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Biomedical subjects

F Koning

Publications and source records attributed to F Koning.

At least 19 recordsLinked to original sources
PubMed2006

An improved RT-PCR method for the detection of killer-cell immunoglobulin-like receptor (KIR) transcripts.

Killer cell immunoglobulin-like receptors (KIRs) are expressed on human natural killer (NK) cells and a proportion of T cells. As the specificity of these NK and T cells is, at least in part, determined by the combination of KIRs they express, it is important to be able to determine the KIR expression pattern of NK and T cell clones to understand their function. However, for most KIR genes, specific reagents to detect expression are currently either unavailable or sensitive to allelic variations. In this study, a reverse transcriptase-polymerase chain reaction (RT-PCR) that uses new primer sets for the gene-specific detection of KIR transcripts is presented and validated. The key advantage of this RT-PCR method over previously published ones is that it was designed to detect transcripts of all confirmed allelic variants of the KIR genes, while remaining gene-specific.

Animals↗
PubMed2006

The impact of single amino acid substitutions in CD3gamma on the CD3epsilongamma interaction and T-cell receptor-CD3 complex formation.

The human T-cell receptor-CD3 complex consists of at least eight polypeptide chains; CD3gamma- and delta-dimers associate with the disulfide linked alphabeta- and zetazeta-dimers to form a functional receptor complex. The exact structure of this complex is still unknown. We now have examined the interaction between CD3gamma and CD3 in human T-cells. For this purpose, we have generated site-directed mutants of CD3gamma that were introduced in human T-cells defective in CD3gamma expression. Cell-surface and intracellular expression of the introduced CD3gamma chains was determined, as was the association with CD3delta, CD3, and the T-cell receptor. Although the introduction of wild type CD3gamma and CD3gamma (78Y-F) fully restored T-cell receptor assembly and expression, the introduction of CD3gamma (82C-S), CD3gamma (85C-S), and CD3gamma (76Q-E) all resulted in an impaired association between CD3gamma and CD3 and a lack of cell-surface expressed CD3gamma. Finally, the introduction of CD3gamma (76Q-L) and CD3gamma (78Y-A) restored the expression of TCR-CD3deltagammazeta2 complexes, although the association between CD3gamma and CD3 was impaired. These results indicate that several amino acids in CD3gamma are essential for an optimal association between CD3gamma and CD3 and the assembly of a cell-surface expressed TCR-CD3deltagammazeta2 complex.

Amino Acid Substitution↗
PubMed2004

A novel and sensitive method for the detection of T cell stimulatory epitopes of alpha/beta- and gamma-gliadin.

BACKGROUND: It is now generally accepted that coeliac disease (CD) is caused by inflammatory T cell responses to gluten peptides bound to HLA-DQ2 or -DQ8 molecules. There is overwhelming evidence that CD patients can mount T cell responses to peptides found in both alpha-gliadin and gamma-gliadin molecules. Assays that would detect the presence or absence of such peptides in food would thus be accurate indicators of safety for consumption by CD patients. AIMS: The development of a sensitive method to detect T cell stimulatory epitopes of alpha-gliadin and gamma-gliadin molecules in food products. METHODS: Monoclonal antibodies (mAb) were raised against peptides encoding the T cell stimulatory epitopes of alpha-gliadin (amino acids (aa) 59-71) and aa gamma-gliadin (aa 142-153 and aa 147-159). These mAb competition assays were developed that quantitatively detect T cell stimulatory epitopes present on both intact proteins and peptides of sizes recognisable by CD4(+) T cells. RESULTS: With the mAb based competition assays, T cell epitopes were detected in pepsin/trypsin digests of wheat proteins and ethanol extracts of various food products, with detection levels lower than those reached with gluten specific T cells. Moreover, the presence of T cell stimulatory epitopes was also detected in preparations of barley, rye, and triticale, other cereals known to be toxic for CD patients. CONCLUSIONS: A new antibody based method has been developed, detecting the presence of T cell stimulatory gluten peptides. This can be used to further ensure the safety of food consumed by CD patients.

Animals↗
PubMed2003

Synaptojanin 2 is recognized by HLA class II-restricted hairy cell leukemia-specific T cells.

