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DC^^^ induced a high level o f IFNy production by responding syngeneic CD4^ and

CD8^ T cells as well as D O ll.lO Tg CD4^ T cells. Such cytokine production was

also evident in T cells stimulated with D C^^^, but the quantity o f IFNy produced was

lower than that induced by DC^^^. This may reflect the difference in the levels o f T

cell proliferation induced by DC^^^ and DC^^^. In addition, production o f IL-4 was

also observed in syngeneic CD4^ T cells and D O ll.lO Tg CD4^ T cells. In the case

o f syngeneic CD4^ T cells, but not mediated IL-4 production (figure

5.13). However, the reverse is true for D O ll.lO Tg CD4^ T cells (figure 5.12).

Theses contrasting results may reflect the differences in signal 1 between the two

IL-12-independent IFNy production mediated by

The mechanisms o f DC^^’^^-induced IFNy production were studied in some detail in

this chapter. Contrary to the literature that suggests a major role o f IL-12 in induction

o f IFNy production by T cells (Trinchieri, 1995), a requirement for IL-12 in D C ^ ^ ^ -

induced IFNy production was not observed (figure 5.16/17). W hile DC^^^®-mediated

IFNy production was partially dependent on IL-12, IFNy induced by was

completely independent o f IL-12.

Recently, a role for IL-18 in amplifying IFNy production independent o f IL-12 was

described (Muller et al., 2001; Xing et al., 2000). However, IL-18 is not likely to be

responsible for the induction o f IFNy, as there was no pronounced IL-18 production

by or either from wildtype DCs (figure 5.15) or IL-12 ko DCs (data not

shown).

To rule out that the possibility that other soluble factors may mediate the IFNy

response by DC^^’^®, the effect o f DC^^^®-T cell culture supernatant in inducing IFNy

production was analysed (figure 5.19). The supernatant o f DC^^Lx cell culture at 24

and 48 hours did not appear to contain soluble factors responsible for induction o f

IFNy production. It is possible that secreted molecules that were crucial in mediating

the IFNy response had been taken up by the T cells, or that the half-life o f such

molecules was insufficient for them to be effective. However, a more likely

explanation is that the IFNy response is mediated by direct physical interaction with

q^ptx/b xhe functional differentiation o f T cells is thought to occur early in their

determining the differentiation o f T cells in response to may have occurred

within the first 24 hours o f DC-T cell contact.

A model: mechanisms of DC**^*^®-mediated T cell responses

Two m ajor questions that have been raised in this chapter are: (1) how do

induce syngeneic T cell activation and differentiation?; and (2) what accounts for the

differences between T cell responses mediated by and W hilst the

answers for neither o f the questions are clear, a number o f clues have arisen to explain

what may be happening between and the T cells.

The absence o f “conventional” signal 1 mediated via M HC-TcR interactions indicates

that M HC-TcR interaction is bypassed by both PTx and PTxB. Given that cell-cell

contact is required to achieve DC^^^^^-mediated T cell responses, signal 1 in this

system is likely to be driven by, and T cell responses induced by, at least one o f the

following: (1) cross-linking o f CDS molecules (and other molecules); (2) direct

mitogenic effect by the toxin [other than by the mechanism in (1)]; and/or (3) an

increase in the overall avidity o f DC^^^® -T cell interaction.

W hether the cell surface-bound toxin was responsible for mediating the subsequent T

cell responses remains unclear. This could be established by further studies using

blocking antibodies against toxin bound to DCs. However, the finding that only a few

minutes o f incubation o f DCs with PTx was sufficient to induce a potent T cell

response indicate that, at least for DC^^^, the toxin bound on the DC surface may be

PTx has been shown to act as a T cell mitogen in a number o f studies (R osoff and

Mohan, 1992; R osoff et al., 1987; Tamura et al., 1983). For example, activation of

splenocytes (Tamura et al., 1983) and Jurkat T cells (R osoff et al., 1987) was

observed following treatment with the toxin. In this chapter, purified CD4^ or CD8^

T cells failed to proliferate (figure 5.2) or to exhibit upregulated expression o f early

activation markers such as CD25 and CD69 (data not shown) in response to

stimulation with PTx/B. However, it is possible that, the toxin bound on the DC

surface may be able to activate T cells, either by cross-linking o f CD3 (and other

molecules), or directly stimulating T cells together with the co-stimulatory molecules

expressed on DCs. Alternatively, given that both DC^^^ and DC^^^® exhibited strong

adherence to each other, it is possible that these DCs may have interacted with T cells

with a higher than normal avidity, resulting in a stronger, and prolonged signalling

from these DCs. This adherence o f DCs mediated by the toxin may be due to (1)

cross-linking by the toxin on the cell surface (Kaslow and Bums, 1992), and/or (2)

upregulation o f adhesion molecules other than those measured in this study (CD 11 a,

CD49d, and CD54).

What accounts for the differences between the effect o f PTx and PTxB on DCs?

Biochemically, the only difference between these molecules is the presence or the

absence o f a catalytic subunit, SI (or A-subunit). The finding that co-administration

o f PTxB and a cAMP elevating agent (dbcAMP) did not compensate for the

difference between the two molecules, together with the observation that PTxB takes

a longer period to exert its effect on DCs than PTx, suggests that the difference may

be explained by the efficiency o f these molecules in binding to receptor(s) on DCs.

The current hypothesis that has emerged from the findings in this chapter is that T cell

activation by is likely to be mediated, at least in part, by toxin bound to the

surface o f DCs. However, how the bound toxin induces its effect is currently

unknown. Despite the long history o f use o f PTx as a mucosal adjuvant, surprisingly

little is known about its mechanisms o f action. DCs, as well as other APCs, have

evolved to recognise “danger” associated with a toxin and initiate potent T cell

responses. However, it is debatable whether the polyclonal T cell responses induced

by the toxin help the immune system to clear infection. It is possible that the bacteria

may use such a toxin as a mechanism to evade a specific immune response. Further

understanding o f the biochemistry o f the toxin and the interaction with its receptor(s)