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The hemoglobin-haptoglobin scavenger receptor CD163 is present in both a membrane-bound form on monocytes and macrophages (mCD163) and a shed soluble circulating form (sCD163). CD163 is a well-described marker of M2-like tumor-associated macrophages, but in patients with metastatic renal cell carcinoma (mRCC), monocyte mCD163 and serum sCD163 levels have not previously been investigated and associated with patient overall survival (OS). Here, we report mCD163 expression on peripheral blood monocytes, as well as sCD163 serum levels, in samples from 89 patients newly diagnosed with mRCC and 20 healthy controls. We found that in mRCC patients, compared to healthy controls, monocyte mCD163 levels were reduced (P

Introduction Natural killer (NK) cells and natural killer T (NKT) cells are implicated in the development and progression of colorectal cancer (CRC). Tumor cells express NK cell receptor ligands that modulate their function. This study aimed to investigate the expression of such ligands in CRC in relation to the phenotype of circulating NK- and NKT cells, and clinical outcome. Methods Primary tumor tissues were analyzed for protein expression of NK cell ligands using immunohistochemistry with automated image analysis in a cohort of 78 CRC patients. For 24 of the 78 patients, RNA expression of NK cell ligands was analyzed in primary tumor tissue using RNA sequencing. Receptor expression on circulating NK- and NKT cells was previously measured by us in 71 of the 78 patients using flow cytometry. Results High Proliferating Cell Nuclear Antigen (PCNA) protein expression in the primary tumor associated with shorter disease-free survival (DFS) of CRC patients (P = 0.026). A trend was observed towards shorter DFS in CRC patients with above-median galectin-3 protein expression in the primary tumor (P = 0.055). High protein expression of galectin-3, CD1d, and human leukocyte antigen (HLA) class I, and high RNA expression of UL16-binding protein (ULBP)-1, -2, and -5, and HLA-E in the tumor tissue correlated with low expression of the corresponding receptors on circulating NK- or NKT cells (P Conclusions Galectin-3 and PCNA expression in the primary tumor may be prognostic biomarkers in CRC patients. Furthermore, our results suggest that NK cell receptor ligands expressed by tumor cells may modulate the phenotype of circulating NK- and NKT cells, and facilitate immune escape of metastasizing cells.

The macrophage-associated molecule CD163 has been reported as a prognostic biomarker in different cancer types, but its role in colorectal cancer (CRC) is unclear. We studied CD163 in the tumor microenvironment and circulation of patients with CRC in relation to clinicopathological parameters. An enzyme-linked immunosorbent assay (ELISA) was used to measure the serum sCD163 levels and multiparameter flow cytometry was used to study the peripheral blood monocytes and their CD163 expression in CRC patients (N = 78) and healthy donors (N = 50). The distribution of tumor-associated macrophages (TAMs) was studied in primary colorectal tumors with multiplex immunofluorescence. We showed that CRC patients with above-median sCD163 level had a shorter overall survival (OS, p = 0.035) as well as disease-free survival (DFS, p = 0.005). The above-median sCD163 remained significantly associated with a shorter DFS in the multivariate analysis (p = 0.049). Moreover, a shorter OS was observed in CRC patients with an above-median total monocyte percentage (p = 0.007). The number and phenotype of the stromal and intraepithelial TAMs in colorectal tumors were not associated with clinical outcome. In conclusion, sCD163 and monocytes in the circulation may be potential prognostic biomarkers in CRC patients, whereas TAMs in the tumor showed no association with clinical outcome. Thus, our results emphasize the importance of the innate systemic immune response in CRC disease progression.

Objective The subset distribution and immunophenotype of circulating immune cells (“peripheral blood immune cell profile”) may reflect tumor development and response to cancer treatment. In order to use the peripheral blood immune cell profile as biomarker to monitor patients over time, it is crucial to know how immune cell subsets respond to therapeutic interventions. In this study, we investigated the effects of tumor resection and adjuvant therapy on the peripheral blood immune cell profile in patients with colon carcinoma (CC). Methods The subset distribution and immunophenotype of T cells (CD3+CD56−), CD56dim NK cells (CD3−CD56dim), CD56bright NK cells (CD3−CD56bright) and NKT-like cells (CD3+CD56+) were studied in preoperative and postoperative peripheral blood mononuclear cell (PBMC) samples of 24 patients with CC by multiparameter flow cytometry. Changes in immunophenotype of circulating immune cells after tumor resection were studied in patients treated with and without (capecitabine-based) adjuvant therapy. Results The NKT-like cell (% of total PBMCs) and CD8+ T cell (% of total T cells) populations expanded in the peripheral blood of non-adjuvant-treated CC patients after surgery. NK- and NKT-like cells showed upregulation of activating receptors and downregulation of inhibitory receptors in non-adjuvant-treated CC patients after surgery. These changes were not observed in the peripheral blood of adjuvant-treated CC patients. Conclusions Our results suggest tumor-induced suppression of NK- and NKT-like cells in CC patients, an effect that could not be detected after tumor resection. In contrast, adjuvant therapy maintained tumor-induced immunosuppression of NK- and NKT-like cells in CC patients.

Tumour-associated macrophages (TAMs) support cancer cell survival and suppress anti-tumour immunity. Tumour infiltration by CD163

Context: We have previously shown that an antidepressant-like effect of probiotics in rats was associated with a higher plasma level of the microbial tryptophan metabolite indole-3-propionic acid (IPA). Objective: We therefore wanted to study the isolated effect of IPA on behaviour and glucose metabolism in rats. Methods: Male Sprague–Dawley rats were fed control or IPA-enriched diet for six weeks (n = 12 per group) and assessed in the elevated plus maze, open field and forced swim test. Blood glucose, metabolic hormones and the white blood cell (WBC) composition were analysed. Results: IPA (mean intake 27.3 mg/kg/day) significantly lowered fasting blood glucose level by 0.42 mM (95% CI 0.11–0.73). Similarly, fasting plasma insulin levels and the homeostatic model assessment (HOMA) index of insulin resistance were reduced, whereas plasma metabolic hormones, behaviour and WBC composition remained unaffected by IPA. Conclusions: Our findings highlight IPA as a promising candidate for treatment of metabolic disorders associated with insulin resistance

Objectives Extracellular vesicles (EVs) are important for intercellular signalling in cancer. Tumour-associated macrophages, expressing the haemoglobin-haptoglobin and mannose receptors CD163 and CD206, are crucial for cancer progression. We recently identified CD163 on EVs in the circulation as a fraction of total soluble CD163 (sCD163). Here, we investigated the presence of CD163 and CD206-positive EVs (EV-CD163, EV-CD206) in patients with multiple myeloma (MM).Methods We enrolled patients with MM (n = 32), monoclonal gammopathy of undetermined significance (MGUS) (n = 8) and healthy donors (n = 16). Plasma protein levels were determined by ELISA before and after vesicle precipitation. Monocytes were examined by flow cytometry, and leucocyte CD163 mRNA by qPCR.Results Fractions of EV-CD163 and EV-CD206 were significantly elevated in patients with newly diagnosed MM (median = 39.8%, 76.5%, respectively) compared to patients with relapse (15.6%, P = .02, 42.5%, P = .003), remission (16.9%, P Conclusions Our results indicate that macrophage-derived EVs may play a role in the late phase of malignant progression of MM, and encourage further EV investigations in functional experiments.

