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D Kabelitz

Publications and source records attributed to D Kabelitz.

At least 91 records · Page 5Linked to original sources
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↗
PubMed1993

Induction of activation-driven death (apoptosis) in activated but not resting peripheral blood T cells.

Signaling via the CD3/TCR complex induces programmed cell death (apoptosis) in immature thymocytes and transformed T lymphocytes (hybridomas or leukemic cells). Accumulating evidence indicates, however, that apoptosis can be triggered also in mature peripheral T cells. Here we show that a significant fraction of cells of a given IL-2-dependent TCR-alpha beta + clone or polyclonal short term line is killed when cultured for 20 h in the presence of PHA, anti-CD3 (OKT3), or anti-TCR (BMA031) mAb. Apoptosis can be triggered by these stimuli in CD4+, CD8+, and CD4-CD8- (double-negative) TCR-alpha beta+ clones. Activation-driven cell death (as quantified by propidium iodide staining and FACS analysis) is associated with fragmentation of DNA into oligonucleosomal bands of approximately 200 bp. Although freshly isolated peripheral blood T cells are largely resistant to apoptosis, the sensitivity to anti-CD3/TCR mAb or PHA-triggered cell death gradually increases upon activation and IL-2-dependent culture of T cells, and reaches a plateau level after 15 to 20 days. These data indicate that stimuli that activate resting T cells initiate death by apoptosis in activated T cells. The implications of these results for the regulation of cellular immune responses and the establishment of peripheral tolerance will be discussed.

Apoptosis↗
PubMed1993

Activation-driven death of human T cell clones: time course kinetics of the induction of cell shrinkage, DNA fragmentation, and cell death.

Signaling via the CD3/T cell receptor complex induces programmed cell death (apoptosis) in IL-2-dependent human T lymphocytes. We have investigated the time course kinetics of the induction of programmed cell death in cloned CD4+ T cells. Morphological changes (cell shrinkage) were noted by flow cytometry as early as 3 hr after stimulation of clone cells with ionomycin, PHA, or anti-T cell receptor antibody BMA 031. Fragmentation of DNA became visible 3 hr after ionomycin stimulation, and 5 hr after PHA or BMA 031 stimulation, and peaked after 8 to 24 hr. Significant cell death (as revealed by flow cytometry analysis of propidium iodide-positive cells) was detected 5 hr after ionomycin treatment and 10 hr after PHA or BMA 031 treatment. With all three stimuli, maximal cell death was recorded after 16 to 18 hr. Taken together, our data indicate that the activation-induced death of mature human T cells is a rapid event which is completed within 18 hr. Induction of DNA fragmentation is preceded by the reduction of cell size which can be readily examined by flow cytometry.

Apoptosis↗
PubMed1993

Human peripheral blood gamma delta T cells are uniformly sensitive to destruction by the lysosomotropic agents leucine methyl ester and leucyl leucine methyl ester.

Treatment of peripheral blood mononuclear cells (PBMC) with the lysosomotropic agent leucine methyl ester (Leu-OMe) eliminates monocytes/macrophages and cytotoxic lymphocytes including CD3- CD16+ natural killer (NK) cells and a fraction of T cell receptor (TcR) alpha beta + CD8+ T cells. We report that freshly isolated peripheral blood gamma delta T cells are highly sensitive to Leu-OMe treatment as well. After incubation of PBMC with 5 mM Leu-OMe or incubation of purified T cells with 50 microM leucyl leucine methyl ester (Leu-Leu-OMe) and subsequent overnight culture, CD3-CD16+ NK cells and gamma delta T cells were no longer detectable by immunofluorescence analysis. The two major gamma delta T cell subsets V gamma 9+V delta 2+ and V gamma 9-V delta 1+ were equally susceptible to Leu-OMe and Leu-Leu-OMe treatment. The elimination of V gamma 9+ T cells by Leu-OMe treatment was confirmed in functional assays. Stimulation of peripheral blood T cells with killed mycobacteria resulted in selective expansion of V gamma 9+ T cells. In contrast, no activation of gamma delta T cells was elicited in Leu-OMe-treated responder T cells stimulated with killed mycobacteria.

Blood↗
PubMed1993

Triggering via the alternative CD2 pathway induces apoptosis in activated human T lymphocytes.

