Unspecific Cells Research Articles

T-cell receptor sequencing specifies psoriasis as a systemic and atopic dermatitis as a skin-focused, allergen-driven disease.

Atopic dermatitis (AD) and psoriasis represent two of the most common inflammatory skin diseases in developed countries. A hallmark of both diseases is T-cell infiltration into the skin. However, it is still not clarified to what extent these infiltrating T cells are antigen-specific skin-homing T cells or unspecific heterogeneous bystander cells. To elucidate this, T cells from lesional skin and from blood of 9 AD and 10 psoriasis patients were compared by receptor (TCR) sequencing. Therefore, peripheral blood mononuclear cells (PBMC) were cell-sorted according to expression of the cutaneous leukocyte antigen (CLA) into skin-homing (CLA+ ) and non-skin-homing (CLA- ) subfractions. Aeroallergen-specific T-cell lines were grown from AD patients' PBMC in parallel. Intra-individual comparison of TCRB CDR3 regions revealed that clonally expanded T cells in skin lesions of both AD and psoriasis patients corresponded to skin-homing circulating T cells. However, in psoriasis patients, these T-cell clones were also detectable to a larger extent among CLA- circulating T cells. Up to 28% of infiltrating cells in AD skin were identified as allergen-specific by overlapping TCR sequences. Our data show that in line with the systemic nature of psoriasis, T-cell clones that infiltrate psoriatic skin lesions do not exclusively possess skin-homing ability and are therefore most probably specific to antigens that are not exclusively expressed or located in the skin. T cells driving AD skin inflammation appear to home nearly exclusively to the skin and are, to a certain extent, specific to aeroallergens.

Identification and Characterization of HLA Class I-Restricted MYD88 L265P-Derived Peptides As Tumor-Specific Targets for Immunotherapy

Non-Hodgkin lymphomas (NHL) are frequent malignancies with considerable mortality. A recurrent somatic and oncogenic driver mutation in the Toll-like receptor adaptor gene MYD88, Leu265Pro (L265P) has been identified in up to 90% of certain NHL subtypes. Genetic alterations affecting a protein-coding region have the potential to generate mutation-derived peptides that are presented by HLA class I proteins and might be recognized by cytotoxic T cells. Because MYD88L265P is a widely occurring and tumor-specific mutation, we investigated the potential of MYD88L265P -containing peptides for CD8+ T cell mediated immunotherapy as a new therapeutic approach for MYD88L265P+ NHL.Based on in silico prediction we identified potential HLA ligands encompassing the MYD88L265P mutation for several HLA class I allotypes. Functional characterization of the candidate HLA class I MYD88L265P-derived HLA class I ligands with regard to induction of T cell responses identified a set of immunogenic peptides for HLA-B*07 and -B*15. In one MYD88L265P-mutated NHL patient, memory T cell responses targeting three different MYD88L265P-derived HLA class I ligands were detected by IFN-γ ELISPOT. Efficient T cell priming was demonstrated in vitro using naïve T cells of healthy volunteers (HVs). In detail, three HLA-B*07 peptides (P1-3B*07) and one HLA-B*15-restricted peptide (P4B*15) were analyzed using artificial antigen-presenting cell-based (aAPC) in vitro priming experiments in three to six HVs, respectively. For all tested peptides proliferation of peptide-specific CD8+ T cells could be detected after in vitro priming. For the HLA-B*07-restricted ligands, peptide-specific CD8+ T cells could be induced in 6/6 (P1B*15), 1/3 (P2B*07) and 3/4 (P3B*07) HVs, respectively, with a maximum frequency of 14.1% peptide-specific CD8+ T cells. For the HLA-B*15-restricted ligand (P4B*15), peptide-specific CD8+ T cells could be induced in 2/3 HVs with a maximum frequency of 9.5% tetramer-positive CD8+ T cells. The functionality and specificity of peptide-specific CD8+ T cells after aAPC-based in vitro priming was validated by intracellular cytokine staining for IFN-γ and TNF-α as well as for the expression of the degranulation marker CD107a. In 3/3 HVs primed with P1B*07 (RPIPIKYKAM) as well as in 1/2 HVs primed with P4B*15 (HQKRPIPIKY), we detected specific and functional CD8+ T cell populations after stimulation with the mutated peptides, but not after stimulation with the corresponding wild type peptides (P1WT: RLIPIKYKAM, P4WT: HQKRLIPIKY). Furthermore, the peptide-specific cytotoxic activity of specific CD8+ T cells was demonstrated in a VITAL assay. The polyclonal P1B*07- and P4B*15-specific CD8+ T cells (0.12% and 0.76% peptide-specific T cells, respectively) lysed autologous peripheral blood mononuclear cells loaded with the mutated peptides, but not cells presenting the wild type peptides. P4B*15-specific CD8+ T cells showed 17.9% (±1.2%) MYD88L265P-peptide-specific cell killing at an E/T ratio of 1:1 compared to 2.6% (±1.2%) of non-specific cell lysis of unspecific effector cells against the same targets in three independent replicates, respectively. The specific lysis showed an E/T ratio-dependent manner as the specific lysis decreases with reducing E/T ratios. P1B*07-specific CD8+ T cells specifically killed 11.4% (±1.7%) of MYD88L265P loaded targets at an E/T ratio of 0.7:1 in comparison to 2.1% unspecific lysis of unspecific effector cells.In this study, we identified and characterized MYD88L265P mutation-derived HLA class I ligands for T cell mediated immunotherapy. The strong immunogenicity of the HLA-B*07 and HLA-B*15-restricted mutation-derived peptides as well as the functionality and specificity of peptide-specific CD8+ T cells, demonstrated by cytotoxicity assays, underline the potential of the MYD88L265P mutation as tumor-specific target. These data highlight the potential of MYD88L265P mutation-specific immunotherapy as a novel broadly applicable and tumor-specific treatment approach for patients with MYD88L265P+ NHL. DisclosuresLangerak:InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics.; DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; Roche: Other: Lab services in the field of MRD diagnostics provided by Dept of Immunology, Erasmus MC (Rotterdam).

