T Cell Signaling Abnormalities in Systemic Autoimmune Disease

The critical contribution of T cells to the generation and perpetuation of systemic autoimmune disease is beyond question. In experimental models they can transfer disease, and genetically engineered antigen-specific autoreactive T cells can promote sustained autoimmunity. In addition, treatment modalities that interfere with T or B cell function ameliorate disease. The discovery by two independent groups (Fields et al., 1996; Li et al., 1996) of the role of the route leading to transcription factor AP-1, as a pivotal mechanism responsible for the maintenance of T cell tolerance, illustrates the importance of postreceptor cell signaling in immune homeostasis. Recent work, showing bidirectional signaling, triggered by the interaction of cells responsible for innate and adaptive immunity, adds a new perspective to the understanding of the interplay between the environment and genes, a subject fundamental to the pathogenesis of autoimmune diseases. Thus, a full understanding of signaling abnormalities in T cells relevant to autoimmunity pathogenesis is not possible without looking at innate immunity cells.

3.1. Signaling Abnormalities in Antigen-Presenting Cells and Autoimmune Disease

Whereas stimulation by immature dendritic cells (DCs) induces tolerization, mature DCs promote full activation and effector functions of T cells (Ohashi and DeFranco, 2002). This is due to the expression of costimulatory molecules in mature DCs, which promote a stable immunological synapse between DCs and adaptive immune cells, allowing a continuous stimulation of T cells (Jacobelli et al., 2004). Some of the critical postreceptor signals needed for efficient antigen-presenting function by APCs have been identified. For example, the coupling of the CD40/CD40L pair triggers bidirectional signaling affecting APCs and T cells (Fujii et al., 2004). The full immunostimulant capability of APC involves augmented expression of MHC and CD80 molecules. Postreceptor signaling in APC influences immune outcomes and may have an impact on immune homeostasis. For example, disruption of STAT3, an adaptor molecule coupling downstream signaling triggered by activation of Janus kinase (JAK) in APCs can break tolerance to antigen in anergic antigen-specific CD4+ cells (Cheng et al., 2003). In other cases, signaling abnormalities may shift the direction of immune responses back to homeostasis. Thus, defective C-Rel expression interfering with normal IL-12 and IL-23 gene expression limits the APC capacity for induction of the Th1

phenotype in T cells, shifting immune responses away from organ-specific autoimmune disease in the genetically susceptible individual (Hilliard et al., 2002). Subtle stimulatory changes, such as those induced by TCR binding of altered peptide ligand presented by APC, induce anergy in autoreactive T cell clones (Quaratino et al., 2000) by aborting the activation process at intermediate steps. The biochemical basis is formation of the p21 (with only two tyrosine-phos-phorylated ITAMs) instead of p23 (with all three tyrosine-phosphorylated ITAMs) Z chains (Sloan-Lancaster and Allen, 1996). DC presenting a self-epitope with an altered peptide can induce anergy in a human autoreactive T cell clone (Quaratino et al., 2000), and short-lived peptides can also have a tolerogenic effect, by diminishing the density of peptide/MHC complexes (Mirshahidi et al., 2004). The basis for these effects is the completion of postreceptor signaling in T cells, from membrane receptors to the nucleus. A better knowledge of these very first steps after TCR ligation may be the basis for future therapeutic approaches in autoimmune diseases (Pugliese, 2003).

