6 vs 4/5, P<0.02, Fisher's exact). Our results suggest that RQ-PCR measurable tumor load >0.01% after SCT may correlate with relapsed/progressive disease. Prospective studies with greater numbers of cases are indicated to better determine the critical tumor load that predicts poor outcome after SCT with RQ-PCR.

Concurrent presence of both patient and donor t(14;18) in a follicular lymphoma patient after undergoing allogeneic BMT: implications for minimal residual disease detection post-transplant.

We report the case of a t(14:18)(+) follicular lymphoma (FL) patient in long-term clinical remission after undergoing an allogeneic bone marrow transplantation (allo-BMT) from a human leukocyte antigen (HLA)-identical sibling donor who was the normal healthy carrier of a t(14:18)(+) B cell clone. Using real-time quantitative PCR (RQ-PCR) and gel electrophoresis, we document the temporal disappearance of the patient's t(14:18)(+) clone early post-transplant with the concomitant emergence and long-term persistence of the donor's t(14:18)(+) clone in the patient's peripheral blood. This report indicates that the use of PCR-based techniques to measure minimal residual disease in FL patients post-alloBMT should incorporate pretransplant screening of the donor for t(14;18). Furthermore, it suggests that healthy individuals with t(14:18) need not be excluded as donors for FL patients treated with allo-BMT.

Factors affecting neutrophil and platelet reconstitution following T cell-depleted bone marrow transplantation: differential effects of growth factor type and role of CD34(+) cell dose.

We have performed univariate and multivariate analysis to determine the factors that affect the kinetics of neutrophil and platelet recovery in 546 recipients of T cell-depleted (TCD) marrow allografts. All patients received marrow depleted of mature CD3(+) T cells by complement-mediated lysis using T(10)B(9)-1A3 (n = 489) or Muromonab-Orthoclone OKT3 (n = 57) monoclonal antibodies. Neutrophil engraftment to 0.5 x 10(9)/1 and platelet engraftment to 20 x 10(9)/l were assessed as endpoints. Factors significantly affecting neutrophil or platelet engraftment in the univariate analysis included patient age, T cell dose, anti-thymocyte globulin use, gender, diagnosis at transplant, CMV serostatus, HLA mismatch, CD34 cell dose (n = 249), and growth factor use and type. These variables were included in the multivariate Cox proportional hazards regression model. The results showed that a faster rate of neutrophil engraftment was independently associated with CD34(+) cell dose > or = 5 x 10(6)/kg and most strongly with growth factor administration. Faster platelet engraftment was associated with transplantation for chronic leukemia, CD34(+) cell dose > or = 2 x 10(6)/kg, an HLA matched related donor, and the absence of growth factor use. G-CSF had a higher relative risk (RR) of enhancing neutrophil engraftment than GM-CSF and significantly delayed platelet engraftment. The combined use of G-CSF + GM-CSF was similar to G-CSF alone. The enhancing effect of G-CSF for neutrophil recovery was most striking for patients who engrafted to 0.5 x 10(9)/1 at or before day 12 (RR = 9.5, P < 0.0001) compared to patients who received no growth factor. Conversely, the delaying effect of G-CSF on platelet engraftment was strongest for patients engrafting on or before day 25 (RR = 0.4, P = 0.0004). Of the independent variables affecting engraftment kinetics in recipients of TCD marrow allografts only growth factor, and to a limited extent, CD34(+) cell dose can be controlled by the clinician. A higher CD34(+) cell dose enhances the rate of both neutrophil and platelet engraftment whereas for G-CSF the benefits of myeloid growth factor use in enhancing neutrophil recovery may be partly offset by a delay in platelet engraftment.

Cytomegalovirus-specific cytolytic T-cell lines and clones generated against adenovirus-pp65-infected dendritic cells.