Newly diagnosed and relapsed follicular lymphoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

Newly diagnosed and relapsed follicular lymphoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

Interleukin-18 enhances the production of interferon-gamma (IFN-gamma) by allergen-specific and unspecific stimulated cord blood mononuclear cells.

IL-18 is a pleiotropic cytokine involved in the polarisation of T-cell response. This study was performed to determine whether or not IL-18 is detectable in phytohemagglutinin (PHA) or betalactoglobulin (BLG) stimulated supernatants of cord blood mononuclear cells (CBMC) and to study the in vitro effect of IL-18 on the interferon (IFN)-gaamma and IL-13 release of CBMC of healthy neonates. CBMC of neonates were isolated by Ficoll density centrifugation. The cytokines IFN-gamma, IL-13 and IL-18 in the cell culture supernatants were measured using the ELISA technique following stimulation with a unspecific (PHA 20 microg/ml) and an allergen-specific stimulus (BLG 25 microg/ml). In order to study the in vitro effect of IL-18, CBMC were stimulated either with medium alone or with IL-18, IL-18 + PHA and IL-18 + BLG. IL-18 levels in supernatants of CBMC were low and did not vary significantly between unstimulated and PHA or BLG stimulated cell cultures (median 21.4; 23.5 and 15.5 pg/ml, respectively). IFN-gamma and IL-13 levels were significantly higher in response to PHA and BLG (PHA: IFN-gamma, 6154; IL-13, 4357; BLG: IFN-gamma, 801; IL-13, 249 pg/ml) compared to unstimulated cell cultures. The addition of IL-18 to PHA or BLG stimulated CBMC significantly enhanced the IFN-gamma release (PHA: 6154; PHA + IL-18: 13474, p = 0.0001; BLG: 801; BLG + IL-18: 1077, p = 0.008). In comparison to incubation without IL-18, the release of IL-13 was invariable or even reduced, when CBMC were stimulated with PHA + IL-18 (4026, p = 0.16) or BLG + IL-18 (124, p = 0.0001) compared to stimulation of CBMC with PHA (4357 pg/ml) or BLG (249 pg/ml) alone. IL-18 is detectable in supernatants of CBMC. We observed a significant effect of IL-18 + PHA as well as IL-18 + BLG on IFN-gamma release in vitro. Based on our findings we conclude that IL-18 could act as a strong TH1-inducing factor on stimulated CBMC also in vivo.

The effect of surgery followed by endotoxin on the unspecific cell mediated immunity

It is especially the cell mediated immunity that is affected in the course of sepsis and following surgical stress. The NK cells, the granulocytes and the monocytes constitute the immediate unspecific cells mediated immunity. We therefore investigated the effect on NK cells, granulocytes and monocytes of surgery, endotoxin induced sepsis and a two-hit model composed of surgery followed by administration of endotoxin.

