PubMed · 4648677
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R Timpl. 1972. [Immunology].. https://pubmed.ncbi.nlm.nih.gov/4648677/
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The stimulation of activated T cells with soluble peptides or peptide-pulsed T-APC in the absence of professional APC can anergize peptide-specific T cells. Here, we studied human T cell clones (TCCs) that either proliferate (T-responder) or do not proliferate (T-nonresponder) to activated T cells as antigen-presenting cells (APC) and investigated the efficacy of anergy induction in these two types of TCCs. The TCCs were specific to the p30 peptide from tetanus toxoid and secreted either a Th0- or a Th1-like cytokine pattern. To induce anergy, the TCCs were first stimulated by addition of the peptides directly to the cell cultures without additional APC (T-APC). Anergy was detected by restimulating these TCCs on professional B-APC. The proliferation, production of cytokines (IL2, IFN-gamma, IL4, IL5, IL10), and the cytotoxicity were measured after the first and second stimulation and compared with nonanergized control cells. Priming of TCCs by T-APC (anergy induction) resulted in an elevated production of IL4. This cytokine shift was also seen in the T-nonresponder TCC despite no induced proliferation. Th1-like TCCs retained their cytotoxicity after anergy induction. In contrast to cells first activated by B-APC, the restimulation of TCCs primed by T-APC lead to a drastic reduction of proliferation and cytokine production for both T-responder and T-nonresponder TCCs. The functional down-regulation of TCCs mediated by soluble peptides could be overcome by addition of IL2, but not by IL1 or IL4. We concluded that the induction of T-cell anergy does not require cell proliferation.
BACKGROUND: Recent observations suggest that thrombosis in vivo is initiated via the tissue factor (TF) pathway. The TF activity of human coronary atheroma has not been reported. METHODS AND RESULTS: Directional coronary atherectomy (DCA) specimens from 63 lesions were analyzed with the use of a quantitative TF-specific activity assay. The median content of TF was 10 ng/g plaque (95% CI, 6 to 13 ng/g; range, 0 to 47 ng/g). After homogenization of the specimens, TF activity was detected in 28 of 31 lesions (90%). With a polyclonal anti-human TF antibody, the use of immunohistochemistry detected TF antigen in 43 of 50 lesions (86%); TF antigen was expressed in cellular and acellular areas of the plaque. Histologically defined thrombus was present in 19 of the 43 lesions with detectable TF antigen and in none of the 7 lesions without detectable TF antigen (19 of 43 versus 0 of 7; P < .02). TF antigen was undetectable with immunohistochemistry in 4 of 13 restenotic lesions (31%) and in 3 of 37 de novo lesions (8%) (P < .05). CONCLUSIONS: TF contributes to the procoagulant activity of most atherosclerotic lesions treated with DCA. The association of immunohistochemically detectable TF with plaque thrombus suggests that TF plays a role in coronary thrombosis. Diminished TF expression in restenotic lesions may in part account for the lower complication rate that has been associated with DCA of restenotic versus de novo lesions. Inhibition of TF may represent a therapeutic goal for the prevention of thrombotic complications associated with percutaneous coronary interventions.
Fixation/permeabilization methods used for the detection of intracellular antigens by flow cytometry often result in the destruction of cellular morphology and surface immunoreactivity, properties useful in flow cytometry for the characterization of cells in heterogeneous populations. In addition, a majority of these methods are incompatible with whole blood and require that peripheral blood leukocytes (PBLs) be purified prior to fixation. This article describes a new technique for the rapid detection of both intracellular and cell surface antigens, while preserving cell morphology, through the use of a single-step fixation/permeabilization reagent, ORTHO PermeaFix (OPF). OPF is compatible with whole blood, allowing for the direct preparation of PBLs without prior cell separation. An additional red blood cell lysing reagent was not required because RBC lysis occurred upon resuspension of OPF-treated whole blood samples in isotonic solution. Discrimination of leukocyte populations by light scatter after OPF treatment was comparable to matched unfixed live cells. In addition, absolute lymphocyte and white blood cell (WBC) counts were not significantly affected when OPF-treated cells were compared with unfixed cells. Treatment of whole blood from 7 normal donors showed no significant difference in percentage of cells positive for CD2, CD3, CD4, CD8, CD16, or CD19 between fixed and unfixed samples when cells were stained before fixation, and no difference in CD3, CD4, CD8, CD16, or CD19 percentages when cells were stained following fixation. Monoclonal antibodies specific for intracellular antigens located at various sites within the cell were tested on fixed samples. OPF-treated peripheral blood lymphocytes showed greater than 95% reactivity for the inner mitochondrial membrane protein bcl-2, and the cytoskeletal cytoplasmic protein vimentin. TIA-1, a cytolytic granule-associated protein, showed differential reactivity within lymphocyte subsets, from a low of 8 +/- 2% in CD4+ cells to 89 +/- 6% in CD16+ cells, when whole blood from five normal donors was fixed and stained. Reh cells treated with OPF showed greater than 95% reactivity for the internuclear protein TdT. A comparison of OPF with two other fixation/permeabilization procedures, 1% paraformaldehyde followed by 45% ethanol and 0.25% paraformaldehyde followed by 0.2% Tween 20, showed that only OPF could be used both prior to or following cell surface staining with no effect on antigen detection while allowing optimal detection of all of the intracellular antigens tested.