Activation of immature thymocytes or transformed (i.e. leukemic) T lymphocytes via CD3/T cell receptor (TcR) signaling can induce programmed cell death (apoptosis). Recent data indicate that anti-CD3/TcR monoclonal antibodies (mAb) also trigger apoptosis in activated (but not resting) mature peripheral LT cells. We now report that interleukin-2 (IL-2) dependent human polyclonal T cell lines as well as T cell clones undergo programmed cell death when triggered via the alternative CD2-dependent activation pathway. In the presence of exogenous IL-2, a pair of mitogenic anti-CD2 mAb suppressed the IL-2-driven proliferative response. Growth inhibition was associated with cell death and DNA fragmentation as revealed by propidium iodide staining and gel electrophoresis, respectively. Induction of apoptosis by anti-CD2 mAb was prevented by cyclosporine A and FK 506. We conclude that programmed cell death can be initiated in activated human T cells by signaling via the CD2 pathway.

Antibodies, Monoclonal↗
PubMed1993

V gamma gene usage in peripheral blood gamma delta T cells.

The majority (50-90%) of gamma delta T cells in the peripheral blood of adult individuals expresses a T-cell receptor (TCR) which uses V gamma 9 and V delta 2 as variable elements. Little is known about the distribution of other V gamma gene elements in the remaining 10-50% of gamma delta T cells. Here we have studied the V gamma gene expression in peripheral blood gamma delta T cells by 3-color flow cytometry analysis applying established monoclonal antibodies (mAb) directed against V gamma 9 and V gamma 4, as well as a novel mAb directed against V gamma 2, V gamma 3 and V gamma 4. On average, 79.9% of gamma delta T cells expressed V gamma 9, 11.9% V gamma 2/V gamma 3, 4.4% V gamma 4, and 7.5% one of the remaining V gamma 5, V gamma 8, V gamma 10 or V gamma 11 elements. There were remarkable variations in the gamma delta subset composition between individual donors. The majority (69.8%) of V gamma 2/V gamma 3/V gamma 4-bearing cells co-expressed V delta 1, while on average only 17.8% of V gamma 2/V gamma 3/V gamma 4-bearing cells co-expressed V delta 2. This is in contrast to V gamma 9-bearing gamma delta T cells, of which 83.1% used V delta 2 and only 12.7% V delta 1. Taken together, this data identifies V gamma 2/V gamma 3 as the second most frequently used set of V gamma elements in human peripheral blood gamma delta T cells.

Adult↗
PubMed1993

Activation-induced cell death (apoptosis) of mature peripheral T lymphocytes.

Programmed cell death (apoptosis) is triggered in immature thymocytes and T-cell hybridomas by signalling via the CD3-T-cell receptor pathway. In this paper, Dieter Kabelitz and colleagues catalogue the recently accumulating evidence that apoptosis can also be initiated in mature peripheral T cells; this may constitute an important aspect of cellular immune regulation.

Allergy and Immunology↗
PubMed1993

Evidence that functional deletion of donor-reactive T lymphocytes in kidney allograft recipients can occur at the level of cytotoxic T cells, IL-2-producing T cells, or both. A limiting dilution study.

The frequencies of circulating donor-reactive cytotoxic lymphocyte precursors (CLP) and Il-2-producing helper lymphocyte precursors (HLP) were determined by limiting dilution analysis in 19 kidney allograft recipients before and at various intervals (up to 2 years) after transplantation. A significant, selective, and stable reduction of the frequencies of donor-reactive (but not of third party-reactive) CLP and/or HLP was observed in some patients beginning 3 to 6 months after transplantation. One patient developed reduced frequency of CLP only, 3 patients reduced frequencies of HLP only, and 2 patients reduced frequencies of both CLP and HLP. The selective reduction of donor-reactive CLP and/or HLP frequencies ranged from 5-25-fold when compared with the pretransplantation level and was associated with stable graft function. These data indicate that functional deletion of circulating donor-reactive T cells can occur at the level of cytotoxic T lymphocytes, Il-2-producing helper T lymphocytes, or both. Implications of these findings for the individualization of immunosuppressive regimens will be discussed.

Humans↗
PubMed1993

Leukaemic T cells from patients with chronic lymphocytic leukaemia of T-cell origin respond to Staphylococcus aureus enterotoxin superantigens.

