1 INTRODUCTION

A healthy adaptive immune system not only requires a robust response to pathogens or to potential oncogenic mutations but also requires the absence of aggressive reactivity to self. This state of unresponsiveness to self generally defines a state of tolerance. Reactivity, of course, depends upon the recognition by the T cell antigen receptor (TCR) of pathogen- or oncogene-derived peptides bound to class I or class II MHC molecules (agonist pMHC). Such recognition of agonist pMHC leads to generation of activation signals dependent upon well-characterized intracellular biochemical events occurring at the TCR (Figure 1). These events generate intracellular downstream signals that contribute to T cell activation and that can lead to clonal T cell proliferation, differentiation, and acquisition of effector functions.

Illustration of ZAP70 activities during TCR recognition of self-pMHC or agonist-pMHC. (A) In the resting state, when the TCR does not interact with any pMHC, ITAMs are not phosphorylated and ZAP70 remains in an autoinhibited state in the cytoplasm. When the TCR recognizes a self-pMHC, the interaction with self-pMHC delivers a sufficient tonic signal to lead to ITAM phosphorylation by the kinase LCK. This then results in the recruitment of ZAP70 to the phosphorylated ITAM motifs but ZAP70 does not become phosphorylated. The degree of self-reactivity is associated with the amount of ζ-chain phosphorylation. Upon recognition of agonist pMHC, coreceptor-associated LCK is brought into the vicinity of engaged TCR and CD3 complexes long enough to phosphorylate ITAMs and recruit as well as phosphorylate ZAP70 that is bound to phosphorylated-ITAMs. Phosphorylation of ZAP70 by LCK on Y315, Y319 and Y493 leads to its activation. (B) Activated ZAP70 subsequently phosphorylates LAT on five key tyrosine residues. Notably, the SH3 domain of LCK (marked with *) interacts with the PIPRSP motif of LAT to actively recruit LAT to the activated ZAP70. This active recruitment of LAT by LCK enhances ZAP70-mediated LAT phosphorylation and augments T cell sensitivity towards weak ligand stimuli. Furthermore, phosphorylated LAT and another ZAP70 substrate, SLP76, can function as scaffold proteins to recruit other downstream signaling proteins and events, which can eventually lead to T cell activation

The robust response to antigenic threats by the host does not imply that there is a complete lack of any recognition of self-pMHC since naive T cells depend upon recognition of self-pMHC for survival.1 Self-pMHC binding to the TCR generates weaker or qualitatively distinct “tonic” signals that ensure T cell survival.2-4 The affinity differences between an agonist-pMHC (typically low μM range) vs a self-pMHC with the TCR is remarkably small (10-15-fold differences).5 Yet, T cells are remarkably sensitive. A single pMHC is sufficient to initiate the response of some T cells and responses are generally very specific.3 Several different models for such ligand discrimination have been suggested to account for the TCR sensitivity and selectivity (reviewed in refs.6, 7). Ultimately, each model depends upon signal transduction events generated by the TCR. These signaling events play roles in all the mechanisms involved in preventing a robust response to self-pMHC and establishing a state of self-tolerance.

Distinct tolerance mechanisms operate centrally or peripherally. Central T cell tolerance occurs during thymocyte development. Cells expressing clonally rearranged and displayed TCRs undergo a quality control assessment to select for a population of thymocytes expressing TCRs that interact appropriately (positive selection) but not too strongly with self-pMHC (reviewed in ref.8). Central tolerance depends upon negative selection which occurs by the elimination of thymocytes with TCRs that interact too strongly with self-pMHC. Such strong reactivity generates potent TCR-dependent signals that lead to apoptosis of these potentially autoreactive cells.