Hairy cell leukemia (HCL) is a chronic mature B-cell leukemia characterized by malignant B cells that have typical hairy protrusions. To characterize possible HCL-associated tumor antigens, we generated an HCL-specific and HLA class II (DPw4)-restricted proliferative CD4+ T-cell clone. To identify the target antigen of these T cells, we constructed a synthetic peptide library dedicated to bind HLA DPw4, and identified a mimicry epitope recognized by the T-cell clone. With this epitope, the recognition motif of the T-cell clone was deduced and a peptide of human synaptojanin 2 (Syn 2) was identified that stimulated the HCL-reactive T-cell clone. Both Northern and Western blot analyses showed that Syn 2 expression was increased in HCL samples compared to other B cells. Besides, the Syn 2-expressing cell line AML193, with the introduced restrictive HLA-DPw4 molecules, was recognized by the HCL-specific T-cell clone. These results indicate that Syn 2 is a target of autoreactive HCL-specific T cells. Since Syn 2 is a phosphatidylinositol 4,5-biphosphatase involved in cell growth and rearrangement of actin filaments, the increased Syn 2 expression may correlate with the disease etiology or the characteristic morphologic alterations caused by the disease.

CD4-Positive T-Lymphocytes↗
PubMed2003

An immunodominant DQ8 restricted gliadin peptide activates small intestinal immune response in in vitro cultured mucosa from HLA-DQ8 positive but not HLA-DQ8 negative coeliac patients.

BACKGROUND: Studies on intestinal T cell clones from the mucosa of patients with coeliac disease have led to the identification of immunogenic gliadin epitopes. One is HLA-DQ8 restricted, its recognition by T cells being increased by introduction of negatively charged residues operated by tissue transglutaminase. AIM: To test HLA-DQ8 restricted epitope in both native (QYPSGQGSFQPSQQNPQA) and deamidated (QYPSGEGSFQPSQENPQA) forms in an organ culture system of treated coeliac mucosa from HLA-DQ8 positive and HLA-DQ8 negative patients. PATIENTS AND METHODS: Jejunal biopsies obtained from 10 patients with coeliac disease (six HLA-DQ8 positive and four HLA-DQ8 negative) were cultured in vitro with a peptic-tryptic digest (PT) of gliadin, or with the native (peptide A) or deamidated (peptide B) peptide. Intraepithelial CD3(+) and lamina propria total CD25(+) and CD3(+)CD25(+) cells were counted, lamina propria intercellular adhesion molecule 1 (ICAM-1) expression was evaluated, as well as that of Fas molecules on epithelial cells. RESULTS: In HLA-DQ8 positive, but not in HLA-DQ8 negative, coeliacs the density of intraepithelial CD3(+) cells, lamina propria total CD25(+), and CD3(+)CD25(+) cells, as well as expression of ICAM-1 and Fas molecules were significantly increased in biopsies cultured with PT, peptide A, or peptide B compared with biopsies cultured in medium alone. CONCLUSION: These data show that the DQ8 restricted gliadin peptide is immunogenic only in the intestinal mucosa of HLA-DQ8 positive coeliac patients in both native and deamidated forms.

Adolescent↗
PubMed2001

T cell responses modulated through interaction between CD8alphaalpha and the nonclassical MHC class I molecule, TL.

The thymus leukemia antigen (TL) is a nonclassical class I molecule, expressed abundantly on intestinal epithelial cells. We show that, in contrast to other major histocompatibility complex (MHC) class I molecules that bind CD8alphabeta, TL preferentially binds the homotypic form of CD8alpha (CD8alphaalpha). Thus, TL tetramers react specifically to CD8alphaalpha-expressing cells, including most intestinal intraepithelial lymphocytes. Compared with CD8alphabeta, which recognizes the same MHC as the T cell receptor (TCR) and thus acts as a TCR coreceptor, high-affinity binding of CD8alphaalpha to TL modifies responses mediated by TCR recognition of antigen presented by distinct MHC molecules. These findings define a novel mechanism of lymphocyte regulation through CD8alphaalpha and MHC class I.

Amino Acid Motifs↗
PubMed2001

Dual HLA class I and class II restricted recognition of alloreactive T lymphocytes mediated by a single T cell receptor complex.

The alloreactive human T cell clone MBM15 was found to exhibit dual specificity recognizing both an antigen in the context of the HLA class I A2 molecule and an antigen in the context of the HLA class II DR1. We demonstrated that the dual reactivity that was mediated via a single clonal T cell population depended on specific peptide binding. For complete recognition of the HLA-A2-restricted specificity the interaction of CD8 with HLA class I is essential. Interestingly, interaction of the CD8 molecule with HLA class I contributed to the HLA-DR1-restricted specificity. T cell clone MBM15 expressed two in-frame T cell receptor (TCR) Valpha transcripts (Valpha1 and Valpha2) and one TCR Vbeta transcript (Vbeta13). To elucidate whether two TCR complexes were responsible for the dual recognition or one complex, cytotoxic T cells were transduced with retroviral vectors encoding the different TCR chains. Only T cells transduced with the TCR Valpha1Vbeta13 combination specifically recognized both the HLA-A2(+) and HLA-DR1(+) target cells, whereas the Valpha2Vbeta13 combination did not result in a TCR on the cell surface. Thus a single TCRalphabeta complex can have dual specificity, recognizing both a peptide in the context of HLA class I as well as a peptide in the context of HLA class II. Transactivation of T cells by an unrelated antigen in the context of HLA class II may evoke an HLA class I-specific T cell response. We propose that this finding may have major implications for immunotherapeutic interventions and insight into the development of autoimmune diseases.

