Dietrich Kabelitz

Generation of Soluble NKG2D Ligands: Proteolytic Cleavage, Exosome Secretion and Functional Implications

The activating natural killer group 2 member D (NKG2D) receptor is expressed on NK cells, cytotoxic T cells and additional T cell subsets. Ligands for human NKG2D comprise two groups of MHC class I‐related molecules, the MHC class I chain‐related proteins A and B (MICA/B) and 6 UL16‐binding proteins (ULBP1‐6). While NKG2D ligands are absent from most normal cells, expression is induced upon stress and malignant transformation. In fact, most solid tumours and leukaemia/lymphomas constitutively express at least one NKG2D ligand and thereby are susceptible to NKG2D‐dependent immunosurveillance. However, soluble NKG2D ligands are released from tumour cells and can down‐modulate NKG2D activation as a means of tumour immune escape. In some tumour entities, levels of soluble NKG2D ligands in the serum correlate with tumour progression. NKG2D ligands can be proteolytically shed from the cell surface or liberated from the membrane by phospholipase C in the case of glycosylphosphatidylinositol (GPI)‐anchored molecules. Moreover, NKG2D ligands can be secreted in exosomal microvesicles together with other tumour‐derived molecules. Depending on the specific tumour/immune cell setting, these various forms of soluble and/or exosome‐bound NKG2D ligands can exert multiple effects on NKG2D/NKG2D ligand interactions. In this review, we focus on the role of various proteases in the shedding of human NKG2D ligands from tumour cells and discuss the not completely unanimous reported functional implications of soluble and exosome‐secreted NKG2D ligands for immunosurveillance.

CARD15 mutations in patients with plaque-type psoriasis and psoriatic arthritis: lack of association

Psoriasis has a strong genetic component in the development of the disease as indicated by familial occurrence and a high concordance rate among monozygotic twins. In genome-wide scans for psoriasis several susceptibility loci have been detected, but the disease-causing genes have not yet been identified. A recent scan, performed on psoriatic arthritis (PsA), which occurs in about 15% of the psoriasis patients showed a significant locus on chromosome 16 in a region that was already described by genome scan for psoriasis. CARD15, a major susceptibility gene for Crohn’s disease (CD) on chromosome 16q, is an interesting candidate gene for psoriasis, because there is a documented clinical association of CD with psoriasis, and recently the association of CARD15 mutations with PsA was reported in Newfoundland population. We investigated the association of this variant with PsA and the overall psoriasis genotype in 59 independent patients with PsA in comparison with 361 age and sex-matched controls. In addition, a second cohort of 89 independent North American PsA patients was included. The diagnosis of psoriasis was made by a dermatologist based on standard clinical criteria. In these patients, PsA was defined as an inflammatory joint disease, negative rheumatoid factor, and lack of another causative condition for arthritis. Using case-control analysis, the G908R mutation was weakly associated with psoriasis and PsA, but due to the low frequency of this mutation statistical significance was not reached. All other variants including leu1007fsinsC and R702W did not show any association with psoriasis or PsA. In conclusion, a disease-causing role for CARD15 mutations could not be confirmed in German or American subjects with PsA.

Effector granules in human T lymphocytes: proteomic evidence for two distinct species of cytotoxic effector vesicles.

Cytotoxic T cells mobilize effector proteins from prestored lysosomal compartments. Since different activation signals result in alternative routes of target cell killing, utilizing either FasL or the granzyme B/perforin pathway, the existence of distinct forms of effector granules was recently suggested. Applying a protocol for the separation of intact organelles from activated T lymphoblasts, we noticed that FasL-associated secretory lysosomes (SL) segregate from vesicles containing larger amounts of granzymes and granulysin. We previously analyzed the proteome of secretory lysosomes from NK and T cells and now describe the proteome of granzyme-containing vesicles. Moreover, intact FasL-associated SL and granzyme-containing vesicles were compared by electron microscopy and respective extracts were characterized by Western blotting. With the present report, we provide a comprehensive proteome map of granzyme-containing granules and unequivocally demonstrate that T lymphoblasts contain at least two distinct types of effector vesicles. Moreover, the overall protein content of the two vesicle populations was compared by 2D difference gel electrophoresis. Interestingly, the observed differences in protein distribution were not restricted to effector proteins but also applied to cytoskeleton-associated elements that could argue for a differential transport or initiation of degranulation. To our knowledge, this is the first comprehensive description of distinct effector granules in T cells.

Effector granules in human T lymphocytes: the luminal proteome of secretory lysosomes from human T cells.

BackgroundCytotoxic cells of the immune system have evolved a lysosomal compartment to store and mobilize effector molecules. In T lymphocytes and NK cells, the death factor FasL is one of the characteristic marker proteins of these so-called secretory lysosomes, which combine properties of conventional lysosomes and exocytotic vesicles. Although these vesicles are crucial for immune effector function, their protein content in T cells has so far not been investigated in detail.ResultsIn the present study, intact membranous vesicles were enriched from homogenates of polyclonally activated T cells and initially characterized by Western blotting and electron microscopic inspection. The vesicular fraction that contained the marker proteins of secretory lysosomes was subsequently analyzed by 2D electrophoresis and mass spectrometry. The proteome analysis and data evaluation revealed that 70% of the 397 annotated proteins had been associated with different lysosome-related organelles in previous proteome studies.ConclusionWe provide the first comprehensive proteome map of T cell-derived secretory lysosomes with only minor contaminations by cytosolic, nuclear or other proteins. This information will be useful to more precisely address the activation-dependent maturation and the specific distribution of effector organelles and proteins in individual T or NK cell populations in future studies.

