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Biomedical subjects

D Kabelitz

Publications and source records attributed to D Kabelitz.

At least 91 records · Page 5Linked to original sources
PubMed1995

Induction of TCR-gamma delta expression on triple-negative (CD3-4-8-) human thymocytes. Comparative analysis of the effects of IL-4 and IL-7.

It is well established that IL-4 and IL-7 control the differentiation of TCR-gamma delta-expressing cells from CD3/TCR-negative thymic precursors. In this study, we have compared the in situ expression of IL-4 and IL-7 mRNA in human postnatal thymus with the in vitro effect of IL-4 and IL-7 on the expansion of TCR-gamma delta + cells from highly purified CD3-4-8- triple-negative thymocytes. IL-4 mRNA expression was restricted to subcapsular regions of the human thymus, whereas cells expressing IL-7 mRNA were distributed throughout the thymic tissue, with some enrichment in subcapsular and cortical regions. Epithelial cells of the outer layer of Hassall's corpuscle strongly expressed IL-7 mRNA. IL-7 but not IL-4 or IL-2, stimulated strong proliferative activity and cellular expansion of triple-negative thymocytes. All three induced the appearance of TCR-gamma delta + cells within 4 days of culture. In the presence of IL-4 or IL-2, 30 to 55% of viable cells were TCR-gamma delta + after 5 to 9 days, whereas only 10 to 15% triple-negative thymocytes cultured with IL-7 expressed TCR-gamma delta. However, comparable absolute numbers of viable TCR-gamma delta + thymocytes were recovered when triple-negative thymocytes were cultured with IL-4 or IL-7, but not with IL-2. Taken together, our results suggest that IL-4 and IL-7 (both of which are produced in situ in thymic tissue) play equally important roles in the in vitro generation of TCR-gamma delta + thymocytes from triple-negative precursor cells.

Gene Expression↗
PubMed1995

Antigen-induced death of alloreactive human T-lymphocytes occurs in the absence of low molecular weight DNA fragmentation.

Stimulation via the CD3/TCR molecular complex induces proliferation of resting T cells, but triggers programmed cell death (apoptosis) in immature thymocytes and preactivated mature T cells. Activation-induced cell death (AICD) triggered by anti-CD3/TCR mAb or by staphylococcus enterotoxin superantigen is associated with fragmentation of genomic DNA into oligonucleosomal fragments of 200 bp length, thus displaying the characteristic features of apoptosis. Here, we show that a fraction (20-50%) of cells in alloreactive CD8 human short-term T cell lines, generated by repeated restimulation with EBV-transformed B cell lines, undergo AICD when restimulated with the appropriate (but not with third party) stimulator cells. AICD of responder T cells is inhibited when stimulator cells are preincubated with anti-HLA class I mAb but not with anti-HLA class II mAb, indicating that T cell death is dependent on alloantigen (HLA class I) recognition by responding CD8 T cells. Importantly, alloantigen-induced T cell death occurs in the absence of detectable DNA fragmentation. Thus, several independent assay systems all failed to reveal low molecular weight DNA fragmentation, even though DNA fragmentation was readily detected in T cell lines exposed to PHA or gamma-irradiation. Alloantigen-induced T cell death was prevented by aurintricarboxylic acid, which has previously been shown to inhibit apoptosis in experimental systems where no DNA fragmentation occurs. Taken together, these results demonstrate that alloantigen can trigger AICD in mature responding T cells in the absence of low molecular weight DNA fragmentation.

Cell Death↗
PubMed1995

Human T-cell receptor variable gene segment families.