Negative selection is not perfect.9 Some potentially autoreactive cells escape into the periphery because signals in the thymus were not strong enough to induce apoptosis or the autoantigen might not have been sufficiently encountered. Several mechanisms account for how autoreactivity is normally prevented in the periphery: induction of T cell anergy—an induced state of unresponsiveness10; suppression of responsiveness of antigen-specific T cells by T regulatory cells (Tregs)11; or by the effects of inhibitory signaling mechanisms.12, 13 Naive T cells normally require signals delivered via their TCR and via a co-stimulatory receptor, typically CD28, to become productively activated.14 CD80 and CD86, are induced on antigen-presenting cells by activation of innate immunity by pathogens or perhaps via inflammation induced by a tumor. Stimulation of CD28 ensures that naive T cells are not induced in the absence of inflammation, that is, so that T cells are not responding to a self-antigen encountered outside of the thymus. In the presence of a strong TCR signal, but in the absence of a co-stimulatory signal, T cells enter a state of unresponsiveness called anergy. Another mechanism to prevent activation of potentially autoreactive cells is via the suppressive influence of Tregs, which may share some level of specificity with stimulated T cells. Finally, sufficient induction of TCR signaling, acutely or chronically, may induce adaptive mechanisms to suppress TCR signaling via ubiquitin ligases or inhibitory receptors. This may be a means to prevent immunopathology during chronic infections.

Discrimination of ligands and induction of central and peripheral tolerance mechanisms depends upon signal transduction by the TCR. Appropriate signaling strength or quality is required for negative selection, anergy induction, Treg generation and activity, as well as induction of inhibitory feedback mechanisms (ie, E3 ubiquitin ligases or inhibitory receptors). Here, we will focus on several systems in which inappropriate or insufficient signaling via the TCR alters the tolerance threshold centrally or peripherally. We will focus our discussion on recent work from our labs that have studied mutants of a single kinase, ZAP70, and its substrate, LAT. ZAP70 is a cytoplasmic tyrosine kinase critically important in proximal events involved in TCR signaling. The mutants we will discuss either decrease or increase TCR signaling function.

1.1 The role and regulation of ZAP70 in TCR signaling

Together with LCK, ZAP70 plays a critical role in proximal events linked to initiating TCR signaling. Engagement of the TCR by pMHC or by anti-receptor (anti-TCRαβ or anti-CD3) monoclonal antibodies (mAbs) initiates critical biochemical events involving LCK and ZAP70 that are summarized for simplicity in Figure 1. ZAP70 plays a critical requisite role. In the absence of ZAP70, distal signaling events leading to calcium increase, Ras and PKC activation, actin cytoskeletal remodeling, and various transcriptional responses do not occur.15 The consequence of loss of ZAP70 function is immunodeficiency in both mouse and man.16

LCK, like other SRC family kinases (SFKs), can autoactivate via trans-autophosphorylation. Thus, in the resting T cell, a pool of active LCK exists.17 This active pool of LCK is established via the combined actions of the inhibitory cytoplasmic tyrosine kinase CSK which phosphorylates Y505 in the C-terminal segment of LCK and the positive regulatory control of the CD45 tyrosine phosphatase which dephosphorylates phospho-Y505.6 A basal homeostatic state in which a relatively stable amount of LCK is active is established through the combined actions of CSK and CD45.18 Fluctuations are presumably controlled through feedback mechanisms that control CSK localization to the plasma membrane via adaptors that serve as substrates for SFKs in order to regulate the amount of CSK at the plasma membrane and thereby negatively regulate LCK activity.

In the non-ligand-bound state, the TCR CD3- and ζ-chains are not tyrosine phosphorylated (see Figure 1, left panel). However, interaction of the TCR with appropriate self-pMHC induces LCK-mediated tyrosine phosphorylation of an evolutionarily conserved sequence motif termed ITAM (for immunoreceptor tyrosine-based activation motif) expressed as a single copy in each of the CD3-chains and is triplicated in the ζ-chain.19 In the case of pMHC stimulation, the co-engagement of CD4 or CD8 with pMHC contributes to the localization of LCK with the stimulated TCR, thereby facilitating ITAM phosphorylation.

ZAP70 and SYK are closely related kinases but are differentially expressed.20 ZAP70 replaces SYK during thymic development. SYK plays a more important role early in thymocyte development, specifically in TCR β-selection, when co-receptors are not involved and pre-TCR expression at the plasma membrane is the main signal for progression of the TCR alpha-beta lineage. SYK-dependent signaling via the pre-TCR is required for its own downregulation and the upregulation of ZAP70.21 Three features of ZAP70 distinguish it from SYK and likely account for the switch to ZAP70 during thymocyte development.16 First, ZAP70 is more tightly autoinhibited than SYK.22 Second, unlike SYK, ZAP70 cannot autoactivate but instead requires phosphorylation input from a SFK.16 Third, ZAP70 has a distinct set of substrates from SYK and from SFKs, whereas the substrate specificity of SYK and SFKs partially overlap.23 To a large extent these differences could reflect the establishment of a hierarchical relationship between LCK and ZAP70 to enforce CD4/CD8 participation in facilitating the TCR preferential recognition of pMHC.

