CD8-depleted Donor Lymphocyte Infusions Research Articles

Tumor Antigen Vaccination of Donors to Augment Graft-Versus-Tumor Effects after Allogeneic Bone Marrow Transplantation with Post-Transplantation Cyclophosphamide

High dose, post-transplantation cyclophosphamide (PTCy) effectively prevents graft-versus-host disease after alloBMT by killing alloreactive T cells recently stimulated by recipient alloantigens. However, remotely primed (>4 days) T cells are resistant to cyclophosphamide-induced immunologic tolerance, raising the possibility that priming BMT donors with tumor antigens could be an effective method to augment anti-tumor immunity after alloBMT with PTCy without exacerbating GVHD.To test this hypothesis, we generated tumor antigen-specific T cells by vaccinating mice with chicken ovalbumin (OVA) expressing Listeria monocytogenes OVA (Lm-OVA) 7 or 30 days before syngeneic or allogeneic BMT, with or without PTCy of recipients bearing the EL4 thymic lymphoma expressing ovalbumin (EL4-mOVA). In a syngeneic BMT model, transferring vaccine primed day -7 or day -30 donor T cells into EL4-mOVA-Luci bearing mice prolonged survival but did not result in cure due to late relapses. To test whether this phenomenon can be found in an MHC matched, minor antigen mismatched alloBMT mouse model, we transferred vaccine primed or unprimed C3H.SW donor splenocytes into tumor bearing B6 recipients. Recipients of alloBMT without PTCy from vaccinated donors had a significant cure rate of 38% (day-30) and 23% (day -7) compared to 0% survival rate for mice receiving unprimed donor splenocytes. Interestingly, PTCy completely abrogated both GVHD and the anti-tumor benefit of donor vaccination, resulting in no prolongation of tumor-free survival of recipients of cells from primed donors compared to recipients of cells from unprimed donors.In light of the abrogation of anti-tumor immunity by PTCy, we tracked the fate of vaccine-primed T cells in alloBMT recipients. Donor cells primed on 7 or 30 days before T cell transfer showed significant numbers of OVA-specific CD8+ effector T cells on day 15 after transplantation in mice that did not receive PTCy. Although PTCy diminished vaccine primed tumor protection, we did detect increased percentages of tumor-specific effector CD8+ T cells in the blood as well as in other compartments on day 15 after transplantation.In an attempt to restore anti-tumor immunity we tested the effects of giving donor lymphocyte infusions (DLI) from tumor antigen-primed donors. DLI depleted of naïve T cells augmented anti-tumor immunity but animals died from graft-versus-host disease. In contrast, CD8-depleted DLI from vaccinated donors given after vaccine-primed BMT plus PTCy prolonged survival from EL4-mOVA without resulting in fatal GVHD. These results are consistent with the hypothesis that vaccine-primed CD8+T cells survive PTCy and their function can be elicited post-transplantation with help provided by tumor-specific CD4+ T cells administered as donor lymphocyte infusion. We plan to test this hypothesis in patients by vaccinating allogeneic BMT donors against a tumor antigen prior to allogeneic BMT + PTCy, with or without CD8-depleted DLI. DisclosuresNo relevant conflicts of interest to declare.

Follow Up On CD8-Depleted Donor-Lymphocyte Infusions After T-Cell Depleted Allogeneic Hemopoietic Stem Cell Transplantation

We applied prophylactic CD8-depleted (CD8depl) donor lymphocyte infusions (DLI) in the setting of T-cell depleted reduced-intensity allogeneic hematopoietic stem cell transplantation (HSCT) in a phase I/II trial. T-cell depletion was carried out by the use of high-dose Alemtuzumab (100 mg or 60 mg for unrelated or sibling donor transplantation, respectively). We have demonstrated the feasibility of this approach after having treated 23 patients in this protocol (Meyer et al. Blood 2007). From 2004 to 2011, 134 patients with different hematologic diseases were included and followed for a median observation time of 1.5 years after transplantation (range, 1.5-9 years). Median age was 56 years (range, 19-71). Stem cell source was peripheral blood from either matched siblings (n=23), matched unrelated (n=62), or donors with single HLA mismatches (unrelated n=48, related n=1). Tapering of cyclosporine A was started in the 6th week after transplantation. Subsequently, CD8depl DLI were administered prophylactically in escalating doses starting with 1x106 CD4 T cells/kg bodyweight. 53 patients received at least one dose of DLI. Among 81 patients who did not qualify for DLI, 60 patients had primary mild GVHD. Following DLI, acute GVHD was the major reason for withholding subsequent DLI-doses (66%), mainly because of acute GVHD ≤ 2°. Extensive chronic GVHD was diagnosed in 9.4% of the patients. Overall survival after 1 and 3 years was 60% and 43%, respectively. Survival significantly differed between the DLI and non DLI group after 3 years (63.8% vs. 30.1%, p=0.002). The inferior outcome of the non DLI group was confirmed when only those patients who did not receive DLI for other reasons than GVHD were considered (28.6%). The largest patient cohort in our trial were patients with AML and MDS (n=21 with and n=33 without DLI). In these patients, the survival benefit for the DLI group after 3 years was significant (75.8% vs 28.9%, p=0.0012). Interestingly, the relapse rate did not differ between DLI and non-DLI patients but the non-relapse mortality. The presence of GVHD at any time was associated with a reduced relapse rate (57.9% vs. 31.4%, p=0.0015), independent of DLI-application. However, GVHD had no impact on overall survival. Decreasing donor T-cell chimerism (TCC) was found in 34 patients who subsequently received DLI and 13 who did not. Following CD8depl DLI, 31 patients (88%) converted to full donor. In contrast, only 2 of the patients with decreasing TCC in the non-DLI group (15.3%) converted spontaneously. In summary, we observed that the application of prophylactic CD8depl DLI was associated with a survival benefit and the re-establishment of full donor T-cell chimerism. In AML/MDS patients the improved survival was associated with lower treatment-related mortality rather than a reduced relapse-rate. Our data strongly suggest randomized trials comparing prophylactic application of CD8depl DLI vs. no DLI as well as CD8depl vs. non-manipulated DLI in a preemptive setting. Disclosures: Off Label Use: MabCampath has been used for T-cell depletion.

