T Cells in Autoimmune Diseases

Chapter 5

T Cells in Autoimmune Diseases Amir Sharabi1,2, George C. Tsokos1 1Division

of Rheumatology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States; 2Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

INTRODUCTION T cells develop in the thymus from bone marrow–derived hematopoietic cell precursor, and during their development they acquire their T cell receptor (TCR) that is educated not to recognize self-antigens. During this selection process, the T cells gain and lose the expression of CD4 and CD8 co-receptors that eventually result in the generation of conventional T cells and T regulatory (Treg) cells that can be sent out to the periphery [1]. However, some autoreactive T cells do leak into the periphery as T cell selection in the thymus is not protected totally from mistakes. Furthermore, in circumstances of deletion of genes that are critical for T cell development in the thymus, such as phosphatase and tensin homolog (PTEN), a potent negative regulator of PI3K signaling [2], and zeta-chain-associated protein kinase 70 (ZAP-70) that is critical for the depletion of autoreactive T cells [3], autoimmune diseases will ultimately develop. The thymic autoreactive T cells, which escape to the periphery, will be later controlled by peripheral tolerance mechanisms able to prevent autoreactive T cells from inducing pathogenic immune responses in target organs. The breakdown of peripheral T cell tolerance to self-antigens enables autoreactive T cells to become active and mediate the effector immune responses through inflammation and support to autoantibody-producing B cells.

GENETIC PREDISPOSITION AND T CELL GENETIC VARIANTS IN AUTOIMMUNE DISEASES Genome-wide association studies have shown that the susceptibility for developing autoimmune diseases is rather polygenic with multiple genetic variants [4]. The highest genetic risk derives from MHC loci, which indicates a key role for antigen recognition by T cells for them to become pathogenic. Additional genetic variants include CD25 (IL-2 receptor, α chain), IL-2, CTLA-4, serine/threonine protein phosphatase 2A (PP2A), and tyrosine protein phosphatase [5,6]. Alternative splicing of T cell genes gives rise to several splice gene variants that are pathogenic. Alternative splicing of the CD3 zeta mRNA in systemic lupus erythematosus (SLE) T cells results in an unstable variant, which contributes to abnormalities with TCR signaling [7]. Furthermore, CD44v3 and CD44v6 are common splice isoforms of the CD44 gene in SLE T cells, which have been shown to correlate with the extent of disease activity, the presence of lupus nephritis, and with positivity for anti-dsDNA antibodies [8]. CD44 variant isoforms are important for the homing capacity of T cells to target organs. In addition, the products of alternative spliced CREM gene, cAMP responsive element modulator alpha (CREMα) and cAMP early repressor I (ICER), represent transcriptional repressors of certain functional repressions. Lupus-prone B6.lpr mice deficient of ICER/CREM are protected from systemic and organ specific autoimmunity [9].

T CELLS IN THE PATHOGENESIS OF AUTOIMMUNE DISEASES There are different subsets of T cells that are defined by their TCR composition (e.g., αβ chains or γδ chains), the expression of co-receptors, namely CD4 for T helper (Th) cells and CD8 for T cytotoxic (Tc) cells, and the expression of master genes, transcription factors, and cytokines. Most of T cell subsets have a role in the pathogenesis of autoimmune diseases (Fig. 5.1), either because of the expression and production of pathogenic cytokines or because of impaired or excessive function.

Th17 Cells CD4 T cells that express the transcription factor ROR-γt produce on stimulation the proinflammatory cytokines IL-17, IL-21, IL-22, IFN-γ, and Granulocyte-monocyte colony-stimulating factor (GM-CSF), all of which promote the development of autoimmune diseases in mice and humans [10,11]. In target organs they generate ectopic lymphoid follicles [12], wherein B cells are activated and become autoreactive [13,14]. Mosaic of Autoimmunity. https://doi.org/10.1016/B978-0-12-814307-0.00005-0 Copyright © 2019 Elsevier Inc. All rights reserved.

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FIGURE 5.1  T cell subsets playing role in autoimmune disease.

