B. Hennemann

Adoptive immunotherapy of cancer using monocyte-derived macrophages: rationale, current status, and perspectives

Adoptive transfer of host defense cells may be able to correct an otherwise defective generation of competent immune cells in patients with cancer. Ex vivo‐grown cytotoxic macrophages (MAC) able to recognize and destroy tumor cells but not normal cells are effective in murine models of metastasizing tumors. After the development of large‐scale technology to generate MAC in vitro from blood monocytes (MO), clinical trials in cancer patients have proven the feasibility and safety of infusing >3 × 109 autologous MO‐derived MAC activated by interferon‐γ or lipopolysaccharide. Various modalities of adoptive immunotherapy with human MAC have been realized: routes of application used were intravenous, intraperitoneal, intrapleural, and through selective hepatic artery perfusion. In addition, MAC have been generated from MO collected after granulyte‐macrophage colony‐stimulating factor treatment in vivo. Biodistribution studies using 111indium‐labeled cells have revealed localization of MAC to sites of bulk tumor growth on regional infusion as well as to liver metastases on systemic application. Malignant ascites disappeared in about 50% of patients after intraperitoneal treatment, yet no other evidence of therapeutic efficacy of MAC could be demonstrated. Further advances of adoptive transfer of MO‐derived cells are developed with emphasis on the generation of antigen‐presenting cells primed in vitro with tumor cells or specific peptides. J. Leukoc. Biol. 64: 419–426; 1998.

Macrophage heterogeneity and differentiation: defined serum-free culture conditions induce different types of macrophages in vitro.

Macrophages (MAC) are important effector cells of the immune system. They arise from circulating blood monocytes (MO), which undergo further maturation upon leaving the vasculature and migrating into the various tissues and body cavities. A similar differentiation process can be followed in vitro when monocytes are cultured in the presence of serum. In this study, different factors and serum proteins, either alone or in combination, were tested for their ability to promote the survival and/or maturation of blood MO in the absence of serum. Elutriation-purified MO cultured for 8 days on hydrophobic teflon foils in the presence of 5% human serum differentiated into large, well-spread MAC, whereas in the absence of serum, MO rapidly died. The serum-induced maturation of MAC was accompanied by a strong expression of CD16, CD14 and MAX antigens. Secretion of TNF-alpha and neopterin increased about 10-fold as compared with freshly isolated MO. The replacement of serum by either M-CSF (100 ng/ml) or immunoglobulin (0.5-5 mg/ml) had a marked effect on MO survival (about 50% of serum-cultured MO), but cells were smaller, less spread out and had low expression of CD16, CD14 and MAX antigens. Their functional competence in terms of TNF-alpha and neopterin release was reduced to 10-20% as compared with MAC cultured in the presence of serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Phase I trial of adoptive immunotherapy of cancer patients using monocyte-derived macrophages activated with interferon γ and lipopolysaccharide

Abstract Cells of the monocyte/macrophage lineage have shown antitumor activity in vitro and in murine models after activation with interferon (IFN) γ. In vitro data suggest an additional effect on macrophage antitumor activity when IFNγ is combined with endotoxin (lipopolysaccharides; LPS). In this study we treated nine cancer patients with a total of 62 MAK infusion cycles with autologous macrophages given intravenously (i.v.) after in vitro activation with IFNγ and LPS. Low-grade fever (WHO I/II) was the commonest side-effect. Chills, nausea, and headache were noted when the number of transfused macrophages exceeded 2×108. One WHO IV toxicity occurred, consisting of hypotension after transfer of 3×108 cells, defining this dose as the maximum cell number tolerated. After pretreatment with ibuprofen, however, the maximum cell number could be increased without reaching dose-limiting toxicity. The highest number of cells reinfused was 15×108. Circulating interleukin(IL)-6 increased in a dose-dependent manner as did IL-1 receptor antagonist (IL-1RA) and IL-8. Tumor response consisted of one case of stable disease (12 weeks) in a patient with formerly progressing colorectal cancer and progressive diseases in eight patients. This study indicates that reinfusion of autologous LPS-activated macrophages upon pretreatment with ibuprofen is feasible and tolerated without major side-effects.

