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Roquinimex, Linomide, is a quinoline derivative with pleiotropic immunomodulatory activities which has been shown to enhance NK function. As part of a phase III placebo-controlled multicenter study patients were randomized to receive Roquinimex, 0.2 mg/kg body weight, or a placebo twice weekly for a duration of 2 yr following autologous bone marrow transplantation for acute myeloid leukemia in remission. At Arhus University Hospital 7 patients were randomized to receive the active drug and 6 to receive the placebo. Surviving patients were followed for 2 yr with immunological monitoring of their natural immune effector cells (NK- and LAK cell activity). Peripheral heparinized blood samples were obtained twice before the onset of conditioning therapy and at several time points after ABMT, and whole blood samples were analyzed by flow cytometry for the detection of leukocyte differentiation antigens as well as by 4 h 51Cr release assays for cytotoxicity. In contrast to previous experience with Linomide, in the present study we found that at 36 wk or later time points Linomide patients exhibited a significant suppression of circulating natural effector cell number and activity when compared with the control group. These observations underline the need for further exploration into novel and manageable immunostimulators.

Silencing of the cyclin-dependent kinase inhibitor gene p15(INK4B) by cytosine methylation of the promoter region has been associated with some types of hematological malignancy. To study in detail the patterns of p15(IN4B) methylation in patients with acute myeloid leukemia, we adopted a novel approach based on PCR amplification of bisulfite-treated DNA followed by resolution of differentially methylated sequences by denaturing gradient gel electrophoresis. This method visually displays the degree and heterogeneity of DNA methylation and enables the isolation and characterization of distinct clonotypic epigenotypes. A surprisingly high degree of intra- and interindividual heterogeneity of p15(INK4B) methylation was observed in the 65 acute myeloid leukemia patients examined. Methylation was detected in 46 (71%) of the patients and was observed more frequently in the French-American-British subtypes M1/M2 than in M4/M5 (P < 0.025). Examination of the same panel of samples using a highly sensitive methylation-specific PCR method showed methylated p15(IN4B) alleles in 61 (94%) of the samples. We present evidence that the higher frequency of p15(INK4B) methylation determined by methylation-specific PCR may, at least in part, be due to the presence of a small fraction of p15(INK4B)-methylated lymphocytes in normal blood.

In our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. The database represents a computer-based replacement for the laboratory notebooks used in the majority of research laboratories worldwide. In addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. Using the commercially available database toolkit software FileMaker Pro, we have developed a relational database solution for management of laboratory information. The system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. Like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. In each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. Thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. Importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. The limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts ‘somewhere’. The often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. Thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline.

Materials and methods: As an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (PBMCs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. Eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. The spontaneous cytotoxicity of the PBMCs was estimated in a 4 h 51Cr-release assay using k562 as NK-sensitive target cells. The PBMCs were characterized, using 4-colour flow cytometry, with special emphasis on the NK-cell subsets. The data were statistically analysed using a multivariate regression model (Stata 8.2). P values

Direct contact between lymphokine-activated killer (LAK) cells and tumour cells is believed to be imperative for initiating tumour cell lysis in vitro as well as in vivo. In order to optimize adoptive immunotherapy (AIT) it is therefore desirable to identify the LAK cell subtype, which ensures maximal infiltration of tumours as well as a substantial cytotoxic reactivity. In this report we have compared short- and long-term cultured murine adherent natural killer (A-NK) cells and mitogen-stimulated, lymphokine-activated T-killer (T-LAK) cells with respect to their proliferative potential, cytotoxicity, requirement for interleukin-2 (IL-2) and ability to infiltrate B16 pulmonary metastases following adoptive transfer. We found that short-term (5 days) cultured A-NK and T-LAK cells both showed a substantial accumulation of tumour tissues. However, A-NK cells gradually lost this ability during in vitro culture whereas T-LAK cells cultured for as long as 20 days retained their ability to infiltrate metastases as efficiently as their short-term cultured counterparts. Moreover, the low requirement of IL-2 by T-LAK cells to achieve maximal infiltration of tumours sharply contrasted with the excessive doses necessary to ensure maximal infiltration by A-NK cells. In conclusion, these data demonstrate that short-term cultured LAK cells of both NK- and T-cell origin are able to infiltrate B16 pulmonary metastases effectively. Importantly, the T cells retain this ability for a considerably longer time and require much less IL-2 support than do A-NK cells, making T-LAK cells attractive for AIT.

We have studied the regulatory capacity of clonal T-lymphocytes from patients undergoing autologous bone marrow transplantation (ABMT) on the generation of CFU-GM from their harvest marrow preparations To this end, T-lymphocytes from peripheral blood from 5 patients undergoing ABMT isolated 10 d before and 7, 14 and 28 d post-ABMT were placed in limiting dilution conditions (384 wells for each patient at each time point) and polyclonally stimulated. From more than 1600 wells with growth from the 5 patients, preparations from more than 900 wells could be expanded (range between patients 33-452) and identified by immunophenotyping (IP) and flow cytometry (FCM) by their exclusive expression of CD4 or CD8. This was significantly fewer than seen in normal donors, especially so at d 7 and 14 post-ABMT. The ratio between CD4 + and CDB + clones varied between 0.6 and 2.8 (median 1.3) and was significantly lower in the patients compared to normal donors (median 3.1; range 3.0-6.5). When the clonal T-cell preparations were co-cultured with autologous bone marrow cells obtained at the time of harvest and depleted for T-lymphocytes, the vast majority of both CD4 + and CD8 + clones exerted a clear enhancement on the CFU-GM growth with no relation to time of blood sampling in its the magnitude. Moreover, a trend seen in the normal donors towards CD4 + clones being more effective in this enhancement was not observed in ABMT patients. We conclude that clonal T-cells from ABMT patients, irrespective of their phenotype and time of isolation, exert an enhancement on the growth of autologous CFU-GM, which is equal to that seen in normal donors.

