Tumor-Activated Human NK Cells Specificall Ylyse Autologous and Allogeneic NK-Resistant Tumor Targets.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3707-3707
Author(s):  
Mark W. Lowdell ◽  
Ismail Bakhsh ◽  
Janet North ◽  
Chloe Marden ◽  
Elena Addison ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Bone Marrow ◽  
Nk Cells ◽  
Tumor Cells ◽  
Mononuclear Cells ◽  
Leukemia Cell ◽  
Lymphokine Activated Killer Cells ◽  
Normal Peripheral Blood ◽  
Significant Difference

Abstract Pre-incubation of resting human NK cells with the leukemia cell CTV-1 primes NK cells to lyse NK-resistant cell lines, primary leukemias and solid tumors, even when HLA-matched; allogeneic or autologous. The primed NK cells remained non-responsive to HLA-C matched and mismatched normal peripheral blood or bone marrow mononuclear cells from multiple donors and did not affect in vitro colony forming unit assays. NK cells isolated from normal healthy donors and co-incubated with irradiated CTV-1 tumor cells synthesized CD69 within 60 min with maximal expression achieved within 6 hr. The tumor-activated NK cells (T-ANKs) lysed NK resistant RAJI and Daudi cells in a 4-hour assay (Fig 1). The degree of lysis was equivalent to that of matched lymphokine activated killer cells (LAK) after non-specific activation with IL-2. CTV-1 cells are HLA-C type 2 homozygous and express HLA-Bw4 alleles. They thus can ligate KIR2DL1 (CD158a) and KIR3DL1 (CD158e1) on NK cells. Co-cultures of HLA-matched or mis-matched NK with CTV-1 showed there was no significant difference in the generation of T-ANK activity in the presence or absence of KIR ligation. T-ANK cells from allogeneic donors were capable of lysis of primary AML cells of all FAB types, the relatively NK-resistant breast cancer cell line, MCF-7, and primary tumor cells isolated from resected tissue from patients with breast cancer and ascities from patients with ovarian cancer. Autologous T-ANK cells were generated from five AML patients in clinical remission; all of whom had low level or undetectable NK activity to their presentation disease. T-ANK cells from all five donors consistently induced significantly greater lysis of autologous AML blasts than the matched NK cells (p<0.05) (Fig 2). To investigate the tumor-restriction of allogeneic T-ANK cells we isolated NK cells from normal donors and either activated them with CTV-1 cells overnight or maintained them in media. These T-ANK cells were then compared with NK cells from the same donor with respect to lysis of normal autologous and allogeneic PBMC. Neither NK nor T-ANK cells lysed autologous PBMC nor did they lyse PBMC from HLA-C mismatched normal donors. To determine the likelihood of bone marrow suppression by T-ANK cells we established hematopoietic colony forming assays with bone marrow from 5 normal donors and added T-ANK from HLA-C mismatched donors at increasing ratios. CFU-GM, BFU-E and CFU-GEMM were not affected by co-incubation with HLA-mismatched T-ANK even at effector:target ratios of 20:1. We believe that this demonstrates a two stage process for NK activation; priming and triggering, the ligands for which are distinct. Tumor priming of NK primes cells capable of lysing a broad spectrum of NK-resistant tumors which may irrespective of KIR ligation which may represent an “off-the-shelf” immunotherapy product. Fig 1 Fig 1. Fig 2 Fig 2.

scholarly journals Detection and viability of tumor cells in peripheral blood stem cell collections from breast cancer patients using immunocytochemical and clonogenic assay techniques [see comments]

Blood ◽  
1993 ◽  
Vol 82 (9) ◽  
pp. 2605-2610 ◽  
Author(s):  
AA Ross ◽  
BW Cooper ◽  
HM Lazarus ◽  
W Mackay ◽  
TJ Moss ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Stem Cell ◽  
Tumor Cell ◽  
Cancer Patients ◽  
Tumor Cells ◽  
Mononuclear Cells ◽  
Peripheral Blood Stem Cell ◽  
Breast Cancer Patients ◽  
Tumor Involvement