Multiple DNA and protein sequence alignments have been constructed for the human T-cell receptor alpha/delta, beta, and gamma (TCRA/D, B, and G) variable (V) gene segments. The traditional classification into subfamilies was confirmed using a much larger pool of sequences. For each sequence, a name was derived which complies with the standard nomenclature. The traditional numbering of V gene segments in the order of their discovery was continued and changed when in conflict with names of other segments. By discriminating between alleles at the same locus versus genes from different loci, we were able to reduce the number of more than 150 different TCRBV sequences in the database to a repertoire of only 47 functional TCRBV gene segments. An extension of this analysis to the over 100 TCRAV sequences results in a predicted repertoire of 42 functional TCRAV gene segments. Our alignment revealed two residues that distinguish between the highly homologous V delta and V alpha, one at a site that in VH contacts the constant region, the other at the interface between immunoglobulin VH and VL. This site may be responsible for restricted pairing between certain V delta and V gamma chains. On the other hand, V beta and V gamma appear to be related by the fact that their CDR2 length is increased by four residues as compared with that of V alpha/delta peptides.

Alleles↗
PubMed1995

Mouse T-cell receptor variable gene segment families.

All mouse T-cell receptor alpha/delta, beta, and gamma variable (Tcra/d-, b-, and g-V) gene segments were aligned to compare the sequences with one another, to group them into subfamilies, and to derive a name which complies with the standard nomenclature. It was necessary to change the names of some V gene segments because they conflicted with those of other segments. The traditional classification into subfamilies was re-evaluated using a much larger pool of sequences. In the mouse, most V gene segments can be grouped into subfamilies of closely related genes with significantly less similarity between different subfamilies.

Amino Acid Sequence↗
PubMed1995

Comparison of human and mouse T-cell receptor variable gene segment subfamilies.

Like the immunoglobulin Igh-V and Igk-V gene families, the human or mouse TCRV gene families may be grouped into subfamilies displaying > 75% nucleic acid sequence similarity among their members. Systematic interspecies sequence comparisons reveal that most mouse Tcr-V subfamilies exhibit clear homology to human TCRV subfamilies (> 60% amino acid sequence similarity). Homologous pairs of TCRV genes in mice and humans show higher sequence similarity than TCRV genes from different subfamilies within either species, indicating transspecies evolution of TCRV genes. Mouse and human homologues show conservation of their relative map order, particularly in the 3' region and a similar sequential and developmentally programmed expression. When the V regions from both species were analyzed together, local length differences and conserved residues in the loop regions were revealed, characteristic of each of the four TCRV families.

Alleles↗
PubMed1995

Differential effects of interleukin-2 and interleukin-7 on the induction of CD4 and CD8 expression by double-negative human thymocytes.

Expression of cell surface CD4 (in the absence of CD3/T cell receptor) characterizes an early stage of intrathymic T cell development. Here, we investigated the appearance of CD4 and CD8 expression on highly purified CD4-8- double-negative human thymocytes in response to interleukin (IL)-7 and IL-2. While IL-7 preferentially promoted the appearance of CD4 single-positive and CD4+8+ double-positive thymocytes, IL-2 primarily induced CD8 single-positive thymocytes. A significant fraction of CD4 single-positive cells generated from double-negative thymocytes via IL-7 lacked cell surface CD3 expression. In contrast, the majority of CD8 single-positive cells generated from double-negative thymocytes via IL-2 coexpressed CD3. We conclude that IL-7 and IL-2 exert differential effects on the differentiation of early human T cell progenitors.

CD3 Complex↗
PubMed1995

Synovial fibroblasts as accessory cells for staphylococcal enterotoxin-mediated T-cell activation.

Rheumatoid arthritis (RA) is thought to be the result of T-cell-mediated autoimmune phenomena. So far, a critical autoantigen has not been identified. Recently, superantigens have been implied in the pathogenesis of RA. In the present study it was tested whether major histocompatibility complex (MHC) class II-positive synovial fibroblast cells (SFC) function as superantigen-presenting cells. SFC were stimulated with interferon-gamma (IFN-gamma) to express class II antigens; then they were cultivated in the presence of T cells with or without staphylococcal enterotoxins (SE). T-cell activation was measured as proliferation and interleukin-2 (IL-2) production. Depending on the dose and type of SE, activation of T-cell clones and also of peripheral T cells was seen. T-cell activation was inhibited by antibodies to MHC class II antigens and also by antibodies to intracellular adhesion molecule type-1 (ICAM-1). The data suggest that class II-positive SFC have the capacity to serve as accessory cells for superantigen-mediated T-cell activation. Thus SFC may participate in the propagation of a T-cell dependent immune response.

