Application Of Donor Lymphocyte Infusions Research Articles

Impact of Donor Chimerism-Guided Immunomodulation after Allogeneic Stem Cell Transplant on the Outcome of Patients with AML and MDS

Impact of Donor Chimerism-Guided Immunomodulation after Allogeneic Stem Cell Transplant on the Outcome of Patients with AML and MDS

Prior Treatment with Alemtuzumab Interferes with T-Cell Engraftment After Allogeneic Stem Cell Transplantation in Patients with Chronic Lymphocytic Leukemia.

Abstract 3351Poster Board III-239 Objectives:The majority of patients with chronic lymphocytic leukemia (CLL) who receive allogeneic hematopoietic cell transplantation (HCT) have fludarabine-refractory disease. The most active single agent in this disease stage is alemtuzumab. Alemtuzumab has a long half-life and induces profound T-cell depletion (TCD). Since TCD may mitigate graft-versus leukemia effects we evaluated „pre-conditioning“ with alemtuzumab followed by a washout period in order to minimize in vivo T-cell depletion of the graft in a phase II study (NCT 00337519). Methods:Patients received cytoreductive treatment with 3 × 30 mg alemtuzumab weekly prior to HCT. The scheduled interval between last dose of alemtuzumab and HCT was increased from two weeks to one month during the study. The antibody level at the day of HCT was measured with an ELISA with a lower limit of detection of 31.25 ng/mL (BioAnaLab lim., Oxford, UK). The conditioning regimen contained fludarabine (150 mg/m2) and busulfan (8 mg/kg). Cyclosporine (CSA) and methotrexate (MTX) were applied as GVHD-prophylaxis. Medically fit patients with relapsed CLL were elible. Results:62 patients with a median age of 57 years were included between April, 2004 and October, 2008. A median of 3 prior regimens had been given. 55% of the patients had fludarabine-resistant disease. Two patients failed to reach HCT due to progressive disease during alemtuzumab therapy. Donors were matched siblings for 26 and matched unrelated donors for 34 patients. The median level of alemtuzumab in peripheral blood after a washout period of two weeks was 62 ng/mL (interquartile range, 45 to 196 ng/mL; minimum below the limit of detection; maximum 490 ng/mL) compared to a median level below the limit of detection after a delay of four weeks (interquartile range, between the limit of detection and 77 ng/mL, maximum 256 ng/mL) (p=0.005). Despite one month time between the last dose of alemtuzumab and HCT 4 out of 30 patients (13%) had alemtuzumab levels greater than 200 ng/mL. No primary or secondary graft failure occurred. A linear relationship between the alemtuzumab level at HCT and the time to complete CD4-T-cell chimerism (TCC) was observed (p=0.003). At day +100 a CD4 positive T-cell-chimerism (TCC) >95% had been achieved by 84% of patients with alemtuzumab levels <100 ng/mL, 83% of patients with antibody levels between 100 and 200 ng/mL and 25% of patients with antibody levels >200 ng/mL (p=0.006). All patients had a complete neutrophil-chimerism at day +100. After early taper of immunosuppression (N=2) or the application of donor lymphocyte infusions in incremental doses (N=5) mixed TCC has been converted to complete TCC in all patients. The median follow-up is 17 months (1 to 61 months). Day +100 non-relapse mortality was 2%. At two years non-relapse mortality and relapse incidence were 21% and 29%, respectively. Two-year overall survival and progression-free survival were 67% (95% CI, 51% to 83%) and 50% (95% CI, 31% to 69%). Conclusions:In patients who received alemtuzumab prior to HCT, residual drug levels may interfere with T-cell engraftment. Lineage specific T-cell chimerism should therefore be assessed prospectively in this group of patients. Persistent mixed T-cell chimerism can be converted by an early taper of immunosuppression and incremental doses of donor lymphocyte infusions. Disclosures:Schetelig:Bayer Schering: Research Funding. Platzbecker:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Reconstitution of CD52-Negative CD4 T Cells after Alemtuzumab-Based T Cell Depleted Allogeneic Hematopoietic Stem Cell Transplantation