Double-Negative T Cells These cells are αβ TCR T cells that do not express CD4 and CD8. Their origin is not clear; however, because they share genes with those of CD8 T cells, it is suggested that at least some of them originate from autoreactive CD8+ T cells that were stimulated continuously [15–17]. In mice, the repression of CD8 locus is triggered by engagement of these cells with autoantigens [18]. In patients with SLE, CREMα mediates CD8 repression through binding to the CD8 locus and the recruitment of HDAC1 and DNMT1, which turn the chromatin inaccessible [19]. In several autoimmune diseases, doublenegative (DN) T cells are expanded and may comprise 1%–5% of peripheral T cells. They produce IL-17 and can help B cells to produce autoantibody. IL-17–producing DN T cells accumulate in the kidney and probably other tissues [20–23].

γδ T Cells These cells represent a minor T cell population that has adaptive and innate-like characteristics important for mucosal immunity [24]. They can present antigen, secrete inflammatory cytokines, promote antibody production, and inhibit Treg cells, thereby playing a pathogenic role in autoimmune diseases [25]. Stress molecules such as those of the HSP60 family augment γδ T cell activity. γδ T cells affect dendritic cells to produce IL-17 resulting in inflammatory cell infiltration in the kidney, and depletion of γδ T cells can diminish this infiltration [26–28].

T Follicular Helper Cells These are CD4 T cells that express the transcription factor Bcl6 and the chemokine receptor CXCR5, which enable them to migrate into germinal centers. Within the germinal centers of lymph nodes and kidneys, T follicular helper (Tfh) cells help the activation of B cells and lead to differentiation of long-lived plasma cells. This process yields the production of high-affinity autoantibodies as it leads to immunoglobulin class switching [29–32]. Tfh cells are frequent in autoimmune

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diseases, and their pathogenic role in SLE has been demonstrated in murine models [31]. In patients with SLE, the number of Tfh cells correlates with both the numbers of plasmablasts and the titers of anti-dsDNA autoantibodies. Interestingly, a subset of Tfh cells in SLE patients was shown to secrete IL-17 and promote B cell activation in the kidney leading to the development of nephritis [29].

Th1 Cells These are CD4 T cells that express the αβ TCR and IFN-γ as their signature cytokine. These are the pathogenic cells that mediate the development of diffuse proliferative lupus nephritis and crescentic glomerulonephritis, anti–GBM-induced glomerulonephritis, and antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis [33–35].

Th2 Cells These are CD4 T cells that express the αβ TCR and produce IL-4 as their signature cytokine. IL-4 activates signal transducer and activator of transcription 6 (STAT6) and leads to the expression of their principle transcription factor, GATA. They also secrete IL-5, IL-6, IL-9, IL-13, and IL-17E that support the humoral response. Th2 cells mediate the development of membranous lupus nephritis in MRL/lpr mice whose Th1 response is genetically deleted [37]. It is noteworthy that in addition to the effects of Th cells on B cells, BAFF and APRIL are B cell survival and growth factors that contribute to the pathogenicity of B cells, and patients with autoimmune diseases produce high levels of these factors [36].

Th9 Cells Th9 cells secrete IL-9 and require TGF-β and IL-4 for their induction [38,39]. They express Foxp3 and IL-4 but at much lower levels than Tregs and Th2 cells, respectively. TGF-β promotes the development of Th9 cells in both mice and humans through the induction of the ETS-transcription factor PU.1 [40,41]. PU.1 binds to the IL-9 promoter and promotes Th9 subset polarization. IL-4 activates STAT6, which stimulates an increase of IL-9 in Th9 cells. MRL/lpr mice with established lupus have expanded Th9 cells in their spleens and kidneys, which correlate with the production of anti-dsDNA antibodies [42]. In addition, experimental autoimmune encephalomyelitis (EAE) could be induced by Th9 cells and IL-9, and the inflammation in the central nervous system was shown to be independent of Th1 and Th17 cells [43].

CD8 T Cells In addition to their cytotoxic role they have against intracellular pathogens and tumors, CD8 T cells are known for their ability to regulate autoimmune and allergic diseases [44]. In experimental autoimmune glomerulonephritis, depletion of CD8+ T cells reduced the severity of disease [45]. Furthermore, in SLE and ANCA-associated vasculitis, an altered gene expression profile in CD8+ T cells was shown to correlate with a negative disease outcome [46]. Also, patients with ANCAassociated nephritis and lupus nephritis have a higher urinary CD8 to CD4 T cell ratio [47].

