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As previously mentioned, AICD, primarily mediated by surface CD95 ligation and CDD are involved in the resolution phase of immune responses. During the activation of mature T cells, transient expression of either CD95 ligand or TNF-a can result in apoptosis via the engagement of the relevant receptor on the same cell or adjacent cells (Figure 1.5.) (90,91). Despite the fact that CD95/CD95L interaction has been demonstrated as the primary death-inducing action in AICD, reports using Ipr

mice, which are deficient in CD95, have shown that other molecules may also be involved in this type of apoptosis. Although Ipr mice do manifest delayed kinetics of T cell apoptosis following the in vivo administration of either superantigens or specific peptide nevertheless activated T cells are eventually deleted by AICD (92,93). Reactive oxygen species and perforin/granzyme-B have also been implicated in AICD (94).

A recent report has proposed that CD95 can also induce AICD in a caspase- independent manner and that this alternative route is mediated by the CD95 associated kinase, RIP (95). This study showed that caspase-8-deficient Jurkat T cells were killed by CD95 ligation and this was still not suppressed by the pan-caspase blocker zVAD. Jurkat T cells lacking RIP were completely protected from CD95-induced death by zVAD suggesting that the caspase-independent route of CD95 mediated death involved RIP (95).

It has also been suggested that the T cell growth factor IL-2, may have a crucial role to play in the progression of AICD (Figure 1.5.). Once activated, T cells are stimulated to divide and secrete IL-2. This abundance of IL-2 in the microenvironment results in activated T cells becoming sensitive to CD95-induced apoptosis. IL-2 secreted by activated T cells binds to its receptor (IL-2R(3) on the T cell surface and subsequently activates the signal transducer and activator of transcription (STAT) 5. Phosphorylation of STAT5 leads to the down regulation of the CD95 inhibitor, FLIP, and the up regulation o f CD95L (96,97). IL-2 is therefore paradoxically required for CD95-induced apoptosis. Antigen stimulation of T cells in IL-2-deficient mice results in diminished CD95-induced cell death and the accumulation of T cells bearing a memory phenotype (98,99). CD95-induced death can occur throughout the immune response and culminates in the death of the majority of activated T cells leading to resolution of the immune response (49).

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CDD results from local depletion of vital cytokines and growth factors. CDD unlike CD95-induced death in T cells does not appear to be dependent on proximal caspases but rather on those more downstream (Figure 1.6.) (49). Mice lacking CD95 or FADD have been shown to display a certain degree of resistance to CD95-induced death but exhibit no reduction of death following cytokine deprivation (49). Transgenic mice that over express Bcl-2 or mice lacking Bim, Bak or Bax expression are susceptible to AICD but not CDD (49). D espite the fact that this body o f evidence points to the mitochondrion as the sole culprit in CDD, T cells bearing null mutations in either Apaf-1 or caspase-9 are not protected from cytokine deprivation death (49).

Apoptosis plays a vital role in culling the majority of activated T cells both during and upon resolution of an immune response. However, factors must exist which are capable of promoting the survival of a proportion of activated T cells from death in order to maintain a viable T cell memory pool.

Chapter 1

CD95

(C)

IL-2

Apoptosis

Caspase activation

CD95L

CD95L

Figure 1.5. CD95-induced apoptosis in activated T cells. (A) A T cell becomes activated when the TCR recognises specific peptide in the context of either MHC class I or II. TCR ligation results in up regulation of CD95 (B), which, once ligated by CD95L on another T cell leads to activation of caspases and ultimately apoptosis. Activation also leads to IL-2 production (C), which can act in both an autocrine and paracrine manner. Binding o f IL-2 to its receptor results in phosphorylation of STATS (D) and subsequent down regulation o f the CD95 inhibitor FLIP (E) and up regulation of CD95L (F). CD95 ligation by CD95 on an adjacent activated T cell results in apoptosis o f that cell (G).

V-

yc Cytokine

Cytokine withdrawal

Bcl-2

IL-2Ry

Apoptosis

Apaf-1

Cytochrome c

/ \

Caspase-9

DIABLO

Mitochondrion

Figure 1.6. Induction o f apoptosis in activated T cells as a result o f cytokine- withdrawal. Cytokine deprivation leads to activation of pro-apoptotic molecules such as Bid, which inhibit the protective action o f both Bcl-2 and B c 1 -x l. The subsequent de­

polarisation of the mitochondrial membrane results in release o f both cytochrome c, which forms the apoptosome with Apaf-l and caspase-9 and DIABLO, which inhibits lAP. Cytokines that signal through the common y chain o f the lL-2 receptor, such as 1L- 2, can prevent CDD (red sun symbol). Type 1 IFN can also inhibit CDD by inducing the up regulation of B c I - x l (red lightning symbol).

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