Immunonutrition - supplementary amino acids and fatty acids ameliorate immune deficiency in critically ill patients

Immunonutrition with omega-3 fatty acids and the "conditionally essential" amino acids arginine, glutamine, cysteine, and taurine can enhance the immune response in critically ill patients. This is due to the immunomodulating properties of these nutrients. Immunonutrition is especially important when a patient's immune response is compromised, as is the case post-operatively or after trauma. Immune deficiency is severely aggravated in sepsis and the systemic inflammatory response syndrome (SIRS). The resulting metabolic stress is characterized by glycolysis, lipolysis, and proteolysis, which may escalate to an hypercatabolic response or "autocannabilism." Catabolic metabolism results in insufficiency of both specific and unspecific immunocompetent cells. Immunonutrition should be started early in such patients for an optimal beneficial effect, preferably via the enteral route. It should include medium chain and long chain triglycerides, polyunsaturated omega-3 and omega-6 fatty acids (in the ratio 1:2), olive oil, and conventional amino acid preparations supplemented with the conditionally essential amino acids arginine, glutamine, cysteine, and taurine.

Therapeutic monoclonal antibodies in oncology: an update: Monoclonal antibodies as enhancers of the host's immunoresponse against the tumour

Monoclonal antibodies (MAb) might induce tumour cell death by a multitude of mechanisms of actions. Principally monoclonal antibodies can be used unconjugated or conjugated to a toxic substance. So far, unconjugated MAb have been most successful and used extensively for treatment of malignant diseases. Several unconjugated therapeutic monoclonal antibodies are on the market and there are a lot more to be launched. Unconjugated MAb have two fundamentally different ways of action. If the MAb recognise a target structure involved in cell proliferation or apoptosis, the MAb may cause programmed cell death or tumour cell cycle arrest by binding to the relevant domain of the tumour antigen and deliver a signal inducing tumour destruction in vivo without activating the immune system [1]. Monoclonal antibodies may also enhance various immune effector functions which can mediate tumour cell destruction. Immune functions which spontaneously might kill tumour cells include antibodies, unspecific cytotoxic cells (monocytes/macrophages, NK cells, granulocytes, eosinophils) and specific killer cells (various T lymphocyte subsets). In this paper, host's immune responses against the tumour enhanced by monoclonal antibodies are reviewed. antigen with a certain degree of specificity even if the normal counterpart is a cell structure of a normal non-transformed cell.

Topographic analysis of proliferative activity in carotid endarterectomy specimens by immunocytochemical detection of the cell cycle-related antigen Ki-67.

On the basis of contradictory results found in animal experiments and coronary atherectomy tissue, there is an ongoing debate about the significance of cellular proliferation in human atherosclerosis. In the present prospective study, the cell cycle-related antigen Ki-67 was detected for topographic determination of cell turnover in distinct regions of human carotid endarterectomy specimens harvested en bloc by surgical biopsy. After en bloc resection, serial sections of 26 consecutive carotid lesions were analyzed by histomorphological examination and immunohistochemistry. Thereby, 319 high-power fields were attributed to separate plaque regions defined as follows: distal boundary of the lesion with normal intima, plaque shoulder, core region, and diffuse intimal thickening. Endothelial cells, smooth muscle cells, T cells, and macrophages were identified by immunostaining of factor VIII-related protein, alpha-actin, CD68, and CD45R0. An overall proliferation index of 0.49+/-1.05% was yielded by positive anti-Ki-67 immunolabeling, predominantly in macrophage-rich areas characterized by high cell density (>1000 cells/mm2) as well as in reparative sites in the perimeter of atheroma, intramural thrombosis, plaque hemorrhage, and neovascularization (P<.01). Few or no signs of proliferation activity were found in normal intima, in areas of dense alpha-actin positivity, or adjacent media. As shown by double immunostaining, macrophages and unspecified mesenchymal cells represented the prevailing proliferating cell type. Our results suggest that proliferation in advanced human carotid lesions is confined to the intima and focally concentrated in central plaque regions negative for alpha-actin. Furthermore, it apparently occurs primarily as part of inflammatory processes and structural repair predominantly involving macrophages, as well as unspecific mesenchymal cells.

Generation of monoclonal antibodies directed against organ-specific endothelial cell surface determinants.

