https://iti.stanford.edu/content/dam/sm/iti/documents/himc/Bendall_etal_Trends-Immunology_2012.pdf

A deep profiler's guide to cytometry.

T lymphocyte mitosis results from the interaction of interleukin 2 (IL-2) with specific receptors that appear only after appropriate immune stimulation. To assess the potential role of IL-2 receptor levels in determining the rate and magnitude of T cell proliferation, the expression of IL-2 receptors by lectin-stimulated human peripheral blood T cells was examined and correlated with T cell growth. Using biosynthetically radiolabeled IL-2 and anti-Tac, a monoclonal antibody that blocks IL-2 receptor binding, IL-2 receptors were found to accumulate slowly and asynchronously among lectin-stimulated T cells and to precede the onset of DNA synthesis. Moreover, a critical threshold of IL-2 receptor density appeared to be required before the commitment to cell cycle progression, as analyzed quantitatively by tritiated thymidine incorporation and flow cytometric analysis of cellular DNA content. Once maximal IL-2 receptor expression occurred, continued proliferation was IL-2 concentration dependent as assessed using homogenous immunoaffinity-purified IL-2. Upon removal of the activating lectin, IL-2 receptor levels progressively declined, and, in parallel, the rate of proliferation diminished. The decay of IL-2 receptors could not be attributed to IL-2-mediated down-regulation. Instead, renewed IL-2 receptor expression was dependent upon the reintroduction of the initial activating signal. Repetitive exposure to lectin resulted in a more rapid reexpression of maximal IL-2 receptor levels, which was then followed by an accelerated resumption of proliferation. Thus, the extent of T cell proliferation after immune stimulation depends upon the interplay of the IL-2 concentration available and the density of IL-2 receptors expressed, both of which are ultimately determined by antigen/lectin stimulation. The awareness of the transience and the antigen/lectin dependence of IL-2 receptor expression, together with the capacity to monitor T cell cultures for IL-2 receptor levels, should facilitate the initiation and maintenance of cloned, antigen-specific T cells in long-term culture. In addition, these findings suggest that, in vivo, the rapidity of acquisition of maximum IL-2 receptor levels by activated T cells and the duration of IL-2 receptor expression may well direct the magnitude of T cell clonal expansion and resultant immune responses.

/pdf/transient-expression-of-interleukin-2-receptors-consequences-2lvammh8c7.pdf

Transient expression of interleukin 2 receptors. Consequences for T cell growth.

Purpose: To review the biologic origin, functional characteristics, and current and potential clinical applications of a novel marker of immune system activation, the soluble interleukin-2 receptor...

The soluble interleukin-2 receptor: biology, function, and clinical application.

Article de synthese sur la regulation de la transcription des genes qui codent les molecules de classe II: regulation de l'expression de ces molecules dans les lymphocytes B et T, les macrophages, les autres cellules qui presentent l'antigene, et dans quelques cellules de divers types dans differents tissus; description des sequences d'ADN impliquees dans l'activite du promoteur de transcription et des sequences activatrices en amont; description des proteines regulatrices qui reagissent avec ces elements d'ADN

Regulation of major histocompatibility complex class-II genes: X, Y and other letters of the alphabet.

Antigen or mitogen-induced activation of resting T cells induces the synthesis of interleukin-2 (IL-2) as well as the expression of specific cell surface receptors for this lymphokine. Failure of the production of either IL-2 or its receptor results in a failure of the T-cell immune response. The receptor is composed of a 33,000-dalton (251-amino acid) peptide precursor that is post-translationally glycosylated into the mature 55,000-dalton form. In contrast to resting T cells, human T-cell lymphotrophic virus I (HTLV-I)-associated adult T-cell leukemia cells constitutively express large numbers of IL-2 receptors. Because IL-2 receptors are present on the malignant T cells but not on normal resting cells, clinical trials have been initiated in which patients with adult T-cell leukemia are treated with a monoclonal antibody that binds to the IL-2 receptor.

https://science.sciencemag.org/content/sci/232/4751/727.full.pdf

The structure, function, and expression of interleukin-2 receptors on normal and malignant lymphocytes.

Cell-surface antigens that are induced to appear on T cells activated by the lectin phytohemagglutinin-P (PHA) can be classified both on the basis of the kinetics of their appearance and on their growth-association properties. Seven distinct T cell activation antigens, defined by monoclonal antibodies, were classified as early, intermediate, or late antigens based on their temporal appearance relative to DNA synthesis. Four antigens, the transferrin receptor, the T cell activation antigen Tac, the 4F2 antigen, and the 49.9 antigen were early antigens, whereas the OKT10 antigen appeared at intermediate times and both HLA-DR and antigen 19.2 appeared late. The use of a dye, Hoechst 33342, which stains DNA stoichiometrically, allowed the simultaneous analysis of immunofluorescence and cell cycle position of individual cells. This analysis unexpectedly revealed that essentially all cells in the proliferative phase of the cell cycle expressed each of the four early-activation antigens. The correlation between expression of the four early-activation antigens and T cell proliferation suggests that these molecules are important for the growth of all T cells. The relationship of two of these activation antigens, known to be the receptors for transferrin and interleukin 2, a T cell growth factor, is discussed with special reference to the roles of their ligands in supporting the growth of T cells.