Recently, the role of pattern recognition receptors (PRRs) serving as links between APCs and adaptive immune cells has shed new light on our understanding of the interplay between innate and adaptive responses, a subject fundamental to the pathogenesis of autoimmune disease. Defective expression or altered molecular configuration of toll-like receptors (TLRs), a highly conserved family of 10 PRRs expressed in innate and adaptive immune cells, may foster sustained responses to self-antigens. For example, the expression of TLR2 by antigen-activated T cells (Komai-Koma et al., 2004) may render them susceptible to activation by bacterial lipopeptide, a ligand for TLR2, and partly explain the triggering of autoimmune disease or its reactivation in the presence of bacterial infection. Another example is the breaking of tolerance to proteolipid protein in mice naturally resistant to experimental encephalomyelitis, when T cells are stimulated in the presence of APCs that have been activated via TLR4 or TLR9, with unmethy-lated CpG oligodeoxy-nucleotides (CpG ODNs) (Waldner et al., 2004). Also, TLRs participate in the interaction between DCs and B cells needed for T cell responses to host-derived DNA (Seibl et al., 2004). Signaling through TLRs is necessary for full maturation of DCs, a role in part played by endogenous ligands HSP60 and HSP70. Ligation of TLRs by these endogenous molecules may precipitate autoimmunity, as occurs with HSP70 (Millar et al., 2003) or with HSPg96, when this molecule is genetically engineered for surface expression (Liu et al., 2003). Recently, the hypothesis of a third signal participating in the regulation of T cell responses has been proposed (Thomas, 2004). Lack of a third signal conveyed by IL-12 and IL1-P may induce anergy in CD8+ T cells (Curtsinger et al., 2003). On the contrary, signals delivered by IL-1P stimulate IL-12 production by DCs, and contribute to the induction of autoimmune myocarditis (Thomas, 2004). Recently, Scheinecker et al. (2000) showed a decreased stimulatory capacity of DCs on T cells from SLE patients, which was attributed to a reduction of the CD11-myeloid-related subset, the mature CD phenotype. Since DCs expressing CD11 correspond to the mature subset (Turley, 2002), a predominance of the tolerogenic CD11-negative DC in SLE, along with intrinsic signaling abnormalities, may further contribute to the paradoxal "anergic" phe-notypic profile shown by lupus T cells (Blasini and Rodríguez, 2004).

3.2. Signaling Abnormalities in T Cells and Autoimmune Disease

To understand abnormalities in T cell signaling that may condition sustained autoimmune responses we need to identify the critical checkpoints in postmembrane signaling responsible for the maintenance of tolerance (Figure 18.2).

3.2.1. Early Signaling Abnormalities of T Cells in Systemic Autoimmunity

The first postmembrane signaling abnormality identified in lupus T cells is altered metabolism of cyclic AMP and defective phosphorylation of protein kinase A (PKA-1), due to a deficiency of the PKA-I and PKA-I isoenzyme activity present in approximately 70% and 37% of SLE patients, respectively (reviewed in Kammer et al, 2004). Another relevant signaling aberration of T cells related to systemic autoimmunity involves disregulated PKB (Akt) activation (Ohashi and DeFranco, 2002). The increased activity of this kinase in trans-genic mice expressing T cells with a constitutively active form of Akt was sufficient for the development of splenomegaly, lymphadenopathy, and autoimmune glomerulonephritis (Rathemell et al., 2003), by a mechanism involving prevention of Fas-mediated death in T cells. Defects in the expression or activity of tyrosine phosphatases may also cause loss of tolerance. Mice heterozygous for Pten and SHIP develop a lymphoproliferative autoimmune syndrome akin to human Sjögren' syndrome, by a mechanism involving overproduction of IL-4 by CD4+ cells (Moody and Jirik, 2004). Another downregulatory mechanism involving tyrosine phosphatases is the coupling of tyrosine phosphatase SHP-2 by the intracytoplasmatic tail of CTLA-4 (Lee et al., 1998). In addition, a molecule expressed after T cell activation and also displaying coinhibitor capabilities is the program death-1 (PD-1) molecule, the receptor for two B7 family members, B7-H1 and B7-DC (Moretta and Bottino, 2004). Like CTLA-4, PD-1 is also able to recruit SHP-2. The importance of CTLA-4 in the maintenance of tolerance is underscored by the predisposition to Graves' disease, autoimmune hypothyroidism, and type I diabetes in patients expressing lower messenger RNA levels due to an allelic variation of CTLA-4 gene (Ueda et al., 2003). The nonobese diabetic (NOD) mouse also exhibits defective CTLA-4 function (Salojin et al., 1998). Besides its role as a negative regulator of downstream signaling in activated T cells, CTLA-4 is also needed to activate CD4+CD25+ regulatory T cells (Takahashi et al., 2000).