The human CD52 molecule is the target of the monoclonal antibody Alemtuzumab, which is used for treating patients with chemo-refractory chronic lymphocytic leukemia as well as for T cell depletion (TCD) in the context of allogeneic hematopoietic stem cell transplantation (HSCT). The molecule is expressed on the surface of lymphocytes, dendritic cells and to a lesser extent on blood-derived monocytes. Previously, investigators have demonstrated that the surface expression of CD52 on T cells is down-regulated after in vitro incubation with Alemtuzumab. By treating purified human CD4 T cells over 4 hours with 10 μg/mL Alemtuzumab in medium supplemented with 10% human AB serum in vitro, we observed a strong decrease of CD52 expression by flow cytometry with a maximum 3–7 days after incubation. The CD52 down-regulation was also found at weaker intensity on CD8 T cells. From previous studies in chronic lymphocytic leukemia patients, it is known that Alemtuzumab treatment also leads to a down-regulation of CD52 on T cells in vivo. However, similar experiments have not been performed in allogeneic HSCT patients receiving Alemtuzumab in vivo for T cell depletion. We therefore analyzed the expression of CD52 on human peripheral blood mononuclear cells isolated at repeated time points from 22 allogeneic HSCT patients after reduced-intensity conditioning with fludarabine and melphalan and in vivo T cell depletion with Alemtuzumab (100 mg). Half of the patients received prophylactic CD8-depleted donor lymphocyte infusions (DLI) to promote immune reconstitution. By flow cytometry, we observed that the CD52 expression on monocytes, B cells, and natural killer cells remained unaltered after transplantation and was not influenced by the application of DLI. In contrast, the majority of CD4 T cells were CD52-negative (median, 72%) after transplantation and they remained CD52-negative in patients who did not receive DLI throughout the first year after HSCT. The permanent lack of CD52 expression could not be explained by a continuous effect of Alemtuzumab, because earlier studies have shown that the antibody is not present in active plasma concentrations beyond day +60 after HSCT. In contrast, patients receiving CD8-depleted DLI demonstrated a significant increase in the proportion of CD52-positive CD4 T cells. In three of our patients (DLI: n=2, non-DLI: n=1) we analyzed the donor chimerism of CD52-positive and CD52-negative CD4 T cells sorted with high purity by flow cytometry. Three months after HSCT (before DLI), the proportion of donor T cells was clearly higher among the CD52-negative compared to the small proportion of CD52-positive cells in all patients (44% vs. 10%, 83% vs. 0%, and 100% vs. 40%). In the patient who did not receive DLI, the donor T cell chimerism remained mixed in the CD52-negative and CD52-positive fractions on days 200 (CD52-negative: 95%; CD52-positive: 15%) and 350 (CD52-negative: 92%; CD52-positive: 65%). In contrast, the two patients receiving CD8-depleted DLI showed a strong increase in the proportion of CD52-positive CD4 T cells that were of complete donor origin. Altogether, CD52 is permanently down-regulated in reconstituting CD4 T cells following HSCT with an Alemtuzumab-based TCD regimen unless DLI are applied. Our data support the idea of an active mechanism for CD52 down-regulation in CD4 T cells that is not related to B cells and natural killer cells and that appears to differently affect donor and host T cells, respectively.

CD8-Depleted Donor Lymphocyte Infusions Convert Mixed into Complete Donor T-Cell Chimerism after T-Cell Depleted Allogeneic Stem Cell Transplantation.