Cardiac and major abdominal surgery are associated with granulocytosis in peripheral blood. The purpose of the present study was to describe the granulocyte and monocyte oxidative burst and the expression of adhesion molecules following cardiac surgery with cardiopulmonary bypass and abdominal surgery. The ability to respond with an oxidative burst was measured by means of flow cytometry using 123-dihydrorhodamine. The adhesion molecules CD11a/CD18, CD11c/CD18, CD44 were measured using monoclonal antibodies. Blood samples from eight patients undergoing open-heart surgery were taken before surgery, 1, 5, 10 and 20 min after aortic clamping, and then 1, 5, 10 and 20 min and 1, 2 and 3 h after declamping. Samples from eight patients undergoing abdominal surgery were taken before surgery, at the end of surgery, and 2 and 3 h post-operatively. A decrease in number of granulocytes and monocytes during cardiopulmonary bypass was observed. The percentage of CD11a-positive granulocytes increased from 30% pre-operatively to 75% following cardiopulmonary bypass, while CD44-positive granulocytes increased from 5% to 13%. Despite the extent of the changes, these were not significant. The oxidative burst of the granulocytes and monocytes decreased after declamping to 15% and 27% of initial values in vitro. Several hours after surgery, there was no significant difference between the two groups. These results can be explained by a granulocyte and monocyte refractory response developing subsequent to an increased per-operative oxidative burst activity, and the induction of adhesion molecules on granulocytes associated with the cardiopulmonary bypass and surgery. In conclusion, open-heart surgery with cardiopulmonary bypass was associated with a rapid and pronounced activation of leukocytes which may play a role in reperfusion injury. Udgivelsesdato: 1998-May

BACKGROUND: Hypogammaglobulinemia may be part of several different immunological or malignant conditions, and its origin is not always obvious. Furthermore, although autoimmune cytopenias are known to be associated with common variable immunodeficiency (CVID) and even may precede signs of immunodeficiency, this is not always recognized. Despite novel insight into the molecular immunology of common variable immunodeficiency, several areas of uncertainty remain. In addition, the full spectrum of immunological effects of the B cell depleting anti-CD20 antibody Rituximab has not been fully explored. To our knowledge this is the first report of development of CVID in a patient with normal immunoglobulin prior to Rituximab treatment.CASE PRESENTATION: Here we describe the highly unusual clinical presentation of a 34-year old Caucasian male with treatment refractory immune thrombocytopenic purpura and persistent lymphadenopathy, who was splenectomized and received multiple courses of high-dose corticosteroid before treatment with Rituximab resulted in a sustained response. However, in the setting of severe pneumococcal meningitis, hypogammaglobulinemia was diagnosed. An extensive immunological investigation was performed in order to characterize his immune status, and to distinguish between a primary immunodeficiency and a side effect of Rituximab treatment. We provide an extensive presentation and discussion of the literature on the basic immunology of CVID, the mechanism of action of Rituximab, and the immunopathogenesis of hypogammaglobulinemia observed in this patient.CONCLUSIONS: We suggest that CVID should be ruled out in any patient with immune cytopenias in order to avoid diagnostic delay. Likewise, we stress the importance of monitoring immunoglobulin levels before, during, and after Rituximab therapy to identify patients with hypogammaglobulinemia to ensure initiation of immunoglobulin replacement therapy in order to avoid life-threatening invasive bacterial infections. Recent reports indicate that Rituximab is not contra-indicated for the treatment of CVID-associated thrombocytopenia, however concomitant immunoglobulin substitution therapy is of fundamental importance to minimize the risk of infections. Therefore, lessons can be learned from this case report by clinicians caring for patients with immunodeficiencies, haematological diseases or other autoimmune disorders, particularly, when Rituximab treatment may be considered.

We have studied the recirculation patterns of leukocyte subpopulations during the first 24 h at 5 min before and 5, 15, 180, and 1440 min after autologous bone marrow transplantation (ABMT) in 14 patients with cancer (6 with AML, 5 with malignant lymphomas, 2 with ALL, and 1 with Ewing's sarcoma) using multiparameter flow cytometry and measurements of myeloid progenitors (CFU-GM). Although the great majority of the injected cell populations were undetectable 5 min after graft infusion, the number of CD3+ T lymphocytes increased at 5 and 15 min and again at 24 h post-ABMT. In contrast, the number of CD56+ natural killer (NK) cells decreased rapidly after ABMT to remain low throughout the observation period. Mature myeloid and monocytic cells (identified by their expression of CD66 and CD14, respectively) were present before, as well as after ABMT in numbers indicating that they were probably of endogenous origin. Immature myeloid cells were identified in a three-color flow cytometric assay as CD13+ CD14- CD66- and tended to increase during the first 15 min after ABMT. Finally, when CFU-GM were followed longitudinally, they were found to be practically absent before ABMT but were clearly detectable in 12 of 14 patients throughout the observation period. We conclude that leukocyte subsets exhibit different recirculation patterns after ABMT, and in light of the increased knowledge about leukocyte-endothelial interactions, these data could provide a platform for attempts to control leukocyte recirculation during stem cell infusion.