In flow cytometry, a compensation matrix is commonly reused over time, especially in clinical laboratories, to save time and reagents. However, generating a same-day compensation matrix is considered the best practice by many experts. BD Biosciences developed Cytometer Setup and Tracking software to deliver proper instrument characterization, performance tracking, and stability. BD's "Application Settings" enable daily cytometer adjustments of photomultiplier tube (PMT) settings to correct for day-to-day variations in instrument performance. Here, we investigated if using Application Settings would improve data stability over time, including the impact on data stability when reusing a compensation matrix. We consecutively analyzed peripheral blood mononuclear cell (PBMC) aliquots from a single healthy donor together with 8-peak Rainbow beads and daily compensation controls (22 runs in total over 6.5 months). We found larger variation within both PBMC subset quantifications and median fluorescence intensity (MFI) levels when using Application Settings (i.e., daily adjusted PMTs) compared to fixed PMT voltages (both with the same Day 0 compensation matrix applied). This larger variation was partly due to errors in compensation, but was also seen for Rainbow beads MFI data (not impacted by compensation), and thus likely produced by imprecise adjustments of PMTs by Applications Settings. Notably, the larger variation observed with Application Settings was most pronounced on a few days of the experiment with very large deviations, whereas on most days Application Settings and Fixed PMTs performed similar. The present results call for caution in using Application Settings in longitudinal studies, especially if also reusing a compensation matrix. In contrast, reusing a compensation matrix over time with fixed PMT voltages yielded stable results comparable with running same-day compensation controls. © 2020 International Society for Advancement of Cytometry.

Objective As the development and progression of colorectal cancer (CRC) are known to be affected by the immune system, cell subsets such as T cells, natural killer (NK) cells, and natural killer T (NKT) cells are considered interesting targets for immunotherapy and clinical biomarker research. Until now, the role of systemic immune profiles in tumor progression remains unclear. In this study, we aimed to characterize the immunophenotype of circulating T cells, NK cells, and NKT-like cells in patients with CRC, and to subsequently correlate these immunophenotypes to clinical follow-up data. Methods Using multiparameter flow cytometry, the subset distribution and immunophenotype of T cells (CD3+CD56−), CD56dim NK cells (CD3−CD56dim), CD56bright NK cells (CD3−CD56bright), and NKT-like (CD3+CD56+) cells were investigated in peripheral blood mononuclear cell (PBMC) samples from 71 CRC patients and 19 healthy donors. Results CRC patients showed profound differences in immune cell subset distribution and their immunophenotype compared to healthy donors, as characterized by increased percentage of regulatory T cells, and reduced expression level of the natural cytotoxicity receptors NKp44 and NKp46 on both CD56dim NK cells and NKT-like cells. Finally, we showed in a multivariate analysis that above-median percentage of CD16+ NKT-like cells was independently associated with shorter disease-free survival in CRC patients. Conclusion The altered phenotype of circulating immune cell subsets in CRC and its association with clinical outcome highlight the potential use of PBMC subsets as prognostic biomarkers in CRC, thereby contributing to better insight into the role of systemic immune profiles in tumor progression. Electronic supplementary material The online version of this article (10.1007/s00262-019-02343-7) contains supplementary material, which is available to authorized users.

Nonspecific binding of monoclonal antibodies (mAbs) to Fc-receptors on leukocytes is an important cause of background fluorescence in flow cytometry, and failing to block such nonspecific binding can lead to erroneous results. A major part of previous studies on blocking reagents for flow cytometry have been done in mice, and published results are not completely in agreement. In humans, Fc-receptors are found on most leukocytes, with highest abundance on monocytes/macrophages. Therefore, in the present study our aim was to thoroughly investigate the efficiency of different commonly used blocking reagents regarding inhibition of nonspecific binding of mouse mAbs to human peripheral blood mononuclear cells (MNCs) and monocyte-derived macrophages (MDMs). Monocytes and MDMs showed strong nonspecific binding of IgG1 and IgG2a isotypes, but not IgG2b. In contrast, B-cells, T-cells, and NK-cells did not substantially bind any of the mouse isotype control antibodies evaluated (IgG1, IgG2a, and IgG2b). Importantly, we show that binding of IgG1 and IgG2a to monocytes and MDMs can be eliminated by blocking, either with a commercial Fc-blocking reagent, with mouse or human serum, or with mouse or human IgG in high concentration. Previously, isotype controls have been widely used in flow cytometry assays. However, we show that such controls may be highly unreliable, and we believe they should not be used as gating controls, or to determine background signal. Based on these results, as well as considerations of price and applicability, our recommendation is not to use isotypes as gating controls in flow cytometry, but instead to use 100 μg/mL of purified human IgG as blocking reagent for elimination of nonspecific binding of mouse mAbs to human MNCs and MDMs. © 2016 International Society for Advancement of Cytometry

Tumor-associated macrophages (TAMs) are of major importance in cancer-related immune suppression, and tumor infiltration by CD163 pos TAMs is associated with poor outcome in most human cancers. Therefore, therapeutic strategies for reprogramming TAMs from a tumor-supporting (M2-like) phenotype towards a tumoricidal (M1-like) phenotype are of great interest. Activation of the transcription factor STAT3 within the tumor microenvironment is associated with worse prognosis, and STAT3 activation promotes the immunosuppressive phenotype of TAMs. Therefore, we aimed to develop a drug for inhibition of STAT3 specifically within human TAMs by targeting the endocytic CD163 scavenger receptor, which is highly expressed on TAMs. Here, we report the first data on a CD163-targeted STAT3-inhibitory drug consisting of corosolic acid (CA) packaged within long-circulating liposomes (LCLs), which are CD163-targeted by modification with monoclonal anti-CD163 antibodies (αCD163)—CA-LCL-αCD163. We show, that activation of STAT3 (by phosphorylation) was inhibited by CA-LCL-αCD163 specifically within CD163 pos cells, with minor effect on CD163 neg cells. Furthermore, CA-LCL-αCD163 inhibited STAT3-regulated gene expression of IL-10, and increased expression of TNFα, thus indicating a pro-inflammatory effect of the drug on human macrophages. This M1-like reprogramming at the mRNA level was confirmed by significantly elevated levels of pro-inflammatory cytokines (IFNγ, IL-12, TNFα, IL-2) in the culture medium. Since liposomes are attractive vehicles for novel anti-cancer drugs, and since direct TAM-targeting may decrease adverse effects of systemic inhibition of STAT3, the present results encourage future investigation of CA-LCL-αCD163 in the in vivo setting.