Mobilization of CD34+ hematopoietic cells, colony-forming cells and long-term culture-initiating cells into the peripheral blood of patients with an acute cerebral ischemic insult.

BACKGROUND In vitro and in vivo data indicate that stem cells found in the bone marrow (BM) are capable of differentiating into neural cells. The aim of this study was to investigate whether potentially pluripotent hematopoietic stem and progenitor cells are recruited from the BM into the peripheral blood as a reaction to ischemic damage of neural tissues. MATERIALS The number of CD34+ cells, colony-forming cells (CFC) and long-term culture-initiating cells (LTC-IC) was measured within 24 h and on day 7 after stroke onset by flow cytometry, or in functional assays in the peripheral blood of 10 patients with acute middle cerebral artery infarct. The National Institute of Health stroke scale, Barthel index and modified Rankin scale were used to monitor the clinical outcome. RESULTS In four patients receiving intravenous thrombolytic therapy (tissue plasminogen activator; TPA), no significant increase of CD34+ cells, CFC or LTC-IC was detected. In six patients without thrombolytic treatment, the mean number of CD34+ cells/mL increased significantly from 1181+/-248 at day 1 to 3001+/-881 at day 7. Accordingly, the numbers of CFC and LTC-IC increased 2.7- and 1.7-fold. Granulocyte colony-stimulating factor and neutrophil elastase were monitored by ELISA and remained unchanged during the study period. DISCUSSION Our results showed a recruitment of hematopoietic progenitor cells from the BM into the peripheral blood after acute ischemic stroke when no thrombolytic treatment was given. Increased progenitor cell recruitment might be caused by so far unknown signaling stimuli of the ischemic penumbra for stem cell mobilization.

Adoptive immunotherapy with tumor-cytotoxic macrophages derived from recombinant human granulocyte-macrophage colony-stimulating factor (rhuGM-CSF) mobilized peripheral blood monocytes

Summary We examined the mobilization of blood monocytes (MO) with recombinant human granulocyte-macrophage colony-stimulating factor (rhuGM-CSF) with regard to the in vitro generation of MO-derived tumor-cytotoxic macrophages (MAC) for use in adoptive immunotherapy of cancer patients. Eleven patients with progressing metastatic cancer received interferon (IFN)-γ activated tumor-cytotoxic MAC generated in vitro from autologous MO with and without pretreatment with rhuGM-CSF. RhuGM-CSF was administered subcutaneously at 10 μg/kg for 7 days. RhuGM-CSF treatment and adoptive cell transfer were well tolerated, and no toxicity greater than WHO II° occurred. Fever was the most common side effect and was seen in all patients during rhuGM-CSF treatment and during 9 of 22 MAC therapies. Bone pain was noted in 5 of 11 patients during rhuGM-CSF therapy. RhuGM-CSF treatment led to a continuous increase in the white blood cell counts and the number of MO within the leukapheresis products. The mean number of transfused MAC was 0.9 x 109 without rhuGM-CSF pretreatment and 1.9 x 109 after rhuGM-CSF administration. The maximum number of MAC that could be generated was 7.3 x 109, but after a dose escalation protocol only up to 2.7 x 109 MAC were transfused. Cytotoxicity against U937 cells increased during MO to MAC differentiation, but was decreased in both MO and MAC on treatment with rhuGM-CSF. This study proves the feasibility of reinfusing MAC generated in vitro from rhuGM-CSF mobilized MO.

Intrahepatic adoptive immunotherapy with autologous tumorcytotoxic macrophages in patients with cancer

Adoptive transfer of cytotoxic macrophages (MAC) may be able to correct for a defective generation of competent effector cells in patients with cancer. Here we report on a Phase I trial of adoptive transfer of autologous MAC by hepatic artery infusion in seven patients with metastatic liver disease. Clinical side effects were mild and consisted of fever and flu-like symptoms. Serum levels of C-reactive protein (CRP) increased within hours after MAC transfer and rose further in the course of repeated therapies. An increase of thrombin-anti-thrombin III complexes occurred in 31% of therapies. Serum neopterin, interleukin (IL)-6, and IL-8 did not change during therapy. In vivo tracing of 111 indium-labeled MAC revealed that on average, 43% of whole-body activity remained in the liver for 7 days. Evidence for tumor response could not be demonstrated. In conclusion, isolated liver perfusion with autologous MAC is well tolerated and induces a profound biological response in the recipient. Regional adoptive immunotherapy might be able to built up, in proximity to metastatic lesions, a potent cytotoxic cell infiltrate that could then be triggered within the patient using exogenous stimuli like endotoxin, cytokines, or other MAC activators.