The Impact of Electrical Charge on the Viability and Physiology of Dendritic Cells

The use of electrical charge for electroporation or electrofusion is widely applied to customize dendritic cells (DC) and their immunological properties as anticancer vaccines. The aim of this study was to evaluate the influence of various electrical field strengths on the recovery, viability and physiology of DC. Immature DC were transferred into low‐conductive medium and electrically charged within a range of 0–1500 V/cm. Viability was assessed by Trypan Blue dye exclusion or staining with impermeant nucleic acid stains and fluorescence‐activated cell sorter analysis. Additionally, apoptosis was determined by flow cytometry after staining with Annexin‐V, endocytosis by uptake of fluorescein isothiocyanate‐dextran and metabolic activity by a standardized fluorescent live/dead assay. There was a strong correlation between the electrical field strength and the viability and physiology of DC. Field strengths ≥1000 V/cm significantly impaired viability, metabolism and endocytotic activity. Dual fluorescence with 7‐7‐amino‐actinomycin D and Annexin‐V demonstrated that loss of viability was predominantly due to necrosis rather than apoptosis. Field strengths ≤500 V/cm allowed to maintain good cell viability and recovery of DC and did not cause alterations of metabolism and endocytosis. Therefore, the frequently used amplification of field strengths to improve the efficacy of electroporation and electrofusion requires critical re‐evaluation.

HLA-DRB1 and anti-cyclic citrullinated peptide antibody production in rheumatoid arthritis.

Anti-cyclic citrullinated peptide antibodies (anti-CCP) are a new diagnostic marker for rheumatoid arthritis (RA), which shows a specificity of 97% and a sensitivity of 81% in the second generation assay. About 61% of RA patients express HLA-DRB1*0401. In a cohort of patients with RA we investigated whether the expression of anti-CCP correlates with the carriage of certain genes on the HLA-DRB1 locus. Our data reveal a highly significant association between anti-CCP and HLA-DR4, and a weaker but still significant association with HLA-DR1. HLA-DRB1*0401 is not a prerequisite for anti-CCP production, but if HLA-DRB1*0401 was present, 90% of our RA patients were anti-CCP positive.

Lysosome-Related Effector Vesicles in T Lymphocytes and NK Cells.

Lysosome‐related secretory organelles combine metabolic functions of conventional lysosomes with an inducible secretory potential. Specialized variants of such bi‐functional organelles are present in several haematopoietic cell types that store, mobilize and/or secrete effector proteins, for example in mast cells, macrophages or cytotoxic effector cells. In the case of T lymphocytes and NK cells, it was believed that secretory lysosomes serve as a common storage and transport compartment for the most relevant cytotoxic effector proteins including FasL, perforin, granzymes and granulysin. However, recent observations suggest that cytotoxic effector cells might be able to mobilize two distinct lysosomal entities in order to react to differential stimulation with either FasL surface appearance or degranulation‐associated release of perforin and granzymes. This assumption is supported by the proteomic characterization of enriched organelles from T and NK cells. FasL‐associated light lysosomes biochemically segregate from morphologically distinct heavy lysosomes that preferentially contain granzymes, perforin and mature granulysin. Here, we briefly summarize the current knowledge about cargo proteins that are stored and transported in secretory vesicles and how these vesicles might be generated and mobilized. In addition, we describe common features and major differences of the two distinct effector organelles and discuss how these observations might expand existing models of cytotoxic effector function.

Experience with Allogeneic Leukocyte Immunization (Ai) for Implantation Failure in the In-Vitro Fertilization Program

Immunological mechanisms might contribute to the repeated implantation failure in the in‐vitro fertilization program. AI might be a valuable therapeutic option in this situation. Here we summarize our extensive experience with this immunotherapy. We conducted a retrospective analysis of all couples who were referred to our outpatients department from 1996 to 1998 for AI after two or more embryo transfers (ET). Six hundred and eighty‐six couples (89%) continued IVF/ICSI and were followed up for 2–3 years. On average patients entered AI after 3–4 IVF–ET at an age of 33 years and completed IVF/ICSI treatment after another 2 ET; 2.6 embryos per ET were transferred according to the German embryo protection law. The cumulative birth rate war 38% after IVF transfer and 42% when spontaneous pregnancies were included. Until the age of 36 years the rates of viable pregnancies and birth were improved by a quarter in the first 6 months as compared to the second half year (33.3% versus 26.3%, 26.0% versus 19.6%) and to the German IVF Registry of 1998. Couples who had undergone more than three ET before AI seemed to benefit most. Our data suggest that AI is a valuable adjuvant therapy with a temporary effect when 3–4 IVF/ICSI transfers have been unsuccessful. The underlying immunomodulatory mechanisms require further investigation.

Inhibition of Human γδ T Cell Proliferation and Effector Functions by Neutrophil Serine Proteases

Human peripheral blood γδ T cells expressing the Vγ9Vδ2 T cell receptor are activated by microbial or endogenous pyrophosphate antigens and indirectly by nitrogen‐containing bisphosphonates. Apart from proliferation, such phosphoantigens induce proinflammatory cytokine production including TNF‐α and IFN‐γ and trigger cytotoxic effector function. Neutrophil granulocytes are known to modulate T cell activation. The neutrophil serine proteases proteinase 3, elastase and cathepsin G have multiple potential targets and promote microbial killing. In this study, we investigated the effect of the three serine proteases on the in vitro proliferation and effector functions of γδ T cells cultured in serum‐free medium. All three proteases inhibited the proliferative activity, suppressed the cytokine production and decreased the cytotoxicity of γδ T cells. Further studies indicated that proteolytic cleavage of IL‐2 and modulation of butyrophilin 3A1 (CD277) expression might contribute to the overall inhibition.