Christoph Frohn

Anti‐myeloma activity of natural killer lymphocytes

Summary. Natural killer (NK) cells are assumed to contribute to a graft‐versus‐leukaemia effect. In vitro experiments have shown that many leukaemic cells are NK‐cell sensitive. Nevertheless, no data concerning the influence of purified NK cells on malignant myeloma (MM) cells exist. We co‐incubated NK cells with three different MM cell lines and fresh bone marrow samples of nine MM patients. The proportion of vital MM cells was determined before and after co‐cultivation by a flow‐cytometry‐based assay. All MM cells tested, with the exception of one cell line (NCI H929), were susceptible to a NK‐cell attack even without exogenous interleukin 2 (IL‐2). The mean killing of the native MM samples was 23·1 ± 5·4% and 34·5 ± 6·5% at 10:1 and 20:1 effector:target ratio respectively, This corresponded to about 2/3 of those values obtained with the highly sensitive line K562. In contrast, CD34‐positive haematopoietic stem cells as well as peripheral mononuclear cells were completely resistant under similar experimental conditions (1·3% killing). To elucidate the underlying triggering mechanisms, we measured human leucocyte antigen (HLA)‐class I expression of the MM cells. No evidence for HLA loss, which could have explained the NK‐cell recognition if it occurred, was demonstrated. These findings may contribute to the understanding of in vivo NK‐cell activation and encourage clinical applications of NK cells for MM patients.

Probability of anti-D development in D- patients receiving D+ RBCs.

BACKGROUND: In some situations, the administration of D+ RBCs to D– patients is necessary. The probability of a subsequent anti‐D formation is assumed to be around 80 percent, a figure based primarily on studies in healthy volunteers. It was hypothesized that patients requiring blood transfusion have a much lower probability of developing antibodies.

Large-scale generation of natural killer lymphocytes for clinical application.

Natural killer (NK) lymphocytes can be used for adoptive immunotherapeutic strategies. Alternatively, they may be employed as adjuvants for stem cell/bone marrow transplantation, either to re-induce remission, or to purge autografts of contaminating malignant cells. We developed a new protocol that enables the generation of NK cells on a clinical scale in a closed system that enables good manufacturing practice (GMP) conformity. Aside from the initial NK cell inoculum, our protocol includes activated feeder cells [irradiated peripheral blood mononuclear cells (PBMC) and no transformed blasts], cytokines [interleukin-2 (IL-2) and IL-15], human serum, and a complex basic media formulation. During the whole expansion period of approximately 14 days, the cells were handled in PTFE (Teflon) bags, whereby fresh medium was added without opening the system. The use of immortalized or virus-transformed feeder cells, as used in many other current research protocols, was completely avoided. A precise controlling of a number of environmental factors was necessary to achieve reproducible results. Increases in NK cell number ranged between 80- and 200-fold. The resulting NK cells were CD56(+), CD3(-), and CD16(+) (75%). They were highly cytotoxic against different malignant target cells and did not produce significant levels of interferon-gamma. Therefore, they belonged to the cytotoxic rather than the immunoregulatory NK subpopulation. No non-specific activation against normal allogenous lymphocytes occurred. This work might permit the realization of future protocols for evaluating the clinical effect of NK lymphocytes in human disease.

Early alterations in the number of circulating lymphocyte subpopulations and enhanced proinflammatory immune response during opioid-based general anesthesia.

The balance between proinflammatory and anti-inflammatory processes is of key importance in the reaction of the body to infection, injury, and surgical trauma. Drugs commonly used in anesthesia and intensive care may modulate immunological reactions by influencing intercellular communication through modification of cytokine response and fluctuation of peripheral immune cells such as natural killer (NK) cells, B cells, and T lymphocyte subpopulations (CD4+ and CD8+ cells). To examine the effects of general anesthesia with the hypnotic agent propofol and the opioid fentanyl, 30 patients undergoing minor elective orthopedic surgery were studied before and 20 min after application of the anesthetic drugs, but before the start of surgery. We found a significant enhancement of TNF-&agr; and IL-1&bgr; release in lipopolysaccharide (LPS)-stimulated whole blood cultures after induction of anesthesia. Similar results were observed with interferon-&ggr; (IFN-&ggr;) in cultures stimulated with phytohemagglutinin (PHA). Conversely, synthesis of the anti-inflammatory cytokine interleukin 10 (IL-10) decreased significantly in LPS-stimulated cultures. During general anesthesia, we found a decrease of circulating lymphocytes, characterized by a significant increase in the percentage of T lymphocytes in favor of CD4+ cells, increased B lymphocytes, and a significant decrease of NK cells. These data suggest that anesthesia with propofol and fentanyl promotes proinflammatory immune responses and influences peripheral lymphocyte composition in patients, which may subsequently affect pathophysiological processes during opioid-based anesthesia.