In a randomized study the effect of whole blood transfusion versus bedside leucocyte-depleted blood transfusion on lymphocyte proliferation, CD4+: CD8+ratio, and levels of soluble interleukin 2 receptor (sIL-2R) and interleukin (IL) 6, as well as on the development of postoperative wound infection and intra-abdominal abscess, was assessed in 60 patients undergoing elective colorectal surgery. Transfusion with whole blood induced a significant decrease in lymphocyte proliferation and CD4+:CD8+ ratio (P< 0·01) as well as a significant increase in sIL-2R and IL-6 levels (P< 0·01). Furthermore, transfusion with whole blood was accompanied by a significant increase in postoperative infectious complications (P< 0·01). In patients transfused with leucocytedepleted blood only slight and transient changes were observed, which were not significantly different from those observed in non-transfused patients.

Slow hematopoietic recovery is a well-known feature in patients undergoing autologous bone marrow transplantation (ABMT), and here we demonstrate that compared with patients with other malignant blood diseases, the thrombocytopenia in acute myeloid leukemia (AML) patients stands out, with the median time to recovery of50 x 10(9) platelets being 101 days for AML patients and 22-37 days for other patient groups. We have consequently evaluated the content and fate of myeloid progenitor cells (BFU-E, CFU-GM, and CFU-GEMM) in bone marrow preparations from cancer patients in an attempt to uncover factors of importance for their slow hematopoietic recovery after ABMT using three experimental setups. First, we analyzed progenitors in marrow samples from 36 patients [18 AML, 5 acute lymphoblastic leukemia (ALL), and 13 non-Hodgkin's lymphoma (NHL)] exposed to multiple doses of severely myelotoxic cytoreductive regimens and observed that AML patients had suppressed numbers of CFU-GEMM and BFU-E (80% that of normal volunteers) but increased levels of CFU-GM. In contrast, the decrease in ALL patients was more pronounced (50% that of normal volunteers) and demonstrable for all progenitors. NHL patients exhibited normal progenitor frequencies of CFU-GM and CFU-GEMM, whereas their BFU-E counts were found to be increased nearly two-fold. Second, we followed the fate of progenitors through the laboratory manipulations of marrow grafts and found no evidence for selective losses in terms of disease (7 AML, 5 NHL, 1 ALL, and 2 testicular germ cell tumor patients) and progenitor type. Third, we retrospectively evaluated the cell yields of grafts from 121 cases [39 AML, 20 ALL, 22 NHL, 9 Burkitt's lymphoma (BL), 18 Hodgkin's disease (HD), and 13 testicular germ cell tumors] and found those of heavily treated AML, HD, and germinal tumors most affected, with a clear negative correlation in AML patients between age on the one hand and mononuclear cell yield and CFU-GM content on the other. We conclude that the amount of previous chemotherapy and age of patients appear to be among the determining factors on the number of myeloid progenitor cells in cancer patients and that thresholds for acceptability of grafts should take these variables into account.

Udgivelsesdato: 22 January

By staining human bone marrow cells with a monoclonal antibody reacting with erythroid precursor cells (AS-E1) and propidium iodide, we have evaluated the proliferative capacity of erythropoiesis in patients with myelodysplastic syndromes (MDS) using flow cytometry. Comparing 36 patients (13 RA/RAS, 13 RAEB, 10 RAEB-t) with 7 normal controls, significant differences in both the percentage of AS-E1 + cells and the fraction of AS-E1 + cells in the S or S-G 2 M-phase between the four groups were found. Since neither the percentage of AS-E1 + cells nor their fraction in S or S-G 2 M alone was found to characterize their proliferative activity, we introduced the proliferative fractions of the erythroid cell, i.e. the number of the AS-E1 + cells in S or S-G 2 M related to all bone marrow cells in S or S-G 2 M. Applying these parameters, we found significantly increased proliferative AS-E1 fractions in the RA/RAS group compared to the normal controls ( p = 0.03 rmand 0.002) respectively, as well as a highly significant decrease with disease progression.

In further studying the mechanism of action of IFN-alpha in HCL, we cultured the HCL cell line JOK-1 and the IFN-sensitive Burkitt cell line Daudi with and without IFN-alpha and investigated the changes in density of a number of surface antigens by use of mAb and flow cytometry analyses. During culture with IFN-alpha, reproducible changes were induced in both cell lines, which were qualitatively similar but differed quantitatively with small and transient changes in JOK-1. Significant decreases in surface antigen expression were observed for CD 19, 23, 37, and for IgM on both cell lines. Moreover, decreases were seen for CD 10, 22, 45, and MHC class II on Daudi, and for CD 20, 21, 27, and 40 on JOK-1. By contrast, only a few antigens increased in density, including CD 39, A96/G8 and SC9, on both cell lines, CD 22 on JOK-1, and CD 21 on Daudi. The increase in CD 39, A96/G8 and SC9 was probably directly related to the mechanism of action of IFN-alpha, whereas the other changes were most consistent with an unspecific inhibition of protein synthesis, possibly due to an accumulation of cells in G0, even though a differentiating effect cannot be ruled out. Thus, the unique in vivo effect of IFN-alpha in HCL was not paralleled by a specific direct effect on JOK-1 in vitro. Our findings therefore do not support the theory that IFN's mechanism of action in vivo is a direct effect on HC, but suggest that indirect effects are involved. Udgivelsesdato: 1992-Mar