Antigen-Presenting Cells↗
PubMed1995

Immunoregulatory effects of the herba Epimediia glycoside icariin.

The in vitro effects of the Chinese Herba Epimediia glycoside icariine (ICA), a chinese herbal extract, on human immune responses were investigated. ICA induced a weak and delayed proliferation of peripheral blood mononuclear cells from healthy donors when compared to phytohemagglutinin. Both T- (TCR alpha beta+) and B cells were the ICA-responding cells. Within T-cells, the relative proportion of CD4-8+ cells increased but that of CD4+8- cells decreased. ICA in certain concentrations could increase lymphokine-activated killer cell (LAK) activity (0.1 to 1.0 microgram/ml) in both tumor patients and healthy donors, and natural killer cell (NK) activity (1.0 to 5.0 micrograms/ml) in tumor patients. Moreover, ICA stimulated the production of tumor necrosis factor-alpha in monocytes from healthy donors. These findings provide evidence that ICA could be applied to adoptive immunotherapy. Generation of LAK cells in presence of an appropriate dose of ICA might be superior to interleukin-2 alone.

Adjuvants, Immunologic↗
PubMed1994

Surface expression of functional T cell receptor chains formed by interlocus recombination on human T lymphocytes.

Structural diversity of lymphocyte antigen receptors (the immunoglobulin [Ig] of B cells and the alpha/beta or gamma/delta T cell receptor [TCR] of T cells) is generated through somatic rearrangements of V, D, and J gene segments. Classically, these recombination events involve gene segments from the same Ig or TCR locus. However, occurrence of "trans" rearrangements between distinct loci has also been described, although in no instances was the surface expression of the corresponding protein under normal physiological conditions demonstrated. Here we show that hybrid TCR genes generated by trans rearrangement between V gamma and (D) J beta elements are translated into functional antigen receptor chains, paired with TCR alpha chains. Like classical alpha/beta T cells, cells expressing these hybrid TCR chains express either CD4 or CD8 coreceptors and are frequently alloreactive. These results have several implications in terms of T cell repertoire selection and relationships between TCR structure and specificity. First, they suggest that TCR alloreactivity is determined by the repertoire selection processes operating during lymphocyte development rather than by structural features specific to V alpha V beta regions. Second, they suggest the existence of close structural relationships between gamma/delta and alpha/beta TCR and more particularly, between V gamma and V beta regions. Finally, since a significant fraction of PBL (at least 1/10(4)) expressed hybrid TCR chains on their surface, these observations indicate that trans rearrangements significantly contribute to the combinatorial diversification of the peripheral immune repertoire.

Antibodies, Monoclonal↗
PubMed1994

Frequency analysis of donor-reactive cytotoxic T lymphocyte precursors in in allograft recipients. Lack of correlation with clinical outcome.

The frequency of donor-reactive and of third party-reactive cytotoxic T lymphocyte precursors (CTLp) was determined by limiting dilution analysis in the peripheral blood of 13 liver allograft recipients before (pre-tpx) and at various intervals (4 to 22 months) after (post-tpx) transplantation. In 6 of 13 patients with stable graft function, no change of donor-reactive CTLp occurred within 9 to 17 months post-tpx, and high levels of donor-reactive CTLp (frequencies ranging from 1/1024 to 1/13,295) were maintained. In contrast, a 4-11-fold reduction of donor-reactive CTLp frequencies was observed in 5 patients within 4 to 22 months post-tpx; in 3 of these patients the measured frequency of donor-specific CTLp dropped to biologically irrelevant levels (1/43,800 to 1/150,000). However, a 5-7-fold increase of the post-tpx frequency of donor-reactive CTLp was observed in two additional patients with stable graft function. Taken together, these results demonstrate that the frequency analysis of donor-reactive CTLp in the peripheral blood does not closely correlate with the clinical outcome. Thus, stable liver allograft function after > 1 year of transplantation can be associated with a specific deletion, unaltered levels, or even an increase in the frequency of circulating donor-reactive CTLp.