CD4 Treg Cells Among the CD4 and CD8 T cells, the most studied Treg cell population resides within the CD4 T cells that express the αβ TCR and the master gene Foxp3. Those CD4 Treg cells develop in the thymus and have typical characteristics that enable them to suppress the immune response [48]. CD4 Treg cells also play a role in tissue repair [49,50]. Defects in several signaling molecules and pathways were shown to hamper Treg cell function, including PP2A, mechanistic target of rapamycin (mTOR) C1, PI3 phosphatase, PTEN, and calcium/calmodulin-dependent protein kinase IV (CaMKIV) [49]. In autoimmune diseases, both decreased numbers and compromised function have been implicated [51,52].

CD8 Treg Cells These are CD8 T cells of multiple subsets that have suppressive capabilities. Their number, function, or both are impaired in patients with autoimmune diseases and in murine models of autoimmunity [53]. Frequent subsets of CD8 Treg cells express low levels of CD28. CD8 knockout mice that were induced with EAE improved their outcome following the adoptive transfer of CD8+CD28low T cells from wild-type animals [54]. Furthermore, in murine SLE, treatment with tolerogenic peptides ameliorated the disease manifestations through the induction of CD8 and CD4 Treg cells [55,56], and CD8 Treg cells were essential for the optimal function of CD4 Treg cells [56,57], suggesting a cross talk between CD8 and CD4 Treg cells.

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MOLECULAR PATHWAYS IN PATHOGENIC AUTOREACTIVE T CELLS Central pathways in T cell development in the thymus, which result in the development of autoimmune diseases, are beyond the scope of this manuscript, but mostly related to interference with the process of educating the TCR recognition of selfantigens and the deletion of autoreactive T cells [1–3]. The main molecular pathways in the periphery that participate in the induction and activation of autoreactive T cells are described here (Fig. 5.2).

CD3 Zeta This T cell surface molecule is part of the CD3 complex, which transduces the signaling from TCR following antigen recognition. Single-nucleotide polymorphisms in the CD3 zeta locus affect its expression, and it is downregulated in several autoimmune diseases [58–60]. Consequently, decreased expression of CD3 zeta can lead to the migration and accumulation of peripheral T cells in tissues where they produce IFN-γ and IL-17 and trigger inflammation [61–63]. T cells with decreased CD3 zeta expression have a higher capacity to infiltrate tissues partly because they overexpress the adhesion molecule CD44 [64]. T cells from patients with SLE also express elevated levels of CD44 and specifically v6 variant of CD44, which has been associated with lupus nephritis [8].

Rho-Associated Protein Kinase This is a serine/threonine kinase that phosphorylates ezrin/radixin/moesin (ERM) protein complex for the regulation of cell migration [65], and the CD44–Rho-associated protein kinase (ROCK)–ERM axis is activated in patients with autoimmune diseases, including SLE [66–68]. Inflammatory conditions for mesangial cells and podocytes also result in ROCK activation [69]. Rock activation can also result in the development of Th17 cells [68].

Protein Phosphatase 2A This is a serine/threonine phosphatase composed of three subunits (each with different isoforms), which is involved in pivotal cellular reactions and responses, including transcription, and cell migration and survival [70]. PP2A-beta subunit plays a role in the survival of autoreactive T cells as it controls T cell apoptosis following IL-2 deprivation, and T cells from patients with SLE have decreased levels of PP2A-beta subunit [71]. In Treg cells, however, it is required for their development through the suppression of mTORC1 [72]. Interestingly, mice whose T cells overexpress PP2A do not develop autoimmune syndrome spontaneously, yet on challenge with anti-GBM antibody, they develop glomerulonephritis that is responsive to treatment with anti–IL-17 antibody [73].

FIGURE 5.2  Molecular pathways in T cells related to autoimmune disease.

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CREM This is a family of transcription factors that become activated following intracellular increase in cAMP. Two isoforms, CREMα and ICER, play a role in T cell differentiation, epigenetic-regulated suppression of IL-2, and regulation of CD8 expression by CREMα [9,19,74–78]. CREMα is increased in SLE T cells and represses IL-2 transcription through at least three pathways: (1) binding to CREB-binding site to withhold the transcription initiation machinery, (2) the recruitment of HDAC1 and DNMT-3a to impose epigenetic closure of the IL-2 locus, and (3) binding and repressing c-fos to reduce AP-1 formation required for IL-2 transcription [74,75]. CREMα together with ICER result in demethylation and acetylation of the IL-17 promoter, processes that upregulate IL-17 transcription [9,74,75].