Although membrane bound and co-receptor-associated LCK is involved in the initiation of TCR signaling, ZAP70 normally resides in the cytoplasm in an autoinhibited conformation (Figure 2). A structural model of autoinhibited ZAP70 is depicted in Figure 2. If both tyrosines in an ITAM are phosphorylated by LCK, the tandem SH2 domains of ZAP70 bind to the doubly phosphorylated ITAM with high affinity (low nanomolar) and a high degree of specificity.24 The binding of the tandem SH2 domains to the doubly phosphorylated ITAM causes a conformational change in the inter-SH2 domain (interdomain A) that partially relieves the autoinhibitory conformation.16 This appears to be the extent to which the initial events involving ZAP70 occur under the influence of self-pMHC since ZAP70 is not phosphorylated under the influence of self-pMHC. Indeed, in freshly isolated thymocytes or T cells from secondary lymphoid tissues, the ζ-chains are tyrosine phosphorylated and non-phosphorylated ZAP70 bound to the doubly phosphorylated ITAMs can be detected.25 The recruitment and limited relief of autoinhibition likely does not proceed further because pMHC is not bound to the TCR long enough to allow for LCK-mediated phosphorylation of key residues in ZAP70 that are required for the further relief of autoinhibition and subsequent kinase activation.

Illustration of ZAP70 structure and signaling domains. (A) Cartoon of ZAP70 structural domains, including the N-terminal and C-terminal SH2 domains, interdomain A and B, and a kinase domain. Some of the tyrosine residues that are phosphorylated by LCK are also shown (red open circles). (B) A crystal structural model (left) or a schematic (right) of autoinhibited ZAP70. The key amino acid residues (illustrated by spheres) at which the mutations in ZAP70 described in this review are also labeled (W131, W163, Y315, Y319 and R360). The red and blue arrows indicate the key interaction among different structural domains of ZAP70 to maintain ZAP70 in the autoinhibitory state

The longer interaction that occurs when the TCR is bound to an agonist pMHC is accompanied by a more prolonged interaction with a CD4- or CD8-coreceptor that may or may not have an active LCK molecule bound. This differential binding of active LCK with co-receptors has been reported in recent work that suggests a co-receptor scanning model may be involved in ligand discrimination.26 Longer duration of binding of co-receptor-associated active LCK with the agonist pMHC bound to the TCR is associated with phosphorylation of Y315 and Y319 in SH2-kinase linker domain (interdomain B) and is also associated with phosphorylation of Y493 in the ZAP70 activation loop (Figure 1). The phosphorylation of Y493 by LCK, thereby stabilizing the kinase active conformation, is a critical step in activating ZAP70 catalytic function.16

The phosphorylation of Y315 and Y319 is also a critically important event. Phosphorylation of these sites stabilizes the open and active conformation of ZAP70 and also allows these phospho-sites to interact with several other proteins including Vav, CrkL, and LCK. Interaction of Vav and CrkL couple ZAP70, and hence the stimulated TCR, to important actin cytoskeleton regulatory events.16 The interaction of phospho-Y319 with the LCK SH2 domain is particularly notable, since it provides several positive feedback functions that serve to enhance ZAP70 activity and TCR signaling.27 First, it serves to maintain co-receptor-associated LCK near the stimulated TCR and active ZAP70, thereby promoting further phosphorylation of ITAMs and ZAP70 molecules. Second, the active conformation of LCK is maintained since the SH2 domain participates in autoinhibition of LCK. Third, the active conformation of ZAP70 is maintained, preventing ZAP70 from returning to an autoinhibited conformation. Fourth, the bridging of the SH2 domain of LCK to phospho-Y319 of ZAP70 facilitates the localization of one of ZAP70's key substrates, LAT, via the SH3 domain of LCK (Figure 1B).28 Thus, the coordinated hierarchical interactions of LCK and ZAP70 serve to not only activate ZAP70 bound to a stimulated TCR but also serve to provide positive feedback functions to the ZAP70 molecule(s) bound to an agonist pMHC-stimulated TCR.