Current studies are showing peculiar accommodations of signaling molecules in lipid rafts of T cells from patients with systemic autoimmune diseases. In normal T cells Lck is enriched within, whereas tyrosine phosphatase CD45 is excluded out of these domains upon antigen binding by the TCR complex (Cannon and Schwartzberg, 2004). This pattern is reversed in T cells from patients with SLE (Jury et al., 2004). In fact, upon activation lupus T cells show diminished levels of raft-associated Lck, persistence of CD45 within these microdomains (Jury et al., 2004), and augmented amounts of GM1 ganglioside in the membrane of lupus T cells (Jury et al., 2004; Krishnan et al., 2004), indi cating a higher density of lipid rafts in T cells from SLE patients. One potential consequence of the latter abnormality is accelerated actin polymerization following T cell activation, probably explaining the increased capping induced by anti-CD3 stimulation in lupus T cells (Krishnan et al., 2004). The diminished expression of Lck in rafts of lupus T cells is apparently due to increased ubiqui-tination, an effect probably related to the effect of sustained oxidative stress in vivo (Jury et al., 2004). We previously reported enhanced activation of p59 Fyn, a src kinase constitutively associated to the CD3 complex in lupus T cells (Blasini et al., 1998a). Enhancement of Fyn activity has also been observed in T cells from NOD mice (Salojin et al., 1997). Fyn is an integral part of signaling from the coreceptor signaling lymphocyte activation molecule (SLAM), a gly-coprotein expressed on activated T lymphocytes and APCs that acts as a coreg-ulator of antigen-driven T cell responses that, after coupling to adaptor protein serum amyloid P component (SAP), fulfills a costimulatory function for T cell activation (Veillete, 2004). This pathway is involved in the pathogenesis of autoimmunity, as evidenced by the protection from hypergammaglobulinemia, production of anti-DNA antibodies, and glomerulonephritis in mice bearing a targeted mutation in the SAP gene (Hron et al., 2004). These defects in critical src kinases, acting at very early stages of the T cell activation process, may explain the abnormal pattern of tyrosine phosphorylation observed in lupus T cells (Matache et al., 1996; Blasini et al., 1998b; Liossis et al., 1998). The abnormal assembly of molecules in the early signalosome formed after TCR ligation may be in part contributed for by the defective expression of TCR Z chains (Liossis et al., 1998; Brundula et al., 1999). These molecules act as a scaffold for downstream signaling by bearing three ITAMs that are tyrosine-phosphorylated upon T cell activation. Tyrosine-phosphorylated ITAMs allow the coupling of ZAP-70 (and its subsequent phosphorylation by Lck), along with SLP-76 and LAT, a complex that activates PLCy1 (Leo et al., 2002). This complex triggers calcium mobilization by generating IP3 from PIP2 in membranes (Figure 18.2). Interestingly, lipid rafts of lupus T cells show absence of ZAP-70 (Krishnan et al., 2004), a fact that may explain the abnormal pattern of calcium mobilization in lupus T cells, possibly due to signaling through an alternate route in the "rewired" cascade in which TCR Z chains are substituted by FCeyRI (Tsokos et al., 2003). The importance of ZAP-70 in the maintenance of tolerance is illustrated by the increased susceptibility to autoimmune arthritis in mice bearing a spontaneous single point mutation of ZAP-70 SH2 domain, which interferes with its coupling to Z chains (Sakaguchi et al., 2003). T cells from ZAP-70 mutants show an altered threshold for T cell activation that hampers thymic deletion of potentially autoreactive clones (Sakaguchi et al., 2003). We have recently observed defective expression of LAT in lipid rafts of lupus T cells (unpublished data), another molecule critical for assembly of the calcium activation complex in T lymphocytes. Displacement of LAT from the signalosome and abnormal calcium mobilization has been observed in synovial T cells from patients with rheumatoid arthritis (RA) (Gringhuis et al., 2000; Cope, 2002).

3.2.2. Intermediate and Late Signaling Abnormalities of T Cells in Systemic Autoimmunity

The role of the Ras/Raf/MAPK signaling cascade in the maintenance of T cell anergy is well established (Williams, 1996). Anergic T cells show diminished activity of Ras that impairs activation of downstream ERK-1 and ERK-2, leading to defective transactivation of AP-1 and inhibition of IL-2 production (Fields et al, 1996). The signals responsible for Ras inactivation in anergic cells are not clear as yet, but possibly involve the triggering of alternate signaling routes when T cells are stimulated in the absence of a second or may be a third signal. Interestingly, altered signaling in T cells from patients with autoimmune diseases resembles the pattern observed in anergic normal T cells (Blasini and Rodriguez, 2004), suggesting that both induction of anergy and rupture of tolerance involve deviation from usual signaling routes.