CD8-Depleted Donor-Lymphocyte Infusions After T-Cell Depleted Allogeneic Hemopoietic Stem Cell Transplantation

CD8-Depleted Donor-Lymphocyte Infusions After T-Cell Depleted Allogeneic Hemopoietic Stem Cell Transplantation

Longitudinal Monitoring of Immune Reconstitution After Allogeneic Peripheral Blood Stem Cell Transplantation (PBSCT): Impact of T-Cell Depletion of the Graft

Patients and methods In this retrospective study, we compared the kinetics of recovery of T-, Band NK-cells compartments in two groups of patients who had undergone allogeneic PBSCT after high dose conditioning: a first group who received CD34-selected PBSC (CD34 group, n= 62), and a second group given unmanipulated PBSC (PBSC group, n= 43). Of note, 45 of 62 patients in the CD34 group received pre-emptive CD8-depleted donor lymphocyte infusions starting at days 40-60 after PBSCT in an effort to prevent disease relapse. Lymphocyte phenotyping was performed by flow cytometry on days 28, 40, 60, 80, 100, 180 and 365, and then yearly thereafter.

Impact of Prophylactic CD8-Depleted Donor-Lymphocyte Infusions After Allogeneic Hematopoietic Stem Cell Transplantation and Alemtuzumab Mediated T-Cell Depletion,

Abstract 4109We have previously demonstrated that the application of CD8-depleted donor-lymphocyte infusions (DLI) is feasible after reduced-intensity conditioning and in vivo T-cell depletion by alemtuzumab. DLI overcome slow lymphocyte recovery associated with alemtuzumab-administration and improve anti-infectious immunity and reliably convert a decreasing T-cell chimerism (Meyer et al. Blood 2007 & BMT 2010).Here we provide clinical follow up data of 117 patients with different hematological diseases and a median observation time of 1 year (range, 1–86 months) post hematopoietic stem cell transplantation (HSCT). The majority of patients either suffered from an acute leukemia / MDS (n=54), lymphoma (n=32), myeloma (n=17), or myeloproliferative neoplasms (n=12). Two patients suffered from non-malignant diseases. The median age of the patients was 56 years (range, 19–71) and none of them qualified for a conventional conditioning regimen. 50 patients had undergone previous transplantations (autologous: n=47, allogeneic: n=3). Donors were matched siblings (n=20), matched unrelated donors (n=55), or unrelated donors with a single HLA mismatch (n=42). Between days 60 and 120 after HSCT, without calcineurin-inhibitors and in the absence of graft-versus-host disease (GVHD), 1×106 CD8-depleted DLI per kg bodyweight were administered. Up to three further DLI were given in escalating doses in 60 to 90 day intervals. Following this procedure, 45 patients received at least one dose of DLI. Among those patients who did not qualify for DLI, 50 patients had primary GVHD. In 22 patients DLI were not administered for other reasons (donor unavailable, infections, relapse). In 64% of DLI induced acute GVHD, which was the major reason for withholding the next DLI-dose step. The rate of acute GVHD > grade 2 was 30%. 10% suffered from extensive chronic GVHD. The 1 and 3 year overall survival was 63% and 43%, respectively. Survival significantly differed between the DLI and the non DLI group after 3 years (63% vs. 27%, p=0.002). Since this trial was not randomized, we also compared the DLI group to only those patients who did not receive DLI for other reasons than primary GVHD and found similar results (62% vs. 28%, p=0.01). As expected, the presence of GVHD at any time was associated with a reduced relapse rate in all patients (55.8% vs 30.8%, p=0.013). Although DLI was associated with a survival benefit, the relapse rate did not differ from that of the no-DLI cohort. AML/MDS patients represented the largest group of patients included in our study (n= 48). Among these, 41 patients achieved the time point for DLI administration, 16 of them received at least one dose of prophylactic DLI. 9 patients developed GVHD after DLI application. 20 patients had primary GVHD as major cause for not receiving DLI. The survival curves differed significantly between the DLI and non DLI group after 1 and 3 years (91.7% vs. 54%, and 82.5% vs 24% p=0.004). The estimated 5 year overall survival for all AML patients was 50.4%. There was no significant difference analyzing the relapse rate (20% vs 18.8%).In summary, the prophylactic application of CD8-depleted DLI in the absence of GVHD was associated with a survival benefit. However, we were not able to relate this benefit to a decreased relapse rate, and we assume a better control of infections. Our data strongly support a randomized trial, comparing prophylactic vs. preemptive / therapeutic DLI application in the context of T-cell depleted HSCT. Disclosures:Meyer:BMS: Membership on an entity's Board of Directors or advisory committees.