Donor lymphocyte infusions (DLI) are increasingly used to treat minimal residual disease or mixed hematopoietic donor-recipient chimerism in T-cell depleted allogeneic stem cell transplantation (SCT). In addition, several clinical trials currently investigate the prophylactic application of DLI to promote donor T-cell reconstitution after transplantation. However, DLI carry a substantial risk of inducing graft-versus-host disease (GVHD). We investigate DLI heavily depleted of CD8 T cells using a clinical grade immunomagnetic in vitro procedure in an ongoing clinical study [Meyer et al., Blood 2007, 109:374]. These DLI are administered in a prophylactic setting to patients with hematological malignancies who are off immunosuppressive treatment and free of GVHD early after allogeneic SCT. The reduced-intensity conditioning regimen consists of fludarabine and melphalan and in vivo T-cell depletion (TCD) by the anti-CD52 antibody Alemtuzumab. Up to now, 24 patients have been treated with 1 to 4 increasing doses of CD8-depleted DLI starting with 1x10^6 CD4+ T cells/kg bodyweight. The median time between SCT and first DLI was 119 days (range, 60–194). Seven of 24 patients (29%) developed acute GVHD of grade 2 to 4 or extensive chronic GVHD following CD8-depleted DLI. We did a longitudinal analysis of lineage-specific T-cell chimerism in 20 patients who received CD8-depleted DLI in comparison to 17 patients who did not qualify for DLI due to spontaneously occurring acute GVHD (n=14) or unavailable donors (n=3). The patients' characteristics in both groups were comparable with a median age of 55 (range, 35–64) years in the DLI group and of 57 (range, 29–67) years in the non-DLI group. The donor types were matched sibling (DLI: n=6; non-DLI: n=2), matched unrelated (DLI: n=8; non-DLI: n=8), and unrelated with 1 HLA-mismatch (DLI: n=6; non-DLI: n=7), respectively. Twelve of the 20 patients in the DLI group and 6 of 17 patients in the non-DLI group showed a secondary decrease of donor T-cell chimerism to a median of 52% (range, 10–90%) between 7 and 35 weeks after transplantation (median, 12 weeks). Only one of the latter spontaneously reconverted to a full donor T-cell chimera. Of the remaining 5 non-DLI patients, 4 patients subsequently relapsed with their underlying disease and one patient still had a mixed T-cell chimerism of 50% two years after transplantation. In contrast, in patients receiving CD8-depleted DLI the proportion of donor T cells significantly increased and 11 of 12 patients converted to durable full donor chimerism. Three patients of the DLI group subsequently developed disease relapse. By monitoring CD52-expression on reconstituting T cells by flow cytometry, we were able to demonstrate the impact of CD8-depleted DLI on post-transplant T-cell reconstitution: In the non-DLI group, the majority of CD4 T cells remained CD52-negative 9 months after transplantation. Simultaneously, the proportion of CD52-expressing CD4 T cells was significantly higher in the DLI group (mean: 42% versus 86%; t-test, p<0.01). A similar difference was not observed in the CD8 T-cell lineage. Our results show that the impact of adoptively transferred CD4 T cells after anti-CD52 antibody-mediated TCD can be readily demonstrated by CD52 staining of reconstituting T cells. In addition, CD8-depleted DLI can be efficiently applied to switch mixed into complete donor T-cell chimerism. On the basis of our data we propose a prospective randomized trial in patients with a secondary decrease of donor T-cell chimerism that investigates if CD8-depleted versus non-depleted DLI lead to an improved outcome with regard to disease control and GVHD.

Involved field radiation therapy and donor lymphocyte infusion for relapsed or refractory non-Hodgkin lymphoma after allogeneic hematopoietic stem cell transplantation

Allogeneic hematopoietic stem cell transplantation (HSCT) is an established treatment for Non-Hodgkin lymphomas that are refractory to or relapse after high-dose chemotherapy. Its beneficial effect on disease-free survival or overall survival has been shown e.g. for follicular lymphoma or diffuse large cell B-cell lymphoma. This is not only mediated by the cytoreductive capacity of the conditioning regimen, but also by the graft-versus-malignancy effect of the donor immune system. However, in some cases the lymphoma relapses or is refractory after allogeneic HSCT and only few treatment options are left for those patients. Amongst these, the application of donor lymphocyte infusions (DLI) [1] and involved field radiation therapy (IFRT) have been shown to be effective and feasible for the local control of lymphoma and even the induction of sustained remission [2]. We treated two patients with allogeneic HSCT for diffuse large B-cell lymphoma and marginal zone B-cell lymphoma, respectively. Both relapsed locally and control of remaining lymphomas was achieved by involved field radiation therapy and subsequent donor lymphocyte infusions. E.g., in one patient paragastric lymphoma masses were reduced from 5 cm (Fig. 1a) to 1 cm (Fig. 1b). Although this treatment had considerable side effects, both patients are alive at day ?280 and ?577 after HSCT and have not relapsed since. This case report demonstrates the feasibility and safety of the combination DLI/IFRT for relapsed lymphomas post HSCT. Thus, this combination should be further evaluated as an alternative treatment option for patient who relapse after HSCT or do not respond to it. We

