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CD19 CAR T cells induce - in part long-lasting - remissions in heavily pretreated patients with relapsed/refractory B-cell malignancies. However, they are associated with unique toxicities, and patient management therefore requires specific expertise.In this review, we outline the basics of their mode of action and present the currently available data on their efficacy in various B-cell and plasma cell malignancies. Currently approved therapies (Tisagenlecleucel, Axicabtagene ciloleucel, Brexucabtagene autoleucel) for patients are outlined as well as indications where approvals are expected in the near future. We discuss practical aspects of CAR T cell therapy from the patient's initial presentation, over leukapheresis, to CAR T cell transfusion. Additionally, we highlight the pathophysiology and principles of the management of the most common toxicities (cytokine release syndrome [CRS], immune cell associated neurotoxicity syndrome [ICANS] and cytopenias).

Introduction Paramunity-inducing-Factors (PINDs) consist of attenuated/inactivated viruses of various poxvirus-genera, used in veterinary medicine as non-antigen-specific, non-immunising stimulators of the innate immune system against infectious and malignant diseases. Their danger-signaling-interactions were tested for their capacity to improve leukemic antigen-presentation on DC generated from AML-patients’ blasts (‘DCleu’) and DC-stimulation/activation of antileukemic T-cells. Methods We analyzed, whether the addition of PINDs during DC cultures (15 healthy, 22 leukemic donors) and mixed lymphocyte culture (MLC, n = 15) with autologous (n = 6), allogeneic (n = 2) or T-cells after stem cell transplantation (SCT; n = 7) would alter the quality and quantity of DC, the composition of T-cell-subsets, and/or their antileukemic functionality (AF) as studied by FACS and functional Fluorolysis-cytotoxicity-assays. Results Effects on 1. DC-cultures: PINDs in DC-cultures lead to increased proportions of mature DC and DCleu, but reduced proportions of viable and overall, as well as TLR4- and TLR9-expressing DC. 2. MLC: PINDs increased early (CD8+) T-cell activation (CD69+), but reduced proportions of effector-T-cells after MLC 3. AF: Presence of PINDs in DC- and MLC-cultures reduced T-cells’ as well as innate cells’ antileukemic functionality. 4. Cytokine-release profile: Supernatants from PIND-treated DC- and MLC-cultures resembled an inhibitory microenvironment, correlating with impaired blast lysis. Conclusions Our data shows that addition of PINDs to DC-cultures and MLC result in a “blast-protective-capacity” leading to impaired AF, likely due to changes in the composition of T-/innate effector cells and the induction of an inhibitory microenvironment. PINDs might be promising in treating infectious diseases, but cannot be recommended for the treatment of AML-patients due to their inhibitory influence on antileukemic functionality.