/pdf/simultaneous-flow-cytometric-analysis-of-human-t-cell-171qt3xs05.pdf

Simultaneous flow cytometric analysis of human t cell activation antigen expression and dna content

In an effort aimed at design of a flow cytometer/sorter system in which light scattering, fluorescence, and extinction measurements could be made in as many as five spatially separated illuminating beams, the performance of different mechanical components, light sources, illumination and collection optics, and flow chambers was compared by changing individual components while maintaining the same basic instrument configuration. A protocol was developed, and is here described, allowing rapid setup of a flow cytometer using a mercury arc lamp and two laser beams for illumination. When two or more illuminating beams are derived from a single laser, it is generally necessary to incorporate automatic gain control in the detector electronics to compensate for source intensity fluctuations and so to achieve acceptable measurement precision. It is, however, possible to use a single laser beam as a source for several flow cytometers without the need for such compensation. Mercury arc lamps provide adequate excitation in the near ultraviolet (uv) for DNA fluorochromes, such as Hoechst 33342, and may be preferable to ion lasers for this purpose. Reported differences in precision and sensitivity between stream-in-air and enclosed flow systems appear owing more to differences in the illumination and collection optics used in different instruments; five flow chamber designs gave equivalent performance when tested in the same instrument. Relatively inexpensive achromatic microscope objectives of numerical aperture (NA) approximately 0.5 may offer the best combination of depth of focus and light gathering power when used as fluorescence collection lenses for flow cytometers; image plane apertures improve system performance in fluorescence measurement.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1002/cyto.990040103

Multistation multiparameter flow cytometry: some influences of instrumental factors on system performance.

Helium-neon lasers are economical and efficient light sources; their utility in flow cytometry to date has been limited by the lack of fluorescent probes that can be excited at 633 nm. Allophycocyanin (APC), a highly fluorescent phycobiliprotein, can be used as an antibody label and has spectral characteristics suitable for use with He-Ne lasers; we undertook to resolve whether a low-power (7 mW) He-Ne laser could provide sufficient excitation to permit flow cytometric detection of APC-labeled antibodies on cell surfaces. We made an APC conjugate of monoclonal antibody 4F2, which reacts with an antigen abundant on the surfaces of activated human T-lymphocytes; APC-4F2 was used to stain blood mononuclear cells that had been cultured with and without phytohemagglutinin (PHA). Cells so stained were examined in a flow cytometer with orthogonal illumination at 633 nm from a 7 mW He-Ne laser; antibody-bearing cells were detectable by fluorescence emission above 665 nm. Cells from the same cultures were stained with fluorescein-labeled 4F2 antibody and examined in a flow cytometer with argon ion laser excitation at 488 nm. Percentages of antibody-bearing cells determined from APC fluorescence and from fluorescein fluorescence were in good agreement. It thus appears that He-Ne lasers and APC-antibodies are usable for immunofluorescence measurements; the sensitivity attainable with this technique remains to be determined.

BRIEF COMMUNICATION Immunofluorescence Measurement in a Flow Cytometer Using Low-Power Helium-Neon Laser Excitation'

https://www.onlinelibrary.wiley.com/doi/pdf/10.1002/cyto.990040314

Immunofluorescence measurement in a flow cytometer using low-power helium-neon laser excitation

In this report we have analyzed cell cycle-related fluctuations of both quantity and density of the T cell differentiation antigens, CD3 (T3), CD4 (T4) and CD8 (T8), as well as the major histocompatibility complex (MHC) antigens on the cell surface of activated T cells. Phytohemagglutinin-activated T cells cultured for 3 days with or without conditioned medium or for 10 days with conditioned medium and mixed lymphocyte culture-derived T cell clones were used for the analysis. Correlated measurements of the surface antigen quantity (immunofluorescence), DNA content (dye Hoechst 33342), and cell size (light scatter), not influenced by synchrony induction methods and cell fixation, were performed by dual-beam flow cytometry. Our results demonstrate that the T cell differentiation antigens, CD3, CD4 and CD8, and class I MHC antigens are increased in density in the G1 phase for all activated T cells tested. In contrast, class II MHC antigens are increased in density in the G2 phase of activated T cells maintained with conditioned medium. Since it is known that the T cell differentiation antigens and class I MHC antigens on activated T cells are necessary for proliferation of T cells, our study suggests that this effect is more significant in the G1 phase. The cell cycle changes in expression of class I and class II MHC antigens, but not of the T cell differentiation antigens, appear to be mediated by soluble factors, probably including interferon-gamma, which could produce a differential increase of class I and class II MHC antigens on G2 phase cells.

Lisa Christenson

Papers

Simultaneous flow cytometric analysis of human t cell activation antigen expression and dna content

Multistation multiparameter flow cytometry: some influences of instrumental factors on system performance.

BRIEF COMMUNICATION Immunofluorescence Measurement in a Flow Cytometer Using Low-Power Helium-Neon Laser Excitation'

Immunofluorescence measurement in a flow cytometer using low-power helium-neon laser excitation

Differential expression of T cell differentiation antigens and major histocompatibility antigens on activated T cells during the cell cycle.