CD8 Antigens↗
PubMed2001

Induction of antigen specific CD4+ T cell responses by invariant chain based DNA vaccines.

In this report, the use of DNA vaccination to induce class II restricted antigen specific proliferative responses was studied. To this end, a construct encoding the invariant chain (Ii) was engineered in which the Class II associated invariant chain peptide (CLIP) sequence was replaced by an immunogenic epitope derived form Heat Shock Protein 60, HSP60 178-186. Transfection studies in vitro showed that this construct can be used to efficiently load MHC class II molecules and present epitopes to MHC class II restricted antigen specific T cells. In addition, we showed that intradermal immunisation of Lewis rats with these constructs induced antigen specific T cells in vivo. Therefore, our Ii-gene constructs can be used to immunise for defined CD4+ T cell epitope sequences.

Amino Acid Sequence↗
PubMed2001

CD4-independent T cells impair TCR triggering of CD4-dependent T cells: a putative mechanism for T cell affinity maturation.

In vivo, T cells expressing low-affinity TCR predominate in primary, but not in secondary responses, a process referred to as T cell affinity maturation. Using CD4 dependence as a measure of the avidity of the interaction between the allospecific TCR and the alloantigen, we show that a similar process occurs in mixed lymphocyte cultures in vitro. Moreover, in coculture experiments high-avidity (CD4-independent) T cell clones inhibited the TCR internalization of low-avidity (CD4-dependent) T cell clones, whereas low-avidity T cell clones had no such effect on high-avidity T cell clones. The extent of inhibition of TCR internalization was dependent on both the avidity of the competing clone and the number of competing cells. Thus, there was a cell dose- and avidity-dependent effect on TCR internalization, an early parameter in T cell activation. These results suggest that low- and high-avidity T cell clones compete for the availability of antigen-presenting cells and that this favors the selective outgrowth of high-avidity T cell clones.

CD3 Complex↗
PubMed2001

Quantity and rigor of qualitative research in four pastoral counseling journals.

Examines the quantity (N = 26) and rigor of qualitative research in The Journal of Pastoral Care, Pastoral Sciences, Journal of Religion and Health, and Pastoral Psychology for 1993-1997. Defines qualitative research using the work of Douglas Sprenkle and Sidney Moon. Uses the eleven criteria developed by Nicholas Mays and Catherine Pope in British Medical Journal for judging rigor. Finds low quantity and mixed quality and discusses implications.

Bibliometrics↗
PubMed2000

Superior tumor protection induced by a cellular vaccine carrying a tumor-specific T helper epitope by genetic exchange of the class II-associated invariant chain peptide.

Efficient loading of MHC class II molecules with a T helper epitope of choice can be achieved through genetic exchange of the MHC class II-associated invariant chain peptide (CLIP) sequence with a sequence encoding the helper peptide. We have now used this method to engineer a cellular vaccine that continuously expresses a tumor-specific helper epitope in a defined costimulatory context. We provide evidence (a) that this cellular vaccine induces peptide-specific helper T cells in vivo that are functional in protecting mice from challenge with a highly aggressive tumor, (b) that this vaccine can directly prime tumor-specific helper T cells in vivo, and (c) that this cellular vaccine is superior compared with similar cells loaded with synthetic T helper peptide in inducing tumor protection. In conclusion, cellular vaccines for activation of antigen-specific helper T cells can be greatly improved by the introduction of invariant chain constructs containing a T helper epitope by class II-associated invariant chain peptide exchange.

Amino Acid Sequence↗
PubMed2000

A Leishmania homologue of receptors for activated C-kinase (LACK) induces both interferon-gamma and interleukin-10 in natural killer cells of healthy blood donors.