The repertoire of HLA-Cw-specific NK cell receptors CD158 a/b (EB6 and GL183) in individuals with different HLA phenotypes.

Over the last few years, natural killer (NK) cells have been shown to express major histocompatibility complex (MHC) molecules recognizing receptors that are thought to function primarily as negative signalling receptors. Much attention has been focused on the NK cell receptors CD158a (EB6) and CD158b (Gl 183), which recognize two alternative epitopes on the HLA‐Cw locus. In order to investigate whether HLA type affects the CD158a/b repertoire, expressed as percentage positive cells for a particular receptor and mean expression on this population of NK cells, peripheral blood lymphocytes of 47 HLA‐typed donors were examined. Peripheral blood samples were examined by flow cytometric analysis to investigate the expression of CD158a and CD158b receptors on the surface of NK cells. In parallel, we determined each individual’s HLA phenotype. There was a great heterogeneity in CD158 expression; nevertheless all individuals had NK cells belonging either to the CD158 a+b−, a−b+ or a−b− populations. No positive or inverse correlations could be shown between either receptor expression intensity or proportion of positive cells, and presence of the appropriate ligand. Thus no association between an individual’s NK receptor repertoire and HLA serotype could be demonstrated. It is concluded that CD158 is expressed on NK cells in a highly redundant fashion. Our data do not support either a positive selection mechanism or the receptor calibration model.

A flow-cytometry based cytotoxicity assay using stained effector cells in combination with native target cells

Flow-cytometry based assays for cellular cytotoxicity have established themselves widely over the last years. Discrimination of target and effector cells is critical for such assays. If scatter properties are not informative, the standard approach until now has been to label the target cells with a suitable fluorescent dye. However, this cannot be applied to a number of experimental settings, e.g. if one effector cell type is tested against several target cells, or if target cells do not incorporate the dye properly. Therefore, our goal was to develop a protocol based on the labelling of effector cells. For this purpose, we came around to using a membrane dye, DIOC18, which is not commonly used for flow-cytometric applications. This dye showed very stable membrane integration properties that allowed long-term coincubation periods (24 h) without leakage to neighbouring cells. The vitality and cytotoxic activity of the effector cells were not altered by staining. For the detection of dead cells, the intercalating DNA-dye 7-AAD was used. The spectral emission wavelengths of this combination also enable the additional use of PE-conjugated antibodies to surface antigens in three-color cytometry devices. Cytotoxicity values obtained by our protocol were highly correlated with values obtained by the chromium release assay at different E/T ratios and using several target cell lines. All in all, we present here an easy to handle protocol, which enables the precise determination of cellular cytotoxicity in various experimental settings.

Kinetics and Organ Distribution of Allogeneic Natural Killer Lymphocytes Transfused into Patients Suffering from Renal Cell Carcinoma

The transfusion of natural killer (NK) lymphocytes into patients suffering from malignant diseases is an approach of current interest in the field of immunotherapy. Little is known about the organ distribution, survival, and clearance of donor immune effector cells in cellular therapy, and no reports exist on these important parameters considering NK cells in particular or any other type of allogeneic lymphocytes in humans. In the context of a clinical Phase I/II study we examined the distribution of transfused allogeneic NK cells in patients suffering from renal cell carcinoma. The NK cells were ex vivo cultivated and activated before transfusion. To assess the circulation of the transfused cells in the peripheral blood, we used a nested PCR technique to detect HLA DRB1 alleles of the NK cell donors. Post-transfusion, all patients showed evidence of circulating donor cells for up to 3 days. After 7 days, all donor cells were cleared from the blood to undetectable levels. To assess organ distribution, (111)In-labeled NK cells were injected and monitored by whole-body scintiscans. A distribution to the whole body, with preference for liver, spleen, and bone marrow, was observed after a short initial uptake in the lungs. No activity was observed in lymphatic nodes. A total of 2/4 evaluable metastases showed a clear accumulation of transfused NK cells. The half-life corrected activity in all body compartments remained almost constant over the 6-day observation period in concordance with the absence of any excretion of radioactivity. This may indicate an extended survival of the transfused cells, despite their foreign nature, in the host organism.

DNA typing for natural killer cell inhibiting HLA-Cw groups NK1 and NK2 by PCR-SSP.