Long-Term Persistence of Functionally Altered GPI-Anchor Negative T-Cell Subsets After Alemtuzumab-Based T-Cell Depleted Hematopoietic Stem Cell Transplantation.

Abstract 2438Poster Board II-415The anti CD52 antibody alemtuzumab is frequently used for in vivo T cell depletion (TCD) in the context of allogeneic hematopoietic stem cell transplantation (HSCT). We have recently demonstrated the persistence of CD52-negative T-cell subsets in patients after HSCT following alemtuzumab-mediated TCD (Meyer, Wagner et al., Bone Marrow Transplantation 2009). The loss of CD52 among lymphocytes was exclusively related to T cells and was more prominent in CD4 compared to CD8 T cells. CD8-depleted donor-lymphocyte infusions (DLI) increased the percentage of CD52-positive CD4 T cells. In patients who did not receive DLI, CD52-negative T cells were detected in significant proportions of up to 40% found even more than 3 years after transplantation. We therefore investigated the regulation as well as the functional consequences of a loss of CD52-expression in T cells of our patients. Peripheral blood T cells of patients with CD52-negative T cells after more than 12 months post allogeneic HSCT following TCD with high-dose alemtuzumab (100 mg) were sorted according to their expression of CD52. RT-PCR showed no difference in CD52 mRNA expression of CD52-positive compared to negative T cells. Since transcriptional regulation was therefore unlikely and CD52 is a glycosylphosphatidylinositol (GPI)-anchored protein, we stained for the presence of further GPI-anchored molecules such as CD55 and CD59 on peripheral blood lymphocytes of our patients. We found that the CD52-negative T cells had also lost expression of CD55 and CD59, whereas CD52-positive cells remained positive for these antigens. We then directly labeled the GPI-anchors using FLAER (fluorescent aerolysin) and thereby confirmed that the loss of CD52 was correlated with a reduced density of the GPI-anchors in the cell-membrane. However, our patients did not exhibit clinical signs of paroxysmal nocturnal hemoglobinuria (PNH), which is in line with the finding that the loss of GPI-anchors was only related to T cells. With the aim to characterize the functional impact of the reduced GPI-anchor density on T cells, we separated CD52-negative from CD52-positive T cells by flow cytometry. The subpopulations were expanded in vitro using low-dose IL2, OKT3, and allogeneic feeder-cells. CD52 expression remained unaltered in CD52-negative as well as CD52-positive cultures for more than 6 weeks. In contrast, when purified T cells of healthy donors were treated with alemtuzumab in vitro (10 μg/mL, 4 h), we only observed a transient down-regulation of the antigen. The growth-kinetics of the non-specifically stimulated T cell cultures did not differ between the CD52-positive and the negative cultures. Yet, when we expanded T cells of a cytomegalovirus (CMV)-positive patient, transplanted from a CMV-positive donor, by subsequent stimulation with overlapping peptides of CMV-pp65, only the proliferation of CD52-positive T cells increased after the addition of peptides. We furthermore applied CD52-positive as well as CD52-negative CD4 and CD8 T cells derived from the antigen-independent culture of this patient in an IFN-gamma ELISPOT assay with autologous dendritic cells (DC) loaded with overlapping peptides of CMV-pp65 and IE1. CMV-specific IFN-gamma spot-production was only evident in the CD52-positive populations. We also conducted IFN-gamma secretion-assays on ex vivo T cells stimulated with autologous DC loaded with CMV-peptides and found a reduced antigen-specific IFN-gamma production in CD52-negative CD4 and CD8 T cells. In addition, we analyzed IFN-gamma secretion of T cells following allogeneic stimulation with DC of a healthy individual and again detected lower levels of IFN-gamma production by CD52-negative compared to CD52-positive T cells. In summary, we demonstrated that the permanent loss of CD52 in a proportion of reconstituting T cells after alemtzumab-based TCD is associated with a loss of GPI-anchors in the cellular membrane. Our data suggest that this loss correlates with reduced T-cell effector-functions in response to antigen-specific stimulation. In addition to a better understanding of the role of alemtuzumab-mediated TCD on T cell reconstitution, further comparison of functional responses in different T-cell subsets in association with the presence or absence of GPI-anchors might help to explore the impact of GPI-anchors and GPI-anchored molecules in this context. Disclosures:No relevant conflicts of interest to declare.