Natural killer (NK) cells from individuals unexposed to Leishmania organisms proliferate with high interferon (IFN)-gamma secretion in response to crude Leishmania antigen preparations. In an attempt to identify the molecules that induce blood cells to proliferate and to secrete cytokines, we tested the effect of a 36-kDa Leishmania homologue of receptors for activated C-kinase (LACK) on peripheral blood mononuclear cells from unexposed individuals. Mainly CD8(+) and NK cells proliferated in response to LACK. At both the mRNA and soluble protein level, the main sources for LACK-induced IFN-gamma and interleukin (IL)-10 were T and NK cells. Furthermore, in the presence of anti-major histocompatibility complex (MHC) class II antibody, there was inhibition of LACK responses in both CD4(+) and CD16/56(+) cells, with a marked decrease in IFN-gamma but with an increase in IL-10 production. We conclude that the response to LACK is part of the response to Leishmania organisms in unexposed donors described elsewhere. That this NK-dominated response is MHC class II sensitive, whether through a direct or indirect effect, is discussed.

Animals↗
PubMed2000

Identification of a novel tumor-specific CTL epitope presented by RMA, EL-4, and MBL-2 lymphomas reveals their common origin.

C57BL/6 mice generate a vigorous H-2Db-restricted CTL response against murine leukemia virus (MuLV)-induced tumors. For many years it has been suggested that this response is directed to an MuLV-encoded peptide as well as to a nonviral tumor-associated peptide. Recently, a peptide from the leader sequence of gag was demonstrated to be the MuLV-derived epitope. Here we describe the molecular identification of the tumor-associated epitope. Furthermore, we show that the CTL response against this epitope can restrict the outgrowth of MuLV-induced tumors in vivo. The epitope is selectively presented by the MuLV-induced T cell tumors RBL-5, RMA, and MBL-2 as well as by the chemically induced T cell lymphoma EL-4. Intriguingly, these tumors share expression of the newly identified epitope because they represent variants of the same clonal tumor cell line, as evident from sequencing of the TCR alpha- and beta-chains, which proved to be identical. Our research shows that all sources of RBL-5, RMA, RMA-S, MBL-2, and EL-4 tumors are derived from a single tumor line, most likely EL-4.

Adoptive Transfer↗
PubMed2000

Chronic schistosomiasis: dendritic cells generated from patients can overcome antigen-specific T cell hyporesponsiveness.

Chronic infection with helminths is associated with down-regulated antigen-specific T cell responses. In order to determine whether schistosome-specific T cells are present, yet functionally unresponsive, or absent in the periphery of chronically infected persons, autologous granulocyte-macrophage colony-stimulating factor and dendritic cells (DC) derived from interleukin (IL)-4 were used as highly efficient antigen-presenting cells (APC). Peripheral blood mononuclear cells (PBMC) from persons harboring Schistosoma haematobium infection and hyporesponsive to parasite antigen were cocultured with autologous DC in the presence of adult worm antigen (AWA). In contrast to PBMC alone, DC-supplemented cultures responded to AWA by proliferation and by IL-4 and IL-5 production and, in some patients, by production of interferon-gamma. Thus, schistosome-specific T helper cells are present in the periphery but are functionally hyporesponsive during active infection with schistosomes. Cytokine responses representing the Th1 and (more importantly) Th2 types can be restored in vitro when DC are used as APC.

Adolescent↗
PubMed2000

The intestinal T cell response to alpha-gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase.

The great majority of patients that are intolerant of wheat gluten protein due to celiac disease (CD) are human histocompatibility leukocyte antigen (HLA)-DQ2(+), and the remaining few normally express HLA-DQ8. These two class II molecules are chiefly responsible for the presentation of gluten peptides to the gluten-specific T cells that are found only in the gut of CD patients but not of controls. Interestingly, tissue transglutaminase (tTG)-mediated deamidation of gliadin plays an important role in recognition of this food antigen by intestinal T cells. Here we have used recombinant antigens to demonstrate that the intestinal T cell response to alpha-gliadin in adult CD is focused on two immunodominant, DQ2-restricted peptides that overlap by a seven-residue fragment of gliadin. We show that tTG converts a glutamine residue within this fragment into glutamic acid and that this process is critical for T cell recognition. Gluten-specific T cell lines from 16 different adult patients all responded to one or both of these deamidated peptides, indicating that these epitopes are highly relevant to disease pathology. Binding studies showed that the deamidated peptides displayed an increased affinity for DQ2, a molecule known to preferentially bind peptides containing negatively charged residues. Interestingly, the modified glutamine is accommodated in different pockets of DQ2 for the different epitopes. These results suggest modifications of anchor residues that lead to an improved affinity for major histocompatibility complex (MHC), and altered conformation of the peptide-MHC complex may be a critical factor leading to T cell responses to gliadin and the oral intolerance of gluten found in CD.

Adult↗