Natural killer T (NKT) cells are a subset of CD1d-restricted T cells at the interface between the innate and adaptive immune system. NKT cells can be subdivided into functional subsets that respond rapidly to a wide variety of glycolipids and stress-related proteins using T- or natural killer (NK) cell-like effector mechanisms. Because of their major modulating effects on immune responsesviasecretion of cytokines, NKT cells are also considered important players in tumor immunosurveillance. During early tumor development, T helper (TH)1-like NKT cell subsets have the potential to rapidly stimulate tumor-specific T cells and effector NK cells that can eliminate tumor cells. In case of tumor progression, NKT cells may become overstimulated and anergic leading to deletion of a part of the NKT cell population in patientsviaactivation-induced cell death. In addition, the remaining NKT cells become hyporesponsive, or switch to immunosuppressive TH2-/T regulatory-like NKT cell subsets, thereby facilitating tumor progression and immune escape. In this review, we discuss this important role of NKT cells in tumor development and we conclude that there should be three important focuses of future research in cancer patients in relation with NKT cells: (1) expansion of the NKT cell population, (2) prevention and breaking of NKT cell anergy, and (3) skewing of NKT cells toward TH1-like subsets with antitumor activity.

Natural Killer (NK) cells are essential in the biological fight against cancer and intracellular pathogens, and their level of activity has in many settings been used as a biomarker for a functional immune response. Currently, NK cell activity is measured using either 51Cr-release assays or flow cytometry based assays revealing the cells´ cytotoxic capacity or by stimulating them to produce cytokines. Although very effective, these are cumbersome techniques not suitable for high volume clinical laboratories. Recently, an assay has been introduced to measure NK cell activity in a simple and standardized manner. Following stimulation of NK cells in whole blood with a recombinant protein, it utilizes the concentration of IFNγ released to the plasma as a surrogate marker for NK cell activity. However, whole blood holds several sources of IFNγ which may blur the results and hamper the interpretation of the test. Therefore, the present study aimed at analyzing how specifically the test is measuring the activity of NK cells. Intracellular flow cytometry showed that NK cells, T cells, and Natural Killer T (NKT) cells were producing IFNγ in the assay, however when analyzing the distribution of lymphocytes in the IFNγ-expressing subset, the proportion of NK cells far exceeded the percentage of T-, and NKT cells (p

Tumor-associated macrophages (TAMs) play an important role in the pathophysiology of human malignancies. They support growth of cancer cells by promoting angiogenesis, and by inhibiting tumour cell apoptosis and anti-tumor immune reactions. Several membrane proteins are well-described markers of human TAMs, including the haemoglobin scavenger receptor CD163 and the macrophage mannose receptor (MR/CD206). Interestingly, both CD163 and MR exist as soluble serum proteins (sCD163 and sMR) that may reflect the activation state of tissue macrophages, including TAMs. Here, we report the first data on sMR as a biomarker in patients with a malignant disease. We have measured concentrations of sMR in peripheral blood serum (n=104) from patients with newly diagnosed multiple myeloma (MM) by an enzyme-linked immunosorbent assay, and examined associations with data from medical records. At diagnosis, sMR levels were elevated in 27% of patients, and decreased after treatment. Further, sMR levels were associated with prognostic markers in MM, and elevated sMR (>0.43mg/L) was an independent marker of overall survival in a multivariate analysis (HR=2.20, P=0.006). Levels of sMR in blood samples showed significant association with sCD163, which may indicate common origin from CD163+MR+TAMs.

The dynamics and role of cytotoxic T lymphocytes (CTLs), natural killer (NK) cells, and NKT cells in the life-threatening inflammatory disease alcoholic hepatitis is largely unknown. These cells directly kill infected and damaged cells through, e.g., degranulation and interferon-γ (IFNγ) production, but cause tissue damage if overactivated. They also assist tissue repair via IL-22 production. We, therefore, aimed to investigate the frequency, functionality, and activation state of such cells in alcoholic hepatitis. We analyzed blood samples from 24 severe alcoholic hepatitis patients followed for 30 days after diagnosis. Ten healthy abstinent volunteers and 10 stable abstinent alcoholic cirrhosis patients were controls. Using flow cytometry we assessed cell frequencies, NK cell degranulation capacity following K562 cell stimulation, activation by natural killer group 2 D (NKG2D) expression, and IL-22 and IFNγ production. In alcoholic hepatitis we found a decreased frequency of CTLs compared with healthy controls ( P < 0.001) and a similar trend for NK cells ( P = 0.089). The NK cell degranulation capacity was reduced by 25% compared with healthy controls ( P = 0.02) and by 50% compared with cirrhosis patients ( P = 0.04). Accordingly, the NKG2D receptor expression was markedly decreased on NK cells, CTLs, and NKT cells ( P < 0.05, all). The frequencies of IL-22-producing CTLs and NK cells were doubled compared with healthy controls ( P < 0.05, all) but not different from cirrhosis patients. This exploratory study for the first time showed impaired cellular cytotoxicity and activation in alcoholic hepatitis. This is unlikely to cause hepatocyte death but may contribute toward the severe immune incompetence. The results warrant detailed and mechanistic studies.

BACKGROUND/OBJECTIVES: The current world-wide obesity epidemic partially results from a vicious circle whereby maternal obesity during pregnancy predisposes the offspring for accelerated weight gain and development of metabolic syndrome. Here we investigate whether low-grade inflammation, characteristic of the obese state, provides a causal role for this disastrous fetal programming in mice.METHODS: We exposed pregnant and lactating C57BL/6JBom female mice to either high-fat diet (HFD), or continuous infusion of lipopolysaccharide (LPS), a potent trigger of innate immunity, and studied offspring phenotypes.RESULTS: Both maternal LPS or HFD treatments rendered the offspring hyperphagic and inept of coping with a HFD challenge during adulthood, increasing their adiposity and weight gain. The metabolic effects were more pronounced in female offspring, while exposed male offspring mounted a larger inflammatory response to HFD at adulthood.CONCLUSIONS: This supports our hypothesis and highlights the programming potential of inflammation in obese pregnancies.International Journal of Obesity advance online publication, 4 July 2017; doi:10.1038/ijo.2017.136.

