[The primary immune response].
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Antigen-Antibody Reactions↗
Biomedical subjects
D Kabelitz
Publications and source records attributed to D Kabelitz.
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↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
Human gamma delta T lymphocytes.
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Animals↗
Differential effects of interleukin-7 and interleukin-2 on T-cell receptor gamma delta-expressing cells within CD4-CD8- postnatal human thymocytes.
Highly purified CD4-8- double-negative (DN) human thymocytes proliferated significantly more in response to interleukin (IL)-7 than to IL-2 in a dose-dependent fashion. Both IL-7 and IL-2 promoted the appearance of T-cell receptor (TCR) gamma delta-expressing cells in single cell suspension culture of DN thymocytes. While IL-7 exerted a vigorous growth-stimulating effect on TCR gamma delta-bearing DN thymocytes, IL-2 preferentially maintained the viability of gamma delta thymocytes without promoting their strong proliferation. In both instances, the major subset of gamma delta thymocytes expressed V delta 1 rather than V delta 2 as a variable delta-chain element on their surface. These results reveal differential effects of IL-7 and IL-2 on TCR gamma delta-bearing human thymocytes and underline the importance of both ILs in the development of DN thymocytes.
CD4-Positive T-Lymphocytes↗
In vitro effects of mistletoe extracts and mistletoe lectins. Cytotoxicity towards tumor cells due to the induction of programmed cell death (apoptosis).
The in vitro effects of therapeutically administered mistletoe extracts (ABNOBAviscum) and pure mistletoe lectins (mainly mistletoe lectin I) on a variety of human and murine tumor cell lines have been investigated. Mistletoe extracts and purified mistletoe lectins inhibited in vitro the growth of all tumor cell lines tested including B cell hybridomas, P815, EL-4, Ke37, MOLT-4 and U937. The mechanism of growth arrest was shown to be due to the induction of programmed cell death (apoptosis). Thus, fragmentation of genomic DNA into oligonucleosomal bands of approximately 200 base pairs in length was observed within 20 h when tumor cells were incubated with mistletoe extracts or lectins. These data point to a rational basis for the direct cytotoxic effects of mistletoe extracts and lectins apart from the postulated immunostimulatory properties of these agents.
Adjuvants, Immunologic↗
A rapid staining procedure for two-color analysis of lymphocyte antigen expression.
Two color immunofluorescence analysis of lymphocyte cell surface antigen expression using an unconjugated plus a biotinylated monoclonal antibody (mAb) requires four incubation steps: (1) unconjugated mAb; (2) fluorochrome-labelled goat anti-mouse Ig; (3) biotinylated mAb; (4) fluorochrome-labelled avidin or streptavidin. We describe a time-saving modification of this procedure which requires only two incubation steps: (1) simultaneous unconjugated and biotinylated mAbs; (2) fluorochrome-labelled avidin/streptavidin followed by fluorochrome-labelled goat anti-mouse Ig. The slightly delayed (5 min) addition of the goat anti-mouse Ig prevents it from binding to the mAb which has already interacted with avidin/streptavidin. Both procedures yield identical results with a variety of different mAbs.
Animals↗
Immunological studies of gamma delta T cells in a case of large granular lymphocyte (LGL) leukemia: leukemic gamma delta+ T cells are resistant to growth stimulation in vitro but respond to interferon-alpha treatment in vivo.
Leukemic T cells from patient BU (WBC 22,000/microliter) resembled morphologically large granular lymphocytes, and expressed a V delta 1-encoded gamma delta T-cell receptor (TCR) on their surface. Upon in vitro activation with various mitogens, IL-2-dependent V delta 1+ cell lines were only obtained if V delta 1+ cells had been positively selected on a cell sorter before culture. However, even under these conditions, the V delta 1+ IL-2-dependent cell lines showed a TCR beta and gamma gene-rearrangement different from that of the freshly isolated leukemic cell population, indicating that they were not derived from the leukemic clone. Thus, known T-cell growth factors (IL-2, IL-4, IL-7) in concert with mitogenic signals failed to induce in vitro proliferation of the V delta 1+ leukemic clone. Interestingly, the patient responded to treatment with interferon-alpha 2c (daily dose of 10(6) I.U.). After 2 1/2 years of continuous interferon treatment, the patient is in partial remission with WBC around 8000/microliter.
Adult↗
Life and death of a superantigen-reactive human CD4+ T cell clone: staphylococcal enterotoxins induce death by apoptosis but simultaneously trigger a proliferative response in the presence of HLA-DR+ antigen-presenting cells.
We report that a human CD4+ T cell clone with specificity for staphylococcal enterotoxin (SE) superantigens A, D, and E can respond to SEs in two seemingly opposite ways. In the absence of antigen presenting cells (APC), SEA, D, and E (but not SEB or C1) strongly inhibited in a dose-dependent manner the responsiveness of clone D894/25 to exogenous IL-2. Growth inhibition was due to SE-induced programmed cell death (apoptosis) as shown by propidium iodide staining and the appearance of the characteristic ladder pattern of DNA fragmentation. Apoptotic cell death was accompanied by significant cell lysis after 4 and 8 h as measured in a 51Cr release assay. In contrast (but as expected), a proliferative response of clone D894/25 was triggered by SEA, D, and E in the absence of exogenous IL-2 but presence of HLA class II-positive lymphoblastoid cell line (LCL) as APC. Moreover, the addition of LCL feeder cells partially prevented the suppression of IL-2 responsiveness by SEs. Surprisingly, however, the latter two culture conditions (i.e. presence of LCL feeder cells with or without exogenous IL-2) were associated with similar levels of induced cell death as in the absence of LCL. At the clonal level, these data demonstrate that SE superantigens induce programmed cell death in a fraction (40-50%) of responsive mature T cells, irrespective of the presence or absence of MHC class II-positive APC. We conclude that the proliferative response of clone D894/25 which is triggered by SEs in the presence of APC and absence of IL-2 must originate from the fraction (50-60%) of clone T cells surviving SE-induced cell death.
Antigen-Presenting Cells↗
Preferential activation of peripheral blood V gamma 9+ gamma/delta T cells by group A, B and C but not group D or F streptococci.
Previous studies have established that inactivated mycobacteria are potent and selective activators of V gamma 9+/V delta 2+ human gamma/delta T cells. Here we have analysed the proliferative response of human gamma/delta T cells to five serologically distinct groups of streptococci. While heat-inactivated streptococci of all five serogroups tested (A, B, C, D and F) induced a strong proliferative response in peripheral blood mononuclear cells (PBMC), only groups A, B and C elicited a selective activation of V gamma 9+ gamma/delta T cells in 10 (serogroup B) or 11 (serogroups A and C) of 11 tested healthy individuals. In striking contrast, groups D and F streptococci failed to activate gamma/delta T cells in nine of 11 donors and induced only a weak gamma/delta T cell response in two additional individuals. Depletion of V gamma 9+ T cells before culture completely eliminated all gamma/delta T cell responses to streptococci. These data indicate that groups A, B and C (but not D or F) streptococci can be included in the growing list of selective ligands for V gamma 9+/V delta 2+ human gamma/delta T cells.
Cells, Cultured↗