Complete remission and successful stem cell mobilization after treatment of refractory plasma cell leukemia with bortezomib

Dear Editor, Plasma cell leukemia (PCL) is a rare form of a plasma cell dyscrasia characterized by the presence of >2×10 plasma cells (PC)/L comprising >20% of circulating PC in the peripheral blood differential count [1]. The prognosis of PCL is generally poor and the median survival for patients with PCL infrequently exceeds 7 months [2]. High-dose chemotherapy followed by autologous stem cell transplantation is currently the most effective therapeutic tool to achieve durable remissions. Prior to the stem cell supported treatment, the collection of an apheresis product containing no or only a few plasma cells is mandatory to pursue this strategy. Here we report the case of a patient in which circulating plasma cells persisted during polychemotherapy and thalidomide treatment and then disappeared after two cycles of treatment with bortezomib allowing the collection of autologous stem cells. The patient was presented to us with multiple fractures of the spine, leukocytosis and a discrete paraproteinuria-type kappa. The peripheral blood smear showed 50% atypical leukocytes with similarity to plasma cells and a CD19CD45CD38CD56CD138 immunophenotype. The trephine bone biopsy showed a hypoplastic marrow with a 50% plasma cell infiltration. After four cycles of orally administered idarubicin and dexamethasone (ID), the proportion of the peripheral plasma cells was 26% (see Table 1). After the mobilization chemotherapy with ifosfamide, epirubicin and etoposide (IEV) and stimulation with granulocyte-colony stimulation factor (G-CSF), the proportion of circulating plasma cells was still 20% and therefore, no stem cell collection was feasible. Next, two cycles of cyclophosphamide, doxorubicine and dexamethasone plus thalidomide (T-CAD) were given. Unfortunately, there was no effective reduction of peripheral plasma cell count or bone marrow infiltration. After one further attempt with cyclophosphamide, adriamycine, dexamethasone, etoposide and cisplatin plus thalidomide (DT-PACE), we found only a marginal decrease of the plasma cell content in the bone marrow. Then, the decision for therapy intensification was made and the patient was treated according to the protocol for patients with acute lymphatic leukemia (ALL). This treatment induced a decrease of the peripheral plasma cell count to 4%, but no significant decrease of the bone marrow infiltration or of the serum parameters was seen. At this point a therapy with bortezomib was initiated as a final attempt. After two cycles of bortezomib the circulating plasma cells disappeared completely, after two further cycles the urinary light chains were no longer detectable. Additionally, the performance status of the patient improved during the therapy with bortezomib, whereas only slight side effects such as discrete lid edema and mild thrombocytopenia were noted. Subsequent mobilization chemotherapy with etoposide and cyclophosphamide followed by G-CSF plus erythropoetin led to a successful stem cell apheresis. The first high-dose chemotherapy with melphalan was made approximately 14 months after the diagnosis. At this point in time, the patient was already in complete remission according to the criteria published by Blade et al. [3]. Bortezomib is a modified dipeptidyl boronic acid that reversibly inhibits the chymotrypsin-like activity of the 26S proteasome of mammalian cells, which degrades specific proteins within the cell, and therefore induces cell death. Richardson et al. reported in 2003 on the use of bortezomib in patients with multiple myeloma, describing an overall response rate of 35% [4]. The use of bortezomib in a concentration of 1.3 mg/m in combination with dexamethasone resulted in an even higher overall response rate >50% [5]. A first report describing the efficacy of bortezomib in the treatment of PCL after high-dose chemotherapy followed by autologous stem cell transplantation J. Grassinger . T. Sudhoff . B. Hennemann (*) Department of Hematology and Oncology, University of Regensburg, 93042 Regensburg, Germany e-mail: burkhard.hennemann@klinik.uni-regensburg.de Tel.: +49-941-9445531 Fax: +49-941-9445511