Abstract Although peripheral blood stem cell collections (PBSC) are thought to have less tumor involvement than bone marrow (BM), the incidence of circulating tumor cells in patients with breast cancer has not been widely investigated. We prospectively investigated the incidence and viability of tumor cell involvement in PBSC and BM collections from breast cancer patients undergoing high-dose chemotherapy/hematopoietic stem cell transplantation. Paired samples of PBSC and BM from 48 patients were analyzed using an immunocytochemical technique that detects one epithelial-derived tumor cell per 5 x 10(5) mononuclear cells. Immunostained tumor cells were detected in 9.8% (13/133) PBSC specimens from 9/48 (18.7%) patients and in 62.3% (38/61) BM specimens from 32/48 (66.7%) patients, a significantly higher rate than in PBSC (P < .005). The geometric mean concentration of tumor cells in contaminated PBSC specimens was 0.8/10(5) mononuclear cells (range 0.33 to 2.0/10(5)) compared with 22.9/10(5) mononuclear cells in BM (range 1 to 3,000/10(5), P < .0001). In culture experiments, clonogenic tumor colonies grew in 21/26 immunocytochemically positive specimens. No tumor colony growth was detected in 30/32 immunocytochemically negative specimens. Immunocytochemical detection of tumor involvement in BM and PBSC correlated significantly with in vitro clonogenic growth (P < .0001). We conclude that PBSC contain fewer tumor cells than paired BM specimens from patients with advanced breast cancer and that these tumor cells appear to be capable of clonogenic growth in vitro.

Synergistic Action of the Human Inhibitory KIR Antibody IPH2102, and the Human CD38 Antibody Daratumumab to Enhance the Lysis of Primary Multiple Myeloma (MM) Cells in the Bone Marrow Mononuclear Cells (MNCs) From Myeloma Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1865-1865
Author(s):  
Inger S. Nijhof ◽  
Michel de Weers ◽  
Pascale Andre ◽  
Berris van Kessel ◽  
Henk M. Lokhorst ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Nk Cells ◽  
Tumor Cells ◽  
Research Funding ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Human Monoclonal Antibody ◽  
Cell Activity

Abstract Abstract 1865 Despite significant improvements in the treatment of multiple myeloma (MM), this progressive malignancy of antibody-producing clonal plasma cells is still considered incurable. New innovative treatments need to be developed to improve long term outcomes. Recent successes of CD20 antibodies in the clinical lymphoma management indicate that targeted immunotherapy can represent a powerful therapeutical strategy for hematological malignancies. Towards developing a similar strategy for MM, we have recently generated a novel human monoclonal antibody, daratumumab (DARA), which targets the CD38 molecule expressed at high levels on MM cells. We have demonstrated that DARA mediates the lysis of CD38+ MM cells via direct apoptosis, complement mediated lysis and antibody-dependent cell mediated cytotoxicity (ADCC). Natural killer (NK) cells appeared important effector cells mediating the ADCC effect. Since NK cell activity against tumor cells is regulated by the balance of signals generated by inhibitory or activating receptors of NK cells (KIRs), we now explored whether blocking the inhibitory KIRs would improve the NK cell mediated DARA dependent lysis of MM cells. Thus, we evaluated the potential benefits of combining DARA with a novel human anti KIR monoclonal antibody, IPH2102, which blocks the inhibitory KIR2DL1/2/3 receptors (HLA-C specific KIRs), and has been shown to augment NK cell function against MM cells. We recently developed FACS-based ex vivo MM cell lysis assays, in which DARA-dependent NK cell-mediated lysis of MM cells can be directly measured in bone marrow MNCs, thus without separating the malignant cells from autologous NK cells and other accessory cells. Using these, we investigated whether the addition of IPH2102 would augment the DARA dependent lysis of MM cells. As expected, DARA induced lysis of MM cells in bone marrow MNCs isolated from MM patients (n=10). Mean lysis at 10 μg/ml DARA was 27.6% (range 11.3–48.1%). IPH2102 showed little or no lysis of MM cells (at 0.3, 1, 3 and 10 μg/ml) in this setting. The combination of 10 μg/ml IPH2102 with 3 and 10 μg/ml DARA significantly enhanced cytotoxicity against primary MM tumor cells compared to DARA alone (p=0.013 and p=0.028 respectively). Mean lysis of MM tumor cells at 10 μg/ml DARA and 10 μg/ml IPH2102 was 38%. These data confirm our previous findings that NK-cell mediated killing is an important mechanism of action of DARA. We demonstrate a clear synergy between DARA and IPH2102 to achieve effective lysis of MM cells directly in the bone marrow MNC of MM patients, indicating that complementary effects may be achieved by combining IPH2102 and DARA in clinical MM management. Disclosures: Weers: Genmab: Employment. Andre:Innate Pharma: Employment. Lokhorst:Genmab: Research Funding. Parren:Genmab: Employment. Morel:Innate Pharma: Employment. Mutis:Genmab: Research Funding.