1.2 ZAP70 substrate selectivity

The substrate specificity of ZAP70 is distinctly different from that of SYK and LCK. Most notable is the inability of ZAP70 to phosphorylate ITAMs, requiring it to depend upon LCK or other SFKs to initiate TCR signaling.23, 29 As mentioned, this substrate specificity conveniently couples LCK-associated co-receptor (CD4 or CD8) involvement to pMHC recognition. The inability of ZAP70 to autoactivate further couples ZAP70 dependency to co-receptor-associated LCK. On the other hand, co-receptor signaling alone does not activate T cells. This is due to the distinct substrate selectivity of LCK and other SFKs, when compared to the substrates preferentially phosphorylated by active ZAP70. The most well-characterized ZAP70 substrates are specific phospho-sites in the adaptors LAT and SLP76.16, 23 These two adaptors are not phosphorylated by SFK family members.

The functions of LAT and SLP76 are both critical to T cell development and T cell function, as delineated in studies of cell lines and deficient mice. LAT is a transmembrane protein that functions, via five ZAP70-mediated tyrosine phosphorylation sites, in the recruitment of effector molecules including PLCγ1 and GRB2/SOS to the plasma membrane in order to activate downstream pathways which lead to calcium, PKC, and Ras activation.6, 30-33 SLP76, via three ZAP70-mediated tyrosine phosphorylation sites and GADS (a GRB2-related adaptor) coordinate the assembly of several signaling complexes including those that involve the TEC family kinase ITK that participates in PLCγ1 phosphorylation and activation.

The ZAP70-mediated phosphorylation sites are notable for the relative absence of basic amino acids near the phosphorylated tyrosines and a relative enrichment of acidic residues, particularly at the −1 position relative to the phosphorylated tyrosine.23 These features are distinctly different from ITAM sequences which have multiple basic residues surrounding phospho-tyrosines and do not have an acidic residue at the −1 position. The selectivity for the sites in LAT and SLP76 is explained by the high concentration of basic residues in the ZAP70 kinase domain substrate-binding segment.23 These basic residues are highly conserved in ZAP70 sequences in more than 40 species sequences identified. Moreover, such a concentration of basic residues in the substrate-binding segment are unusual in the other cytoplasmic tyrosine kinases. Among the 32 cytoplasmic tyrosine kinases, the kinase domain of ZAP70 is one of the three most basic and is distinguished from others since the basic residues are unusually concentrated in the ZAP70 kinase domain substrate-binding region. Molecular dynamic simulations support the notion that the acidic substrate peptides dock with the basic residues in the ZAP70 catalytic domain.23 These findings suggest that the substrate-binding region of ZAP70 serves as an electrostatic filter in finding its substrate tyrosines in LAT and in SLP76.

One feature of LAT that is noteworthy is a highly conserved proline-rich region that plays a role in localizing LAT to the stimulated TCR complex (see Figure 1B).28, 34 We showed that this proline-rich sequence in LAT interacts with the SH3 domain of LCK, presumably while activated LCK is bound to the stimulated TCR and to ZAP70 as discussed above.

It is clear that ZAP70 plays a critical role in TCR signaling. In previous studies, we have used a PP1 analog-sensitive inhibitor model system to show that the catalytic activity is critically important for T cell responses in vitro and in thymocyte developmental transitions in thymic slices.35, 36 Titrating the degree of ZAP70 catalytic activity in a thymic slice system had impacts on the percentages of double positive thymocytes able to make developmental transitions. This suggests that strength of signaling has a direct influence on thymocyte selection, and likely on mature T cell responses. Here, we review recent studies from our laboratories that highlight the impact of well-characterized human and mouse mutations on T cell development, mature T cell responses, and tolerance.