Regardless of chronic lymphocytic leukaemia (CLL) being the most common adult leukemia, therapeutic approaches remain mainly palliative. Particularly in relapsed and refractory malignancies lenalidomide demonstrated promising efficacy and it is a therapeutic approach especially in elderly patients. Lenalidomide is a second generation analog of thalidomide and as such an immunomodulating drug (IMID) with various effects on the microenvironment of tumor cells. Direct cytotoxicity in CLL has been negated so far. The presented work investigates effects of lenalidomide on the tumor cell in vitro. Therefore peripheral blood mononuclear cells (PBMC) of CLL patients and CD19 sorted tumor cells have been treated and compared. Beside cell viability, intracellular pathways and cell activation have been measured. In contrast to the present postulate lenalidomide had a direct cytotoxic effect on CLL cells. An effect that can only be observed after a few days though. After 96 hours lenalidomide reduced cell viability in 24 of 27 patients by an average of 25 % compared to the negative control (p < 0,0001) and reduced ATP in all measured patients. It had an even greater effect on the sorted tumor cells compared to the unsorted environment. Particularly high risk patients (unmutated IgVH, 17p deletion, TP53 mutation) showed good treatment response. An elevated caspase-3 activity suggests that apoptosis might play a role. However the fact that the effect could not be antagonized by caspase inhibition (z-VAD-FMK) indicated that caspase independent mechanisms are involved too. A suppression of the canonical NF-κB pathway as with the predecessor thalidomide could not be demonstrated. A direct cytotoxic effect of lenalidomide in CLL represents a completely new approach. Although underlying mechanisms remain to be revealed, the good response of high risk subtypes could help allocating therapy to patients. Furthermore understanding the role of pathways such as PI3K/Akt or NF-κB in this context could help to understand the pathophysiology of the malignancy itself., Trotzdem die Chronisch Lymphatische Leukämie (CLL) die häufigste Leukämie beim Erwachsenen darstellt, bleiben die verfügbaren Therapien weiterhin von vorwiegend palliativem Charakter. Besonders bei refraktären Patienten und Rezidiven hat Lenalidomid vielversprechende Ergebnisse erzielt und stellt gerade beim älteren Patienten einen Therapieansatz dar. Als Zweitgenerationsanalogon von Thalidomid gehört es zur Gruppe der Immunmodulatoren (IMID) mit einem vielseitigen Wirkungsspektrum auf das Mikroenvironment der Tumorzelle. Direkte zytotoxische Effekte wurden in der CLL bisher verneint. In der vorliegenden Arbeit wurden der Einfluss des Medikaments auf die Tumorzelle in vitro untersucht. Dazu wurden periphere mononukläre Zellen (PBMC) von CLL-Patienten und CD19-angereicherte Tumorzellen der selben Patienten behandelt und verglichen. Untersucht wurde neben der Viabilität, intrazelluläre Signalpathways sowie die Zellaktivierung. Im Gegensatz zum bisherigen Postulat konnte hierbei gezeigt werden, dass Lenalidomid einen direkten zytotoxischen Effekt auf die CLL-Zelle besitzt. Ein Effekt, der jedoch erst nach einigen Tagen zum Tragen kommt. Nach 96 Stunden Behandlungszeit zeigte sich bei 24 der 27 untersuchten Patienten eine Reduktion der Viabilität um durchschnittlich 25 % im Vergleich zur Negativkontrolle (p < 0,0001) sowie eine ATP-Reduktion bei allen untersuchten Patienten. Dieser Effekt war zudem ausgeprägter, als bei der Behandlung der Tumorzelle in PBMC-Umgebung. Besonders Hochrisikopatienten (fehlende IgVH-Mutation, 17p-Deletion, TP53-Mutation) zeigten ein gutes Ansprechen. Eine erhöhte Caspase-3-Aktivität in den behandelten Zellen lässt zudem vermuten, dass die Induktion von Apoptose dabei eine Rolle spielt. Eine fehlende Antagonisierbarkeit durch Caspase-Inhibition (z-VAD-FMK) legt jedoch nahe, dass weiter caspaseunabhängige Mechanismen beteiligt sind. Für eine Suppression des kanonischen NF-κB -Pathways, wie bei der Vorgängersubstanz Thalidomid, gab es indes keinen Hinweis. Ein direkter zytotoxischer Effekt in der CLL stellt einen völlig neuen Ansatz dar. Auch wenn die zugrunde liegenden Mechanismen zu klären bleiben, kann das gute Ansprechen prognostisch ungünstiger Subtypen ein Anstoß bei der Zuordnung von Patient und Therapie sein. Welche Rollen PI3K/Akt oder NF-κB dabei spielen bleibt zu klären, kann aber auch dabei helfen die Pathophysiologie der Erkrankung selbst zu verstehen.

Infection with Epstein-Barr virus (EBV) affects most humans worldwide and persists life-long in the presence of robust virus-specific T-cell responses. In both immunocompromised and some immunocompetent people, EBV causes several cancers and lymphoproliferative diseases. EBV transforms B cells in vitro and encodes at least 44 microRNAs (miRNAs), most of which are expressed in EBV-transformed B cells, but their functions are largely unknown. Recently, we showed that EBV miRNAs inhibit CD4(+) T-cell responses to infected B cells by targeting IL-12, MHC class II, and lysosomal proteases. Here we investigated whether EBV miRNAs also counteract surveillance by CD8(+) T cells. We have found that EBV miRNAs strongly inhibit recognition and killing of infected B cells by EBV-specific CD8(+) T cells through multiple mechanisms. EBV miRNAs directly target the peptide transporter subunit TAP2 and reduce levels of the TAP1 subunit, MHC class I molecules, and EBNA1, a protein expressed in most forms of EBV latency and a target of EBV-specific CD8(+) T cells. Moreover, miRNA-mediated down-regulation of the cytokine IL-12 decreases the recognition of infected cells by EBV-specific CD8(+) T cells. Thus, EBV miRNAs use multiple, distinct pathways, allowing the virus to evade surveillance not only by CD4(+) but also by antiviral CD8(+) T cells.

