Primeros acercamientos

8.2.3.1. DC sensitisation / epicrine signals

In the introduction of this thesis, the role o f T cells in back-signalling to DCs has been reviewed. Two pairs o f ligands from the TNF-R family appear to play an important role. Activated T cells up-regulate CD40L and TRANCE, which bind to CD40 and RANK on DCs respectively. CD40 signalling enhances the expression of class II MHC, B7.1, B7.2 and ICAM-1 (Celia et aL, 1996). Thus activated T cells promote the recruitment and effective activation o f further T cells. CD40 ligation also up-regulates RANK (Anderson et aL, 1997). Both CD40 and RANK signalling result in IL-12 secretion, which in conjunction with ICAM-1, favours Thl differentiation (Yang and Wilson, 1996; Celia et aL, 1996; Josien et aL, 1999 ; Salomon and Bluestone, 1998; Luksch et aL, 1999). TRANCE ligation also enhances IFN-y production by T cells (Chen et aL, 2001). Finally, the expression of CD40L is sustained longer in Thl than Th2 cells (Lee et aL, 2002b). These reports are supported by data from Chapter 7, which show that, in neutral in vivo conditions (that do not involve injection of polarised T cells), the proportion of Thl compared with Th2 cells increases with the number of activated cells. Although CD40L initially favours the development o f Thl cells, both CD40L expression (Roy et aL,

1993) and IL-12 secretion (Langenkamp et aL, 2000) are limited in duration. CD40L also initiates Th2 differentiation by the up-regulation of B7 molecules on DCs, because costimulation via CD28 is essential for Th2 development (King et aL, 1995; Rulifson et aL, 1997). This signal may progressively counterbalance the strong Thl stimulus initially delivered by CD40L. Finally, CD40 signalling also up-regulates 4- IBBL (Diehl et aL, 2002) and OX40L (Brocker et aL, 1999) expression on DC, both of which provide survival signals to T cells. Although 0X 40 and ICOS, both induced on activated T cells, have been implicated in the maintenance of Th2 functions more than Thl functions, it is unclear, for example, whether 0X40 is important for the early development o f Th2 cells; it has been reported to costimulate either Thl (De Smedt et aL, 2002) or Th2 (Ohshima et aL, 1998; Tanaka et aL, 2000) in a primary response. B cells, however, deliver a strong Th2 polarising signal via 0X40 stimulation, which enhances IL-4 and represses IFN-y expression (Flynn et aL,

Chapter 8: General discussion

DC activation by CD40L is the key epicrine signal in cooperation between activated and naïve/activated T cells. The overall contribution o f CD40L on Thl versus Th2 commitment may be evaluated in our model by using T cells from CD 154'^' TCR-Tg mice. The relative contribution o f each CD40L-induced epicrine signal may be more difficult to dissect because some o f these signals (e.g. IL-12) may also be induced by PAMPs or play a role in processes other than just T cell differentiation (e.g. B7, OX40L). Defining the role, both quantitatively and temporally, of these epicrine signals will allow a more rational choice for immunotherapeutic targets. Help given by T cells at high frequency to naïve T cells at low frequency progressively disappeared when the number o f those at low frequency was increased (Chapter 7). If two naïve T cell populations were both at high frequency, no or little help was observed between them (Chapters 6 and 7). This help is likely to be a combination of epicrine and paracrine signals, and their relative contribution needs to be addressed. In contrast to naïve T cells, polarised T cells were able to deliver to naïve T cells further signals, which appeared to have a greater paracrine component (Chapter 6). These polarised T cells, present at lower frequency than naïve T cells, are indeed less likely to influence naïve T cells via CD40L expression. Although CD40L expression is only transient, it would be interesting to check whether polarised T cells retain other markers such as TRANCE, which can signal back to DCs.

S.2.3.2. Cytokines

Cytokines secreted by activated T cells may directly affect other T cells. Such cytokines may support T cell proliferation (e.g. IL-2, IL-4), modulate T cell differentiation (e.g. IFN-y, IL-4) or inhibit T cell proliferation and/or function (e.g. IL-10). For example, activated CD4+ T cells that have acquired a polarised phenotype influence naïve CD4+ T cells to adopt a similar profile, both in vitro

(Schuhbauer et aL, 2000) and in vivo (Chapter 6). The production of IFN-y by activated CD8+ T cells, in addition to that secreted by Thl cells, also importantly contributes to the generation of more Thl cells (Mailliard et aL, 2002). These data all suggest that DC play an important role for two reasons: (i) DC gather T cells in a more confined area so that cytokines secreted by some T cells may benefit others, (ii) DC function may be modulated by cytokines produced by some T cells so that they adjust the activation of other T cells accordingly.