We have recently shown diminished activation of ERK-1 and ERK-2 in T cells from SLE patients, possibly due to abnormal coupling of Ras nucleotide exchange factor hSos to adaptor protein Grb2 in cells activated through the TCR/CD3 complex in vitro (Cedeno et al., 2003). Also, diminished ERK activation was demonstrated by Deng et al. (2003) in lupus T cells, and was proposed as the mechanism for diminished DNA methylation and enhanced gene activation in lupus T cells (Oelke and Richardson, 2004). In a model of in vitro induced anergy, Yi et al. (2000) showed upregulated expression of CD40-L that was caused by lack of phosphorylation of Cbl/Cbl-b, and sustained phosphorylation of ERK. Cbl-b, an ubiquitin ligase, functions as a negative regulator of receptor clustering and raft aggregation in T cells. Loss of the molecular adaptor Cbl-b frees antigen receptor-triggered receptor clustering, lipid raft aggregation, and sustained tyrosine phosphorylation from the requirement for CD28 costimula-tion, diminishing the threshold for antigen responses in T cells. Introduction of the Cbl-b mutation into a Vav1-/- background relieved the functional defects of Vav1T cells and caused spontaneous autoimmunity (Krawczyk et al., 2000). Cbl-b-deficient mice develop anti-DNA antibodies (Bachmaier et al., 2000), further illustrating the importance of this downregulatory molecule in the maintenance of tolerance. Finally, Cbl-interacting proteins Sts-1 and Sts-2 negatively regulate TCR signaling. T cells from mice lacking Sts-1/2 are hyperresponsive to TCR stimulation, show increased ZAP-70 activity, augmentation of cytokine production, and increased susceptibility to autoimmunity in a mouse model of multiple sclerosis (Carpino et al., 2004; Kowanetz et al., 2004). These data suggest abnormal signaling involving the Ras/Raf/MAPK pathway in T cells from SLE and other autoimmune conditions. Clearly, further studies are needed to understand the status of this critical pathway in the breakdown of immune homeostasis.

Late effects of upstream signaling abnormalities in autoimmune T cells are defective activation of transcription factors. T cells from SLE patients show defective nuclear translocation of NFkB, due to abnormal expression of the p65 subunit (Wong et al., 1999). Two other transcription factors, AIRE and FOXP3, are involved in induction of tolerance in humans and their mutation could induce severe autoimmune disease (Ramsdell and Ziegler, 2003).

One of the most relevant consequences of impaired signaling in T cells is abnormal regulation of calcium mobilization, a critical signal for IL-2 gene activation. Defective IL-2 production is a mechanism for disruption of tolerance. Responses mediated through the IL-2 receptor p are needed to maintain homeostasis and prevent autoimmunity (Suzuki et al., 1995; Malek et al., 2000), and gene-targeted mice lacking the IL-2 gene develop various forms of autoimmune manifestations, including autoimmune colitis (Ludviksson et al., 1997). T cells from SLE patients have diminished production of IL-2 due to limited transcriptional activity of the IL-2 gene promoter (Wong et al., 1999; Solomou et al.,

2001). Also, IL-2 deficiency favors autoimmunity by compromising the generation of regulatory CD4+CD25+ T cells (Shevach, 2000; De Lafaille and Lafaille,

2002). In addition, SLE patients show diminished numbers of peripheral blood CD4+CD25+ regulatory T cells (Liu et al., 2004) and mice prone to lupus show early diminishment of CD4+CD25+ cells (Wu and Staines, 2004). The generation and function of CD4+CD25+ regulatory T cells depend on normal signaling involving STAT molecules, including STAT5 (Antov et al., 2003) and STAT1. STAT 1-deficient mice become susceptible to experimental autoimmune encephalomyelitis (Nishibori et al., 2004) and STAT5A/5B-deficient mice show autoimmunity affecting multiple organs (Snow et al., 2003).

Some of the above-discussed signaling abnormalities can alter cell cycle control and disrupt biological responses in the T cell compartment that could promote autoimmunity. For instance, resistance to Fas-mediated apoptosis related to high expression of the cyclin kinase inhibitor p21 (WAF-1/CIP-1) can be observed in T and B cells from lupus-prone mice (Lawson et al., 2004). This defect may explain the accumulation of the activated/memory type CD44highCD4+ cells arrested in the G0/G1 phase of the cell cycle. Table 18.1 summarizes several identified abnormalities of key signaling molecules in T lymphocytes that may be involved in the pathogenesis of autoimmune disease.

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