The extra-chromosomal virulence plasmid Spv (Salmonella plasmid of virulence) plays an important role in Salmonella enterica pathogenicity encoding amongst others for the ADP-ribosyltransferase SpvB. The SpvB protein catalyzes the modification of G-actin with mono-ADP-ribose resulting in the depolymerization of the actin cytoskeleton. In infected cells, SpvB is translocated into the host cell cytosol from intracellular Salmonella-containing vacuoles. To study the effects of SpvB we used three different S. enterica strains: wild type (WT), a SpvB-deficient strain (Mvp 528) and a strain with an additional plasmid carrying a HA-tagged SpvB (p2967). The aim of our project was primarily to characterize SpvB in these strains in terms of expression and activity and to analyze the secretion of SpvB in more detail. First, all three S. enterica strains were characterized with respect to their growth properties and showed a similar exponential growth. In addition, SpvB was detected in strain p2967 by a polyclonal anti-SpvB antibody, whereas strain Mvp revealed no SpvB expression. The presence of HA-tagged SpvB in strain p2967 was confirmed by immunodetection with an anti-HA antibody. To check the biological activity of the expressed SpvB, we performed an in vitro ADP-ribosylation assay. Modification of G-actin was observed in WT strain and, to a higher extent, in p2967 strain. In contrast, the Mvp strain did not catalyze the modification of G-actin. Afterwards, we established an infection model in mammalian cells. Therefore, we tested the suitable multiplicity of infection (MOI) by using MOIs between 0.1 and 100 and observed the morphological changes during 24 h. Next, we showed the successful uptake of Salmonella into the macrophages by plating lysates from infected J774A.1 cells on LB-Agar. To gain more insight into the translocation of SpvB and to address a possible participation of host cell chaperons, we finally analyzed the fate of SpvB in infected cells.

Interferon alpha (IFN-α) and interferon beta (IFN-β) are naturally occurring cytokines, which belong to the type I interferons and share the same receptor leading to very similar therapeutic effects. The immunomodulatory effect of type I interferons includes modulation of antibody production, inhibition of lymphocyte proliferation, inhibition of delayed-type hypersensitivity and enhancement of T-cell and NK-cell cytotoxicity. An increasing number of open clinical studies and case reports have demonstrated the efficacy of IFN-α for severe ocular inflammation in patients with Behçet's disease and of interferon-β, which has been used mainly for the treatment of multiple sclerosis.

Immunotherapeutic strategies may be a treatment option in patients with refractory acute myelogenous leukemia (AML) or, in cases of complete remission after conventional therapy regimens, may help to reduce disease recurrence or delay time to progression. Evidence suggests a key role of dendritic cells (DCs) in cancer immunotherapy due to their capacity to present tumour antigens to effector cells. We generated cytokine-induced killer (CIK) cells from healthy donors and examined their responses in vitro in an LDH release assay against three cell lines and allogeneic HLA non-matched blasts from three patients with de novo AML after coincubation with autologous peripheral blood monocyte-derived DCs. Although DCs were unable to enhance CIK cell effects against all three cell lines tested, the cytotoxic activity against the patients’ AML cells increased after coculture with mature DCs, which was significant in two of three patients. However, neither prior pulsing of the DCs with blast cell lysates nor with leukemic cell-derived total RNA further enhanced the lytic capacity of the CIK cells. On the contrary, pulsing reduced or even reversed the cytotoxic activity of the effector cells. This decrease of allogeneic cytotoxicity led us to conclude that monocyte-derived DCs may be useful in autologous or allogeneic vaccine strategies for the treatment of AML or in priming donor lymphocytes in vitro, but unfractionated antigens as pulsing agents may have inhibitory effects on T cell efficiency and their employment in immunotherapeutic strategies for AML seems questionable., GMS German Medical Science; 9:Doc18; ISSN 1612-3174

Formaldehyde (FA) is an important industrial compound. It has also applications as a disinfectant and preservative. Furthermore, FA is naturally occurring in cells, tissues and body fluids. It is known to be genotoxic; the primary DNA alterations induced by FA are DNA-protein crosslinks (DPX). The aim of this work was to characterize various aspects of the genotoxicity of FA in vitro and in vivo. The concentration-effect relationship of genotoxic effects of FA in three genotoxicity tests was characterized by fitting different curves to the data. The results suggest a thresholded mechanism of FA-induced genotoxic effects. The induction and the repair of FA-induced DPX were investigated in A549 human lung cells and primary human nasal epithelial cells. The susceptibility of both cell types for the induction of DPX is very similar. DPX are repaired by these cells within about 8 hours. The expression of FA-metabolizing enzymes (Glutathion-dependant FA dehydrogenase, aldehyd-dehydrogenase 1A1, aldehyd-dehydrogenase 2) in A549 cells was analyzed after FA-exposure for 1,4 and 24 hours by real time PCR. No significant alterations were measured. The induction of genotoxic effects by FA was investigated in human blood cultures. The results show that high amounts of DPX are needed at the start of the culture to induce cytogenetic effects in lymphocytes. Although several biomonitoring studies reported genotoxic effects in the blood of subjects exposed to FA, the results shown here suggest that it is very unlikely that such effects are actually related to FA-exposure. An inhalation study with human volunteers was conducted to assess local mutagenic effects of FA by the micronucleus test with exfoliated buccal mucosa cells. FA peak exposure was up to 1 ppm and the cumulative dose was 13,5 ppm h over two weeks. The results indicate that under these exposure conditions, FA does not induce micronuclei in buccal mucosa cells.