Major depressive disorder (MDD) is highly associated with dysmetabolic conditions, such as obesity and diabetes mellitus type 2, and the gut microbiota may interact with both disease entities. We have previously shown that a high-fat diet (HFD) exacerbated depressive-like behaviour uniquely in Flinders Sensitive Line (FSL) rats that inherently present with an increased level of depressive-like behaviour compared with Flinders Resistant Line (FRL) rats. We therefore investigated whether multispecies probiotics possessed anti-depressant-like effect in FSL rats or protected against the pro-depressant-like effect of HFD. We also examined blood and cerebral T cell subsets as well as plasma cytokines. Lastly, we investigated the effect of HFD in outbred Sprague-Dawley (SD) rats to substantiate the association between depressive-like behaviour and any immunological measures affected by HFD. HFD exacerbated the depressive-like behaviour in FSL rats in the forced swim test, whereas SD rats remained unaffected. Probiotic treatment completely precluded the pro-depressant-like effect of HFD, but it did not affect FSL rats on control diet. Cerebral T lymphocyte CD4/8 ratios closely mirrored the behavioural changes, whereas the proportions of Treg and Th17 subsets were unaltered. No association between blood and brain CD4/8 ratios were evident; nor did plasma cytokine levels change as a consequence of HFD of probiotic treatment. Our findings suggest that MDD may hold a dysmetabolic component that responds to probiotic treatment. This finding has wide implications owing to the high metabolic comorbidity in MDD. Furthermore, the close association between depressive-like behaviour and cerebral T cell populations demonstrate lymphocyte-brain interactions as a promising future research area in the field of psychoneuroimmunology.

The gut microbiota has recently emerged as an important regulator of brain physiology and behaviour in animals, and ingestion of certain bacteria (probiotics) therefore appear to be a potential treatment for major depressive disorder (MDD). However, some conceptual and mechanistical aspects need further elucidation. We therefore aimed at investigating whether the habitual diet may interact with the effect of probiotics on depression-related behaviour and further examined some potentially involved mechanisms underlying the microbe-mediated behavioural effects. Forty male Sprague-Dawley rats were fed a control (CON) or high-fat diet (HFD) for ten weeks and treated with either a multi-species probiotic formulation or vehicle for the last five weeks. Independently of diet, probiotic treatment markedly reduced depressive-like behaviour in the forced swim test by 34% (95% CI: 22-44%). Furthermore, probiotic treatment skewed the cytokine production by stimulated blood mononuclear cells towards IFNγ, IL2 and IL4 at the expense of TNFα and IL6. In addition, probiotics lowered hippocampal transcript levels of factors involved in HPA axis regulation (Crh-r1, Crh-r2 and Mr), whereas HFD increased these levels. A non-targeted plasma metabolomics analysis revealed that probiotics raised the level of indole-3-propionic acid, a potential neuroprotective agent. Our findings clearly support probiotics as a potential treatment strategy in MDD. Importantly, the efficacy was not attenuated by intake of a "Western pattern" diet associated with MDD. Mechanistically, the HPA axis, immune system and microbial tryptophan metabolism could be important in this context. Importantly, our study lend inspiration to clinical trials on probiotics in depressed patients.

Although most cancer types have been viewed as immunologically silent until recently, it has become increasingly clear that the immune system plays key roles in the course of tumor development. Remarkable progress towards understanding cancer immunogenicity and tumor-immune system interactions has revealed important implications for the design of novel immune-based therapies. Natural immune responses, but also therapeutic interventions, can modulate the tumor phenotype due to selective outgrowth of resistant subtypes. This is the result of heterogeneity of tumors, with genetic instability as a driving force, and obviously changes the immunogenicity of tumors. In this review, we discuss the immunogenicity of colorectal cancer (CRC) in relation to tumor development and treatment. As most tumors, CRC activates the immune system in various ways, and is also capable of escaping recognition and elimination by the immune system. Tumor-immune system interactions underlie the balance between immune control and immune escape, and may differ in primary tumors, in the circulation, and in liver metastases of CRC. Since CRC immunogenicity varies between tumors and individuals, novel immune-based therapeutic strategies should not only anticipate the molecular profile, but also the immunological profile of a specific tumor.

Nonspecific binding of monoclonal antibodies (mAbs) to Fc-receptors on leukocytes is an important cause of background fluorescence in flow cytometry, and failing to block such nonspecific binding can lead to erroneous results. A major part of previous studies on blocking reagents for flow cytometry have been done in mice, and published results are not completely in agreement. In humans, Fc-receptors are found on most leukocytes, with highest abundance on monocytes/macrophages. Therefore, in the present study our aim was to thoroughly investigate the efficiency of different commonly used blocking reagents regarding inhibition of nonspecific binding of mouse mAbs to human peripheral blood mononuclear cells (MNCs) and monocyte-derived macrophages (MDMs). Monocytes and MDMs showed strong nonspecific binding of IgG1 and IgG2a isotypes, but not IgG2b. In contrast, B-cells, T-cells, and NK-cells did not substantially bind any of the mouse isotype control antibodies evaluated (IgG1, IgG2a, and IgG2b). Importantly, we show that binding of IgG1 and IgG2a to monocytes and MDMs can be eliminated by blocking, either with a commercial Fc-blocking reagent, with mouse or human serum, or with mouse or human IgG in high concentration. Previously, isotype controls have been widely used in flow cytometry assays. However, we show that such controls may be highly unreliable, and we believe they should not be used as gating controls, or to determine background signal. Based on these results, as well as considerations of price and applicability, our recommendation is not to use isotypes as gating controls in flow cytometry, but instead to use 100 μg/mL of purified human IgG as blocking reagent for elimination of nonspecific binding of mouse mAbs to human MNCs and MDMs. © 2016 International Society for Advancement of Cytometry.

