Quantitative studies on T cell diversity. III. Limiting dilution analysis of precursor cells for T helper cells reactive to xenogeneic erythrocytes.

Abstract

Splenic T cells exposed to concanavalin A (Con A), and subsequently to factors produced by rat spleen cells in response to Con A (Con A sup), acquire the ability to function as helper T (TH) cells in response to xenogeneic erythrocytes (RBC). Help is measured as the reconstitution of the plaque-forming cell response of a spleen cell population depleted of T cells by treatment with anti-Thy-1 serum and complement. We propose that precursor TH cells differentiate during the in vitro treatment into mature TH cells. As differentiation occurs under limiting dilution conditions, an estimation of the precursor frequency should in principle be possible. However, a single-hit Poisson distribution does not fit our data. Instead, we observe, dependent on the T cell concentration, three separate "peaks" of response. In many experiments, using sheep, horse, and chicken RBC as antigens, we reproducibly find these "peaks" at 40-190, 600-3,000, and 20,000- 100,000 T cells, placed into limiting dilution cultures, respectively. By various experiments we can show that the helper activity is not due to passively transferred rat factors, but to the titrated cells themselves. The active cell is a T cell that appears to function in an antigen-specific way and to require direct cell contact to do so. It thus resembles the classical helper T cell. As we find precursor TH cells already at very low concentrations of T cells, we titrated the range between 0 and 100 T cells/well carefully. The bent shape of the titration curves does not always allow a statistically satisfying regression analysis, and we therefore cannot estimate precise precursor frequencies from every experiment. However, a common sense argument can be made that these frequencies must be on the order of 1/10-1/100 T cells. We propose that the limiting dilution curves obtained in this system most likely reflect fundamentally important cellular interactions that regulate immunological effector functions. We favor a concept of independently interacting sets of helper and suppressor T cells of various frequencies, but other models are possible.

Immunologic tolerance in lymphatic filariasis. Diminished parasite-specific T and B lymphocyte precursor frequency in the microfilaremic state.

“…This nonlinear relationship between T cells in limiting dilution and the fraction of positive cultures has been increasingly observed, especially when T cell concentrations are examined over a broader range in response to heterogenous antigens. These types of findings are felt to result from multiple limiting populations of interacting T cells (23,24). Our results are consistent with the concept of several populations of antigen-reactive T cells and suggest the presence of both modulating and proliferating T cells responding to the parasite antigens.…”

Characteristic Cytokine Products of Th1 and Th2 Cells in Hemodialysis Patients

“…These findings support the hypothesis of a Th1-Th2 balance [7]. Our findings that IFN-γ production by Th1 cells or NK cells is elevated, whereas the IL-4 and IL-10 production by Th2 cells is decreased, are in agreement with the notion that IFN-γ selectively inhibits Th2 cell proliferation [6, 57]and suppresses expression of IL-10 mRNA [58], while IL-10 inhibits cytokine synthesis by Th1 cells [59]. IL-2 is the most potent human T cell growth factor, and IFN-γ is the most powerful activator of macrophages [60].…”

Network regulation among T cells; Conclusions from limiting dilution experiments

“…The experimental details of this assay have been described [19,20,44]. Briefly: T cells from normal mice are subjected to limiting dilution, exposed to concanavalin A (Con A) for 1-2 days, and thereafter for seven days to TCGF-containing media that induce differentiation and clonal expansion.…”

“…Basically, the experiments are concerned with the determination by limiting dilution experiments [42] of precursor frequencies for effector and regulatory T cells involved in the response to a number of commonly used laboratory antigens, and with the alteration of such frequencies after exposure of the immune system to these antigens [19,20,26,28,44]. The results of these experiments were surprising in two respects: first, they suggest that the clonal selection theory in a strict sense is not applicable to T cells because, on the one hand, antigen-recognizing T cells occur at greater frequencies than would be compatible with clonal selection and, on the other hand, clonal expansion is not a necessary requirement in the establishment of T cell immunity.…”