Suppression of NK cell activity is considered to be an unfavourable prognostic factor for tumour progression. There is proof that mistletoe extracts may increase NK cell activity. However, the inverse relation between an increase of NK cell activity and clinical progress of cancer has not been investigated.The relation of NK cell activity and progress of cancer in patients under therapy with mistletoe extracts was examined in a prospective, monocentric, cohort study. At the same time the in vitro killing of K562 cells and autologous tumour cells was compared.40 patients with operable cancer of the breast or colon were included. The patients did not receive any immunologically relevant therapies except for mistletoe extracts. The absolute NK cell count in peripheral blood as well as the in vitro NK cell activity were monitored for up to 2 years and compared with clinical outcome as well as quality of life.The absolute NK cell count in peripheral blood increased within the observation period. Patients without progression had a significantly higher mean activity of NK cells against K562 cells than patients with progression. In the latter group, only stage IV patients showed reduced NK cell activity. The killing activity against autologous tumour cells was5% in about 77.5% of the patients and could not be evaluated further. The NK cell activity against K562 cells was not related to the number of NK cells.We found a relation between NK cell activity and the progression of malignant disease. In further studies the causality of this relation has to be clarified. The establishment of NK cell activity against autologous tumour cells as a suitable parameter during follow-up was not successful.

The expression of B7-1/2 molecules in addition to TAA in APC significantly improves the generation of specific CTL in vitro. Moreover, human fibroblasts, usually devoid of APC capacity, can be engineered into effective APC upon infection with recVV encoding costimulatory molecules together with specific epitopes. The generation of artificial APC by infection of non professional APC with appropriately engineered recVV is a new concept that may be useful in the implementation of immunotherapy strategies.

Cytotoxic T-Lymphocytes were generated from tumor-infiltrating lymphocytes of a newly established pancreatic cancer cell line, which lysed autologous tumor cells significantly and HLA-class I restricted. ICAM-1+ tumor clones were significantly higher lysed than an ICAM-1- clone. Stimulation with IFN-gamma resulted in a higher expression of ICAM-1 and significantly higher lysis, which was inhibited by anti-ICAM-1 monoclonal antibodies.

Bispecific antibodies (bsAbs) can efficiently mediate tumor cell killing by redirecting preactivated or costimulated T cells to disseminated tumor cells, especially in a minimal residual disease situation. This study demonstrates that the trifunctional bsAb BiLu is able to kill tumor cells very efficiently without any additional costimulation of effector cells in vitro and in vivo. Remarkably, this bsAb also induces a long-lasting protective immunity against the targeted syngeneic mouse tumors (B16 melanoma and A20 B-cell lymphoma, respectively). A strong correlation was observed between the induction of a humoral immune response with tumor-reactive antibodies and the survival of mice. This humoral response was at least in part tumor specific as shown in the A20 model by the detection of induced anti-idiotype antibodies. Both the survival of mice and antitumor titers were significantly diminished when F(ab′)2 fragments of the same bsAb were applied, demonstrating the importance of the Fc region in this process. With the use of T-cell depletion, a contribution of a cellular antitumor response could be demonstrated. These results reveal the necessity of the Fc region of the bsAb with its potent immunoglobulin subclass combination mouse immunoglobulin G2a (IgG2a) and rat IgG2b. The antigen-presenting system seems to be crucial for achieving an efficient tumor cell killing and induction of long-lasting antitumor immunity. Hereby, the recruitment and activation of accessory cells by the intact bsAb is essential.

Dendritic cells play an important role within the mucosal immune system of the upper aerodigestive tract. They process exogenous and endogenous antigens and are able to induce a cytotoxic Tc/s lymphocyte reaction against tumors. Recently published data indicate that the in vitro application of tumor lysate incubated dendritic cells can provide a defense against melanoma. Before these new therapeutic strategies are available for the therapy of laryngeal cancer, basic studies have to be performed concerning the distribution of dendritic cells and other subpopulations, such as T- and B-lymphocytes and macrophages. In the present study the distribution of these subpopulations were examined within the epithelial and connective tissue compartments ("tumor front") of 20T2 squamous cell carcinomas of the supraglottis and glottis. The number of dendritic cells was compared with clinical parameters to check whether a high number of dendritic cells could be correlated with a better prognosis. In contrast to T-lymphocytes, results showed that dendritic cells were mainly located within the epithelial compartment of the tumors, their number ranging from 20 cells/mm2 to700 cells/mm2. By comparing each patient's clinical course with the number of dendritic cells, findings showed that those patients who died within the first postoperative year were characterized by a very small number of dendritic cells within their tumor tissue (100 cells/mm2). Although the number of patients was low, results indicate that a high number of dendritic cells within tumor tissue suggest a better prognosis.