Haploidentical stem cell transplantation after a reduced-intensity conditioning regimen for the treatment of advanced hematologic malignancies: posttransplantation CD8-depleted donor lymphocyte infusions contribute to improve T-cell recovery

AbstractHaploidentical hematopoietic stem cell transplantation provides an option for patients with advanced hematologic malignancies lacking a compatible donor. In this prospective phase 1/2 trial, we evaluated the role of reduced-intensity conditioning (RIC) followed by early add-backs of CD8-depleted donor lymphocyte infusions (DLIs). The RIC regimen consisted of thiotepa, fludarabine, cyclophosphamide, and 2 Gy total body irradiation. Twenty-eight patients with advanced lymphoproliferative diseases (n = 24) or acute myeloid leukemia (n = 4) were enrolled. Ex vivo and in vivo T-cell depletion was carried out by CD34+ cell selection and alemtuzumab treatment. The 2-year cumulative incidence of nonrelapse mortality was 26% and the 2-year overall survival (OS) was 44%, with a better outcome for patients with chemosensitive disease (OS, 75%). Overall, 54 CD8-depleted DLIs were administered to 23 patients (82%) at 3 different dose levels without loss of engraftment or acute toxicities. Overall, 6 of 23 patients (26%) developed grade II-IV graft-versus-host disease, mainly at dose level 2. In conclusion, our RIC regimen allowed a stable engraftment with a rather low nonrelapse mortality in poor-risk patients; OS is encouraging with some long-term remissions in lymphoid malignancies. CD8-depleted DLIs are feasible and promote the immune reconstitution.

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.

CD8-Depleted Donor Lymphocyte Infusions Can Improve Both T- and B-Cell Reconstitution after Allogeneic Stem Cell Transplantation from Haploidentical Family Donors

Purpose: Haploidentical hematopoietic stem cell transplantation (haplo-SCT) provides an option for cancer patients lacking a compatible donor. However, the delayed immune reconstitution increases incidence of opportunistic infections and disease relapse. We conducted a phase I-II prospective trial to evaluate the effect of escalating doses of CD8-depleted (Miltenyi Device) donor lymphocyte infusions (DLIs) following RIC haplo-SCT. Here, we present the data on the immune reconstitution.Patients and Methods: Twenty-eight patients (pts) with advanced lymphoproliferative diseases (n=24) or acute myeloid leukaemia (n=4) were enrolled in this study. Fifteen (54%) of 28 pts had refractory disease. All pts received a thiotepa-fludarabine based RIC regimen, with an ex vivo and in vivo T-cell depletion performed by CD34+ cell selection and alemtuzumab infusion. Fifty-four CD8-depleted DLIs were administered to 23 pts [dose level 1 (n=4): 1×104/kg on days + 45, +75, +105; dose level 2 (n=11): 5×104/kg on days + 45, +75, +105; dose level 3 (n=8): 1×104/kg on day+ 45, 5x104/kg on days +75 and +105].Results: At a median follow-up of 29 months, 12 pts are alive (n=3 Hodgkin Lymphoma, n=6 Non-Hodgkin Lymphoma, n=1 Multiple Myeloma, n=2 Acute Myeloid Leukemia) and 16 died from any cause [n=6 for non-relapse mortality (NRM), n=10 for disease progression] with a 2-year estimates of overall survival of 39%. The 2-year cumulative incidence of NRM and relapse were 26% and 51%, respectively. Six pts (26%) developed grade II-IV acute graft-versus-host disease (GVHD) (dose level 1: no GVHD; dose level 2: 5 cases; dose level 3: 1 case). Following CD8-depleted DLIs, the major findings were:(i) a significant expansion of memory CD4+ cells and CD19+ cells (median value 107/μL and 135/μL, respectively) at 120 days after haplo-SCT; (ii) de-novo T-cell responses to cytomegalovirus (CMV) peptides performed by activation-induced CD137 expression: the median frequency of CMV-specific CD8+/CD137+ and CD4+/CD137+ cells were 2% and 0.4% at 200 days after haplo-SCT, respectively, as evaluated in a subset of pts at risk for CMV reactivation (R CMVpos/D CMVpos/neg); this frequency was associated to a clearance of CMV antigen; (iii) recovery of thymopoiesis: 10 of 20 (50%) pts showed measurable TREC at 1 year after haplo-SCT; (iv) the increase of CD19+ cells was associated to reconstitution of B lymphocyte repertoire as analysed by Immunoglobulin heavy chain (IgH) CDR3 spectratyping: median number peaks in normal donors and pts, at day 360 after haplo-SCT, were 17 (range, 14–21) versus 12 (range, 7–14), respectively.Conclusions: Our study showed that CD8-depleted DLIs are a feasible procedure with low acute GVHD when using dose level 3. Long-term disease remissions can be observed in advanced lymphoid malignancies. Escalated doses of CD8-depleted DLIs exert complex effects on immune reconstitution: (i) provide help to expansion of CD8 T-cell clones, as suggested by the occurrence of the anti-CMV reactivity; (ii) support B-cell recovery, as demonstrated by a partial recovery of CDR3 polyclonality.

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.