scholarly journals Hepatic Niche Leads to Aggressive Natural Killer Cell Leukemia Proliferation

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3309-3309
Author(s):  
Kazuaki Kameda ◽  
Yuji Miyatake ◽  
Yoshinobu Kanda ◽  
Ai Kotani
Keyword(s):  
Bone Marrow ◽  
Natural Killer Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Rna Sequencing ◽  
Research Funding ◽  
Killer Cell ◽  
Cell Leukemia

Abstract Aggressive natural killer cell leukemia (ANKL) is a rare form of natural killer (NK)-cell neoplasm with median survival of less than 2 months. Recently, the genomic mutation analysis using tumor cells reveled that the mutational profile of ANKL was similar to that of extranodal NK / T-cell lymphoma, which has relatively better prognosis than ANKL, explaining no causative mutations with a dismal prognosis. Here, using patient-derived xenograft model (PDX) mouse, we show that hepatic niche plays an important role in the ANKL biology. We established PDX mouse by intravenously injecting ANKL cells derived from patient peripheral blood or bone marrow samples to immunocompromised mice, which enables comprehensive analysis for tumor cells as well as tumor microenvironment. In total, we obtained four PDX strains derived from different patients. Time series pathological and flowcytometric analyses revealed that the ANKL cells initially engrafted and proliferated in sinusoidal or peri-portal area of the liver. This sinusoid or peri-portal distribution of ANKL in the liver was also confirmed with the patient liver specimen. To further determine the feature of ANKL in the liver, we selected liver or spleen tropic cells by serial adaptive transfer from each organ to the next mice. The liver-tropic ANKL cells proliferated more rapidly than splenic ANKL cells, which was evident by the significantly shorter survival of PDX mice injected liver-tropic cells (Figure). We performed RNA-sequencing using liver-tropic ANKL cells, spleen-tropic ANKL cells and NK-cells derived from healthy donors. These three types of cells showed distinct populations in principal component analysis. To further clarify the interaction between ANKL and liver niche, we performed additional RNA sequencing using total liver of mouse with or without bearing leukemic cells. In the cell-cell interaction analysis, we used two computational methods, mixed-species RNA-seq (Komura, et al. BMC Genomics 2016), which can distinguish transcripts derived from human (cancer) with mouse (non-cancer niche cells), and NicheNet (Browaeys, et al. Nat Methods 2020), which is a computational algorithm to model intercellular communication by linking ligands to target genes. These two methods allowed us to investigate the interaction between liver niche ligands and ANKL receptors. Among the listed ligand-receptor interactions, we focused on the macrophage migration inhibitory factor (MIF) and its receptor, CD74 axis. While CD74 was upregulated in ANKL cells compared with normal NK cells, MIF was highly expressed in the liver mainly liver sinusoid and Kupffer cells. Although we failed to culture primary ANKL cells in vitro, ANKL cells treated with MIF showed improved viability in vitro compared with untreated cells. Deletion of CD74 on the ANKL cells using CRISPR-Cas9 system attenuated the tumor formation in the liver as well as in bone marrow and spleen of PDX mouse compared with the wild type ANKL cells. These findings highlight that the liver, non-canonical hematopoietic organ in adults, is a principal niche where the liver specific components are required for survival and proliferation of ANKL cells. MIF-CD74 axis might play an important role in the communication between ANKL and hepatic niche. Figure 1 Figure 1. Disclosures Kanda: Otsuka Pharmaceutical: Honoraria, Research Funding; Sanofi: Research Funding; MSD: Honoraria.

scholarly journals Three-Dimensional Reconstructed Bone Marrow Matrix Culture Improves the Viability of Primary Myeloma Cells In-Vitro via a STAT3-Dependent Mechanism

2021 ◽  
Vol 43 (1) ◽  
pp. 313-323
Author(s):  
Yung-Hsing Huang ◽  
Meaad Almowaled ◽  
Jing Li ◽  
Christopher Venner ◽  
Irwindeep Sandhu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Viability ◽  
Mononuclear Cells ◽  
Three Dimensional ◽  
3D Culture ◽  
Trypan Blue Exclusion ◽  
Conventional Culture ◽  
Significant Difference ◽  
Dependent Mechanism