Drug-induced toxic epidermal necrolysis (TEN) is a rare but frequently lethal disease of the skin and mucosa of unknown etiology. Toxic metabolic and immunologic products in the serum have been discussed as possible causes. For this reason we have performed plasmapheresis on these patients for many years. Results regarding survival and healing of the lesions are quite favorable, as exemplified with two patients described here who benefited from the procedure despite their old age. In order to gain insight into possible disease mechanisms that would explain the beneficial effect of plasmapheresis, we did immunohistochemical studies on these patients before and in one case after the procedure. In dermis and epidermis, there was a striking increase of macrophages and a relative increase of CD8-positive lymphocytes which might act as cytotoxic effector cells; these findings can be interpreted as the result of an immunological reaction. An increased staining of lesional epidermis with TNF alpha, as previously noted by other authors, should be interpreted with caution since necrotic cells are involved. On the basis of the clinical results, the pathogenetic considerations, and in view of the lack of specific treatment modalities, clinical studies concerning plasmapheresis for the treatment of toxic epidermal necrolysis are recommended, particularly since this procedure is widely available and generally well tolerated.

In this study the cytotoxic activity of natural killer cells (NK) and lymphokine activated killer cells (LAK) in the peripheral blood was examined in ten patients with AIDS and in ten patients with Crohn's disease. The results were compared to the values of ten healthy patients. Cells of the lamina propria of the colon were isolated and cultivated to LAK-cells after the in vitro stimulation with interleukin-2 from patients belonging to these three groups. The highest NK-activity of the monoclonal cells in the peripheral blood (PBMC) was found in the control group. In patients with AIDS and Crohn's disease the activity of the LAK-cells of PBMC was significantly higher than the activity of the NK-cells. The PBMC activity of NK- and LAK-cells in the control group showed no important differences. The spontaneous cytotoxic activity of mononuclear lamina propria cells was very low or not verifiable. In lamina propria LAK-cells only a very low cytotoxic activity was found too. These results show that an in vitro stimulation of PBMC with interleukin-2 in patients with AIDS or Crohn's disease lead to a significant increase of the cytotoxic cell activity.

In this study we examined a combined immunotherapy using lymphokine activated tumor infiltrating lymphocytes and active specific immunotherapy with virus-modified tumor cells in disseminated breast and ovarian cancer patients. During the therapy a significant increase in cytotoxicity could be observed, which nevertheless could not be maintained after the end of therapy. In an ongoing prospective randomized study we are using antigen specific stimulated cytotoxic T-lymphocytes to extend this effect.

Immunopathological reactions may determine the pathogenesis of some viral infections of cats and dogs. Three pathomechanisms may aggravate the viral disease or may ultimately cause death. Some viruses cause transient or persistent immunosuppression (Feline Immunodeficiency, Feline Leukemia, Feline Panleukopenia Virus, Canine Parvovirus-2, Canine Distemper Virus). In other viral infections cells and tissues are destroyed as a sequela of cell-mediated cytotoxicity reactions (demyelinating encephalitis in distemper). The third example of a pathogenic immune reaction is virus-induced immune complex diseases (infections with FeLV, FIPV and CAV-1).

The Reticuloendothelial System (RES) consists of cells descending from the monocytes which are able to perform phagocytosis of foreign materials and particles. 90% of the RES are located in the liver. The most important function of the RES ist phagocytosis, but it also takes part in cytotoxicity against tumor cells and has a function in the regulation of the immune system. The importance of the RES is obvious in diseases of the bile ducts. An obstruction can lead to a decrease in the function of the RES. Beside a mathematical model there are also a few experimental methods to measure the phagocytic capacity and thus the efficiency of the RES. They are based on a clearance study of the amount of particles absorbed by the RES. This study can e.g. be done with Nanocoll which has been marked with 99mTc. But instead of the bloody clearance, a scintigraphic study using a gamma camera can be performed. With that, the increase time, the liver extraction fraction and the steepness of increase can be determined. Such a test could be of clinical relevance as a parameter for the prognosis of patients with trauma, sepsis or shock. The function of the RES in the pathogenesis of the biliary pancreatitis could also be examined closer with the help of such a test. The requirements a test of the RES should meet are reproducibility, repeatability and practicability.