Background and purpose: High-dose intravenous immunoglobulin (IVIg) is an established treatment for chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). Although Fc receptors on natural killer cells have been suggested as a target for IVIg, the pharmacological effects are not yet clarified. We hypothesize that IVIg therapy, dependent on the plasma IgG level, suppresses the cytotoxic capacity by a reduction in numbers of NK cells and their Fc receptor CD16. Patients and methods: Ten consecutive patients with CIDP in maintenance therapy with IVIg were studied before and immediately after the infusion of 0.7–2.0 g/kg IVIg. Peripheral blood mononuclear cell samples from these patients were analyzed immediately after isolation using flow cytometry and cytotoxicity assays. Results: We found that following IVIg treatment, the cytotoxic activity of NK cells in CIDP patients was suppressed, partly caused by a dose-dependent decline in the number of circulating NK cells. In addition, a dose-dependent blockage of CD16 occurred. Conclusions: The study implies that IVIg infusion induces a substantial decline in the number of peripheral NK cells and a suppression of NK-cell-mediated cytotoxicity. We propose that these impairments of the NK cells contribute to the therapeutic effect of IVIg in CIDP.

We have investigated the role of the Th17-related cytokines interleukin-17A (IL-17A), IL-21, and IL-23 in spondyloarthritis (SpA) by examining their association with disease activity and magnetic resonance imaging (MRI) findings in patients with SpA (n = 80). Furthermore, to investigate the cellular origins of the cytokines, paired mononuclear cells from blood and synovial fluid were examined for the expression of IL-17A, IL-21, and IL-23R using multicolor flow cytometry. Both IL-21 and IL-23 levels were increased in plasma from SpA patients compared with healthy volunteers (P < 0.05), whereas IL-17A was not. A significant correlation was observed between individual levels of IL-21 and IL-23 (r = 0.7, P < 0.001). No association between individual levels of IL-17A, IL-21, and IL-23 with C-reactive protein (CRP), MRI changes, and clinical scoring (BASMI, BASFI, and BASDAI) were observed. The frequency of CD4+CD45RO+ T cells expressing IL-21 and IL-23R was increased in the inflamed SpA joint compared to peripheral blood (P < 0.05). This study demonstrate that the plasma levels of the Th17-related cytokines IL-21 and IL-23, but not IL-17A, are increased in SpA patients, but we did not find evidence that the level of these cytokines reflect disease activity in SpA.

Udgivelsesdato: 2010-May INTRODUCTION: Diving, hyperbaric oxygen, and decompression have been described as inducers of alterations in various components of the human immune system, such as the distribution of circulating lymphocytes. Hypothetically, the monitoring of specific lymphocyte subsets during hyperbaric exposure, including T- and NK-cell subsets, can serve as biomarkers of hyperbaric stress. METHODS: Eight experienced saturation divers and eight reference subjects, naive to deep saturation diving, were examined. Peripheral blood mononuclear cells were isolated before and at different points during a 19.3-d dry heliox saturation dive to 2.64 MPa (254 msw). The NK cell cytotoxicity was estimated in a 4-h 51Cr-release assay using the NK cell sensitive tumor cell-line K562 as target cells. The major lymphocyte subpopulations, with special emphasis on the NK cell subsets, were phenotypically delineated by the use of 4-color flow cytometry. RESULTS: Although NK cell cytotoxicity increased significantly in the divers during the compression phase and the reference subjects who remained in normoxic conditions outside the chamber, the NK cell cytotoxicity was significantly higher in the divers. DISCUSSION: This finding, together with augmentation in the absolute number of circulating NK cells in the divers due to a possible activation of specific parts of the innate cellular immune system during hyperbaric exposure, suggests the monitoring of specific immune functions can be useful as biomarkers of hyperbaric-induced inflammatory stress.

Human herpesvirus 6B (HHV-6B) is the causative agent of the common childhood febrile illness, exanthema subitum. The virus is predominantly regarded as a T-cell tropic virus, although in reality it has the ability to infect a wide variety of cell types including monocytes, macrophages and dendritic cells (DC). Although DC are important immune regulators, the modulating effects of HHV-6B on DC are controversial. Here, we examine the phenotypic and functional consequences of HHV-6B infection of DC. The addition of HHV-6B to immature DC led to expression of the nuclear viral p41 protein and cell surface expression of the viral glycoprotein gp60/110 consistent with HHV-6B infection. Nevertheless, HHV-6B did not induce noticeable cytopathogenic effects or cell death in infected DC. Importantly, HHV-6B infection induced a partial phenotypic maturation of immature DC as demonstrated by a substantial increase in the expression of HLA-DR, CD86 and CD40, whereas only a minor increase in CD80 and CD83 was observed. This phenotypic maturation was, however, not followed by functional maturation, because HHV-6B infection did not induce IL-10 and IL-12p70 production in immature DC. However, infected DC were still able to react to bacteria-derived stimuli such as lipopolysaccaharide by an even more pronounced production of IL-10 and IL-12p70 when compared to that of uninfected DC.

Udgivelsesdato: 2010-Jan Natural killer (NK) cells are essential components of the immune response against human cytomegalovirus (HCMV). As NK cells are part of the innate immune system providing an immediate defense against pathogens, short-term exposure to HCMV-infected cells may induce changes in the phenotype and function of these cells. To identify immediate reactions of NK cells to HCMV, we co-cultured peripheral blood mononuclear cells with HCMV-infected fibroblasts for 24 and 72 hours. A distinct, HCMV-mediated, proportional enlargement of a subset of NK cells expressing CD94/NKG2A was sustained throughout the period of incubation. As preceding studies have shown that HCMV can cause an increase in CD94/NKG2C(+) NK cells, our results were surprising. The NK cells showed intense upregulation of the early activation marker CD69 in response to HCMV. The CD94/NKG2A(+) NK cells demonstrated the highest expression of CD69. Studies of HCMV-induced interferon-gamma expression after 24 hours of co-culture showed that this cytokine was almost exclusively produced by the CD94/NKG2A(+) subset of NK cells. In summary, our data demonstrate that HCMV induces an immediate proportional enlargement of a functionally active CD94/NKG2A expressing subset of NK cells.