We investigated the in vitro responsiveness of peripheral blood lymphocytes from two patients with T-cell chronic lymphocytic leukaemia (T-CLL) to Staphylococcus aureus enterotoxin (SE) superantigens. T-cell receptor (TcR) alpha beta (V beta 7.1)-expressing CD4+ leukaemic T cells from patient HE (white blood cell count 480,000/microliters) proliferated in response to SEA and, only at 1000-fold higher concentrations, to SEB, SED, and SEE. CD4+CD8+ TcR alpha beta (V beta 12.1)-expressing leukaemic T cells from patient KO (white blood cell count 120,000/microliters) were activated by SEB but not by the other tested SEs. In both instances, the activation of leukaemic T cells by SE was dependent on the presence of HLA-DR+ cells. Southern blot analysis of TcR beta gene rearrangement confirmed that the proliferating cells were derived from the leukaemic T-cell clone and not from contaminating normal T cells. These data indicate that leukaemic T cells from patients with T-CLL exert a clonally variable responsiveness to SE superantigens. We conclude that recognition of specific antigen and subsequent signal transduction can be initiated via the TcR of leukaemic T-CLL cells.

Antigens, Bacterial↗
PubMed1993

Clonal specificity of human gamma delta T cells: V gamma 9+ T-cell clones frequently recognize Plasmodium falciparum merozoites, Mycobacterium tuberculosis, and group-A streptococci.

Peripheral blood gamma delta T cells expressing a V gamma 9/V delta 2 T-cell receptor are stimulated by killed bacteria including Mycobacterium tuberculosis (m.tb.) and group-A streptococci (strep A). In addition, recent data indicate that V gamma 9/V delta 2 T cells from unexposed individuals also respond to Plasmodium falciparum (P. falcip.) merozoites. Here we analyzed the reactivity to these ligands of 23 V gamma 9/V delta 2, 3 V gamma 9/V delta 1, and 4 V gamma 9-/V delta 1 clones derived from 8 healthy individuals after phytohemagglutinin stimulation of cell sorter-selected gamma delta T cells. Upon restimulation in the presence of irradiated antigen-presenting cells, the majority of V gamma 9/V delta 2 clones recognized m.tb. and strep A (but not strep D), and about one third of the clones also recognized P. falcip. Some clones, however, recognized only one or two of the tested ligands, and 4 V gamma 9/V delta 2 clones did not react at all. Interestingly, 2 of 3 V gamma 9/V delta 1 clones proliferated in response to m.tb., P. falcip., strep A and strep D, while V gamma 9-/V delta 1 clones were not activated by any of the tested ligands. Nucleotide sequence analysis indicated a broad diversity of V gamma 9 N regions in V gamma 9/V delta 2 clones. At the clonal level, our results demonstrate that individual V gamma 9/V delta 2 T cells can recognize m.tb., strep A, as well as P. falcip.-infected erythrocytes, with no influence of the expressed V gamma 9 N region.

Animals↗
PubMed1993

Interleukin-4 augments production of the third complement component by the alveolar epithelial cell line A549.

The elements of allergic inflammation and the involvement of helper T lymphocytes are increasingly being recognized in the immunopathogenesis of asthma. Allergen exposure leading to the activation of allergen-specific T cells present in the lung can result in the release of cytokines which in turn can locally stimulate the cellular constituents of the lung. The airway epithelial cells may be the key participants in such an interaction. Therefore, we examined the ability of T-cell-derived IL-4 to modulate the production of C3 and C5 by the human type-II pneumocyte cell line A549, which is known to produce all the components and the regulatory proteins of the complement system. For estimation of C3 an ELISA detecting native C3 was used. Following stimulation of A549 with hrIL-4 a dose-dependent (1-50 U/ml) enhancement of C3 production was observed, which reached its maximum (5-fold of unstimulated cells) at 48 h and gradually declined thereafter. Concentrations of hrIL-4 higher than 50 U/ml did not further increase C3 production. In parallel experiments hrIFN-gamma at concentrations between 10 and 50 U/ml stimulated the C3 production to more than twice the quiescent state level within 24 h. In the pneumocyte cell line A549 we demonstrated the expression of a gene for the IL-4 receptor which appears to mediate the biological effect of this lymphokine. A diminution in the functionally active C5, estimated by ELISA at the same time, was observed in supernatants of A549 cultures following stimulation with hrIL-4 as well as with hrIFN-gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line↗