Assessment of the tissue distribution of adoptively transferred adherent lymphokine-activated killer A-LAK) cells by use of 51Cr indicated that these effector cells, after an initial phase in the lungs, distributed in high numbers to liver and spleen (30% and 10% of injected dose, respectively). However, when this experiment was repeated with 125IdUrd as cell label, fewer than 2% and 0.5% of the injected cells distributed into liver and spleen respectively. To analyse this discrepancy, we compared the tissue distribution of 51Cr- and 125IdUrd-labelled A-LAK cells with that indicated by alternative direct visual methods for identification of the injected cells, such as fluorescent dyes (rhodamine and H33342) or immunohistochemical staining of asialo-GM1-positive cells. The number of i.v. injected A-LAK cells found in the liver by all visual methods ranged from 1% to 5% of the injected dose, supporting the data obtained with 125IdUrd, whereas 25%-30% of the 51Cr label was consistently found in this organ. Autoradiography of the liver 24 h after i.v. injection of 51Cr-labelled cells revealed a background activity that was four- to fivefold higher than the control level, indicating substantial non-specific accumulation in the liver of 51Cr released from A-LAK cells. We conclude that 51Cr cannot be reliably used in investigations of cell traffic to the liver because of non-specific accumulation of the 51Cr label, particularly in this organ. In contrast, labelling with 125IdUrd or rhodamine and immunohistochemical staining of asialo-GM1-positive cells appear to be reliable and essentially equivalent methods for investigations of the fate of adoptively transferred A-LAK cells. Using these methods, we found that only few A-LAK cells redistribute to the liver upon i.v., i.e. systemic, injection, whereas 40%-50% of locally (intraportally) injected A-LAK cells remain in the liver for at least 24 h.

We here describe a method for estimation of the absolute number of NK cells in various organs of C57BL/6 mice. Using anti-asialo-GM1 heteroantisera to identify NK cells in tissue sections routinely stained by two-layer immunoperoxidase techniques, scoring was performed using light microscopy. Extrapolation to the absolute number of NK cells/organ was done after counting 10 systematically sampled sections of each organ. Due to the thinness of the sections (8 microns) many cells are cut and will appear in at least two sections, and a correction factor was constructed to eliminate this bias. With this method we show that spleens of eight-week-old C57BL/6 male mice contain about 4 x 10(6) NK cells, which is consistent with previous findings obtained by other methods. However, application of this stereological method to liver and lungs revealed the existence of as many as 0.75 and 2.5 x 10(6) NK cells in these organs, respectively, i.e. three to 10 times as many as previously found in single-cell suspensions obtained by enzymatic dissociation of liver and lung tissue. This stereological method for enumeration of the absolute number of small subpopulations of cells is generally applicable to other organs and to other antigens, and does not require the application of cumbersome single-cell isolation procedures, during which an unknown number of cells might be lost. Udgivelsesdato: 1992-Mar

In a longitudinal study of a 32-year-old male with Ph1+ hybrid leukemia we have followed the immunophenotype and configuration of Ig- and TCR genes during the course of different chemotherapy regimens directed first against the myeloid and later against the lymphoid components of the disease. We identified changes in all parameters, interpretable as an evolution of the malignant clone resulting in a leukemic switch towards a more lymphoid character. Thus, while the expression of the myeloid antigens CD13 and CD33 decreased, that of CD10 (CALLA) and CD20 (B1) increased. Moreover, while the configuration of the Ig heavy and light chain lambda genes remained constant during the whole period of treatment, that of the Ig light chain kappa gene and TCR beta gene displayed extensive rearrangements after initiation of ALL therapy. Since this patient represents a de novo acute leukemia as evaluated by location of the translocation-breakpoint on chromosome 22, our data clearly indicate that Ig- and TCR gene rearrangements might prove a valuable addition in monitoring Ph1+ hybrid leukemias, providing guidelines for optimizing chemotherapy. Udgivelsesdato: 1992-Feb

Abstract 3680 Poster Board III-616 Therapeutic antibody preparations are used in the treatment of several diseases. Thus, human immunoglobulin is administered as high dose intravenous immunoglobulin (IVIg) to patients suffering from a wide range of disorders. Moreover, various monoclonal antibodies (MoAbs) are approved for therapy in cancer and immune diseases. Natural Killer (NK) cells exert important immune effector functions. As these cells express the IgG receptor, CD16 (FcγRIIIa), it is of clinical importance to investigate how these cells react to therapeutic antibody preparations. We found that cytotoxic effector functions of NK cells were significantly inhibited by therapeutic antibody preparations in vitro. Natural cytotoxicity and antibody dependent cell-mediated cytotoxicity (ADCC) were inhibited by treating peripheral blood mononuclear cells (PBMCs) with the polyclonal immunoglobulin preparation Beriglobin, as well as the MoAb preparation Rituximab, prior to standard 51Cr-release assays compared to untreated cells. Moreover, our data showed that freshly isolated NK cells had IgG bound to the cell surface. The level of bound IgG increased following short-time exposure to both therapeutic antibody preparations, and the level of CD16, which is the key receptor for inducing ADCC, decreased. To investigate whether the NK-inhibitory effects of IVIg were demonstrable in vivo too, we analyzed blood from patients diagnosed with chronic inflammatory demyelinating polyneuropathy (CIDP), a disorder in which high-dose IVIg as monotherapy has shown striking effects. Comparing peripheral blood samples obtained from patients before treatment and 3 days following high-dose IVIg using multiparameter flow cytometry, we demonstrated a substantial decrease in the number of CD56+/CD3- NK cells. Moreover, as shown in vitro, the expression of CD16 on the NK cells was markedly reduced following high-dose IVIg treatment due to IgG engaged by the FcγRIIIa receptor (CD16). Finally, we found that NK mediated cytotoxicity decreased significantly when comparing peripheral blood samples from patients before and 3 days following IVIg treatment. In conclusion, our data presented in this study show that high-dose immunoglobulin therapy induces NK inhibitory effects in vitro as well as in vivo and thus may have important consequences for the overall therapeutic efficiency. Disclosures: No relevant conflicts of interest to declare.