Although identified over a hundred years ago, the precise role of the eosinophil in eosinophil-associated diseases still remains undefined. However, the results of modern cellular, immunological, and biochemical investigations have suggested that eosinophils contain unique constituents and functional adaptions through which eosinophils gain a profound cytotoxic capacity. This property may not only be crucial for the host defence mechanism against helminthic infections but may also be involved in the pathogenesis of chronic inflammation associated with allergic reaction and other eosinophilic disorders. There is increasing evidence available that the eosinophil may cause damage to host tissue and elicit tissue fibrosis. The eosinophil appears to adapt certain general cellular characteristics during its ontogeny and participation in inflammatory reactions, such as hypodensity, expression and induction of surface markers and adhesion molecules. In addition, there is evidence that the eosinophil may undergo a shift between hypo- and hyperreactivity states. Factors regulating the level of eosinophil activation may include both lipid mediators and cytokines released by macrophages, mast cells, platelets, endothelial cells, and T lymphocytes. The rapidly evolving understanding of the functional properties of the eosinophil will further help to define the pathogenesis of certain eosinophilic disorders and may provide the basis for new therapeutic strategies in eosinophilic disorders.

HLA class I lymphocytotoxicity testing is strongly complement-dependent. We tested 13 commercially available rabbit complement batches (7 distributors) using a panel of 14 donors with decisive patterns of HLA antigens. We defined a scoring system that weights the different possible faulty reactions. The rating score ranged from 12.45 to 46.55 (median 16.09). Comparing the reactions in batches with low and high rating scores gave statistically significant differences (chi 2 test, p0.05). Our complement reaction score may be helpful to find the optimal complement batch for HLA class I lymphocytotoxicity testing.

ICAM-1-mediated cell-cell adhesion is essential for various immunologic functions, including non-MHC-restricted cytotoxicity. Shedding of ICAM-1 occurred after stimulation of melanoma cells with TNF alpha and IFN gamma. Soluble forms of ICAM-1 inhibited the conjugate formation of NK clones with K562 and of LAK cells with melanoma cells. Furthermore, the non-MHC-restricted cytotoxicity mediated by NK clones or LAK cells could be abrogated by soluble ICAM-1.

We have previously shown that social confrontation between aggressive fish (e.g., Tilapia) produces a suppression of several immunological parameters--nonspecific cytotoxicity and mitogen-stimulated proliferation in pronephric lymphocytes--in the subordinate fish. By using the opioid antagonist, naltrexone, we now demonstrate indirectly that this immunosuppression is in part mediated by the endogenous opioid system. Evidence is presented that naltrexone-mediated reversal of immunosuppression may be limited to the populations of the cytotoxic and T-cell lineages. The proliferative response to lipopolysaccharide is unaffected by naltrexone. Our data also demonstrate that serum from subordinate (immunosuppressed) fish is immunosuppressive in normal fish: an effect that can be reversed by naltrexone. These results support a link between the neuroendocrine and immune systems in these animals.

As compared with the lymphatic system, the mononuclear phagocyte system (MPS) represents the older defense system in the course of evolution. Organisms with defective or with no lymphocytic function are able to live for a certain time while metazoa cannot develop and exist without cells capable of phagocytosis. It is known that up to 80% of the mass of malignant experimental tumors may consist of macrophages. The biological relevance of these tumor-associated macrophages (TAM), however, is contested to date. Besides well-known tasks in specific and non-specific defense, the cells of the MPS possess nutritive functions. The different functions of TAM have been essentially examined in animal experiments in vivo and in vitro up to now. These studies have shown, e.g., that TAM of different phenotypes to some extent are assigned to different functions and that each tumor or tumor cell line has a specific TAM pattern. The available number of monoclonal antibodies (MAB) able to recognize different groups of human macrophages has only increased in the recent past. This also provided evidence of the phenotypic heterogeneity of macrophages in the human system. On the other hand, there is little knowledge to date of the possible biological cause of the presence of numerous macrophages also in the stroma of malignant tumors in man and on the functional relevance of particular macrophage phenotypes. In the present studies, the phenotypic pattern of tumor-associated cells was examined by immunohistochemistry in the model of human mammary carcinoma, using 18 monoclonal antibodies (Ki-M1-8, Leu-M1-3, Leu-M5, EBM11, CD1, anti-transferrin receptor [TFR], anti-MHC I, anti-MHC II or anti HLA-DR) and one polyclonal antibody (anti protein-S100). The first part of the study contains a semiquantitative evaluation of 216 cases of mammary carcinoma. The analytical results are correlated with established prognostic factors available in the case of malignant tumors in general and in that of mammary carcinomas in particular (age, menopausal status of patients, axillary lymph node and estrogen/progesterone receptor status, size of tumor, tumor type according to WHO, degree of malignancy according to histopathological and nuclear grading). As tumor parameters of absolute biological relevance, proliferating activity (Ki67) and MHC phenotype of tumor cells and amount or types of tumor-associated lymphocytes (TAL) are examined in order to analyze their correlation with prognostic factors and their importance in the tumor-macrophage system.(ABSTRACT TRUNCATED AT 400 WORDS)