Prophylactic transfer of CD8-depleted donor lymphocytes after T-cell–depleted reduced-intensity transplantation

AbstractAllogeneic hematopoietic stem cell transplantation (SCT) regimens incorporating the lymphocytotoxic CD52 antibody alemtuzumab demonstrate efficient engraftment and reduced graft-versus-host disease (GVHD). However, these protocols substantially impair posttransplantation antiviral and antitumor immunity. To accelerate immune reconstitution after alemtuzumab-based reduced-intensity SCT, we administered prophylactic CD8-depleted donor lymphocyte infusions (DLIs) starting on days 60 and 120 after transplantation. DLIs were processed in an immunomagnetic good manufacturing practice depletion procedure resulting in a 2.5- to 6-log reduction in CD8 T cells. Of 23 high-risk patients with hematologic malignancies, 11 received a total of 21 CD8-depleted DLIs. Five patients developed transient grade I acute GVHD following transfer. Only 2 patients with HLA-C–mismatched donors showed grade II and III acute GVHD and subsequently progressed to limited chronic GVHD. Following DLIs, 4 patients with declining hematopoietic donor chimerism converted to full chimeras. A 2.1-fold median increase of circulating CD4 T cells was observed within 2 weeks after infusion. Non-DLI patients did not show a comparable rise in CD4 counts. Four patients demonstrated enhanced frequencies of cytomegalovirus-specific CD4 and CD8 T cells following transfer. Our results suggest that prophylactic CD8-depleted DLIs accelerate immune reconstitution after lymphodepleted HLA-matched SCT and carry a low risk of inducing severe GVHD.

CD8-Depleted Donor Lymphocyte Infusions Can Boost Immune Reconstitution after Haploidentical Stem Cell Transplantation Following Reduced-Intensity Conditioning Regimen.

Haploidentical stem cell transplantation (SCT) can be used in relapsed haematological malignancies for patients lacking a matched sibling or unrelated donor. Major barriers of this strategy are the poor immune reconstitution and the high risk of relapse. Here, we report results of a phase I–II trial evaluating early add-backs of CD8-depleted donor lymphocytes (DLIs) (from 1x10^4 up to 1x10^5 cells/kg starting at day+45 up to day +105 at monthly intervals) after a reduced intensity conditioning (RIC) regimen [thiotepa (10 mg/kg), fludarabine (120 mg/sqm), cyclophosphamide (60 mg/kg) and TBI (2 Gy)]. Ex-vivo and in-vivo TCD were carried out by CD34+ cell selection using the CliniMACS device and alemtuzumab (15mg/m2, day-2), respectively. Twenty-one patients [n= 10 NHL (n=5 CLL, n=5 high-grade NHL), n=7 HL, n=1 MM, n=1 ALL, n=2 AML] were transplanted with advanced disease: 16 (76%) failed a previous autograft and 13 (62%) had refractory disease. A median of 10.4x10^6/Kg CD34+, 1x10^4/kg CD3+, 10x10^4/kg CD19+, 0.9 x10^4/kg NK+ were infused. All patients engrafted with full donor chimerism from day +90. At a median follow-up of 12 months (range, 4–41 months), 12 of 21 pts are alive (7 CR, 2 PR and 3 PD) and 9 died [n=3 infection with GVHD (+610, +187, +253), n=6 disease]. The estimated 2-year overall survival was 49%: pts transplanted in remission had better outcome (83% versus 31%, p=0.13). The estimated 2-year cumulative incidence of TRM and relapse were 27% and 58%, respectively. CMV reactivation and hospital readmissions for opportunistic infections occurred in 76% and 57% of patients, respectively. For CD8 depletion of donor leukaphereses, a new depletion protocol using clinical grade CD8 microbeads (Miltenyi) was applied. This procedure is efficient to reduce the content of CD8 T cells by 3 logs while the median cell recovery of CD3+, CD4+, CD56+/CD3+, CD 20+ was 60%, 86%, 54%, 72%, respectively. Before DLIs, only 2 of 21 patients (10%) developed acute GVHD (no grade III–IV). A total of 36 CD8-depleted DLIs were administered to 17 pts without any acute toxicity. Following DLIs, 6 pts (35%) developed acute GVHD (grade II) and 5 (30%) chronic GVHD (n=2 limited, n=3 extensive). Overall, the incidence of acute GVHD is higher (50% vs 22%, p=0.33) in pts receving larger numbers of donor cells (10–15x10^4/kg versus 3–5x10^4/kg CD8-depleted DLIs). The median values of CD4+/uL, CD8+/uL and NK+ were 100, 280 and 680 at 4 months and 220, 200, 500 at 6 months after SCT in patients receiving CD8-depleted DLIs. Measurable TREC/ucg DNA (mean value 316; mean value donors 3740) and polyclonal T cell repertoire, evaluated by spectratyping, were observed at 9 months in patients younger than 40 years and/or without GVHD. Our results suggest that:haploidentical SCT with RIC regimen provides high engraftment rateT-cell addback allows the achievement of more than 100/uL CD4+ at 4 months after SCT in the majority of patientsSurvival rate is promising in patients who had transplantation in remission suggesting that this strategy should be evaluated earlier in high risk haematological diseases.