Materials and methods: Isolated cryopreserved human peripheral blood mononuclear cells (PBMCs) were stimulated with IL-2 and IL-12. This stimulation has previously been shown to activate NK cells. Cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. This method was compared to the current standard 51Cr release assay. Cells were treated with BFA to accumulate IFN-γ, stained for surface markers, permeabilized and stained for intracellular IFN-γ. Flow cytometry was then performed to measure intracellular IFN-γ production in PBMC, especially in NK cells. Results: We have demonstrated that stimulation with IL-2 and IL-12 is effective in increasing the number of IFN-γ-positive cells. There is a distinct difference between the CD3-CD56dim and the CD3-CD56bright subsets, with a much greater proportion of IFN-γ-positive cells in the CD3-CD56bright subset. The effects of stimulation with IL-2 and IL-12 on cytotoxicity will be presented, as will the relation between IFN-γ production and cytotoxicity. In addition, we will present results of these assays applied to porcine cells. Discussion: In combination, these tests will address NK cell function by combining cytotoxicity with IFN-γ production in NK cell subsets. The results will demonstrate whether this could serve as a useful tool in describing NK-cell function, which could be of value in clinical and experimental settings.

Tracking adoptively transferred antigen-specific T lymphocytes is an important prerequisite for devising better protocols for cellular therapy. To this end we have developed a highly sensitive method for “in situ” visualization of labelled lymphocytes in vivo by combined PET and magnetic resonance imaging (MRI) to monitor the distribution of adoptively transferred tumour-specific T cells in a mouse model system. Moreover, quantitation of the adoptively transferred cells in tumor was performed by flow cytometry. C57BL/6J mice carrying subcutaneous tumours of the ovalbumin (OVA)-expressing malignant melanoma cell line B16-OVA were adoptively transferred with OVA-specific CD8+ T cells labelled with 124IdU. Five days after transfer of T cells, mice were killed and subjected to PET and MR imaging. Using a newly developed method for co-registration of the two image modalities, the anatomical localisation of the transferred cells was visualised and the amount of radioactivity in various anatomical locations very accurately determined. For quantitation of tumor infiltrating non-labelled OVA-specific CD8+ T cells by flow cytometry (using AbsoluteCount Beads), tumors were removed from mice day 1 until day 8 following adoptive transfer (6 mice/group) and prepared for single cell suspension before labeled with anti-CD8-FITC and SIINFEKL-Tetramer-PE. Results showed a clear tumor localization of the adoptively transferred OVA-specific T cells in the tumours. In two independent experiments comprising 12 and 13 evaluable mice, respectively, we found a mean value of 0.909 +/− 0.468 Bq and 0.926 +/− 0.553 Bq in the tumours, and only 0.182 +/− 0.479 Bq and 0.026 +/− 0.480 Bq in the corresponding contralateral control volumes. The difference in activity between the tumour regions and the control regions was statistically highly significant with 2p-values of 0.002 and 0.006 for the two experiments. Using flow cytometry it was shown that the number of OVA specific T lymphocytes accumulating in tumor gradually increased until day 5 after transfer when an average of 3.3 million SIINFEKL-specific cells per gram tumor tissue was found. From day 5 until day 8 the number of SIINFEKL-specific cells per gram tumor tissue fluctuated at a fairly constant level. This method presented for tracking adoptively transfered tumor specific T lymphocytes represent a significant advancement for studies of adoptively transferred specific T cells, and could potentially be developed for diagnostic purposes. Moreover, since these studies show that tumor-specific T cells home to subcutaneous tumours in substantial numbers, we suggest that these migrating cells could be employed in a new form of therapy as carriers of toxic substances to tumors.

The effect of human interferons on different types of lymphocyte-mediated killer assays was explored. Killing by T cells generated through mixed lymphocyte cultures as well as antibody-dependent lymphocyte-mediated cytotoxicity was not influenced by the addition of interferon. Enhancement of cytolysis produced by natural killer cells was observed when interferon was added during the assay, but enhancement could also be induced if the effector cells were pretreated with interferon for 2 hr prior to the lytic reaction. Killing of a cell line susceptible to natural killing was increased and a cell line which is normally relatively resistant to this type of killing became a susceptible target.

Normal human peripheral blood lymphocytes were incubated in the presence of different concentrations of interferon for various incubation periods. Subsequently, the amount of beta 2-Microglobulin and HLA-A, B and C surface antigens was estimated by means of quantitative immunofluorescence (flow cytofluorometry) and by a radioimmunoassay for beta 2-Microglobulin. It was found that the amounts of these MHC antigens increased in a dose and time-dependent way after interferon treatment. Furthermore, the influence of different temperatures on this IFN-induced increase in beta 2-Microglobulin was gradually enhanced after incubation at 37 degrees C to 39 degrees C incubation mostly suppressed the beta 2-Microglobulin increase observed at 39 degrees C. The total amount of membrane associated beta 2-Microglobulin was estimated by a radioimmunoassay. After interferon treatment a beta 2-Microglobulin increase was observed ranging from 39% to 122% in different experiments. It is shown that interferon is the substance responsible for the effects on lymphocytes reported here, because completely pure interferon-proteins had the same activity as has been shown for partially purified interferon preparations.