Cytomegalovirus (CMV) infection is a serious complication of allogeneic bone marrow transplantation (BMT). CMV disease can usually be prevented by passive immunization with donor-derived CMV-pp65-specific T-cell clones if provided early post-BMT. The classic method of generating CMV-specific T-cell clones requires donor-derived fibroblast lines infected with CMV as stimulators, thus limiting the availability of CMV immunotherapy to those patients for whom a donor skin biopsy can be obtained 6 to 8 weeks pretransplantation. To overcome this limitation we have used monocyte-derived dendritic cells (DCs) to induce donor anti-CMV cytotoxic T lymphocytes (CTLs). Matured, adeno-pp65-infected DCs were added at day 0 and at day 7 of a 2-week culture of donor peripheral blood mononuclear cells. DC-primed cultures were compared with cultures stimulated in an identical fashion with CMV-infected fibroblasts or with adeno-pp65-infected freshly isolated blood monocytes. Specific killing of CMV-infected fibroblasts was detected in all except the culture stimulated with pp65-infected monocytes. DCs infected after maturation elicited greater CTL activity than did DCs matured after infection. A series of 5 CD8+ clones from a fibroblast-stimulated culture and 7 CD8+ clones from a mature-DC-stimulated culture derived from a single HLA-A*0201+ individual were characterized. All 12 clones lysed autologous CMV-infected fibroblasts. All except 1 clone from the CMV-infected fibroblast arm (fibroblast arm) lysed vaccinia-pp65-infected B-lymphoblastoid cell lines (BLCLs); none lysed vaccinia-pp150-infected or noninfected BLCLs. Ten of 10 CD8+ clones tested were restricted by HLA-A*0201. Seven of the 12 clones were Vbeta6+ (2 from the fibroblast arm and 5 from the DC arm) with an identical Vbeta6.1-J1.4 sequence. Three clones from the fibroblast arm and 5 clones from the DC arm recognized the pp65 peptide NLVPMVATV (amino acids [aa], 495-503). These data show that CMV-specific T-cell clones with similar restriction patterns, T cell-receptor usage, and specificity can be generated using monocyte-derived pp65-infected-DC or CMV-infected-fibroblast stimulators. This approach should broaden the applicability of CMV-specific T-cell immunotherapy to a wider spectrum of patients by reducing the time required to generate CMV-specific T-cell clones.

Loss of renal function following bone marrow transplantation: an analysis of angiotensin converting enzyme D/I polymorphism and other clinical risk factors.

Chronic renal failure is an acknowledged late complication of BMT. It is related to the intensive chemotherapy, radiation and supporting medications. Polymorphism in the angiotensin converting enzyme (ACE) gene is associated with progression of nephropathy caused by diabetes and IgA nephropathy. We sought to determine whether ACE genotype and other clinical factors were associated with loss of renal function after BMT. We determined the genotype of 106 adult allogeneic BMT recipients, who received a similar preparative regimen, survived 1 year, and had assessment of renal function up to 3 years after BMT. We found that the distribution of genotypes was similar to the general population; 29%, 51%, and 20% for the DD, DI, and II genotypes, respectively. There was no statistical difference in patient survival between the three groups. Among all patients, the average creatinine clearance declined from 124 (95% CI 117, 131) to 89 (95% CI 78, 100) ml/min over the 36 months after BMT. Decline in renal function over time was less for patients with the DD compared to the II genotype (P = 0.040). Renal function in patients with the DD genotype was also better than those with the DI genotype, but this was of borderline statistical significance (P = 0.055). Renal shielding reduced decline in renal function compared to no shielding (P = 0.026). We conclude that the ACE genotype does not seem to influence survival, but the DD genotype may be protective against renal injury after BMT. Furthermore, we confirm that renal shielding during TBI reduces the renal injury after BMT.

Complement-mediated T-cell depletion of bone marrow: comparison of T10B9.1A-31 and Muromonab-Orthoclone OKT3.