2 DECREASED ZAP70 FUNCTIONAL EFFECTS ON T CELL FUNCTION AND THE IMMUNE SYSTEM 2.1 ZAP70 loss of function leads to immune deficiency

It is well-established that impaired TCR signaling leads to immune deficiency. Yet, ZAP70 deficiency in humans is a rare cause of combined immunodeficiency (CID) or severe CID (SCID) caused by recessive homozygous/compound heterozygous loss-of-function mutations in the ZAP70 gene37-39 and as reviewed in40). Laboratory evaluation of patients is notable for a paucity of CD8 peripheral blood T cells and the presence of normal numbers of non-functional CD4 T cells. Individuals with ZAP70 mutations can present with varied clinical manifestations. Patients with ZAP70 deficiency are particularly susceptible to respiratory infections including viral, bacterial, fungal, and protozoal infections,40 and can also present with cutaneous disorders.37, 40, 41 Many of these manifestations have been comprehensively reviewed in a systematic review of 49 ZAP70-deficient patients.40

2.2 A link between immunodeficiency and autoimmunity in humans

The link between immunodeficiency and autoimmunity has long been appreciated in humans. Perhaps it is most evident in patients with primary immunodeficiency where up to 20%-25% may develop autoimmune cytopenias, rheumatoid arthritis (RA), or other autoimmune diseases.42-44 The greatest risk has been associated with partial T cell immunodeficiencies and common variable immunodeficiency.44 The reverse association has also been described. In the setting of primary autoimmunity, despite the presence of activated immune cells mediating disease, observational studies have found an increased susceptibility to serious infections (independent of immune suppressive medications) and premature aging of the immune system, suggesting that impaired immune function might be a primary defect that, in turn, subverts tolerance.45-48 For example, RA CD4 T cells have the paradoxical ability to differentiate into pathogenic effector cells despite their impaired response to TCR engagement.49-58 Importantly, this paradox is observed in the Zap70 mutant SKG mouse model of arthritis (discussed below), making it one of the few mouse models to capture the contribution of an autoreactive TCR repertoire with impaired TCR signal transduction to the pathogenesis of arthritis.

Likewise, a subset of the ZAP70-deficient patients described by Sharifinejad et al40 developed non-infectious clinical manifestations, such as autoimmune disease (n = 7, 19.4%), including autoimmune cytopenias, autoimmune nephritis, and autoimmune enteropathy. The heterogeneity in ZAP70 mutations, clinical manifestations, and frequent need for treatment with a hematopoietic stem cell transplant (HSCT), can make it difficult to achieve a comprehensive immunologic and molecular view of ZAP70 deficiency in humans. However, mouse models (eg, SKG mouse model) can provide additional insight into the importance of ZAP70 function for immune cell development, T cell repertoire selection, and the propensity for autoimmunity in Zap70 murine mutants.

2.3 Impairments in TCR signaling can result in autoimmunity via disparate mechanisms

That T cells with severely impaired signaling can effectively differentiate into pathologic effector cells that mediate an inflammatory, autoimmune syndrome seems counter-intuitive. Yet, there are multiple reports linking immune deficiency with immune dysregulation and autoimmunity.52, 59-65 It has been suggested that the partial reduction in the number or function of T cells can disturb a “tolerogenic balance” and thereby generate a combination of immunodeficiency and hyper-immune dysregulation.62 The precise mechanism remains elusive, although murine models suggest multiple factors are at play, including selection of an autoimmune TCR repertoire, lymphopenia-induced exaggerated homeostatic proliferation precipitating autoimmune disease, deficient Treg function, resistance to Treg suppression, and T cell dysregulation causing failed induction of anergy in autoreactive T cells despite chronic antigen stimulation.60, 63 Notably, ZAP70-deficient mice, as well as LAT-mutated mice (the latter discussed in more detail later in this review), reveal a causal link between impaired TCR signaling associated with immune dysregulation and autoimmunity.66-70 Similarly, mouse models with cytokine signaling defects such as in STAT5A/5B-deficient mice can also serve as examples of immunodeficiency and immune dysregulation and suggest a synergistic role for cytokine signaling in the activation of self-reactive T cells.62, 71

2.4 ZAP70 loss of function mutations can lead to autoimmunity in murine models

Impairments in T cell signaling are linked to the pathogenesis of certain T-cell-mediated autoimmune diseases in humans,40, 72 and are also described in murine models. Previously, Goodnow and colleagues identified two hypomorphic Zap70 mutants from an ENU mutagenesis screen. Studies from these Zap70 mutants demonstrated that impaired TCR signaling and thymic selection can produce a potentially autoreactive repertoire of TCRs, and revealed a signaling threshold below which autoimmunity develops.62, 67 Compound heterozygotes were generated and studied on the C57BL/6 genetic background in which these mice develop dsDNA autoantibodies, a characteristic of SLE, and hyper-IgE syndrome.