Mononuclear cells from the blood of healthy normal humans were kept in cultures under nonstimulating conditions for 16 hr in the presence or absence of human interferon. The relative quantities of HLA antigens and β 2 -microglobulin on the cultured cells were determined by quantitative immunofluorescence (fluorescence-activated cell sorter) and by the capacity of cells to absorb out cytotoxic antibodies against the relevant antigens. Interferons of different origin and purities enhanced the expression of HLA antigens and β 2 -microglobulins, whereas membrane immunoglobulins and antigens recognized by antiserum raised against human brain and T cells were the same on interferon-treated and control cells. Similar interferon effects were observed on an Epstein-Barrvirus-negative Burkitt lymphoma cell line. The enhanced expression of histocompatibility antigen subsequent to intereferon treatment was observed on B- and T-enriched lymphocyte populations and was found to be dose dependent with the optimum with “physiological” concentrations of interferon. Pretreatment of lymphocytes with interferon for 2 hr was found to be as effective as having interferon present during the total culture period. The interferon-induced enhancement of antigen expression on cells was dependent on active protein synthesis.

Fetal hematopoietic cells that express the common acute lymphoblastic leukemia antigen (CALLA) were purified from both fetal liver and fetal bone marrow by immune rosetting with sheep erythrocytes coated with rabbit anti-mouse immunoglobulin and by fluorescence-activated cell sorting. Dual fluorescence techniques disclosed that these cells were heterogenous with respect to the expression of a series of differentiation and activation antigens defined by monoclonal antibodies. Thus, whereas all CALLA+ cells were Ia+ and expressed two activation antigens, J2 and T10, only 30-50% expressed B1 antigen. Furthermore, using methanol-fixed cells, it could be shown that approximately 20% contained intracytoplasmic mu chains (cyto-mu) and that approximately 15% were positive for the terminal transferase enzyme (TdT) marker. The CALLA+ fetal cells thus closely resemble the childhood acute lymphoblastic leukemia cell with respect to surface marker phenotype. A population of CALLA- cells devoid of mature erythroid and myeloid surface markers was found to contain higher numbers of TdT+ cells but lower numbers of cyto-mu, B1, and Ia+ cells than the CALLA+ subset. In vitro analysis of normal, purified CALLA+ cells demonstrated that incubation at 37 degrees C with J5 monoclonal antibody specific for CALLA resulted in the specific modulation of surface antigen. Similar results have previously been obtained with CALLA+ tumor cells. Although phenotypic analysis of CALLA+ cells suggests that these cells are relatively immature lymphoid cells, CALLA+ cells do not appear to contain either myeloid precursor cells (CFU-G/M) or the earliest lymphoid stem cells.

Udgivelsesdato: 1980-null

By means of simple rosette sedimentation methods two subsets from human peripheral blood lymphocytes have been isolated: (1) (E, Fc)- and (2) (E, Ig)-. The first subset was obtained by centrifuging suspensions of macrophage-depleted PBL in which E and EA rosettes had been allowed to form simultaneously. The dominant marker of these E- Fc- cells was surface Ig, and during 4 days of culture this population did not alter its surface markers. Subset 2 was obtained in two ways following rosette centrifugation with AET-treated SRBC and rabbit anti-human Ig-coated autologous RBC. This ‘Null cell population’ was shown to be highly variable as judged by the surface markers applied after 4 days of culture, and it is suggested that Null cells contain a number of immature lymphoid cells that may acquire their surface marker during culture. It is concluded that the methods described for purification of small subsets of human lymphocytes are effective and easy to perform and might be used to purify cells for functional studies.

Human leukocytes treated with Sendai virus yield interferon predominantly of the alpha-type (HuIFN-alpha). Successful attempts to purify these "native" species have been performed and the final analysis, which included an SDS-PAGE disclosed 13 stained and separated IFN-proteins in the molecular weight-range of 16.6-23.5 kD. These stained IFN proteins were eluted individually from the gel slices and assessed for antiviral activity in human, monkey, and bovine cells, as well as for immunomodulatory effects (in vitro) on human lymphocytes. Based on equal amounts of (human) IFN units, as determined by IFN titration on human cells, the "immunological efficacies" of the 13 different HuIFN-alpha species were determined in three different immunological systems with the following results: (1) Augmentation of the NK function was a property of all species, although the two lower species (16.6 kD, 16.9 kD) were clearly less efficient with "titers" in the NK system reduced 25-fold. (2) Enhanced expression of HLA on lymphocyte membranes was induced by all the HuIFN-alpha species to the same extent. (3) Addition of IFN to mixed lymphocyte reaction (MLR) augmented the CML outcome of the cultures. In this system all 13 species exerted their effect equally well; no clear inferiority or superiority of individual species were seen. It is concluded that the fractionation of the IFN-alpha into 13 species does not give rise to IFN species which are specific only for some functions and not for others. All species exert all functions, although the relatively "immunological" titers in the NK system varied within the species. Udgivelsesdato: 1983-null

Natural killer cell (NK) activity in peripheral blood (PB) was followed longitudinally for up to 2 yr after initiation of low-dose IFN-alpha-2b therapy in nine hairy cell leukemia (HCL) patients. A whole blood NK (WB-NK) assay was employed in order to measure the NK activity per unit blood. The pretreatment WB-NK activity was consistently low, indicating that the patients' total NK activity in PB is decreased. Striking differences in WB-NK activity were observed between splenectomized and non-splenectomized patients, whereas no consistent patterns were found when using the conventional NK assay. Thus the WB-NK activity of splenectomized patients showed an immediate increase after initiation of treatment, while the activity in non-splenectomized patients decreased and remained low during the first 3-6 months. Subsequently, after reduction in spleen size, the WB-NK activity began to increase. In splenectomized patients, a second rise in WB-NK was observed after 3-6 months of therapy, coinciding with the normalisation of the peripheral blood counts. In both groups of patients incubation with IFN in vitro induced a rise in NK activity before start of treatment, which was abrogated promptly after initiation of therapy, indicating a maximal in vivo boosting of the NK cells. These differences observed indicate that the response of splenectomized and non-splenectomized HCL patients to IFN treatment should be evaluated separately.