Udgivelsesdato: 1987-Mar

A hairy cell leukaemia (HCL) patient is presented in whom the peripheral blood mononuclear cells (PBMCs) carried suppressor T-cell markers (CD3+, CD2+, CD8+/CD4-, CD38+). Analysis of genomic DNA of PBMNC showed the presence of a monoclonal population of T cells, the T-cell receptor (TCR) beta-chain gene being rearranged on both alleles (DR/DR), while the immunoglobulin (Ig) heavy chain-genes were in germline configuration. The neoplastic cells were found to react with the monoclonal antibody RAB-1 - originally described as belonging to the B lineage-restricted monoclonal antibodies - and to carry RAB-1/CD-8 in a double marker assay. Natural killer activity of PBMNCs against K562 target cells was severely reduced, while the cells were found to exert strong antibody-dependent cellular cytotoxicity. Udgivelsesdato: 1989-Oct

We have delineated the erythropoietic compartment in normal and malignant bone marrow (BM) by using the monoclonal antibody (mAb) AS-E1 directed against the transferrin receptor by flow cytometric (FCM) analysis. In normal BM we found a bimodal expression in antigen density with a minor subset (approximately 3%) expressing AS-E1high and a larger subset (approximately 15%) expressing AS-E1low. By fluorescence activated cell sorting, morphological examination of smears stained by immunocytochemistry and by BFU-E assays the AS-E1high fraction was shown to contain cells of erythroid origin (proerythroblasts, basophilic erythroblasts and polychromatic erythroblasts), whereas the AS-E1low fraction consisted mainly of promyelocytes and myelocytes. In patients with malignant hematological disorders we found a more pronounced heterogeneity in the density and the degree of AS-E1low expression compared to normal BM, and to further characterize the AS-E1low cells in patients and to exclude that this broad reactivity interfered with the identification of the AS-E1high cells, we employed triple-color FCM assays with mAbs directed against the myeloid surface markers CD13 and CD66 in addition to AS-E1. In all patients we found that 80-90% of the AS-E1low cells co-expressed CD13 and/or CD66 and thus were of myeloid origin. Finally, we evaluated 2 methods for determination of the AS-E1high subset and found an assay involving forward light scatter and logAS-E1 density to be sufficient. We conclude that AS-E1high is a valid FCM marker for the normal erythropoiesis.

Udgivelsesdato: 2008-May Systemic administered lipopolysaccharide (LPS) induces a cytokine response in peripheral blood without correlations with cytokine content at the organ level. We hypothesised (1) that cytokine mRNA expression in peripheral blood mononuclear cells (PBMCs) preceded the plasma cytokine increase during endotoxaemia and (2) that statins as anti-inflammatory agents modified the LPS-induced cytokine responses. 30 pigs were randomised into 3 groups: placebo (I) or atorvastatin 80 mg (II) for 21 days, followed by LPS-infusion on day 22, or controls (III). LPS was infused at increasing concentrations (2.5 to 15 mug/kg/h) for 30 min, followed by sustained infusion (2.5 mug/kg/h) for 330 min. We measured plasma IL-6, IL-10, and TNF-alpha, and their mRNA expression in PBMCs during the LPS-infusion, and the cytokine content in kidney and heart biopsies at 360 min. LPS reduced TNF-alpha mRNA in PBMCs at 60 min, whereas IL-6 mRNA increased at 240 min. There were no correlations with plasma cytokines, which peaked at 60 min (IL-10 and TNF-alpha) and 240 min (IL-6). Cytokine content did not increase in organs, and no effects of statins could be demonstrated. In conclusion, LPS-infusion reduced leukocyte TNF-alpha mRNA and increased IL-6 mRNA, whereas plasma TNF-alpha, IL-6, and IL-10 increased markedly.

Multiple sclerosis (MS) is an inflammatory disease where phagocytic cells infiltrate the nerve tissue and act as terminal agents in destruction of the myelin sheath. However, the mechanism that triggers the ability of these cells to recognize myelin remains obscure. We show that myelin basic protein (MBP), a major autoantigen in MS, is a potent and specific ligand for the integrin αMβ2 (Mac-1, CD11b/CD18) expressed mainly on phagocytic cells. MBP undergoes a dramatic conformational change when liberated from the lipid-rich environment of the myelin sheath. The MS drug glatiramer acetate mimics the conformationally labile regions of MBP, interacts in the unfolded state strongly with αMβ2, and inhibits the MBP binding to αMβ2. Our study reveals a link between MBP, glatiramer acetate, and the αMβ2 integrin, and suggests a new model for MS pathogenesis based on the recognition of unfolded MBP by the αMβ2 integrin.

// Andreas D. Orskov 1, * , Marianne B. Treppendahl 1, * , Anni Skovbo 2, 3 , Mette S. Holm 4 , Lone S. Friis 1, 5 , Marianne Hokland 2 , Kirsten Gronbaek 1 1 Department of Hematology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark 2 Department of Biomedicine, Aarhus University, Aarhus, Denmark 3 FACS Core Facility, Aarhus University, Aarhus, Denmark 4 Department of Hematology, Aarhus University Hospital, Aarhus, Denmark 5 Department of Hematology, Odense University Hospital, Odense, Denmark * These authors have contributed equally to this work. Correspondence to: Kirsten Gronbaek, e-mail: kirsten.groenbaek@regionh.dk Keywords: myelodysplastic syndromes, hypomethylating agents, DNA methylation, programmed death-1, T cells Received: November 29, 2014 Accepted: February 09, 2015 Published: March 18, 2015 ABSTRACT The hypomethylating agents (HMAs) are standard therapy for patients with higher-risk myelodysplastic syndrome (MDS); however, the majority of the patients will lose their response to HMAs over time due to unknown mechanisms. It has recently been shown that T cell expression of the immunoinhibitory receptor PD-1 is regulated by DNA methylation. In 12 of 27 patients (44%) PD-1 promoter demethylation was observed in sorted peripheral blood T cells isolated over consecutive cycles of treatment with 5-azacytidine (5-aza). The PD-1 promoter demethylation correlated with an increase in PD-1 expression. Moreover, demethylation of the PD-1 promoter correlated with a significantly worse overall response rate (8% vs. 60%, p = 0.014), and a trend towards a shorter overall survival ( p = 0.11) was observed. A significantly higher baseline methylation level of the PD-1 promoter was observed in T cells of non-responding patients compared to healthy controls ( p = 0.023). Accordingly, in addition to their beneficial function, HMAs induce PD-1 expression on T cells in the MDS microenvironment, thereby likely hampering the immune response against the MDS blasts. Thus, we suggest that activation of the PD-1 checkpoint during HMA treatment can be a possible resistance mechanism, which may be overcome by combination therapy with a PD-1 pathway inhibitor.

B-cell chronic lymphocytic leukemia (B-CLL) is recognized as the most prevalent leukemia in the Western world. Conventionally, it is believed that B-CLL cells, as a result of failed apoptosis, accu...