Primary myeloma (PM) cells are short-lived in conventional culture, which limited their usefulness as a study model. Here, we evaluated if three-dimensional (3D) culture can significantly prolong the longevity of PM cells in-vitro. We employed a previously established 3D model for culture of bone marrow mononuclear cells isolated from 15 patients. We assessed the proportion of PM cells, viability and proliferation using CD38 staining, trypan blue exclusion assays and carboxy fluorescein succinimidyl ester (CFSE) staining, respectively. We observed significantly more CD38+ viable cells in 3D than in conventional culture (65% vs. 25%, p = 0.006) on day 3. CFSE staining showed no significant difference in cell proliferation between the two culture systems. Moreover, we found that PM cells in 3D culture are more STAT3 active by measure of pSTAT3 staining (66% vs. 10%, p = 0.008). Treatment of IL6, a STAT3 activator significantly increased CD38+ cell viability (41% to 68%, p = 0.021). In comparison, inhibition of STAT3 with Stattic significantly decreased PM cell viability in 3D culture (38% to 17% p = 0.010). Neither IL6 nor Stattic affected the PM cell viability in conventional culture. This study suggests that 3D culture can significantly improve the longevity of PM cells in-vitro, and STAT3 activation can further improve their viability.

scholarly journals Identification of a subset of murine natural killer cells that mediates rejection of Hh-1d but not Hh-1b bone marrow grafts.

1989 ◽  
Vol 170 (1) ◽  
pp. 191-202 ◽  
Author(s):  
C L Sentman ◽  
J Hackett ◽  
V Kumar ◽  
M Bennett
Keyword(s):  
Bone Marrow ◽  
Natural Killer Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Bone Marrow Cells ◽  
Immune Functions ◽  
Infected Cells

NK cells demonstrate many immune functions both in vitro and in vivo, including the lysis of tumor or virus-infected cells and the rejection of bone marrow allografts. However it remains unclear whether or not all NK cells can mediate these various functions or if NK cells exist in functionally distinct subsets. We have developed a new NK-specific mAb, SW5E6, which binds to approximately 50% of murine NK cells. The 5E6 antigen identifies a distinct and stable subset of NK cells and is expressed on about one-half of fresh or rIL-2-activated murine NK cells. Both 5E6+ and 5E6- NK cells are capable of lysing YAC-1 tumor cells in vitro and in vivo. By treating animals with SW5E6, we demonstrate that the 5E6+ subset is necessary for the rejection of H-2d/Hh-1d but not H-2b/Hh-1b bone marrow cells. Thus NK cells exist as functionally separable subsets in vivo.

634 Production and testing of a novel bispecific nanobody construct targeting NK cells and EGFR expressing malignancies

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A670-A670
Author(s):  
Elisa Toffoli ◽  
Abdolkarim Sheikhi ◽  
Roeland Lameris ◽  
Lisa King ◽  
Jurriaan Tuynman ◽  
...  
Keyword(s):  
Nk Cells ◽  
Tumor Cells ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Cell Activity ◽  
Nk Cell Activity ◽  
Peripheral Blood Mononuclear ◽  
Mutational Status ◽  
Tumor Cell Lysis

BackgroundThe ability to kill tumor cells with an acceptable toxicity profile, makes Natural Killer (NK) cells promising assets for cancer therapy. However, strategies to enhance the preferential accumulation and activation of NK cells in the tumor microenvironment would likely increase the efficacy of NK cell-based therapies.MethodsIn this study, we show a novel bispecific nanobody-based construct (biVHH) targeting both CD16A (low-affinity Fc receptor: FcRγIIIA) on NK cells and EGFR on tumors of epithelial origins.ResultsHigher levels of NK cell activity and subsequent tumor cell lysis were found in vitro in the presence of the biVHH and were dependent on the expression of both CD16A and EGFR while they were independent of the KRAS mutational status of the tumor. Increased NK cell activity was found in NK cells derived from colorectal cancer (CRC) patients when co-cultured with the biVHH and EGFR expressing tumor cells. Finally, higher levels of cytotoxicity were found against patient-derived metastatic CRC cells in the presence of the biVHH and autologous peripheral blood mononuclear cells or allogeneic NK cells.ConclusionsBased on our results, the bispecific CD16A and EGFR targeting VHH construct could be a useful tool in combination with various NK cell-based therapies.