The SKG genetic mouse model of arthritis susceptibility on the BALB/c background, is yet another example of autoimmunity in the setting of immune deficiency.64 This may also reflect the importance of genetic background in driving specific manifestations of autoimmune disease, as these mice develop joint specific autoimmunity rather than features associated with SLE. The SKG mouse, first described in 2003, harbors a missense mutation (W163C) in ZAP70 (Figure 3).64 This mutated Zap70 variant has a functionally impaired C-SH2-binding domain that prevents optimal interaction with the stimulated, phosphorylated TCR complex. This causes severely impaired downstream TCR signaling events resulting in altered positive and negative thymic selection. As a consequence, SKG T cells exhibiting the strongest affinity for self-pMHC ligands are positively selected instead of undergoing deletion, giving rise to a more autoreactive TCR repertoire.64, 65, 73, 74 This allows for the escape of arthritogenic CD4 T cells, which are responsible for spontaneous T-cell-mediated arthritis, immunopathologically similar to human RA. Disease features include RA-associated autoantibodies, destructive synovitis, and interstitial lung disease.64, 75 Studies of TCR transgenic mice crossed to the SKG mutation established that altered arthritogenic TCR specificity and altered TCR signaling are both essential for disease,59, 64, 65 and suggest that non-specific T cell expansion in a lymphopenic host is not solely responsible for arthritis development in SKG mice. This highlights the importance of dysregulated peripheral tolerance due to altered TCR signaling in addition to TCR specificity in disease development.

Allelic ZAP70 mutations directly impact T cell signaling strength. Hypomorphic and hypermorphic ZAP70 mutants and the resulting signaling defects are summarized. The stars represent the location of individual amino acid mutations: the blue stars represent hypomorphic mutants, the pink star represents weaker hypermorphic mutant and the red star represents the stronger hypermorphic mutant. All the signaling effects resulting from individual mutations are illustrated (black arrows and cross symbols). Red-filled circles represent phosphorylated tyrosine residues

The SKG mice not only exhibit a loss in central and likely peripheral tolerance that can be molecularly dissected but they also recapitulate the paradoxical ability of RA CD4 T cells to differentiate into pathogenic effector cells despite impaired TCR signaling.49-58, 64, 76 In this model SKG CD4 T cells are sufficient and necessary to cause arthritis.64 We have shown that adoptive transfer of naive SKG CD4 T cells are sufficient to trigger disease.76

2.5 Impaired TCR signaling reveals a threshold for autoimmunity in the thymus

Our lab previously generated another Zap70 mutant mouse (YYAA) in which two tyrosines, Y315 and Y319 (see Figure 3), are both mutated to alanine.65 The YYAA mutation resulted in an unanticipated partial loss of ZAP70 function—revealing the importance of the scaffolding roles of these two tyrosines. Like the SKG mouse, this mouse also has impaired T cell development, diminished TCR signaling (but not quite as severe), and defective negative and positive selection. These Zap70 mutations were sufficient to drive rheumatoid factor (RF) production in BALB/c mice challenged with zymosan but did not produce frank arthritis in response to identical environmental challenges that produced arthritis in the SKG line.65 Direct comparison to SKG mice on the BALB/c background did not reveal substantial differences in IL-17 production by T cells following zymosan treatment, and Treg function appears to be defective in both mice.65 The basis for the failure of this mouse to have a CD4 T cell response that leads to joint inflammation is not clear. We suspect that the SKG and YYAA mutants have differentially altered basal signaling (the homeostatic state of unstimulated T cells) and/or modification of their signaling circuitry (up or down regulation of signaling regulators) to alter the response potential in these two mice. Thus, these two Zap70 mutations in YYAA and SKG mice may differentially skew their TCR repertoires and have T cells with altered TCR signaling in ways that manifest as different autoimmune syndromes.