Calcium/Calmodulin-Dependent Protein Kinase IV This is a serine/threonine kinase that regulates gene expression through transcription factors associated with activation and development of T cell subsets. In SLE patients and lupus-prone mice, the expression of CaMKIV is increased in T cells following their activation [77,78]. This results in decreased IL-2 production and compromised Treg cell function [77]. Specifically, in SLE T cells, CaMKIV enhances the binding of CREMα to the IL-2 promoter and reduces its activity [78]. Depletion of CaMKIV in T cells from both mice and human with SLE recovers IL-2 production and Treg cell differentiation [77]. CaMKIV also promotes Th17 cell differentiation through the Akt/mTOR pathway [79] and the recruitment of these cells into tissues, including the kidneys [80], where it affects the function of mesangial cells [81] and podocytes [82].

Mechanistic Target of Rapamycin Pathway mTOR is a serine/threonine kinase that regulates T cell activation, proliferation, and survival, and it is frequently activated in autoimmune diseases [83]. There are two major mTOR complexes, namely mTORC1 and mTORC2, and the former plays a role in Th1 and Th17 cell differentiation, whereas the latter is essential for Th2 differentiation [84]. In SLE, the mTORC1 is activated by PI3K/Akt pathway, CaMKIV, and ROCK [68,79,85], which impairs Treg cells and increases the development and tissue recruitment of Th1 and Th17 cells to target organs [86].

Signal Transducer and Activator of Transcription This is a family of signaling molecules downstream the cytokine receptors that dictate the differentiation and function of immune cells, including T cells. Following the engagement of a cytokine receptor, STAT molecules are phosphorylated by janus kinases and become activated. For instance, STAT4 promotes Th1 cell differentiation in response to IL-12; STAT6 promotes Th2 cell differentiation in response to IL-4; STAT3 promotes Th17 or Tfh cells in response to IL-6, IL-21, IL-23, and TGF-β; and STAT5 promotes Treg cells in response to IL-2 and TGF-β. These differentiation events of T cells are regulated by mTOR pathway [87]. SLE T cells express high levels of STAT3 [88], and STAT3-induced IL-17 production and Tfh cell differentiation are required for infiltration and accumulation of immune complexes in murine lupus nephritis [89,90].

EPIGENETICS AND AUTOIMMUNITY Epigenetic pathways enable cells to make functional adjustments through the expression or repression of genes in response to a variety of stimuli ranging from cell surface molecules to signaling molecules. Methylation of DNA CpGs and histone modifications are common epigenetic mechanisms, and gene expression is increased when DNA is hypomethylated [91]. In SLE, genes that are hypomethylated and play a pathogenic role include CD70, CD11A, CD40L, IL-4, IL-10, IL-13, and IL-17 [91,92]. Furthermore, PP2A gene is hypomethylated in SLE T cells [93], and increased activity of PP2A results in ERK/MEK dephosphorylation and decreased DNMT1 activity with a resultant hypomethylation of the pathogenic molecules, CD70 and CD11a [94].

CONCLUDING REMARKS The study of T cells in patients with systemic autoimmunity and lupus-prone mice has generated novel insights into the pathogenesis of the disease and more importantly in the expression of organ damage. Study of T cell subsets has demonstrated the poor function of Treg cells and the poor control of the autoimmune and the inflammatory responses. Recognition of the expansion of DN T cells has provided insight into their ability to help B cells to produce anti-dsDNA antibodies and to produce the proinflammatory cytokine IL-17 in the periphery and when they infiltrate the kidney. Study of the CD8

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cell subset and their poor cytotoxic function has provided a better understanding of the inability of SLE patients to fend off infections. More importantly, detailed studies of the biochemistry of T cell signaling events and gene transcription processes have unveiled a number of plausible treatment targets and biomarkers of disease activity. For example, a ROCK inhibitor is in Phase II trial, and mTOR inhibitors have been considered enthusiastically along with Syk and CaMKIV inhibitors. Low-dose IL-2 has been extolled in uncontrolled reports, whereas an anti-p40 (IL-23) monoclonal antibody has completed a successful Phase II trial. In parallel, a number of surface molecules (CD44v3 or C6) or gene expression profiles have been shown to have biomarker value. Yet, T cells in patients with SLE and other autoimmune diseases present enormous complexity, and continuous intense studies will reveal not only novel targets and biomarkers but also potential to develop tools toward personalized medicine.

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36  SECTION | II  Cellular and Molecular Mechanisms

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