Improved Immune Reconstitution Following Pre-Emptive CD8-Depleted Donor Lymphocyte Infusions (DLIs) after Haploidentical Stem Cell Transplantation (SCT) Using a Reduced-Intensity Conditioning (RIC) Regimen.

Allogeneic stem cell transplantation (SCT) from an haploidentical family donor has been reported as a viable option in acute leukemias when matched donors are unavailable. However, the extensive T-cell depletion (TCD), required to prevent graft-versus-host disease (GVHD), is associated to delayed immune recovery and high transplant-related mortality. In an ongoing phase I–II trial for patients (pts) with advanced hematological malignancies, we combined a RIC regimen, including thiotepa (10 mg/kg), fludarabine (120 mg/ms), cyclophosphamide (60 mg/kg) and TBI (2 Gy), with pre-emptive administration of CD8-depleted DLIs (starting from 1x104 up to 1x105 cells/kg). Ex-vivo and in-vivo TCD were carried out by CD34+ cell selection using the CliniMACS device and alemtuzumab (30 mg day −2), respectively. The aim of the study was to investigate in a dose finding study the safety and the impact on immune-reconstitution of CD8-depleted DLIs. Six-teen pts with hematological malignancies (n=14 NHL/HD, n=1 ALL, n=1 AML) were included, median age was 35 years (range, 15–65), 10 (63%) were chemorefractory, and 68% and 75% had failed a previous auto or at least 3 CT lines, respectively. Pts received a median of 10.6 x106/Kg CD34+ and 1x104/kg CD3+. All pts engrafted with full donor chimerism from day +30. At a median follow-up of 9 months, 12 pts were alive and 4 died (n=1 infection, n=3 disease). The estimated OS at 2 years was 58%; 7 of 16 (44%) relapsed at median time of 100 days after SCT. CD8-depletion of 14 donor lymphocyte aphereses was performed with a new depletion protocol (Clinimacs CD8-Microbeads) that reduces the content of CD8+ cells by at least 3 logs. The median CD3+, CD4+, CD56+/CD3+, CD20+ cell recovery were 62% (range, 35–91%), 88% (63–128%), 51% (8–78%), 76% (33–128%), respectively. Before DLIs, only 1 of 16 pts (6%) developed de novo acute GVHD (grade II). A total of 22 CD8-depleted DLIs were administered to 9 of 16 pts without any engraftment problem. The first cohort of pts (n=5) received a total dose of 3–6x104/kg CD8depleted DLIs starting at day +45 divided in 3 monthly infusions: none of them developed aGVHD. Given no toxicity, we escalated doses and the second cohort (n=4) received a total dose of 10–25x104/kg CD8-depleted DLIs divided in 3 monthly infusions: 3 pts had acute GVHD (grade II). Overall, the incidence of acute GVHD was higher (75% vs 0%, P<0.04) in pts receving larger numbers of donor cells. Interestingly, the median values of CD4+/ul and CD8+/ul were 98 (range, 8–612) and 150 (range, 15–988) at 4 months; 247 (range, 55–333) and 235 (range, 3–1000) at 5 months after SCT. The median value of CD19+/ul cells were 134 (range, 0–292) and 160 (range, 0–256) at 4 and 5 months, respectively. NK cells remained between the value of 394/ul and 569/ul in the first 6 months after SCT. Our results suggest that: (1) haploidentical SCT with RIC regimen is feasible with a high rate of engraftment and a low acute GVHD incidence; (2) pre-emptive CD8-depleted DLIs are feasible without GVHD until the total dose of 6 x104/kg; 3) higher doses can induce acute GVHD, but no grade III–IV was observed; 4) despite the limited number of pts, we observed a faster immune recovery and a relatively low mortality rate for infections.

Preemptive Transfer of GMP-Grade CD8-Depleted Donor Lymphocytes after T-Cell Depleted Reduced-Intensity Transplantation.