NK cells have been shown to play an important role in the lungs with regards to tumor cell clearance and resistance of this organ to metastases. Here, we have investigated whether NK cells play a similar role in organs other than the lungs. We conclude that while organ-resistance to metastases correlates well with the NK activity of the host, a clear correlation between NK activity and clearance of tumor cells is found only in the lungs. We also demonstrate that activation of NK cells with the TLR 3 ligand poly I:C results in a substantial increase in the number of organ-associated NK cells. This increase may explain the increased resistance to metastasis seen in many organs after poly I:C treatment. Finally, we present data showing that NK cells activated ex vivo with IL-2 are able to localize to lung tumors following iv adoptive transfer and to significantly reduce the tumors they infiltrate. We conclude that NK cells, which currently are under intense investigation owing to their newly discovered immunoregulatory functions, remain very potent antitumor killer cells capable of killing not only circulating tumor cells, but also well-established micro metastases.

Histamine (HDC) inhibits formation and release of phagocyte-derived reactive oxygen species, and thereby protects natural killer (NK) and T cells against oxidative damage. Thus, the addition of histamine may potentially improve the efficacy of interleukin-2 (IL-2). We have explored this potential mechanism clinically in two randomised phase II trials in metastatic renal cell carcinoma (mRCC). In parallel with the clinical trial in Denmark (n=63), we obtained serial blood samples and tumour biopsies searching for a potential histamine effect in situ. At baseline and on-treatment weeks 3 and 8, we monitored the 'good guys' (i.e. NK and T cells) and 'bad guys' (i.e. monocytes/macrophages and neutrophils) simultaneously in blood (n=59) and tumour tissue (n=44). Patients with high number of monocytes and neutrophils in peripheral blood had very poor survival, with apparently no benefit from either IL-2 alone or IL-2/HDC treatment. Blood monocytes (r=-0.36, P=0.01) and neutrophils (r=-0.46, P=0.001) were negatively correlated with cytotoxicity, whereas blood NK cells were positively correlated with cytotoxicity (r=0.39, P=0.002). Treatment with IL-2 alone resulted in a significantly higher number of circulating monocytes (P=0.037) and intratumoral macrophages (P=0.005) compared with baseline. In contrast, IL-2/HDC resulted in an unchanged number of circulating monocytes and intratumoral macrophages, and in addition, a significantly increased number of intratumoral CD56(+) NK cells (P=0.008) and CD8(+) T cells (P=0.019) compared with baseline. The study provides evidence that circulating monocytes and neutrophils are powerful negative prognostic factors for IL-2-based immunotherapy and establishes a biological rationale for the potential use of histamine in conjunction with IL-2 in mRCC.

Autoimmune lymphoproliferative syndrome (ALPS) is caused by a nonmalignant defective Fas-mediated apoptosis. The main clinical manifestations are chronic lymphadenopathy, splenomegaly, and autoimmune cytopenia. Most patients with ALPS have a FAS germline mutation. ALPS has occasionally been associated with glomerulonephritis and we present the first report of tubulointerstitial nephritis associated with probable ALPS. A 5-year-old girl presented with fever, vomiting, hypertension, and azotemia. No autoantibodies, viral, or streptococcal antibodies were detected. A renal biopsy showed small-vessel vasculitis with normal glomeruli and inflammation in the interstitium. The patient responded to prednisolone treatment and obtained a full renal recovery. Symptoms of connective tissue disorder supervened and after the development of more pronounced splenomegaly, a diagnosis of ALPS was confirmed.

Purpose: It has been hypothesized that tumor cells expressing Fas ligand (FasL) might be able to counterattack and neutralize tumor-infiltrating lymphocytes. We assessed the effect of FasL tumor counterattack on the clinical outcome of interleukin-2 (IL-2)–based immunotherapy in metastatic renal cell carcinoma.Experimental Design: Tumor core needle biopsies were obtained before IL-2–based immunotherapy in 86 patients and repeated within the first cycle in 57 patients. Tumor cells expressing FasL and intratumoral lymphocyte subsets expressing CD4, CD8, CD56, and CD57 were analyzed by immunohistochemistry.Results: At baseline, negative FasL staining in tumor cells was seen in 10 of 86 (12%) biopsies, whereas intense FasL staining was seen (a) in fewer than 10% of tumor cells in 26 (30%) biopsies; (b) in 11 to 50% of tumor cells in 25 (29%) biopsies; (c) in 51 to 90% of tumor cells in 18 (21%) biopsies; and (d) in >90% of tumor cells in 7 (8%) biopsies. On treatment, tumor FasL expression did not change from baseline levels. Moreover, tumor FasL expression was not correlated with objective response or survival whereas the absolute number of CD4+, CD8+, CD56+, and CD57+ cells per mm2 tumor tissue at baseline was significantly higher in responding patients compared with nonresponding patients (P = 0.01, P = 0.008, P = 0.015, and P < 0.001, respectively). During the first course of immunotherapy, the absolute number of CD4+, CD8+, and CD57+ cells per mm2 tumor tissue was significantly higher in responding patients compared with nonresponding patients (P = 0.034, P < 0.001, and P < 0.001, respectively). However, no correlation was observed between the number of intratumoral lymphocytes and tumor FasL expression level.Conclusion: These observations do not support the hypothesis that FasL tumor “counterattack” has an effect on the clinical outcome in metastatic renal cell carcinoma during IL-2–based immunotherapy.

Flow cytometric measurement of intracellular cytokines in T cells exposed to antigen is a widely used method for quantification of an antigen-specific T-cell response. As the frequency of antigen-specific T cells is often very low, any improvement in signal to noise ratio is of great importance. Thus, in this study, the ability of antigen-pulsed dendritic cells (DCs) to increase the number of antigen-specific, interferon-gamma (IFN-gamma)-producing CD4+ T cells measurable both in fresh peripheral blood and in reconstituted frozen blood mononuclear cell (MNC) samples was evaluated. Cytomegalovirus (CMV) was used as antigen in a 10 h assay, using cells from both CMV-seropositive and -seronegative donors. When reconstituted frozen samples were analysed, the general response towards CMV lysate in CMV-seropositive donors was 23-86% lower compared to the corresponding fresh blood samples. Antigen-pulsed DCs could not improve the sensitivity of the intracellular cytokine-detection assay when fresh peripheral blood samples were used. Interestingly, however, the addition of CMV lysate-pulsed DCs to cryopreserved MNC samples substantially increased the frequency of specifically induced IFN-gamma-producing cells to a level comparable to the frequency found in the corresponding fresh blood samples.