Human proto- and metazoa, which spend a part of their life-cycle within erythrocytes or extracellularly in human blood or in monocyte-derived macrophages, are considered as blood parasites. In spite of their individually widely differing habitat, they elicit a common hematological syndrome essentially consisting in hemolytic anemia and splenomegaly. Furthermore, some basic factors of host/parasite interaction are very similar: (1) Specific cell attachment through receptor/ligand mechanisms such as the integrin/RGD-oligopeptide system, (2) competition for essential nutrients such as iron, which may be influenced therapeutically to the disadvantage of the parasite by the use of iron chelators, and (3) provocation of host defense through T-cell dependent, cytokine-mediated macrophage activation.

Interleukin 2 (IL-2) is predominantly produced by T-helper cells (TH1) having the phenotype CD4+, and by subpopulations of thymocytes after antigenic or mitogenic stimulation. IL-2 causes an indefinite growth of T-cells, and its function depends on binding to IL-2 receptors (IL-2R alpha and IL-2R beta). Thus the immune response of T cells is controlled through the expression of the IL-2 receptors and the IL-2 binding. IL-2 receptors are expressed not only by T-cells but also by B-cells, NK cells, monocytes, thymocytes, thymic stroma cells, oligodendrocytes and endothelial cells. This explains the various functions of IL-2, such as increased immunoglobulin production, growth of certain B-cell subpopulations, macrophage-dependent cytotoxicity, growth and differentiation of oligodendrocytes and proliferation of lymphokine activated killer (LAK) cells. Abnormal production of IL-2 may lead to autoimmune diseases, immunodeficiencies and, under certain circumstances, to T-cell leukemia. With antibodies against the IL-2 receptors the binding of IL-2 may be blocked to avoid auto-aggressive destruction in autoimmune diseases. LAK cells increase the growth of NK cells and T-cell cytotoxicity against transformed cells. LAK cells, especially those from tumor infiltrating lymphocytes, in conjunction with IL-2 have already been used with promising initial results in the treatment of distant metastases. In the future LAK cell therapy with IL-2 may be adopted to prevent metastases and second primary tumors in high-risk patients with head and neck cancer.

Cytokines are mainly produced by monocytes and lymphocytes and influence a broad spectrum of physiological processes within the hematopoietic and the immune system. Clinical trials have mainly been initiated with a subgroup of cytokines namely the hematopoietic growth factors. Interleukin 3 (IL-3) was recently introduced into phase I and II trials. This factor was also termed "multipoietin" because of its ability to stimulate in vitro the hematopoietic stem cell and thus all three hematopoietic cell lines. The latest in vivo data, however, demonstrate that IL-3 stimulates leukopoiesis and megakaryopoieis with only marginal effects on erythropoiesis. In large clinical trials the therapeutic effect of this factor is investigated in patients with myelodysplastic syndromes. Further indications will be other primary bone marrow failures and aplasia caused by bone marrow transplantation, aggressive chemotherapy and radiation. Potential applications offered by combinations with other cytokines can hardly be foreseen.

The immunomodulator Uro-Vaxom (an immunoactive fraction of E. coli, FEC) is a therapeutic agent used to control bacterial infections. FEC was applied to macrophages of C57BL/6 mice to investigate the in vitro activation of these cells of the unspecific immune system. Experiments were designed to test the secretory, immuno-regulatory and cytotoxic function of macrophages after application of FEC. As presented here, production of Interleukin-6 and tumor-necrosis-factor were significantly and dose-dependent way increased, whereas it was not possible to induce a secretion of Interleukin-1. In addition, FEC activated macrophages kill Leishmania donovani promastigotes, Candida albicans and Staphylococcus aureus. In comparison to a pressed echinaceae-preparation, FEC activated mouse macrophages secrete Interleukin-6 and tumor-necrosis-factor and kill protozoa, fungi and bacteria, with higher efficiency.