The combination of fludarabin (150mg/m2) / melphalan (140mg/m2) based reduced-intensity allo-transplantation (RIT) with in vivo T-cell depletion (TCD) by the anti-CD52 antibody alemtuzumab (100mg) has demonstrated efficient engraftment and reduced graft-versus-host disease (GVHD) (Kottaridis et al., Blood 2000). However, the slow lymphocyte recovery following this protocol is associated with reduced anti-infectious and antitumor immunity. In an attempt to improve immune-reconstitution after TCD / RIT, we investigated the early preemptive use of CD8-depleted donor lymphocyte infusions (DLIs). For CD8 depletion of donor leukaphereses, a new depletion protocol using clinical grade CD8 microbeads was applied. This procedure was efficient in reducing the content of CD8 T cells by 2.5 to 4 logs, while NK cells, B cells and CD4 T lymphocytes remained largely unchanged. Up to now, 13 high-risk patients with hematological malignancies were treated. In 4 patients, persistent cutaneous GVHD prevented the preemptive DLI application. These patients exhibited a spontaneous increase in T-cell numbers to > 200/μl in the peripheral blood before day +120 after transplantation. Neither increasing T-cell numbers nor persistent acute GvHD were seen in the 9 patients who qualified for preemptive DLIs. Animal data suggest that the local persistence of host antigen-presenting cells might have initiated or triggered the early onset cutaneous GVHD. Therefore, Langerhans cells (LCs) were isolated from skin biopsies for prospective chimerism analyses. In the analyzed patients, LCs demonstrated a delayed switch to full donor status beyond day +100 after transplantation.Nine patients without early GVHD received a total of 13 CD8-depleted DLIs starting on day +60 after sibling and day +120 after unrelated donor transplantation, respectively. CD4 T-cell numbers demonstrated a median increase of 2.2-fold within 2 weeks. Transient DLI associated grade-I GVHD occurred in 5 patients after a median of 32 days (range, 21–59). Only one HLA-C-mismatch patient developed temporary grade-III disease that was associated with a detectable level of mismatch-directed CD8 T cells in peripheral blood. Two patients developed limited chronic GvHD. In cases of decreasing donor-chimerism, the transfer of CD8-depleted DLIs were sufficient to reinduce full donor status. In 3 patients suffering from multiple CMV reactivations, anti-cytomegalovirus CD4 and CD8 responses were markedly enhanced following DLIs. We hereby present the transfer of GMP-grade CD8-depleted donor lymphocytes. In contrast to previous studies, DLIs were applied preemptively to overcome the long-lasting immunosuppression after TCD / RIT-based allo-transplantation. Our results suggest that the preemptive use of clinical-grade CD8-depleted DLIs accelerates immune reconstitution after alemtuzumab-based RIT and thereby leads to an increase in virus-specific immunity and maintenance of donor chimerism with a low risk of GVHD.

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.

CD8+ cell depletion of donor lymphocyte infusions using cd8 monoclonal antibody-coated high-density microparticles (CD8-HDM) after allogeneic hematopoietic stem cell transplantation: a pilot study

A CD8 murine monoclonal antibody-coated high-density microparticle (HDM) has been developed, which allows for the rapid depletion of CD8+ T cells from apheresis products by gravity sedimentation. We conducted a study to determine the efficacy and safety of CD8 depletion of donor lymphocyte infusions (DLI) to treat relapse after stem cell transplantation using the Eligix CD8-HDM Cell Separation System. Patients were targeted to receive 3 x 10(7) CD4+ T cells/kg. Nine patients were enrolled, three with CML, three myeloma, two CLL, and one NHL. A median of 1 x 10(10) mononuclear cells were obtained by apheresis and processed. The median depletion of CD8+ cells was 99.3% (97.8->99.5%). CD8 depletion was highly specific, with a median recovery of CD4+ cells of 75%. A median of 2.9 x 10(7) CD4+ cells/kg was infused. No infusional toxicity was noted. All CML patients achieved a complete molecular remission. A CLL patient demonstrated a complete response. One patient developed GVHD (grade II acute GVHD and subsequently chronic GVHD). The CD8-HDM Cell Separation System appears to be highly selective and effective in depleting CD8+ T cells from DLI apheresis products, and CD8-depleted DLI is capable of mediating a graft-versus-leukemia effect while minimizing GVHD.

Graft-versus-host-reactive donor CD4 cells can induce T cell-mediated rejection of the donor marrow in mixed allogeneic chimeras prepared with nonmyeloablative conditioning

AbstractMurine mixed hematopoietic chimerism can be achieved following nonmyeloablative conditioning with cyclophosphamide, T cell–depleting monoclonal antibodies, and thymic irradiation. Donor lymphocyte infusions (DLIs) 35 days after bone marrow transplantation (BMT) convert mixed to full donor chimerism and mediate graft-versus-lymphoma effects without graft-versus-host disease. We evaluated the role of T-cell subsets in DLIs in converting mixed to full donor chimerism in a fully major histocompatibility complex–mismatched strain combination. Whereas DLIs administered on day 35 converted 100% of mixed chimeras to full donor chimerism, conversion was less frequent when either CD4 or CD8 cells were depleted, indicating that both subsets contribute to the conversion. Surprisingly, administration of CD8-depleted DLIs led to complete loss of donor chimerism in a high proportion (54%) of recipients compared with CD4-plus CD8-depleted DLIs (15%) or CD4-depleted DLIs (0%) (P < .05). DLIs administered at early time points after BMT (eg, day 21) also precipitated rejection of donor marrow by recipient αβ T cells, in association with donor CD4 cell expansion and high production of interleukin 2 (IL-2), IL-4, and interferon-γ. Thus, DLIs can paradoxically induce marrow rejection by residual host αβ T cells. These results have implications for the timing of and use of subset depletion of DLIs in recipients of nonmyeloablative transplants.