This is the first report describing natural killer (NK) and antibody- dependent cellular cytotoxicity (ADCC) of malignant monoblasts. Pure acute monoblastic leukemia was diagnosed in bone marrow aspirations from two patients by use of conventional cytochemical methods as well as multiple immunologic techniques including detection of ALL antigens and terminal transferase. The malignant cells were subsequently found to be potent effectors in NK and ADCC assays. Addition of partially purified alpha-interferon to the in vitro cultures was found to have an enhancing effect on NK activity, whereas no modulation was seen in ADCC. These findings are discussed in the light of our present knowledge of lymphoid NK cells.

The standard E rosette method and two previously described methods claimed to give improved E rosetting for enumeration of human T lymphocytes have been compared with respect to the speed of rosette formation, and the mechanical stability of the rosettes formed. Following rosette formation with the three methods, the lymphocyte-erythrocyte suspensions were sedimented on Ficoll-Isopaque to deplete them of rosette-forming cells and red cells. The purified lymphocyte preparations were tested for B and T cell markers to determine the degree of contamination with T cells. One of the improved rosetting methods was clearly better than the others, and led to the recovery of B lymphocytes with a contamination of only 2.0+/-1.9 per cent of T lymphocytes. Udgivelsesdato: 1976-null

The effect of alpha interferon (alpha IFN) on the expression of histocompatibility--as well as differentiation antigens on normal and malignant hematopoietic mononuclear cells--were investigated by cell cytofluorometry using a panel of monoclonal antibodies (MoAbs). An increase in the expression of HLA-antigens detected by beta 2-Microglobulin (beta 2-M) could be demonstrated for peripheral blood mononuclear cells, non-T cells, Null cells, activated T cells, fetal thymocytes, adherent cells, and on four malignant non-T lymphoblastoid cell lines. In contrast, no significant differences were observed in the expression of antigens specific for B-lymphocytes (B1), T-lymphocytes (T3, T4, T6, T8, T11), NK-cells (901) and adherent cells (Mo1-4). Likewise, the expression of Ia-antigens remained unaltered on non-T cells, Null cells, and monocytes. The only other effect of IFN was an increase in the number as well as the amount of lymphocytes expressing the T10 antigen. It thus seems that the enhancing effect of IFN on resting cells of the immune system is highly selective. On the four lymphoblastoid cell lines, the expression of the common acute lymphoblastic leukemia antigen (CALLA) was significantly decreased concomitantly with the increase in MHC-antigens. On the other hand, the density of both a HLA-D related Ia antigen (I2) and a B-lymphocyte differentiation antigen (B1) remained unaltered following IFN treatment. The implications of these findings are discussed. Udgivelsesdato: 1983-null

Publisher Summary This chapter discusses the measurements of changes in histocompatibility antigens induced by interferons. A major nonantiviral effect, among others, of interferon is the modulation of histocompatibility antigens on the surface of a variety of cells. Concerning major histocompatibility complex (MHC) class 1 antigens (HLA-A, -B, and -C), all three types of human interferons (Hu-IFNs) (alpha, beta, and gamma) induce an increase in expression and synthesis of these antigens. When looking at MHC class II antigens, Hu-IFN-γ is the only type of interferon able to induce a substantial increase in the expression of these antigens, and cells from the monocyte/macrophage lineage are, especially sensitive to this effect of immune IFN. Several methods have been reported for the demonstration of MHC enhancement, but an indirect immunofluorescence method with subsequent measurement in a fluorescence-activated cell sorter (FACS-II) is found to be easy, convenient, and sensitive to very small changes. By storing the data in the FACS, it is possible to recall them in a computer system for further calculations.

During a phase II trial of recombinant IFN-alpha given in doses of 50 X 10(6) units/m2 three times per week to lung cancer patients, 13 patients were evaluated longitudinally in NK and ADCC assays and in immunofluorescence tests enumerating the number of cells reactive with the new N901 NK-cell antibody. An increase in NK-cell activity could be demonstrated when values before and 24 h after the first injection of IFN were compared, but simultaneously the enhancing effect of IFN-alpha on NK-cells added to in vitro cultures was abolished, probably as a result of preactivation of NK cells in vivo. After 2-4 weeks of treatment, the majority of patients exhibited a pronounced decrease in NK-cell activity while still retaining the inability to be boosted by IFN added in vitro. Investigations with the NK antibody N901 showed that the initial increase in NK activity appeared concomitantly with an increase in the number of N901 positive cells, indicating that the mechanism behind this increase was an increase in the number of circulating NK cells. In contrast, the decrease in NK activity mentioned above was not followed by a similar decrease in the number of N901+ cells, and it was concluded that this decrease might be attributable to either an exhaustion phenomenon or to an induction of a refractory state of peripheral blood NK cells. When measuring ADCC activity, increases in lytic activity were seen only in patients in whom they could be attributed to non-IgG-dependent (NK-like) mechanisms. These data are discussed in relation to other clinical trials using leukocyte or recombinant IFN-alpha. Udgivelsesdato: 1984 Fall