Organ-specific determinants expressed on the luminal surface of vascular endothelia are often unstable when cells are removed from their normal tissue environment and grown in culture. Unspecific endothelial cells of large vessel origin [e.g., bovine aorta (BAEC)] can be modulated to express and preserve such determinants when they are grown on the extracellular matrix of the desired organ. Lung matrix-modulated BAEC were used here to generate MAb against lung-specific vascular endothelia. Immunization was accomplished with outside-out membrane vesicles obtained by incubating BAEC monolayers grown on lung matrix with a low-strength paraformaldehyde solution. In four of the six fusions performed, this active immunization was preceded by passive immunization with mouse antiserum directed against membrane vesicles from BAEC grown on plastic. Among the growing hybrids, 7.6% secreted MAb that bound efficiently to both BAEC grown on lung-derived matrix and BAEC grown on plastic, while 3.5% (50) secreted MAb that bound primarily to BAEC grown on lung matrix. The fusion data show that only a passive/active immunization protocol yielded MAb directed against lung-specific endothelia. For example, MAb 6D3 and 5F5 selectively recognized endothelia from small- and medium-sized venules of bovine lungs, but failed to react with endothelial cells in other organs and tissues.

Protection against Experimental Allergic Encephalomyelitis with Complete Freund’s Adjuvant Is Unaffected by Ionizing Irradiation

Long-lasting resistance to experimental allergic encephalomyelitis (EAE) was obtained in Lewis rats either by an immunization with complete Freund's adjuvant blended with aluminum hydroxide (ALU-CFA) or by convalescence from EAE. Total body irradiation at a dose of 500 R given both 1 day after ALU-CFA (or disease induction) and EAE challenge did not abrogate the protection. No antibodies against myelin basic protein could be detected in irradiated animals. In contrast high antibody titers were measured in non-irradiated controls recovered from EAE and subsequently resistant to the disease. Therefore it is unlikely that antibodies against myelin basic proteins are of any importance in protection from EAE. The studies suggest that unspecific radioresistant suppressor cells play a role in resistance to EAE.

Immunocytochemical identification and localization of peptide hormones in the gastro-entero-pancreatic (GEP) endocrine system of the mouse and a stomachless fish, Barbus conchonius.

A large number of antisera mainly raised against mammalian hormones are tested immunocytochemically on the GEP-endocrine system of mouse and fish (Barbus conchonius). The endocrine pancreas of mouse and fish appeared to contain the same four endocrine cell types; insulin-, glucagon-, PP- and somatostatin-immunoreactive cells. In mouse about 13 GEP endocrine cell types are distinguished: 1. insulin-, 2. somatostatin-, 3. glucagon-, 4. PP-, 5. (entero)glucagon-/PP-like, 6. CCK-like, 7. substance P-, 8. neurotensin-, 9. VIP-, 10. gastrin-, 11. secretin-, 12. beta-endorphin-, 13. serotonin-immunoreactive cells. Based on this and a previous study at least 13 GEP endocrine cell types seems to be present in stomachless fish: 1-9 as described for mouse, 10. (entero)glucagon-like, 11. met-enkephalin, 12. VIP-like, 13. unspecific immunoreactive endocrine cells. Coexistence of glucagon and PP-like peptides is found in the gut and pancreas of mice and in the gut of B. conchonius. In mouse pancreas and fish gut, endocrine cells showing only PP- or glucagon-like immunoreactivity are found too. In mouse stomach some endocrine cells showing only PP-immunoreactivity are demonstrated. In the same region coexistence of C-t-gastrin- and FMRF-amide-immunoreactivity is found in endocrine cells. The importance of these phenomena are discussed. Enteric nerves immunoreactive with antisera raised against substance P and GRP are found in mouse, against somatostatin and met-enkephalin in both mouse and fish and against VIP in fish.

Ultrastructural characterization of 6 immunoreactive enteroendocrine cells in Barbus conchonius (Teleostei, Cyprinidae).

Using the semi-thin/ultra-thin technique six different immunoreactive endocrine cell types are ultrastructurally identified in 0.5% glutaraldehyde fixed gut of B. conchonius. In addition two of them (gastrin- and PP-immunoreactive cells) are also characterized with the immunogold method, showing that the immunoreactivity is only restricted to the secretory granules. Size distribution histograms and the average diameters of 30% (d30) of the largest granules are given, showing a gradual increase in granule size from unspecific immunoreactive cells, (d30 = 110 nm) via gastrin- (119 nm), VIP-like- (127 nm), met-enkephalin- (143 nm) and PP- (174 nm) to glucagon-immunoreactive cells (178 nm). The presence of PP- and glucagon-immunoreactivity in the same cells and the consequence for their granule size is discussed. In the distal part of the gut endocrine cells are found showing no immunoreactivity with the antisera used; their granules (d30 = 144 nm) were, although not significantly, larger then those of VIP-like-immunoreactive cells, also found in that part of the gut. It is supposed that they represent substance P-immunoreactive cells. Unfortunately, secretory granules of several cell types showed about 20% more shrinkage in 0.5% glutaraldehyde fixed tissue, than in osmicated tissue.

