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The T-cell receptor provides T cells with specificity for antigens of particular molecular structure (the "epitope"); the T-cell pool in an individual responds to the presence of many different antigenic epitopes, but any particular T cell will respond preferentially to one defined epitope. After stimulation of a T cell by the binding of its receptor to its cognate antigen in the context of a major histocompatibility complex (MHC) molecule on an antigen-presenting cell, the T cell will begin to proliferate and synthesize cytokines. Tetramer binding and the enzyme-linked immunospot (ELISPOT) method have been used to determine what proportion of cells in the T-cell pool can bind to a defined antigenic peptide or will secrete cytokines in response to a particular antigenic stimulation. The method described here uses tracking dyes to determine what proportion of T cells will proliferate in response to stimulation. As a flow cytometric "single-cell" method, it can be combined with tetramer and cytokine staining to determine the precursor frequencies of cells in the T-cell pool able to bind tetramer, to synthesize cytokines, and to proliferate in response to antigen.

Fluorescent dyes that stain cell membranes or cytoplasm and then partition between daughter cells at division have been used in conjunction with flow cytometry to measure the proliferation of cells. In this paper, using peripheral blood mononuclear cells responding to tetanus toxoid, we describe an extension of this dye methodology to calculate the precursor frequency of antigen-specific T-cells. With mathematical deconvolution of the fluorescence histograms providing information about the proportion of cells in each of the daughter generations, information can be derived about the precursor frequency of cells in the original population that responded to the specific stimulus. Data from a model system with different proportions of fixed and viable cells indicate that the flow method returns accurate values for precursor frequency. Based on the characteristics of flow cytometric data acquisition, it is estimated that the flow method could detect proliferation of cells that represented, before addition of the stimulus, approximately 1/10(5) of the population. When comparing results to those from the limiting dilution technique, the flow cytometric method returns values that indicate higher precursor frequencies. Possible reasons for this discrepancy are discussed. The flow cytometric method offers the advantage of simplicity as well as the additional ability to phenotype the responding cells and determine their rate of proliferation. The flow method may find use as a simple, routine assay in the fields of allergy, transplant rejection, and autoimmunity and for quantitating responses to vaccination and cancer immunotherapy.

A novel in vitro assay of renal epithelium tight junction function was used to assess the efficacy with which rabbit anti-thymocyte globulin (ATG) blocks epithelium damage mediated by lymphokine-activated killer (LAK) cells. It was found that LAK cells lysed renal epithelial cells poorly in standard chromium-release assays but that they caused a rapid, and almost total, reduction in trans-epithelium monolayer resistance, indicating tight junction failure and, hence, loss of tissue function. LAK cell-mediated cytolysis of the sensitive K562 cell line was completely blocked in the presence of ATG at a concentration of 200 micrograms/ml. Addition of ATG at this concentration to damaged renal cell monolayers in the presence of LAK cells allowed the trans-monolayer resistance to recover rapidly to levels approaching the values recorded before initial addition of LAK cells. On this basis it seems likely that the rapid restoration of renal function frequently observed after appropriate "rescue" therapy during episodes of acute rejection may reflect subtle changes in tissue function rather than recovery from widespread graft cell cytolysis.

The ability of lymphokines to affect the development and differentiation of mouse thymocytes in vitro was evaluated in a carefully controlled 3-day organ culture system. Concanavalin A (Con A)-induced supernatant (SN) from the T-cell clone D10.G4, which contains high concentrations of interleukin-3 (IL-3), IL-4 and IL-5, but lacks IL-1, IL-2 and interferon (IFN), markedly increased the proportion of CD4+CD8- cells, and decreased the proportion of CD4+CD8+ cells. These effects were unaffected by dialysing the SN, showing them to be caused by macromolecular factors. Highly purified recombinant IL-3 and IL-4 could exert similar effects, rIL-3 and rIL-4 both increasing the proportion of CD4+CD8- cells, and rIL4 in addition reducing the proportion of CD4+CD8+ cells. In conjunction with the findings of other investigators, these results indicate that at least four lymphokines (IL-1, IL-2, IL-3 and IL-4) can control T-cell development in the thymus.

In many transplant centers, the T lymphocytes of transplant patients are routinely monitored, both to predict and diagnose the cellular events that result from transplantation and to evaluate the effectiveness of immunosuppressive therapy. During the course of this monitoring, we have observed anomalous results: the number of T cells said to be present varies greatly depending on the surface marker used for the assay. It is shown, in this study, that these "anomalous" results can be predicted from a knowledge of the spectrum of specificity of the anti-lymphocyte antibody therapy being administered. Because polyclonal antibodies have varied and unstated specificities, it is difficult to interpret results obtained from monitoring T-cell numbers or subset ratios without some knowledge of the specificities of the drugs being used.