Chapter 8: General discussion

IL-2 is a potent T cell growth factor, which may act in an autocrine or a paracrine manner depending on the amount secreted per cell. Paracrine IL-2 may contribute importantly in the help from CD4+ to CD8+ T cells (Keene and Forman, 1982; Malek, 2002). In the CAT model, the help provided by other activated cells resulted in enhanced cytokine production, but IL-2 was the least enhanced cytokine. Moreover, the clonal expansion was not affected by help, presumably because clonal expansion, for each precursor frequency group, has reached levels that cannot be boosted further.

Thl cells were found to express high levels of IFN-y, which correlated with large spots in IFN-y ELISPOT assays. However, Thl cells (OVA-specific) did not augment the number of naïve T cells (PCC-specific) that express IFN-y, presumably because at the precursor frequency examined (3x10^ per mouse) the propensity to differentiate into Thl is already high and close to a maximum. Usually, the number o f IFN-y+ T cells in the lymph nodes drops quite rapidly because these cells either leave the tissue or die. Interestingly, OVA-specific Thl cells sustained the number of IFN-y+ PCC-specific cells, either by stimulating the differentiation o f more IFN-y+ cells that replace those which disappeared, or by preventing emigration or death of Thl cells. IFN-y can enhance both the secretion of IL-12 by DC (Snijders et aL,

1998) and the responsiveness to IL-12 by T cells (Smeltz et aL, 2002). However, at the time when the higher number o f IFN-y+ cells were observed (day 5), it is unclear whether DCs have all disappeared (Ingulli et aL, 1997), or no longer produced IL-12 (Langenkamp et aL, 2000), or on the contrary, had their function and survival sustained, for instance by RANK signalling (Wong et aL, 1997; Josien et aL, 1999). Thl cells may maintain DCs function better, because they preferentially express TRANCE (Chen et aL, 2001). This hypothesis merits further investigation, because TRANCE may be a useful target to sustain Thl responses in tumour models. At similar precursor frequency, polarised Thl cells, but not naive cells, were able to facilitate Thl differentiation in naïve T cells of different specificity upon concomitant activation. However, naïve clones that were activated in higher numbers were able to enhance Thl differentiation in other clones at low precursor frequency. Whether this help resulted from the accumulation of IFN-y in the vicinity or a better sensitisation of DCs to produce IL-12 requires further study. However, the role of

Chapter 8: General discussion

IFN-y was suggested by the observation that linkage was not always necessary for help, perhaps because high levels of IFN-y produced may diffuse between DC clusters.

Early studies demonstrated the role o f accumulating autocrine IL-4 after sufficient exposure to antigen in inducing Th2 differentiation (Croft and Swain, 1995; Demeure et al., 1995). This autocrine IL-4 production is sufficient to promote Th2 development in the absence o f any other source o f IL-4 (Noben-Trauth et aL, 2000), but this production may remain very modest (Ben-Sasson et aL, 2000). Repetitive stimulation or exogenous IL-4 appears to be required for eliciting a more vigorous Th2 response. CD4+ T cells, in the model used in this thesis, express lower levels of IL-4 than IFN-y and this limited production may explain why Th2 cells constitute a smaller population than Thl cells (a fact also associated with the genetic background). It is unclear whether CD4+ T cells are repeatedly stimulated during a primary response in vivo. Alternatively, these cells may rely on IL-4 secreted by other cells. Other freshly activated CD4+ T cells have an important role as a source of IL-4 in this respect (Gollob and Coffman, 1994; Croft and Swain, 1995). If these cells remain within a confined microenvironment, then limiting autocrine IL-4 may act as a paracrine stimulus, by accumulating over time. Th2 cells also constituted a source o f paracrine IL-4 in this model and significantly improved Th2 differentiation in naïve T cells (Chapter 6). The requirement for a local concentration of IL-4 may explain why linkage was preferred, since Th2 cells had no effect if they were not recruited to the same DC as the naïve T cells (Chapter 6). As was observed for IFN- y, IL-4 production by naïve T cells of one antigen specificity was not enhanced by the presence of naïve T cells o f another, unless the difference in frequency between the two was high (Chapter 7). Thus, the local concentration o f IL-4 is more limited if few cells are activated, but may be increased to some extent in the presence o f a larger population, which is concomitantly activated. Finally, a study by Ismail and Bretscher (2001) also reflected the need for close CD4+ T cell cooperation for efficient Th2 development. Irradiated mice reconstituted with low numbers of splenocytes elicit an exclusively Thl-type of response after immunisation with xenogeneic red blood cells. Th2 cells progressively emerged after reconstitution with higher numbers of splenocytes, resulting in a mixed Thl/Th2 response.