Over the last few years, natural killer (NK) cells have been shown to express MHC molecule recognizing receptors which are thought to function primarily as negative signaling receptors. HLA-Cw seems to play a key role as the corresponding ligand. Two distinct HLA-Cw groups which differ in amino acid residues 77 and 80 inhibit separate subsets of NK cells. In order to classify target cells with respect to their expression of HLA-Cw groups we established a group specific PCR-SSP which directly amplifies the relevant epitope coding sequences. The PCR protocol was validated by retyping cell lines obtained from the International Histocompatibility Workshop and by comparing those results with those acquired from allele-specific genotyping and serotyping on 80 donor-recipient pairs from our kidney transplantation unit. In the context of inhibitory HLA-Cw receptors, our protocol which definitively discriminates the two alternative epitopes is the more direct and thus more reliable approach, and is less labor intensive compared to an allele specific PCR or serotyping. In addition serotyping does not detect at all certain alleles. Basic NK cell research and clinical transplantation immunology may benefit from this newly established PCR SSP technique.

Feasibility of the Adoptive Transfusion of Allogenic Human Leukocyte Antigen-matched Natural Killer Cells in Patients With Renal Cell Carcinoma

Patients with metastasized renal cell carcinoma have a poor prognosis with conventional therapies. The feasibility and safety of donating purified natural killer (NK) cells without additional cytokines were evaluated. In contrast to all previous studies, the NK cells were derived from allogenic donors. The NK cell donors were HLA-C matched to enable NK cell inhibition via killer cell inhibitory receptors and HLA-C. This should obviate a graft-versus-host reaction against nonmalignant HLA-expressing tissues in the allogenic constellation. The average number of cells applied per transfusion was 1.02 ± 0.265 × 109. The purity of the NK cells was 85% to 95%, and most of the contaminating cells were monocytes. Twenty-six transfusions given to 11 patients did not cause any minor or major adverse effects, with the exception of one episode of transient fever. One patient had an objective regression of his lung metastases that had been progressing continuously before. No cytotoxic HLA antibodies could be detected 3 weeks after the transfusions. The observed tolerance to this therapeutic regimen suggests the need for further studies with increased doses of cytokine-activated NK cells.

β-(1→3)-D-glucan modulates DNA binding of nuclear factors κB, AT and IL-6 leading to an anti-inflammatory shift of the IL-1β/IL-1 receptor antagonist ratio

Backgroundβ-1→3-D-glucans represent a pathogen-associated molecular pattern and are able to modify biological responses. Employing a comprehensive methodological approach, the aim of our in vitro study was to elucidate novel molecular and cellular mechanisms of human peripheral blood immune cells mediated by a fungal β-1→3-D-glucan, i.e. glucan phosphate, in the presence of lipopolysaccharide (LPS) or toxic shock syndrome toxin 1 (TSST-1).ResultsDespite an activation of nuclear factor (NF)κB, NFinterleukin(IL)-6 and NFAT similar to LPS or TSST-1, we observed no significant production of IL-1β, IL-6, tumor necrosis factor α or interferon γ induced by glucan phosphate. Glucan phosphate-treated leukocytes induced a substantial amount of IL-8 (peak at 18 h: 5000 pg/ml), likely due to binding of NFκB to a consensus site in the IL-8 promoter. An increase in IL-1receptor antagonist(RA) production (peak at 24 h: 12000 pg/ml) by glucan phosphate-treated cells positively correlated with IL-8 levels. Glucan phosphate induced significant binding to a known NFIL-6 site and a new NFAT site within the IL-1RA promoter, which was confirmed by inhibition experiments. When applied in combination with either LPS or TSST-1 at the same time points, we detected that glucan phosphate elevated the LPS- and the TSST-1-induced DNA binding of NFκB, NFIL-6 and NFAT, leading to a synergistic increase of IL-1RA. Further, glucan phosphate modulated the TSST-1-induced inflammatory response via reduction of IL-1β and IL-6. As a consequence, glucan phosphate shifted the TSST-1-induced IL-1β/IL-1RA ratio towards an anti-inflammatory phenotype. Subsequently, glucan phosphate decreased the TSST-1-induced, IL-1-dependent production of IL-2.ConclusionThus, β-1→3-D-glucans may induce beneficial effects in the presence of pro-inflammatory responses, downstream of receptor binding and signaling by switching a pro- to an anti-inflammatory IL-1RA-mediated reaction. Our results also offer new insights into the complex regulation of the IL-1RA gene, which can be modulated by a β-1→3-D-glucan.