Although it is clear that natural killer (NK) cells have the ability to recognize and kill tumour cells in vitro, their potential as a highly effective treatment for tumours has not yet been realized in the clinical setting. Following activation, endogenous and adoptively transferred NK cells can be found in tumours. However, not all tumours are equally well-infiltrated, and many of the infiltrating cells do not make target-cell contact but rather reside in the tumour stroma. New insights into the migration of NK cells, their activation status and production of matrix-degrading proteases might help to overcome this localization defect, with implications for the treatment of human cancer.

Heat shock proteins are potent regulators of apoptosis, and so they may also be involved in normal cellular differentiation and cancerogenesis. We used quantitative two-dimensional gel electrophoresis for determining whether either the constitutive chaperonic heat shock cognate protein 70 (hsc70) or heat shock protein 60 (hsp60) contribute to B-cell differentiation and leukemogenesis. We compared the expression of these hsps in normal peripheral blood (PB) CD19+ B-cells, in pediatric bone marrow (BM) CD19+ CD10+ B-cell precursors (BCPs) from normal donors, and in BCPs from common acute lymphoblastic leukemia (c-ALL) patients at diagnosis and at relapse. We found that the mean levels of hsc70 in c-ALL BCPs at initial presentation and at relapse failed to differ significantly. Likewise, they failed to differ significantly from the level in high-expressing normal BM BCPs or from that in low-expressing PB B-cells. Mean levels of hsp60 expression in c-ALL BCPs at initial presentation and at relapse were similar and not distinguishable from that in normal BM BCPs, however, elevated (by a factor of 2–3) compared with that in PB B-cells. Hsc70 and Hsp60 expressions were increased (by a factor of 2 of mean levels) in populations of normal BM BCPs as compared with populations of PB B-cells. Thus, no abnormal levels of hsc70 and hsp60 were detectable in populations of pediatric c-ALL BCPs neither at diagnosis nor at relapse. In contrast, our data were in support of developmentally regulated levels of hsc70 and hsp60 expression during B-cell ontogenesis.

Low‐dose total body irradiation (LTBI) is known for its antitumor immune modulatory effects. Moreover, there is theoretical ground suggesting that combining LTBI with interleukin‐2 (IL‐2) will have a synergistic immune‐mediated antitumor effect. However, the use of LTBI in combination with IL‐2 or other forms of immunotherapy has not been tested before. Aim of the work: To test the efficacy of combining LTBI and IL‐2 in controlling lung metastases in a murine model for malignant melanoma compared to IL‐2 alone. Material and methods: Ten‐week‐old female C57BL/6 mice were inoculated intravenously (on day 0) with 1 million B16F1 malignant melanoma cells. The mice received either no treatment (control group), LTBI alone (single fraction of 0.75 Gy), IL‐2 treatment alone (30,000 CU × 2 daily for 5 consecutive days), or a combination of LTBI and IL‐2. LTBI was given on day +7 and IL‐2 treatment started day +8. On day +14, the mice were sacrificed and the lungs were removed and analyzed for tumor burden. Lung sections were also tested for tumor‐infiltrating cells using immunohistochemical staining. Peripheral blood and splenic cells were collected and tested for the percentage of the various lymphocytic subsets using immunostaining and flow cytometry. Results: Tumor burden expressed as the percentage of lung area occupied with metastases (±1 SD), was the same in the control group (8.1 ± 4.9%), and in the group receiving LTBI alone (8.3 ± 4.5%). Tumor burden was reduced to 6.4%(±3.4%) in the IL‐2 alone group (P = 0.3) and further reduced to 3%(±1%) in the combined treatment group (P = 0.004). The difference in tumor burden between the IL‐2 alone group and the combined treatment group was statistically significant (P = 0.006). The combined treatment caused a significant increase in the number of natural killer (NK) cells and macrophages infiltrating the metastatic sites. This was associated with a significant increase in the percentage of CD122+(IL‐2Rβ) cells and NK cells in both peripheral blood and spleens. Conclusion: We conclude that combining LTBI and IL‐2 treatment is synergistic and therapeutically more effective than IL‐2 alone. The data points to NK cells and macrophages as likely major effectors of the synergistic outcome of the combined treatment. This observation may have important clinical implications in the treatment of patients with metastatic malignant melanoma.

We have shown previously that i.v. injection of interleukin-2-(IL-2) activated natural killer (A-NK) cells together with IL-2 leads to a substantial localization of the A-NK cells into most, but not all, well-established B16 lung metastases in C57BL/6 mice within 12-24 hr. We demonstrate that the morphology of the lung metastases, (loose or more compact in appearance), and their location in the lungs (on the surface or deep in the lung parenchyma) are closely tied to the infiltration-permissiveness of the metastases as well as their sensitivity to treatment with A-NK cells. Although more than 1100 A-NK cells/mm(2) were found in deep metastases with a "loose" morphology (D-L), only 534, 90 and 89 cells/mm(2) were found in surface-loose (S-L), surface-compact (S-C) and deep-compact (D-C) metastases, respectively. The best infiltrated metastases responded best to the A-NK cell therapy. Thus, metastases of the D-L phenotype became reduced by 65-90% after treatment with 2 x 10(6) A-NK cells and IL-2 (120000 IU Peg-IL-2 every 12 hr for 3 days) compared to similar lesions in animals treated with PEG-IL-2 alone. In contrast, poorly infiltrated metastases, that is lesions of the compact phenotype (D-C and S-C) as well as loose metastases on the lung surface (S-L), did not react significantly to this treatment. We conclude that adoptively transferred A-NK cells are able to eliminate even well-established metastases. The existence of metastases that are resistant to infiltration by the transferred effector cells at time of treatment might reduce the efficacy of cell-based immuno-therapeutic ventures.

Background: Natural killer (NK) cells constitute an essential component of the innate immune system in the defence against infected and malignant cells. In this study the in vitro effect on NK cell activity of three different local anesthetics with different lipid solubility was investigated. Methods: Venous blood from seven healthy volunteers was incubated with three amide local anesthetics with three different concentrations of lipid solubility: lidocaine 0.50, 1.00 and 2.00 mg/ml, ropivacaine 0.375, 0.75 and 1.50 mg/ml, and bupivacaine 0.25, 0.50 and 1.00 mg/ml. After 1 h of incubation, mononuclear cells were isolated and cryopreserved until tested for NK cell cytotoxicity in a 4-h 51Cr-release assay against K-562 target cells. Natural killer cell cytotoxicity of mononuclear cells incubated with isotonic saline was used as the control. Results: A significant suppression in NK cell cytotoxicity was demonstrated for all three local anesthetic agents when the NK cell cytotoxicity was compared with the cytotoxicity estimated after incubation with the isotonic saline (P