2.6 Nur77-TCR signaling reporter permits identification of arthritogenic CD4 T cells in RA-prone SKG mice

One specific arthritogenic TCR directed against a ubiquitous self-antigen was identified in the SKG model,59 but it was not known whether rare antigen-specific T cell clones drive disease or whether the entire pre-immune TCR repertoire has arthritogenic potential because it is highly autoreactive. It is also not clear how tolerance of such clones is broken in the face of profoundly depressed TCR signaling in SKG mice. Therefore, we combined the SKG mice with a fluorescent reporter to read out the relative strength of TCR signaling (termed by us SKGNur mice) to examine antigen-specific TCR signaling strength in pathologic CD4 T cells.76 This reporter tethers GFP expression to the regulatory region of Nr4a1 (encoding the orphan nuclear hormone receptor Nur77), which is rapidly and selectively upregulated in response to antigen, but not inflammatory stimuli.77-79 Using the reporter, we identified a subpopulation of CD4 T cells that is greatly enriched for their arthritogenic potential (enriched in the GFPhi relative to GFPlo fractions, see Figure 4).76 We found that higher levels of Nur77-eGFP in SKG CD4 (GFPhi) T cells marked their reactivity to self-pMHC in an autologous mixed lymphocyte reaction (AMLR). We established that these SKG GFPhi naive CD4 T cells were more arthritogenic than the GFPlo subset using an established adoptive transfer model in which SKG CD4 T cells are sufficient and necessary to cause arthritis in a lymphopenic host.64, 76 The T cells with increased autoreactivity nonetheless had diminished ex vivo-inducible TCR signaling, perhaps reflective of adaptive inhibitory mechanisms induced by chronic autoantigen exposure in vivo. This enhanced autoreactivity was associated with their ability to more readily differentiate into interleukin-17 (IL-17)-producing cells. Furthermore, the more arthritogenic SKG CD4 T cells were associated with upregulation of IL-6 cytokine receptor signaling machinery, which might be attributable, in part, to a reduced amount of expression of suppressor of cytokine signaling 3 (SOCS3)—a key negative regulator of IL-6 receptor signaling. As a result, the more arthritogenic GFPhi CD4 T cells from SKGNur mice were hyperresponsive to IL-6 receptor signaling.

Properties of SKGNur77 GFPhi T cells. Impaired TCR signaling in SKG mice and an altered T cell repertoire along with chronic endogenous antigen encounter confer increased arthritogenicity of selfreactive T cell clones in SKG mice. The most self-reactive clones (depicted in purple) that have escaped negative selection are marked by the highest levels of Nur77-eGFP expression (GFPhi). These cells respond more robustly to self-antigen, are hypersensitive to cytokines like IL-6, and differentiate more readily into IL-17 producing T cells. As a consequence, they cause more severe joint tissue damage during early stages of arthritis (Ashouri et al, 2019)

Similar to our findings in the SKGNur model, analogous antigen-activated CD4 T cells (enriched for the TCR signaling marker Nur77) were present in RA synovium compared to matched PBMC's from the same donor.76 In addition, Nur77 protein expression in CD4 T cells obtained from the RA joints far exceeded that in CD4 T cells isolated from synovial tissue of patients with a nonautoimmune form of arthritis.76 These results support the hypothesis that pathogenic CD4 T cells are enriched in RA joints and are likely recognizing an intraarticular autoantigen(s). Consistent with findings from SKGNur mice, SOCS3 expression was similarly downregulated in RA synovium and suggests a similar mechanism of IL-6 hyper-responsiveness in RA joints. Taken together, our findings suggest that despite impaired TCR signaling, autoreactive T cells exposed to chronic antigen stimulation exhibit heightened sensitivity to IL-6 receptor (IL-6R) signaling, which contributes to their arthritogenicity in SKG mice (Figure 4), and perhaps in patients with RA.

Recently, we have characterized these arthritogenic SKG CD4 T cells in greater detail. We have performed single-cell RNA and TCR sequencing analyses and have identified genes that are up- or downregulated and have also identified TCRs that are enriched in this subset of pathogenic T cells even prior to the development of arthritis. Our unpublished data suggest that impaired negative selection of of thymocytes with TCRs that recognize endogenous antigens in SKG mice creates a biased repertoire of autoreactive naive CD4 T cells that are poised to contribute to disease pathogenesis through their altered transcriptome as they encounter antigen in the periphery.