PO2-43 PROBUCOL DOSE-DEPENDENTLY PROMOTES REVERSE TRANSPORT OF CHOLESTEROL FROM MACROPHAGES TO FECES IN VIVO

Background and objectives: Donor lymphocyte infusions (DLI) have become widely used for prevention or treatment of relapse after allogeneic hematopoietic stem cell transplantation. Increasing use of reduced intensity conditioning regimens (RICR) and subsequent application of DLI forced the hemapheresis centers to collect donor lymphocytes in certain quantity and quality. The place of growth factors especially granulocyte colony stimulating factor (rhG-CSF, filgrastim) in allogeneic hemapoietic stem cell (HSC) collection is established, but there is no consensus about the role of rhG-CSF. We aimed to clarify the dose effect of rhG-CSF on lymphocyte subpopulations (CD3+, CD3+4+, CD3+8+, CD19+, CD3−16+56+) cells and CD34+ HSC.Donors and methods: Major indications for DLI (mean volume: 180 ± 52 ml) were for relapse or transplants using RICR mainly in patients with acute leukemia (n=20) or chronic myeloid leukemia (n=15). In four years we performed 40 lymphocyte apheresis (LA) on 30 healthy (med. age 28, M/F 21/9) donors using continuous flow cell separators by processing 2–2.5 times of their total blood volume (TBV). The apheresis data is divided into three groups according to rhG-CSF dose used for priming. Donors in Group I (n=18), Group II (n=9) and Group III (n=13) received no rhG-CSF (steady state), rhG-CSF 5 μg/kg/d sc × 5 days and rhG-CSF 10 μg/kg/d sc × 5 days, respectively. There was no difference within groups concerning TBV processed and recipient body weight.Results: A total of 11,565 ml (±3700) of blood was processed in 216 min (±36.5) at an inlet of 56.8 ml/min (±10.6) using 999 ml (±307) ACD. The CD34+ HSC increased with increasing rhG-CSF dose as expected. Median CD3+ lymphocyte yield per recipient body weight in Group I, II and III were 0.9 × 10e8/kg (range: 0.1–2.1), 2.9 × 10e8/kg (range: 1.6–4.3) and 2.1 × 10e8/kg (range: 0.6–6.9), respectively. The primed donors T lymphocyte yield was 2–3-fold more in comparison to Group I. This gain was most significant between Group I and III in terms of mean CD3+ (1.09 × 10e8/kg vs 2.41 × 10e8/kg, p=0.02), CD3+4+ (0.64 × 10e8/kg vs 1.44 × 10e8/kg, p=0.02) and CD3+8+ (0.42 × 10e8/kg vs 0.89 × 10e8/kg, p=0.03) cells, respectively.Conclusion: Though the yield of lymphocyte subsets in G-CSF primed donors exceeds the non-primed donors, the target range of 1 × 10e7–1 × 10e8/kg CD3+ lymphocytes could be achieved in the majority of the apheresis procedures without rhG-CSF priming. The yield of T and B lymphocyte subsets are increased by G-CSF stimulation but not on a logarithmic scale, which did not correlate into a clinical relevance.

Adoptive Donor T-Cell Infusions Are Necessary and Efficient To Induce Lasting Molecular Remissions after Highly Purified CD34+ Sibling Donor Transplants for First Chronic Phase Chronic Myeloid Leukemia - Final Report and Long-Term Follow-Up of a Phase II Study.