BACKGROUND: T10B9.1A-31 (T(10)B(9)) and Muromonab-Orthoclone OKT3 (OKT3) are both murine MAb with a narrow specificity for T lymphocytes. Over the past 10 years, we have used each for T-cell depletion (TCD) of BM. In this report we describe similarities and differences using these antibodies, as well as their effects on patient outcome. METHODS: We compared BM mononuclear cells (BMMC) prepared using a Cobe Spectra apheresis machine with density gradient (DG) separation to remove RBC and enrich for CD34(+) cells prior to TCD. FACS and limiting dilution assays (LDA) were used to measure the efficiency of TCD, the subsets of cells removed and CD34 content. Univariate statistics were used to assess graft outcome, including GvHD, graft failure, post-transplant lymphoproliferative disease (PTLD), relapse, DFS, and TRM. RESULTS: BMMC preparation on the Cobe Spectra resulted in superior recovery of CD34(+) cells. However, this method could not be used with OKT3 due to inhibition of T-cell lysis. Optimal TCD required two rounds of complement at room temperature for OKT3, compared with one or two rounds for T(10)B(9). TCR(gamma delta)(+) T-cells, but not natural killer cells, were spared to a greater degree with T(10)B(9). Further T-cell loss occurred during culture with T(10)B(9) but not with OKT3. Overall efficiency of TCD was superior using T(10)B(9). The risk of acute GvHD was higher with OKT3-mediated TCD, independent of T-cell content, and may have led to a higher incidence of PTLD. A decreased risk of relapse for patients with high-risk disease was seen with OKT3-treated grafts, but engraftment, TRM and DFS did not significantly differ. DISCUSSION: TCD using OKT3 results in higher T-cell content and higher rates of acute GvHD and PTLD compared with T(10)B(9). Cobe Spectra cannot be used for BMMC processing with OKT3, fewer CD34(+) are therefore infused. Technical, as well as biological, differences between narrow specificity MAbs can affect graft outcome.

Analysis of risk factors for the development of GVHD after T cell-depleted allogeneic BMT: effect of HLA disparity, ABO incompatibility, and method of T-cell depletion.

Multivariate analysis was performed to determine the independent factors affecting the risk of acute GVHD (aGVHD) grades II to IV and extensive chronic GVHD (cGVHD) and the rate of survival in 481 recipients of T cell-depleted (TCD) marrow allografts who received transplants at a single center between 1991 and 2000. All patients received grafts partially depleted of CD3+ T cells by complement-mediated lysis using 2 narrow-specificity monoclonal antibodies (MoAbs), T10B9.1A-31 (n = 400) or Muromonab-Orthoclone OKT3 (n = 81). Factors considered in the analysis included patient/donor sex, age, cytomegalovirus (CMV) status, and ABO blood group along with T-cell dose, disease, and disease status, donor relationship, HLA antigen (Ag)mismatch (MM), growth-factor use, anti-thymocyte globulin use, year of transplantation, and the MoAb used for TCD. The results showed an association of HLA with an increased relative risk (RR) of aGVHD for recipients of grafts from relateddonors that were > or =2 Ag MM (n = 73, RR = 2.09, P = .005), matched unrelated (UR) donors (n = 130, RR = 1.98, P = .004), and > or =2 Ag MM UR donors (n = 34, RR = 2.68, P = .003) compared with the baseline matched-sibling group (n = 121). No increased risk of aGVHD was seen for 0 to 1 Ag MM family donors (n = 24) or 1 Ag MM UR donors (n = 99). aGVHD risk was increased with minor, but not major or major-minor, ABO disparity (RR = 2.0, P = .003) compared with that of ABO-identical pairs. We found less effective TCD and resultant higher T-cell dose for recipients of grafts that were T cell depleted using OKT3. However, the use of OKT3 and not the T-cell dose was associated with increased aGVH-D risk (RR of 1.84, P = .001). Increased risk of extensive cGVHD was associated with patient age of >20 years (RR = 2.2, P < .0001) and with CMV status (positive patient/negative donor, RR = 1.9, P = .002). Decreased survival was associated with older age (>20 years), a > or =2 Ag MM related donor, a 1 or > or =2 Ag MM UR donor, risk group, and a CMV-positive patient/-negative donor pair. There was no difference in survival for 0 to 1 Ag MM related or matched UR donors compared with the baseline group. These data indicate that there are quantitative as well as potential qualitative differences in outcome depending on the TCD method. Expected and unexpected risk factors for GVHD and survival were associated with partial TCD. Our data support the consideration of ABO match in donor selection, the preferential selection of CMV-positive donors for CMV-positive recipients, and the acceptance of 1 but not > or =2 Ag HLA MM donors.