Adolescent↗
PubMed1994

Rapid quantification of lymphocyte subsets in heterogeneous cell populations by flow cytometry.

Determination of the number of viable cells or quantification of lymphocyte subsets in heterogeneous cell populations is critically important for cytotoxicity assays, apoptosis assays, or the analysis of differential activation of T-cell subsets by distinct stimuli. In this report, we describe a rapid flow cytometry method termed Standard Cell Dilution Analysis (SCDA) specifically to quantify any subset of phenotypically definable, viable cells in heterogeneous populations using a FACScan flow cytometer. This method combines: (1) specific detection of lymphocyte subsets by phycoerythrin-conjugated monoclonal antibodies, (2) electronic exclusion of dead cells or cell debris by propidium-iodide staining and gating on forward vs. sidescatter, respectively, and (3) admixture of a known amount of fixed, fluorescein isothiocyanate stained cells immediately before analysis as a constant parameter to allow for calculation of cell quantity. We have used SCDA to analyze the in vitro growth characteristics of various human T-lymphocyte subpopulations in response to different activation stimuli.

Antibodies, Monoclonal↗
PubMed1994

Primary activation of V gamma 9-expressing gamma delta T cells by Mycobacterium tuberculosis. Requirement for Th1-type CD4 T cell help and inhibition by IL-10.

Purified peripheral blood gamma delta T cells proliferated vigorously in response to killed Mycobacterium tuberculosis (M. tb.) in the presence of PBMC but not in the presence of T cell-depleted (E-) feeder cells. Addition of graded numbers of autologous CD4 T cells to E- feeder cells reconstituted in a dose-dependent fashion the response of V gamma 9-expressing gamma delta T cells to M. tb. IL-2 was identified as the major CD4 T cell-derived helper factor required for gamma delta T cell proliferation after stimulation with M. tb. In addition, neutralizing anti-IFN-gamma but not anti-IFN-alpha Ab inhibited the responsiveness of V gamma 9 T cells, suggesting that endogenously produced IFN-gamma was involved in the activation of gamma delta T cells by M. tb. Although gamma delta T cells could not proliferate on their own in the absence of CD4 T cells (or exogenous IL-2), the appearance of IL-2 receptors (CD25) was triggered in the absence of CD4 T cells. Furthermore, IL-10 strongly inhibited the activation of V gamma 9 T cells among unfractionated PBMC responder cells. Similarly, the responsiveness of purified gamma delta T cells to M. tb. occurring in the presence of CD4 T cells was strongly inhibited by IL-10, whereas the activation occurring in the presence of exogenous IL-2 was not impaired. These results show that interactions with Th1-type CD4 T cells are required for efficient activation of peripheral blood gamma delta T cells by M. tb. In addition, our results have practical implications for creating experimental conditions aimed at identifying V gamma 9-selective (myco)bacterial ligands.

Antigen-Presenting Cells↗
PubMed1994

New monoclonal antibody (23D12) recognizing three different V gamma elements of the human gamma delta T cell receptor. 23D12+ cells comprise a major subpopulation of gamma delta T cells in postnatal thymus.