Microelectrophoretic application of metabolic modifiers of the phosphofructokinase-hexosediphosphatase-system onto various types of bulbar respiratory neurons.

Three metabolic modifiers of the PFK-HDPase system were applied to bulbar respiratory neurons and, for comparison, also to unspecific cells. F-6-P did not alter the spike density of the majority of the respiratory and about half of the unspecific neurons. When responding, IE units were activated and unspecific cells were inhibited. Citrate did not alter the spike density of about half of all neurons tested. When responding, excitation prevailed in IE and E units, while unspecific cells were inhibited. AMP did not alter the spike density of the majority of the respiratory and about half of the unspecific cells. When responding, I neurons were activated, inhibition preponderated in IE units and both effects occurred in unspecific cells. The large number of zero effects suggests that the activity of only part of the cells is governed by the PFK-HDPase system.

Microelectrophoretic application of antagonists of putative neurotransmitters onto various types of bulbar respiratory neurons.

Seven antagonists of putative neurotransmitters were applied to bulbar respiratory neurons and, for comparison, also to unspecific cells. The antagonists exerted distinct effects when released alone, permitting to draw conclusions about receptor properties of the various cell types. With strychnine, specific antagonist of glycine, excitation prevailed in EI, I and E neurons. With bicuculline, specific antagonist of GABA, excitation preponderated in EI and E cells. About half of the unspecific neurons were activated and the remainder were unresponsive. GDEE (glutamatediethylester), antagonist of glutamate, excited part of the IE neurons and inhibited part of the E units, while the remainder of both types as well as 2 EI cells tested were not affected. With flupentixol, antagonist of dopamine, excitation prevailed in I neurons. About half of the IE and E units remained unaffected, while in the remainder E cells inhibition preponderated over excitation. With yohimbine, an alpha-adrenoceptor blocker, inhibition prevailed in E units. The two EI as well as the majority of the I neurons remained unaffected, with two cells of the latter type being activated. Propranolol, a beta-adrenoceptor blocker, inhibited about half of the E neurons, while the remainder as well as most IE and the 2 EI cells tested were not affected. Cyproheptadine, an antagonist of 5-HT, excited most E neurons. As concerns NE-receptors, those of the alpha-type might be involved in activation of part of the E cells only, whereas all other NE effects (inhibition or activation) are mediated by CNS-specific receptors different from the alpha- and beta-type. 5-HT effects apparently are mediated by two different receptor types.

Effects of pentylenetetrazol on respiration and bulbar respiratory neurones and its interaction with K + ions and citrate

Interactions between pentylenetetrazol (PTZ) and the metabolic modifier citrate and also with K + ions were examined. When superfused onto the bulb, at low concentrations PTZ increased tidal volume and slowed down respiration; the opposite was true for higher concentrations. Addition of citrate or K + ions did not essentially alter PTZ effects. Applied microelectrophoretically, PTZ influenced the spike density of three-quarters, and K + ions and citrate of two-thirds of all cells tested. When effective, PTZ excited the majority of the inspiratory (I) and inspiratory-expiratory neurones (IE), both excited and inhibited expiratory units (E) and inhibited the majority of the non-respiratory neurones. Citrate inhibited the majority of IE and E neurones; when combined with PTZ, effects of PTZ were abolished in most respiratory but not in unspecific cells. K + ions predominantly excited IE and E units. When combined with PTZ, inhibition exerted by PTZ on unspecific neurones was reversed into activation. This was not true for respiratory cells. Since activation of respiratory cells mediated by PTZ was neutralized by the metabolic inhibitor citrate and was mimicked by K + ions, PTZ may well act as modifier of the oxidative metabolism of glucose and of the passive efflux of K + ions.

Proliferation of an unspecific esterase positive (monocytopoietic) cell system in human bone marrow as observed from nucleic acid synthesis.

In order to study the proliferation activity of the unspecific esterase positive cells (EPC) of the bone marrow, which are claimed to be monocytic precursor cells, bone marrow aspirate of a normal human being was incubated with H3-thymidine. The smears were stained for demonstration of the Naphthol-AS-acetate esterase and with the Pappenheim stain. In the EPC's the DNA synthesis was investigated by combined application of cytophotometric measurement of the DNA content (Feulgen photometry) and of autoradiography.