The role of Interferon-gamma (IFN-gamma) in the immunotherapy of neuroblastoma was investigated. In vitro experiments showed that IFN-gamma augments the cytotoxicity of Natural Killer (NK) cells and of interleukin 2 (IL-2)-activated NK (LAK) cells against neuroblastoma target cells. Incubation of the neuroblastoma cells with IFN-gamma resulted in an increased susceptibility of these target cells to NK and LAK cells. Additionally, the IFN-gamma-treated neuroblastoma cells showed an increased susceptibility to the antibody-dependent cellular cytotoxicity (ADCC). In patients who have been treated with continuous infusions of IL-2, IL-2-induced secretion of IFN-gamma was detected by measuring the elevation of the 2-5 A synthetase activity in peripheral mononuclear cells or the 2-5 A oligoadenylates in the serum, although IFN-gamma itself was not detectable. From these results we conclude that IFN-gamma may play an important role in the immunotherapy of neuroblastoma in combination with IL-2 and/or with monoclonal antibodies.

The cytokine interleukin 2 (IL 2) has proved to be an effective agent in immunotherapy of human cancers. Natural killer cells and cytotoxic T-lymphocytes are activated via membrane receptors by IL 2. IL 2-activated natural killer cells and cytotoxic T-lymphocytes develop high cytolytic activity against a variety of tumor cells in vitro. After extensive animal experiments, IL 2 was used in humans either alone or in combination with lymphokine-activated killer cells or tumor-infiltrating lymphocytes. Its application in humans is still experimental, although initial results are very promising. Further studies are necessary to establish treatment protocols with high therapeutic efficacy and tolerable side effects.

Immunohistochemical investigations on skin lesions of lichen ruber planus indicate HLA-DR-expression by keratinocytes, Langerhans cells (LC), and infiltrated mononuclear cells. In the early stage of this condition, the lymphocytic infiltrate consists of a considerable part of (CD8+) cytotoxic/suppressor- and (LGL+) natural killer cells. Evidently, the cytotoxic potential of these lymphocyte subpopulations is directed against basal keratinocytes and epidermal LC. As a result of cytotoxic/cytolytic activities, non-cell associated CD 1-reactivity accumulates in the dermoepidermal junction zone, thus indicating damaged LC.

There were anti-B-lymphocyte toxins in 40 samples (= 7.58%) from 527 examined sera of pregnant women and polytransfused persons without HLA cytotoxins and 84 samples (= 33.2%) from 253 anti-HLA sera. Of 49 specific anti-B sera 27 were suitable for typing B-lymphocyte antigens; with the help of 13 of these sera 5 specific antigens of B-lymphocytes called 1-5 could be determined. These 27 sera produced a positive cytotoxic reaction, mainly with the strength of +++ or ++. Attempts of absorbing HLA antibodies from anti-B lymphocyte sera led to unsatisfactory results. Sometimes the content of anti-B-lymphocyte toxins could also be diminished by a platelet absorption, in other cases the absorption was insufficient and had to be repeated therefore.

Thrombocytopenic patients refractory to random-donor platelet support can usually be substituted by platelets obtained from donors identical with respect to HLA-A and -B antigens [1]. Since such "full-house" identity is rarely available, reliable cross-match tests (CM) are needed in order to pick out donors compatible in spite of HLA non-identity. By use of the thrombocyte migration test (TMT), DUQUESNOY et al. have shown that antiplatelet antibodies may exert an inhibitory effect on platelet migration. Therefore, we have explored this test system for its efficiency as CM compared with the "long-time" lymphocyte cytotoxicity test (LLT). Of 8 sera from polysensitized patients, serial dilutions were performed and simultaneously tested by TMT and LLT with cells obtained from the same control individuals. The sensitivity of TMT exceeded LLT by 2-4 dilution steps in all sera tested. Moreover, 9 patients with incompatible transfusion responses to single-donor platelets in spite of negative LLT had the following results in TMT with pre-transfusion sera: 5 positive (i.e. migration inhibition), 1 negative: enhanced migration was observed 3 times. This phenomenon of enhancement remains to be clarified, since low titers of anti-HLA-antibodies do not cause enhanced platelet migration. Repeated transfusion of platelets obtained from the same donor may cause antibody production against leukocytic antigen not shared by platelets, leading to "false-positive" LLT. 2 patients were successfully substituted with platelets from HLA/MLC-identical siblings in spite of positive LLT, but with negative TMT; in 2 cases with unrelated HLA-A and -B identical donors, TMT results were inconsistent.

It was shown by means of radioactive labelling that adult animals and humans are also able to absorb protein intact in its biological active form from the gastro-intestinal tract. A large part of these macromolecules retains their original biological activity. Depending on size, particles (polls, synthetic particles) can also be absorbed. The energy supply is not influenced by this phenomenon. It is postulated that a rudimentary mechanism present in the lymphatic system of the gastro-intestinal tract is involved. This absorbation mechanism receives information from the immune system about foreign and the body's own substances and about useful and unuseful nutriment. On the basis of investigations, carried out by us, it is speculated that immunological information takes place over the lymphatic tissue of the gastro-intestinal tract and is passed on the lymphatic system.