6.1
Introduction
The presence and persistence of IL-12 and molecules required for IL-12 signalling are necessary for Thl cell differentiation. For example, healing Thl cell responses against L major infection in IL-12p40 null mice require addition o f exogenous IL-12 not only at the beginning but also throughout the course of infection [605]. Similarly, sustained expression of the IL-12Rp2 chain is essential for developmental commitment to the Thl phenotype in both mouse and humans [77, 8 6, 87, 523, 606].
The timing and duration of the IL-12/ STAT4 response depend on the functional expression of the molecules that participate in STAT4 activation. Therefore, it is important to understand the mechanisms involved in the regulation o f IL-12, IL-12R and STAT4 expression. IL-12 is produced by antigen presenting cells, primarily monocytes and DCs by T cell-dependent and independent mechanisms. Bacteria, viruses, yeast, intracellular parasites and IFN-y act in a T cell-independent manner, while activated T cells induce the production o f IL-12 through CD40/ CD40L and MHCII-TcR interactions during antigen presentation (see Chapter 1, section 1.4.2).
STAT4 expression is also tightly regulated and restricted to activated T lymphocytes (Chapter 3, Figure 3.2 B and [76]). In monocytes and mature DCs, STAT4 expression is induced in response to LPS and IFN -y and is negatively regulated by the Th2 cytokines IL-4 and IL-10 [73, 607]. STAT4 is constitutively expressed in freshly isolated NK cells but it has been suggested that IL-2 is necessary for sustained expression of STAT4 in cultured NK cells [85].
IL-12 signalling requires the functional expression of both IL-12RP1 and IL-12RP2 [6 6, 71, 75]. T cells only express IL-12RP1 and IL-12RP2 following antigen receptor mediated activation. Experimentally this is usually achieved by T cell activation with mitogens or following TcR triggering with anti-CD3 antibodies (Chapter 3, Figure 3.3 and [63, 95, 565]). TcR triggering alone is not sufficient for maximal expression of IL-12R, rather CD28/ B7 derived signals are also required for optimal induction apparently through the production of regulatory cytokines (see Chapter 1, section 1.4.4 and [78-81]). For example, it has been reported in murine T cells that the enhancement of IL-12RP1 and IL-12RP2 by CD28 co-stimulatory signals is likely to be mediated by induction of IL-2 [79-81]. After initial induction by antigen directed signals the magnitude and maintenance o f functional IL-12R is then regulated by cytokines. Most studies have concentrated on the regulation of IL-12RP2, because its sustained expression proved to be essential for Thl cell commitment [77, 8 6]. IFN-a, IL-12, IFN-y and IL-18 induce IL-12RP2 expression in human and/ or mouse systems, respectively [9, 40, 77, 79, 8 6, 89, 516, 523, 539,
606, 608]. IL-12Rp2 regulation by IL-12 and I F N -a has been proposed to be mediated through STAT4 [286]. However, little is know about the regulation of the IL-12RP1 expression.
Synergistic interactions between IL-12 and IL-2 are important in Thl cell differentiation, the regulation of NK cell function and the maturation/ differentiation of cytotoxic CD8^ T cells (see Chapter 1, section 1.5.3 and [104, 108, 122, 259,
260, 491]). In NK cells the regulatory effects of IL-2 on IL-12R expression may explain the role of IL-2 as a modulator of IL-12 responses. IL-2 up-regulates the constitutive expression of IL-12R(3l and IL-12RP2 in NK cells [85]. The experiments presented in this chapter were developed to investigate the influence of IL-2 on the IL-12/ STAT4 response in human T lymphocytes.
6.2
Results
6.2.1 Long term kinetics of the IL-12/ STAT4 response
We have shown that IL-12 activation of STAT4 is sustained for several hours (Chapter 5). However, T cell differentiation requires days. The kinetics of STAT4 activation over several days has not been studied. Accordingly, PBL-T cells were stimulated or not with IL-12 over a 24 hour period (Figure 6.1). Oligonucleotide affinity precipitation was performed using equal amounts o f total protein from the different cell populations. Active DNA-bound STAT4 molecules can be detected at both 18 minutes and 24 hours after IL-12 stimulation, but not in unstimulated cells (Figure 6.1). Both electrophoretic mobility forms of STAT4 (STAT4pl and STAT4p2) are present at 18 minutes, while only STAT4p2 is present at the 24 hours time point. However, the amount of DNA-bound STAT4 is strikingly reduced following 24 hours o f IL-12 stimulation (Figure 6.1).
6.2.2 IL-12 stimulation down-regulates the cell surface levels of the IL-12R
A decrease in expression o f the IL-12R could explain the termination of the IL-12/ STAT4 response over a period of 24 hours. To test this possibility, PBL-T cells were stimulated or not with IL-12 for up to 48 hours. The cell surface levels of both chains o f the IL-12R were determined by specific immuno-staining and flow cytometry analysis. The pattern of expression of the IL-12R|3l chain and the IL- 12RP2 chain are different in T cells maintained in IL-12 (Figure 6.2). IL-12 stimulation induces rapid down-regulation of IL-12RP2. The cell surface levels of IL-12R(32 are reduced after 18 minutes of exposure to IL-12 and decay even further by 50-60% between one and four hours of IL-12 stimulation. This down-regulation
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