Retrospective Comparison of CD34-selected Allogeneic Peripheral Blood Stem Cell Transplantation Followed by CD8-depleted Donor Lymphocyte Infusions with Unmanipulated Bone Marrow Transplantation

We have previously reported the feasibility of allogeneic CD34-selected PBSC transplantation followed by pre-emptive CD8-depleted DLI (study group). In this report, we retrospectively compare the clinical outcome of the 24 patients included in this study with an historical group of 35 patients receiving unmanipulated marrow (BMT group). Patients in the study group had significantly faster neutrophil and platelet recovery and were discharged earlier than BMT patients. The actuarial 150-day (after DLI) probability of developing grade II-IV acute GVHD was 28% for the study group versus 62% for the BMT group (p=0.002). The actuarial 2-year probability of developing chronic GVHD was similar (37 versus 36% (NS)) but chronic GVHD was significantly delayed in the study group (p=0.003). The actuarial 2-year probability of relapse was 30% in the study group versus 33% in the BMT group (NS). The actuarial 2-year probability of survival was 45% in the study group versus 43% in the BMT group (NS). We conclude that allogeneic transplantation of CD34-selected PBSC followed by pre-emptive CD8-depleted DLI is feasible with rapid engraftment and minimizes the risk of severe GVHD. Large prospective trials are required to confirm these results.

Long-term follow-up of recipients of CD8-depleted donor lymphocyte infusions for the treatment of chronic myelogenous leukemia relapsing after allogeneic progenitor cell transplantation

Donor lymphocyte infusions (DLIs) are an effective treatment for relapsed Chronic myeloid leukemia (CML) after allogeneic transplantation but are limited by the occurrence of GVHD. CD8+ T lymphocytes are involved in the pathogenesis of GVHD but may not be essential for the graft-versus-leukemia (GVL) effect in CML. We have treated 26 CML patients with posttransplantation relapse with CD8-depleted DLI. Thirteen of 15 patients (87%) who relapsed in early-phase CML achieved complete cytogenetic response, but only 1 of 11 who relapsed in advanced-phase disease achieved complete response. Acute GVHD occurred in 2 patients (8%), and extensive chronic GVHD occurred in 2 patients (11%). Treatment-related mortality was 11.5%. Responses were durable; with a median follow-up of 4.2 years (1-7.5 years), only 1 responding patient relapsed (7%). CD8-depleted DLI was equally effective and safe after unrelated donor transplants and sibling transplants. Cytogenetic clonal evolution at the time of DLI was not predictive of treatment failure unless associated with hematologic criteria for disease acceleration. CD8 depletion is an effective method to separate GVL from GVHD for posttransplantation relapsed CML. This strategy is associated with durable complete remissions and a low rate of complications and therefore merits further investigation in larger-scale comparative trials. Biol Blood Marrow Transplant 2001;7(10):568-75.

Cellular therapy of cancer with natural killer cells: will it ever work?

Journal of Hematotherapy & Stem Cell ResearchVol. 10, No. 1 An Hematotherapist's Point of ViewCellular Therapy of Cancer with Natural Killer Cells: Will It Ever Work?Hans-G. KlingemannHans-G. KlingemannSearch for more papers by this authorPublished Online:9 Jul 2004https://doi.org/10.1089/152581601750098174AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited byEffect of frequently used chemotherapeutic drugs on the cytotoxic activity of human natural killer cells16 February 2007 | Molecular Cancer Therapeutics, Vol. 6, No. 2Natural killer cell-based immunotherapeutic strategiesCytotherapy, Vol. 7, No. 1Kinetics and Organ Distribution of Allogeneic Natural Killer Lymphocytes Transfused into Patients Suffering from Renal Cell Carcinoma J.-M. Brand, B. Meller, K. Von Hof, J. Luhm, M. Bähre, H. Kirchner, and C. Frohn9 July 2004 | Stem Cells and Development, Vol. 13, No. 3Ex vivo expansion of natural killer cells for clinical applicationsCytotherapy, Vol. 6, No. 1Role of Immunotherapy in Stem Cell Transplantation1 January 2003 | International Journal of Hematology, Vol. 77, No. 1Nonmyeloablative Allogeneic Hematopoietic Stem Cell Transplantation F. Baron and Y. Beguin9 July 2004 | Journal of Hematotherapy & Stem Cell Research, Vol. 11, No. 2Nonmyeloablative Stem Cell Transplantation with CD8-Depleted or CD34-Selected Peripheral Blood Stem Cells Frédéric Baron, Etienne Baudoux, Pascale Frère, Soraya Tourqui, Nicole Schaaf-Lafontaine, Roland Greimers, Christian Herens, Georges Fillet, and Yves Beguin9 July 2004 | Journal of Hematotherapy & Stem Cell Research, Vol. 11, No. 2Retrospective Comparison of CD34-selected Allogeneic Peripheral Blood Stem Cell Transplantation Followed by CD8-depleted Donor Lymphocyte Infusions with Unmanipulated Bone Marrow Transplantation4 September 2013 | Hematology, Vol. 7, No. 3Cellular Immunotherapy of Malignancies Using the Clonal Natural Killer Cell Line NK-92 Torsten Tonn, Sven Becker, Ruth Esser, Dirk Schwabe, and Erhard Seifried9 July 2004 | Journal of Hematotherapy & Stem Cell Research, Vol. 10, No. 4 Volume 10Issue 1Feb 2001 To cite this article:Hans-G. Klingemann.Cellular Therapy of Cancer with Natural Killer Cells: Will It Ever Work?.Journal of Hematotherapy & Stem Cell Research.Feb 2001.23-26.http://doi.org/10.1089/152581601750098174Published in Volume: 10 Issue 1: July 9, 2004PDF download