We have cloned common acute lymphoblastic leukemia (CALLA)-positive cells from human fetal bone marrow containing less than 1 in 10,000 E-RFC in round-bottomed microtiter wells (one cell per well) using the autocloning unit of an EPICS-V cell sorter. Expansion of such cells (with IL-2 and heavily irradiated autologous thymocytes as feeder cells) resulted in growth in 6-14% of the wells (mean, 11%) with cells with mature T lymphocyte phenotype. Two-color fluorescence analysis of outgrowing cultures furthermore ascertained that these cells had differentiated through a phase of simultaneous expression of T4 and T8 antigens and at the same time expression of the thymocyte-associated T6 antigens. Thus, given the fact that 10-20% of T cell acute lymphoblastic leukemia (T-ALLs) are CALLA+, we have been able to identify a human prethymic T lymphocyte population that might be the normal counterpart of precursor cell to the CALLA+ T-ALL cell. Udgivelsesdato: 1987-Jun-1

We have investigated the interaction between human platelets and the NK-system, with special emphasis on the action of secretory products from platelets in an NK assay with 51Cr-labelled K562 as target cells. Supernatants from thrombin-stimulated platelets added to the NK assay consistently decreased the NK-cytotoxicity by 40% +/- 4.3%, indicating the existence of secreted products from platelets as a source of NK-inhibiting substances. In contrast, no direct cytotoxic effect of these secretory products on the target cells (K562) was seen. Thus, normal human platelets, when stimulated with thrombin, are capable of secreting different, yet undefined factors, which significantly inhibit NK activity in vitro. The results also suggest that the role of products from contaminating in vitro activated platelets should be borne in mind when performing conventional NK assays. Udgivelsesdato: 1986-Oct

Publisher Summary This chapter focuses on size distribution and β2-microglobulin content of human natural killer (NK) cells analyzed by fluorescence-activated cell sorting (FACS). In a study described in the chapter, normal peripheral blood mononuclear cells were isolated by Isopaque–Ficoll method and stained with a FITC-conjugated rabbit anti-β2-M antiserum. The cells were subsequently processed on a FACS II machine. In the chapter, the dot plot screen of the FACS II from a typical experiment is shown, each cell being represented by a dot giving its size (or forward light scatter, x-axis) and the fluorescence intensity (y-axis). Both lymphocytes and monocytes—as verified by Giemsa-stained smears—can be delineated and lymphocytes can be further subdivided into four lymphocyte subsets according to their β2-M content and size. The FACS was set up to sort the 20% least and the 20% most fluorescing cells, and it was found that cells with low β2-M content, irrespective of size, always exhibited lower NK than the high β2-M containing cells and that IFN-inducible NK cells moreover were found in the β2-M rich cell fractions, indicating that the association between β2-M and NK might be stronger than that between NK and cell size.

IFN proteins are a family of lymphokines with anti-viral effects. Several other effects of IFNs have also been described, including enhancement of natural killer (NK) cell activity, enhancement of cytotoxic T-lymphocyte activity, and enhancement of the expression of major histocompatibility complex (MHC) antigens. The latter effects have been characterized as immunomodulatory, whereas the well-known inhibition of growth of malignant cells has been termed anti-proliferative. This review summarizes the current knowledge of the enhancement of MHC products by IFNs. Whereas the basic methodologies for demonstrating the enhancement are simple and reliable, especially when using flow cytometry (FCM), the biological relevance of this reaction is largely unknown. Based on recent findings, however, we have hypothesized that the above-mentioned diverse effects of IFNs are all - in some way or other - related to the classical anti-viral mechanism. This concept proposes that the MHC-enhancing effect of IFNs is a vital part of the immunological defense against virus infections and an integral part of the anti-viral effects of IFN proteins. Udgivelsesdato: 1988-Nov

By E-rosette sedimentation procedures and fluorescence activated cell sorting of monoclonal antibody-coated T lymphocytes, we have investigated the relationship between the human T lymphocyte subsets defined by the monoclonal antibodies against the T4 and T8 antigens and those defined by avidity differences to sheep erythrocytes. Through a correlation was demonstrated between the T4+ (inducer) cells and the high avidity ("active") T cells and between the T8+ (suppressor) cells and low avidity T cells, these subsets were far from identical, and it is concluded that the application of monoclonal antibodies represents another valuable and standardized approach for estimating T cell subsets. Udgivelsesdato: 1982-Apr

Fractionation of human peripheral blood lymphocytes according to either their size or their amount of β 2 -microglobulin ( β 2 -M) or HLA molecules in the fluorescence-activated cell sorter has enabled us to test the possible relationship between the NK activity, on the one hand, and the amount or the density of surface antigens on the other. Generally, large lymphocytes exhibited higher NK activity than the small ones, and likewise, lymphocytes with high content of β 2 -M were better NK effectors than the low β 2 -M containing cells. However, since small lymphocytes with high β 2 -M content were better NK effectors than large lymphocytes with low β 2 -M content, the above-mentioned findings were relative rather than absolute. In contrast to the close relationship between β 2 -M and NK activity, the amount of HLA antigens (measured with a monoclonal anti-HLA framework antibody) was not found to be correlated to NK activity. The notion that β 2 -M was important in the NK reaction was supported by experiments using papain-treated effector cells which showed that a selective removal of β 2 -M from the cell surface was paralleled by a decrease in the NK reactivity. However, since F(ab′) 2 fragments of the anti- β 2 -M antibody did not inhibit NK activity, the β 2 -M molecules itself cannot be a part of the NK receptor. Finally, regarding the enhancement of NK by interferon, it was shown that lymphocytes with high amounts of β 2 -M were more prone to this effect than lymphocytes with low amounts of β 2 -M.