Since September 1998, we prospectively studied the feasibility of transplantation using purified peripheral blood CD34+ cells from HLA-identical sibling donors in first chronic phase chronic myeloid leukemia (CML). A total of 60 patients (pts) with a median pretransplant risk score of 2 (range 1–4) has been included in this study. One patient received an unmanipulated graft due to poor CD34+ donor cell mobilization, while three pts (5%) were successfully retransplanted with an unmanipulated graft from the primary donor after secondary graft failure (n=2) or from an unrelated donor after hematologic relapse (n=1). As part of the study protocol, all pts were closely monitored for BCR-ABL transcripts using real-time RT-PCR analysis of peripheral blood cells as well as BCR-ABL-interphase FISH and metaphase karyotyping of marrow cells. Of the 60 pts, 56 were eligible for the application of donor lymphocyte infusions (DLI), but 7 pts did not receive DLI due to sustained molecular remission and complete chimerism. Thirty-one pts (52%) received DLI because of increasing BCR-ABL transcript levels or hematologic relapse, and 18 pts (30%) as programmed T-cell add-back. The median starting dose was 0.33 (0.01 – 10) x 106 CD3+ cells per kg with a median maximum dose 3.3 (0.17 – 100) x 106 CD3+ cells per kg. DLI induced a lasting reduction of median BCR-ABL transcript levels (BCR-ABL/GAPDH ratio) of more than 3 log10 and the estimate of being in a complete molecular remission at 7 years is 83% ± 5%. Six pts. (10%) did not respond to DLI, but 4 of these pts. attained a cytogenetic and molecular response by imatinib and/or interferon treatment. The cumulative risk of grades II-IV acute GvHD is 15% ± 5% for all study pts, and the risk of chronic GvHD is 25% ± 6%, respectively. After a median follow-up period of 46 (range 6 – 86) months for all pts, the cumulative 7-year survival estimate is 91% ± 4% (survival rate 92%). Causes of death were disease progression, secondary malignancy, liver failure, septicemia, and systemic capillary leak syndrome in one patient each. In conclusion, the concept of highly purified peripheral blood CD34+ cell transplantation in conjunction with adoptive DLI is associated with a particularly low risk of non-relapse mortality and allows induction of lasting molecular disease control in the majority of first chronic phase CML patients.

Sustained Complete Molecular Remissions After Treatment With Imatinib-Mesylate in Patients With Failure After Allogeneic Stem Cell Transplantation for Chronic Myelogenous Leukemia: Results of a Prospective Phase II Open-Label Multicenter Study

In the era of molecular therapy of chronic myelogenous leukemia (CML) applying BCR-ABL tyrosine kinase inhibitors, the usefulness of molecular end points, in particular, quantitative polymerase chain reaction (PCR) for BCR-ABL in monitoring responses has been broadly accepted. Therefore, we have designed a prospective phase II trial in CML, which, for the first time, evaluated the feasibility and safety of molecular end points as surrogate markers to guide through a stratified treatment algorithm within a multicenter trial. As a clinical model, we adopted minimal residual disease (MRD) found in relapse after allogeneic stem cell transplantation (SCT) in CML. Forty-four patients were enrolled and received the BCR-ABL tyrosine kinase inhibitor imatinib (IM) at a starting dose of 400 mg/d. The quality of molecular responses achieved then decided on discontinuation of IM or dose escalation up to 800 mg/d, and finally, on application of donor lymphocyte infusions. Results Seventy percent of patients achieved a complete molecular response (CMR), defined as nested PCR-negativity for BCR-ABL in three consecutive samples. Interestingly, in four out of 10 patients who discontinued IM, CMR was durable even after cessation of IM with a median follow-up of 494 days. This suggests the possibility of long-term tumor control in a subset of patients. The treatment strategy showed that IM treatment was well-tolerated and highly efficacious in MRD after allogeneic SCT. Moreover, this study demonstrated that evaluation of a molecular end point within a multicenter trial can be a safe and effective tool for clinical decision making.