The gamma delta TCR is expressed on 1 to 5% of CD3+ human peripheral blood T lymphocytes. The majority of peripheral blood gamma delta T cells expresses V gamma 9 paired with V delta 2; this subset strongly responds to certain microbial ligands. Other gamma delta T cell subsets with unknown Ag specificity expressing different V gamma elements are present in peripheral blood and lymphoid tissue. We describe a new anti-human V gamma mAb termed 23D12 with unusual specificity. As revealed by analysis of a large number of T cell clones and transfectants expressing molecularly well-defined gamma delta TCR, mAb 23D12 recognized several, but not all, members of the human V gamma 1 family, specifically V gamma 2, V gamma 3, and V gamma 4 but not V gamma 5 or V gamma 8. In combination with available mAb against V gamma 4, mAb 23D12 was used to identify V gamma 2- or V gamma 3-bearing cells. On average, 23D12+ cells accounted for 18% of peripheral blood gamma delta T cells and 56% of postnatal gamma delta thymocytes. In combination with anti-V gamma 9 mAb, mAb 23D12 23D12 identified gamma delta cells expressing V elements other than V gamma 2, V gamma 3, V gamma 4, or V gamma 9. Such cells are detectable in peripheral blood and postnatal thymus. Using mAb 23D12, we also confirmed the appearance of two distinct TCR gamma-chains on the surface of some gamma delta T cells.

Animals↗
PubMed1994

T cell receptor gamma delta repertoire in HIV-1-infected individuals.

While V gamma 9/V delta 2 cells dominate among peripheral blood gamma delta T cells in healthy adults, the majority of gamma delta T cells in most HIV-1-infected individuals express V delta 1. We asked whether these elevated levels of V delta 1 T cells were due to clonal expansion. Three-color flow cytometry with monoclonal antibodies against V gamma 2/V gamma 3/V gamma 4, V gamma 4 and V gamma 9 was used to investigate V gamma usage in 27 patients with elevated numbers of V delta 1 T cells. While the relative proportion of V gamma 9 cells among gamma delta T cells was significantly reduced in HIV-1+ individuals (10 +/- 11% vs. 80 +/- 17%, p < 0.001), the fraction of gamma delta T cells using V gamma 5 or V gamma 8 was significantly increased (54 +/- 15% vs. 7 +/- 11%, p < 0.001). In 1 patient, 76% of the V delta 1 cells expressed V gamma 2 or V gamma 3, suggesting clonality of the V delta 1 population. In line with this assumption, analysis of the V delta 1-J delta junctional regions by reverse transcription-polymerase chain reaction (RT-PCR) resulted in products of only one junctional length, as demonstrated by electrophoresis on denaturing gels, and 12 out of 16 (75%) in-frame junctional sequences were identical in this patient. In other HIV-1+ patients, RT-PCR resulted in products of several distinct sizes, also indicating a highly restricted repertoire. After sequencing the V delta 1-J delta junctional regions of 3 additional patients, we found repeated but patient-specific in-frame junctions accounting for 10-30% of the sequenced clones. However, limited V delta 1-J delta junctional diversity was also seen in healthy donors. RT-PCR products from 10 healthy individuals resulted in distinct bands on denaturing gels. In 1 of them exhibiting a single prominent band, 10 out of 17 (58%) sequenced junctions were identical. Two other healthy donors displayed 2/14 and 5/18 identical junctional sequences, respectively. Taken together, our results reveal significant alterations of V gamma usage in HIV-1+ patients, while the V delta 1 junctional repertoire is similarly restricted in HIV-1+ and HIV-1- individuals. Therefore, these data argue against an obligatory clonal expansion of V delta 1-expressing cells during HIV-1 infection.

Base Sequence↗
PubMed1994

Cytokines involved in intrathymic T cell development.

Cytokines play an important role in intrathymic T cell development. Over the past several years, genes of newly discovered cytokines have been cloned, and the respective recombinant proteins have become available for functional studies of T cell development. In this paper we have attempted to review the respective roles of interleukins 1, 2, 4, 6, and 7. Special emphasis was given to interleukin 7 which has been shown to play a key role in early T cell differentiation.

Animals↗