3 INCREASED ZAP70 FUNCTIONAL EFFECTS ON THE MAINTENANCE OF T CELL TOLERANCE

Recent studies on two hypermorphic ZAP70 mutations, R360P and W131A, illustrate how increased ZAP70 function associated with disrupting ZAP70 autoinhibtion impacts T cell function and immune tolerance (Figure 3).80-83

3.1 Weakening ZAP70 autoinhibition results in increased ZAP70 function

While both R360P and W131A mutations destabilize ZAP70 autoinhibition and render the kinase partially constitutively active, structural analyses suggest that the mechanisms of interference are distinct (Figures 2 and 3).84, 85 W131A disrupts the interdomain A-interdomain B interaction, which is regulated by phospho-ITAM binding. In contrast, the R360P mutation appears to relieve the interdomain B and kinase domain interaction (Figures 2 and 3), a role regulated by LCK-mediated phosphorylation of Y315 and Y319. The activating effects of these two mutations are further supported by biochemical analyses. When we co-expressed LAT with the full-length ZAP70 W131A mutant in human embryonic kidney (HEK) 293 cells, we observed a moderate degree of spontaneous LAT phosphorylation.86 In contrast, spontaneous LAT phosphorylation was initially not detected in HEK293 cells transfected with the full-length R360P mutant.80 In follow-up studies when HEK293 cells were transfected with LAT and the ZAP70 R360P mutant lacking the SH2 domains and interdomain A segment, we observed spontaneous LAT phosphorylation by the R360P mutant.83 Together, these results support the notion that both the W131A and R360P mutations directly weaken ZAP70 autoinhibition, with the R360P mutant behaving as a weaker hypermorphic allele compared to the stronger W131A hypermorphic mutant allele in TCR signaling.

3.2 ZAP70 R360P contributes to human autoimmunity by interfering with peripheral tolerance mechanisms

Notably, the R360P mutant is associated with a recently discovered complex familial autoimmune syndrome characterized by bullous pemphigoid, glomerulonephritis, colitis, and autoantibody to factor VIII.80 In this family, two out of three children born to healthy parents were affected by this autoimmune syndrome without evidence of immunodeficiency. Both affected children carried compound heterozygote missense mutations, R192W and R360P, in ZAP70. Structural modeling and biochemical studies revealed that the R192W is a loss-of-function allele due to reduced binding of mutant ZAP70 to the phosphorylated ζ-chain.80 Consistent with our findings in HEK293 T cells, transient overexpression of the ZAP70 R360P mutant in ZAP70-deficient P116 Jurkat cells resulted in increased TCR-induced signaling and expression of the activation marker CD69. Additionally, the activating effects of the R360P allele were only revealed in the presence of the hypomorphic R192W allele, but were neutralized by wildtype ZAP70.80 This is consistent with the clinical observations that both the father and a sister who carry only the R360P heterozygote mutation are healthy. Together, these results suggest that the hypermorphic R360P mutation is most likely responsible for the autoimmunity seen in these patients.

Our recent work on ZAP70 R360P homozygote knock-in mice provides further insights into the mechanism by which the R360P mutation contributes to the complex autoimmune syndrome in humans.83 In the context of a polyclonal TCR repertoire, the R360P mutation has no apparent impact on T cell development. However, we observed a substantial increase in the percentage of central memory CD8 T cells in the lymph nodes of 8-week-old R360P mice, suggestive of enhanced TCR tonic signaling in R360P T cells in vivo. Biochemical analyses confirmed that the R360P mutation was a weak hypermorphic allele that increased TCR signaling through weakened ZAP70 autoinhibition, as evidenced in enhanced ZAP70 phosphorylation at Y319 and faster calcium mobilization following anti-CD3 stimulation.83 The impact of R360P on TCR signaling was further revealed when the ovalbumin peptide-reactive OTI TCR transgene was introduced into the R360P mice, eliminating a possible compensatory TCR repertoire shift. Our utilization of weak peptide agonists for this transgene as TCR stimuli was quite revealing. When CD8 OTI T cells were stimulated by antigen-presenting cells loaded with a panel of well-characterized altered chicken ovalbumin peptides (OVA257-264) presented by H-2Kb with varied agonist affinity, the R360P CD8 OTI T cells exhibited selectively enhanced CD69 upregulation and proliferation in response to weak peptide agonists.83 Importantly, in the presence of IL-2, the R360P CD8 OTI T cells exhibited a stronger proliferative response to Catnb,83 a naturally occurring, positively selecting self-peptide for the OTI TCR