scholarly journals Effect of Campath-1H antibody on human hematopoietic progenitors in vitro

Blood ◽  
1993 ◽  
Vol 82 (3) ◽  
pp. 807-812
Author(s):  
MH Gilleece ◽  
TM Dexter
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Fluorescein Isothiocyanate ◽  
Lymphoid Cells ◽  
Human Complement ◽  
Hematopoietic Progenitors ◽  
Pilot Studies ◽  
Significant Difference ◽  
And Control

The humanized antibody CAMPATH-1H has been shown in pilot studies to be beneficial in the treatment of lymphoid malignancy and other lymphoproliferative diseases. The antigen recognized by this antibody is not confined to lymphoid cells, and work with rat antibodies of similar specificity has not eliminated the possibility of damage to human hematopoietic progenitors, particularly those capable of repopulating bone marrow and sustaining hematopoiesis. This study aimed to discover if hematopoietic progenitor cells were affected by treatment with CAMPATH-1H, with or without human complement. Bone marrow mononuclear cells from healthy volunteers were treated with saturating concentrations of CAMPATH-1H, human complement, or CAMPATH- 1H plus human complement. The CD34-positive fraction of the mononuclear cells was treated similarly. Residual progenitor activity was measured in the colony-forming unit-granulocyte, erythroid, monocyte, megakaryocyte assay and compared with untreated controls. There was no significant difference (at the 5% level) between treated and control cells. Mononuclear cells were divided into CAMPATH-1H-positive and CAMPATH-1H-negative fractions by fluorescein isothiocyanate-CAMPATH-1H labeling and fluorescence-activated cell sorter separation. Hematopoietic progenitors were predominantly found in the CAMPATH-1H- negative fraction. Furthermore, mononuclear cells treated with CAMPATH- 1H and complement were equivalent to controls in experiments that investigated the capacity of these cells to form hematopoietic foci in long-term cultures.

Alloreactive NK-Cells Cure Tumor Stem Cell Containing Breast Cancer.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4082-4082
Author(s):  
Michel Van Gelder ◽  
Peter Frings ◽  
Catarina Matos ◽  
Gerard Bos ◽  
Harry Schouten ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Stem Cells ◽  
Bone Marrow ◽  
Nk Cells ◽  
Bone Marrow Cells ◽  
Nk Cell ◽  
Tumor Stem Cells ◽  
4T1 Breast Cancer