Campath-1H-Based Reduced-Intensity Conditioning Followed by Allogeneic Blood Stem-Cell Transplantation and Preemptive CD8-Depleted Donor Lymphocyte Infusions.

Recent reports have demonstrated the feasibility of using the anti-CD52 antibody Campath-1H for in vivo T-cell depletion (TCD) in the context of a fludarabine/melphalan-based reduced-intensity conditioning regimen (Kottaridis et al. Blood 2000, 96:2419). Major disadvantage of this protocol is the severe immunosuppression which results in an increased rate of opportunistic infections and disease relapses. Donor lymphocyte infusions (DLI) are frequently used to overcome this limitation. The application of DLI, however, is associated with a profound risk of GvHD. Depletion of CD8+ lymphocytes from either the allotransplant or from DLI has proven feasible to reduce the incidence of GvHD (Nimer et al. Transplantation 1994, 57:82; Giralt et al. Blood 1995, 86:4337).We set up a phase-I allotransplantation study which combines the Campath-1H (100mg)/fludarabine (150mg/m2)/melphalan (140mg/m2) preparative regimen with the preemptive administration of CD8-depleted DLI in patients with hematological malignancies not eligible for myeloablative conditioning. After early withdrawal of CsA immunosuppression, CD8-depleted DLI are scheduled in a dose-escalating regimen starting on day +60 after HLA-identical sibling transplantation and on day +120 after unrelated donor transplantation, respectively. For CD8-depletion of donor leukaphereses a new CliniMACS-based protocol using clinical grade CD8 Microbeads is used. Our data show that this procedure is efficient in reducing the content of CD8+ T-cells by at least 2.5 to 3.5 logs, while CD4+ cells remain largely unchanged.In this ongoing study, 8 patients (4 myeloma, 2 CMPD, 1 ALL, 1 NHL) were included and received peripheral blood stem cells (PBSC) from HLA-identical sibling (1), HLA-matched (5) or DRB1-mismatched unrelated donors (2), according to high-resolution HLA typing. Chimerism analyses on total bone marrow-derived cells as well as on purified CD4, CD8, CD19, CD14 and CD15-positive peripheral blood cells showed durable complete donor chimerism in all but one patient (see below). After successful withdrawal of CsA, 2 patients already received CD8-depleted DLI without any acute toxicity. In the first patient (UPN 3), who was transplanted from an HLA-identical sibling, DLI were given on days 60 and 120 (1 and 5 x 106 CD4+ T cells, respectively). The second patient (UPN 4), who was transplanted from a matched unrelated donor, received the first dose of 1 x106 CD8-depleted DLI on day 120. DLI were regularly followed by a more than 2-fold increase of CD4+ cells during the first two weeks after application, while CD8+ cells remained unchanged. After the second DLI, patient UPN 3 developed a grade 2 GvHD of the skin (d+22 after DLI). Both patients were at high risk for CMV reactivation (donor: CMV negative, host: CMV positive) and repeatedly received preemptive treatment. Immediately prior to the second DLI, the CD8+ cell count in patient UPN 3 increased during an ongoing CMV reactivation. Line-specific chimerism analysis revealed that more than 50% of these CD8+ cells were of host origin. As expected, transfer of CD8-depleted DLI from CMV-negative donors was unable to entirely prevent CMV reactivation in seropositive patients. Nevertheless, our preliminary data suggest that the preemptive transfer of CD8-depleted DLI is able to accelerate immune reconstitution after reduced intensity TCD allogeneic PBSCT without rapid increase of CD8+ donor cells.