Chapter 8: General discussion

Th2 cells not only enhanced the number of IL-4+ cells, but also diminished the number o f IL-2+ cells. The presence o f Th2 cells surprisingly did not significantly affect the number o f IFN-y+ cells. Thus, this effect may well be mediated by IL-10, which is reported to inhibit the transcription of IL-2 without affecting that of other cytokines such as IFN-y or IL-4 (de Waal Malefyt et aL, 1993). Confirmation o f this hypothesis may be obtained, for example, by measuring by ELISPOT the number of IL-10+ cells within the Th2 inducer population after in vivo restimulation.

The relationship between DC / T cell ratio and CD4+ T cell differentiation is complex. An in vitro study showed that 1:4 and 1:300 DC-T cell ratios favour Thl and Th2 responses respectively (Tanaka et aL, 2000). An earlier report from the same group suggested that a high number of DC per T cell favoured Thl differentiation because of more IL-12 per T cell from these DCs (Ohshima and Delespesse, 1997). These results indicate that the DC-T cell ratio may subtly modulate Thl/Th2 response depending on the signal that DCs receive. On the one hand, more T cells may have to compete for the utilisation of IL-12 secreted by DCs, so that T cells at lower number benefit more from IL-12. On the other hand, more T cells may also induce more IL-12 secretion by DCs via CD40L to compensate. The amount o f IL-4 produced by T cells after activation may also alter this balance, because IL-4 can inhibit IL-12 production (Koch et aL, 1996; Ohshima and Delespesse, 1997; Ria et aL, 1998). Thus, if T cells are low IL-4 and high IFN-y producers, such as in our mouse in vivo model, a high number o f T cells per DC may favour Thl differentiation. In contrast, if T cells are high IL-4 and low IFN-y producers, such as in a human in vitro model (Ohshima and Delespesse, 1997; Tanaka et aL, 2000), then the secretion of IL-12 may be more limited and Thl differentiation may preferentially occur at low number o f T cells per DC.

The contribution of the IL-2, IFN-y, IL-4, IL-10 and other cytokines in the cooperation between T cells may be further investigated in the CAT model, where the inducer/helper population would come from TCR-transgenic cytokine-deficient mice. This may be useful in estimating the contribution of epicrine signals and in understanding why cooperation between CD4+ T cells leads to better differentiation into cytokine-producing cells without affecting activation and proliferation.

Chapter 8: General discussion

S.2.3.3. Optimising the function o f DCs

Dendritic cells provide a site for both competition and cooperation between T cells (Figure 8.1). The ability to express regulatory genes as well as antigens in DCs may also be exploited to improve vaccination efficacy. For example, the recruitment and activation of T cell clones may be limited by the length o f time DCs remain in lymph nodes. Thus, signals that prolong survival or delay apoptosis may be used in conjunction with vaccines. Sustaining Bcl-2 expression in DCs, for instance, resulted in greatly enhanced CTL responses (Nopora and Brocker, 2002). PMDD is a method o f choice with which to introduce both antigen and regulatory genes inside the same cell. Vaccination using ex vivo manipulated and antigen-pulsed DCs is a method that has also attracted considerable attention, in particular for the treatment of cancer (Jefford et aL, 2001 ; Steinman and Pope, 2002).

The cooperative effect of linked epitope presentation by APCs may be exploited to enhance the response of low responsive individuals. The greater ability of pre­ activated/memory over naïve T cells to improve the response of other naïve T cells was evidenced by the study described in Chapter 6 and by another study by Cheng et al. (1998), where anti-tumoral immunity has been successfully boosted by in vivo

priming of OVA-specific CD4+ T cells prior to injection o f OVA-pulsed tumour cells. The linkage between OVA and the tumour antigens thus allowed the development of an effective anti-tumour cytotoxicity that was not achieved with the immunisation with tumour cells alone. Multivalent vaccines may offer significant advantage over monovalent vaccines by increasing cooperation between T cells. However, such epitopes should be carefully selected and tested in order to avoid possible interference. The influence that different antigenic determinants may have on each other is discussed in the last section below.