CD84 expression on human hematopoietic progenitor cells

OBJECTIVE CD84 is a member of the CD2 subgroup of the immunoglobulin receptor superfamily. Members of this family have been implicated in the activation of T cells and NK cells. Expression of CD84 was originally described on most mononuclear blood cells as well as platelets. To elucidate its presence on other blood cell types, we analyzed the expression pattern of CD84 on human immature CD34+ and mature hematopoietic cells. METHODS Expression analysis was carried out by flow cytometry. The differentiation potential of CD84+ progenitor cells was assessed by colony-forming assays and long-term cultures. RT-PCR was used to analyze CD84 mRNA isoforms. RESULTS In addition to monocytes, macrophages, B cells, and some T cells, CD84 is expressed on the cell surface of the majority of granulocytes. In addition, 64%+/-5% of CD34+ progenitor cells isolated from peripheral blood and 30.5%+/-5% from bone marrow of healthy volunteers also express CD84. The majority of CD34+ cells coexpressing lineage antigens were CD84+. In methylcellulose CD34+CD84+ cells formed primarily erythroid colonies, whereas myeloid or mixed colonies were scarce. The frequency of long-term culture-initiating cells in peripheral blood was approximately fivefold higher in CD34+CD84- vs CD34+CD84+ cells. In short-term cultures, 95% of the initially CD34+CD84- cells became CD84+ after 72 hours. CONCLUSIONS CD84 is expressed on cells from almost all hematopoietic lineages and on CD34+ hematopoietic progenitor cells. The proliferative potential of CD34+ cells decreases with increasing CD84 expression, suggesting that CD84 serves as a marker for committed hematopoietic progenitor cells.

Granulocyte–macrophage colony‐stimulating factor modulates lipopolysaccharide (LPS)‐binding and LPS‐response of human macrophages: inverse regulation of tumour necrosis factor‐α and interleukin‐10

Granulocyte–macrophage colony‐stimulating factor (GM‐CSF) is a well‐known stimulus for the activation, differentiation and survival of monocytes (MO). Up to now most investigations focused on the short‐term effects of GM‐CSF. In this study we investigated the effects of GM‐CSF on the long‐term differentiation of human MO in the presence of serum. We found that MO‐derived macrophages (Mφ) cultured with serum plus GM‐CSF (GM‐Mφ) were different from control Mφ (SER‐Mφ) in terms of lipopolysaccharide (LPS)‐stimulated cytokine release: GM‐Mφ showed an increased tumour necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6) production, especially at lower LPS concentrations, but the secretion of IL‐10 was diminished. In addition, GM‐Mφ secreted TNF‐α but not IL‐6 and IL‐10, spontaneously. The spontaneous TNF‐α production was not due to LPS contamination as it could not be blocked by anti‐CD14 antibody. Flow cytometry revealed, however, that the receptor for LPS, CD14, was up‐regulated on GM‐Mφ and those Mφ released twice as much soluble CD14 into the supernatant as compared with SER‐Mφ. The higher CD14 expression also resulted in an enhanced LPS‐binding capacity of GM‐Mφ. Furthermore, the LPS‐response of GM‐Mφ could only be blocked by about fourfold higher concentration of anti‐CD14 antibody compared with SER‐Mφ. In summary, GM‐CSF promotes the generation of a pro‐inflammatory type of Mφ in two different ways: first, the down‐regulation of autocrine IL‐10 production increases the release of cytokines such as IL‐6 and TNF‐α and second, the up‐regulation of membrane and soluble CD14 expression leads to a higher sensitivity towards LPS‐stimulation.

111In-oxine labelling of tumour-cytotoxic macrophages generated in vitro from circulating blood monocytes: an in vitro evaluation.

SUMMARYThe labelling of ex-vivo activated macrophages with doses of 111In-oxine sufficient for gamma camera imaging does not damage cell viability or the functional competence of cells (secretion of tumour necrosis factor-alpha and interleukin-6). The mean labelling efficiency was about 84%. Release of 111In equivalent to 15% occurred over 24 h under cell culture conditions, indicating good stability of the radioactive cell label. 111In-oxine-labelled macrophages are a suitable tool for biokinetic studies of adoptive immunotherapy.