Dose-escalated donor lymphocyte infusions following reduced intensity transplantation: toxicity, chimerism, and disease responses

AbstractData on the application of donor lymphocyte infusions (DLIs) following reduced-intensity transplantation (RIT) remain limited. Persistence of host antigen-presenting cells might increase the efficacy or toxicity of cellular immunotherapies. We report the results of dose-escalating DLIs in 46 patients undergoing RIT, who received a total of 109 infusions to treat mixed chimerism or residual or progressive disease. Diagnoses were myeloma (n = 19), Hodgkin lymphoma (n = 13), non-Hodgkin lymphoma (n = 10), and other (n = 4). Thirty-two had an HLA-matched family donor and 14 an unrelated donor. Grades II to IV graft-versus-host disease (GVHD) occurred in 5 sibling and 7 unrelated donor recipients. GVHD was more common (P = .002), occurred at lower T-cell doses, and was more severe in the unrelated donor cohort. Conversion from mixed to multilineage full donor chimerism occurred in 30 of 35 evaluable patients. Presence of mixed chimerism in the granulocyte lineage at the time of DLI did not predict for chimerism response or GVHD. Disease responses occurred in 63% of patients with myeloma and 70% of those with Hodgkin lymphoma and were not predicted by changes in chimerism. These data support the presence of clinically relevant graft-versus-Hodgkin activity and indicate that DLI may be associated with a significantly increased toxicity in unrelated compared to sibling donor transplant recipients receiving identical treatment protocols.

The impact of granulocyte colony stimulating factor at content of donor lymphocytes collected for cellular immunotherapy

Background and objectives: Donor lymphocyte infusions (DLI) have become widely used for prevention or treatment of relapse after allogeneic hematopoietic stem cell transplantation. Increasing use of reduced intensity conditioning regimens (RICR) and subsequent application of DLI forced the hemapheresis centers to collect donor lymphocytes in certain quantity and quality. The place of growth factors especially granulocyte colony stimulating factor (rhG-CSF, filgrastim) in allogeneic hemapoietic stem cell (HSC) collection is established, but there is no consensus about the role of rhG-CSF. We aimed to clarify the dose effect of rhG-CSF on lymphocyte subpopulations (CD3+, CD3+4+, CD3+8+, CD19+, CD3−16+56+) cells and CD34+ HSC. Donors and methods: Major indications for DLI (mean volume: 180 ± 52 ml) were for relapse or transplants using RICR mainly in patients with acute leukemia ( n=20) or chronic myeloid leukemia ( n=15). In four years we performed 40 lymphocyte apheresis (LA) on 30 healthy (med. age 28, M/F 21/9) donors using continuous flow cell separators by processing 2–2.5 times of their total blood volume (TBV). The apheresis data is divided into three groups according to rhG-CSF dose used for priming. Donors in Group I ( n=18), Group II ( n=9) and Group III ( n=13) received no rhG-CSF (steady state), rhG-CSF 5 μg/kg/d sc × 5 days and rhG-CSF 10 μg/kg/d sc × 5 days, respectively. There was no difference within groups concerning TBV processed and recipient body weight. Results: A total of 11,565 ml (±3700) of blood was processed in 216 min (±36.5) at an inlet of 56.8 ml/min (±10.6) using 999 ml (±307) ACD. The CD34+ HSC increased with increasing rhG-CSF dose as expected. Median CD3+ lymphocyte yield per recipient body weight in Group I, II and III were 0.9 × 10e8/kg (range: 0.1–2.1), 2.9 × 10e8/kg (range: 1.6–4.3) and 2.1 × 10e8/kg (range: 0.6–6.9), respectively. The primed donors T lymphocyte yield was 2–3-fold more in comparison to Group I. This gain was most significant between Group I and III in terms of mean CD3+ (1.09 × 10e8/kg vs 2.41 × 10e8/kg, p=0.02), CD3+4+ (0.64 × 10e8/kg vs 1.44 × 10e8/kg, p=0.02) and CD3+8+ (0.42 × 10e8/kg vs 0.89 × 10e8/kg, p=0.03) cells, respectively. Conclusion: Though the yield of lymphocyte subsets in G-CSF primed donors exceeds the non-primed donors, the target range of 1 × 10e7–1 × 10e8/kg CD3+ lymphocytes could be achieved in the majority of the apheresis procedures without rhG-CSF priming. The yield of T and B lymphocyte subsets are increased by G-CSF stimulation but not on a logarithmic scale, which did not correlate into a clinical relevance.