Abstract Abstract 4082 Poster Board III-1017 Background Patients with metastasized solid tumors can not be cured by current standard treatment options. One hypothesis is that slow cycling chemo-resistant tumor stem cells give rise to new tumors after cytoreductive treatment, ultimately leading to chemoresistant tumors. Acute myeloid leukemia tumor stem cells can be killed by alloreactive T- or NK-cells, as patients have a higher survival chance after allogeneic transplantation. In the past we published the curative effectiveness of allogeneic spleen and bone marrow transplantation in breast cancer bearing mice. Our current aim was to study if either alloreactive T- or NK-cells are able to cure these mice. In addition we wanted to know if the tumors contain slow cycling tumor stem cells. Methods The 4T1 breast cancer cell line was cultured under standard conditions. Fifty thousand 4T1 cells were injected s.c. in the flank of CB6F1 (H-2b/d) mice. At day 12 mice were treated with 2× 2Gy total body irradiation and 200 mg/kg cyclophosphamide (CY), followed by transplantation of spleen and bone marrow cells. B6CBAF1 (H-2b/k) mice were used as allogeneic donors. These donors harbour alloreactive NK-cells towards CB6F1 recipients as determined by an in vivo NK-cell alloreactivity assay. NK-cell or T-cell depleted grafts were obtained from donors injected with anti-AsialoGM1 or a combination of anti-CD4 and anti-CD8 respectively some days before spleen and bone marrow harvest. Tumor stem cells were defined as slow cycling (i.e. label retaining) Hoechst extruding cells. 4T1 tumor cells were labelled with Vybrant CM-Dil (Dil) and then injected s.c. in CB6F1 mice. Fourteen days later tumors were excised. Part of the tumor was prepared as single cell suspension. Subsequently the presence of Dil retaining tumor stem cells was determined by fluorescence microscopy. The other part of the tumor was snap frozen and studied by histochemistry and fluorescence microscopy. For in vitro cytotoxicity testing 4T1 cells were again labelled with Dil and then seeded in 96 well plates at 30 cells/well. Chemotherapeutics were added in the different plates: 4-OH-CY (the active metabolite), cisplatine and doxorubicin, in concentrations far above the lowest maximal lethal dose as determined in MTT assays. Wells were checked weekly for growth and presence of tumor stem cells. Hoechst staining was used in these cultures to underscore that Dil retaining chemorefractory cells are indeed stem cells. Results Ninety percent of CB6F1 mice injected with 4T1 breast cancer cells developed a tumor growing progressively to a size that made it necessary to sacrifice them. Growth in mice treated with radio- and chemotherapy only or additionally with CB6F1 spleen and bone marrow cells was delayed for 10 days compared with tumor growth in untreated mice, but the incidence was equally high (90% and 80% respectively). In contrast, but in concord with our previous published results, only 10% of 4T1 breast cancer bearing mice transplanted with haploidentical spleen and bone marrow cells did show progressive tumor growth (follow-up 99 days at the time of abstract submission, p<0.004 log-rank test)). Similarly, mice transplanted with spleen and BM cells from T-cell depleted donors did not develop tumors at all (p<0.004 log-rank test), in contrast to recipients of in vivo NK-cell depleted grafts (90% tumor incidence). Label retaining tumor stem cells are unmistakably present in tumors at the day of transplantation, evidenced by the presence of a minute number of Dil retaining cells in single cell suspension of the tumor and in tumor tissue sections. More direct evidence for chemo-resistance of 4T1 tumor stem cells is derived by results of in vitro co-culture experiments. When Dil labelled 4T1 cells are plated in 30 cells/well concentration, approx. 25% of the wells contain one or two label retaining cells while the other wells comprise non-label retaining cells only. After incubation with chemotherapeuticals growth is observed only in wells containing a label retaining cell. These label retaining cells are also Hoechst negative. Conclusions This report provides the first evidence that tumor stem cells can be eliminated in vivo by alloreactive NK cells resulting in cure of chemorefractory breast cancer. It is also shown in vitro that only tumor stem cell containing tumor colonies resist the effect of various chemotherapeuticals commonly used in oncology as they do in vivo. These results strongly encourage the exploration of clinical alloreactive NK therapy in patients with metastasized solid tumors. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Effect of Campath-1H antibody on human hematopoietic progenitors in vitro

Blood ◽  
1993 ◽  
Vol 82 (3) ◽  
pp. 807-812 ◽  
Author(s):  
MH Gilleece ◽  
TM Dexter
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Fluorescein Isothiocyanate ◽  
Lymphoid Cells ◽  
Human Complement ◽  
Hematopoietic Progenitors ◽  
Pilot Studies ◽  
Significant Difference ◽  
And Control

Abstract The humanized antibody CAMPATH-1H has been shown in pilot studies to be beneficial in the treatment of lymphoid malignancy and other lymphoproliferative diseases. The antigen recognized by this antibody is not confined to lymphoid cells, and work with rat antibodies of similar specificity has not eliminated the possibility of damage to human hematopoietic progenitors, particularly those capable of repopulating bone marrow and sustaining hematopoiesis. This study aimed to discover if hematopoietic progenitor cells were affected by treatment with CAMPATH-1H, with or without human complement. Bone marrow mononuclear cells from healthy volunteers were treated with saturating concentrations of CAMPATH-1H, human complement, or CAMPATH- 1H plus human complement. The CD34-positive fraction of the mononuclear cells was treated similarly. Residual progenitor activity was measured in the colony-forming unit-granulocyte, erythroid, monocyte, megakaryocyte assay and compared with untreated controls. There was no significant difference (at the 5% level) between treated and control cells. Mononuclear cells were divided into CAMPATH-1H-positive and CAMPATH-1H-negative fractions by fluorescein isothiocyanate-CAMPATH-1H labeling and fluorescence-activated cell sorter separation. Hematopoietic progenitors were predominantly found in the CAMPATH-1H- negative fraction. Furthermore, mononuclear cells treated with CAMPATH- 1H and complement were equivalent to controls in experiments that investigated the capacity of these cells to form hematopoietic foci in long-term cultures.

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