{'fraction_non_alphanumeric': 0.04193061840120663, 'fraction_numerical': 0.028355957767722473, 'mean_word_length': 5.013301088270858, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
CD8+ T Regulatory Cells in Lupus

Abstract T regulatory cells (Tregs) have a key role in the maintenance of immune homeostasis and the regulation of immune tolerance by preventing the inflammation and suppressing the autoimmune responses. Numerical and functional deficits of these cells have been reported in systemic lupus erythematosus (SLE) patients and mouse models of SLE, where their imbalance and dysregulated activities have been reported to significantly influence the disease pathogenesis, progression and outcomes. Most studies in SLE have focused on CD4+ Tregs and it has become clear that a critical role in the control of immune tolerance after the breakdown of self-tolerance is provided by CD8+ Tregs. Here we review the role, cellular and molecular phenotypes, and mechanisms of action of CD8+ Tregs in SLE, including ways to induce these cells for immunotherapeutic modulation in SLE.


Introduction
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by widespread inflammation, autoantibody production, and immune complex deposition. SLE affects major organ systems in the body, with lupus nephritis as a leading cause of death. [1][2][3] In SLE, immune homeostasis is impaired. Many investigations have attempted to modulate the abnormal immune regulation in SLE, having as a therapeutic goal the restoration of immune self-tolerance and the suppression of the activity and number of pathogenic cells and the production of autoantibodies by inducing T regulatory cells (T regs ) . [4][5][6][7][8][9][10] Many biotechnology and pharmaceutical companies are also currently working to translate the knowledge on the biology of T regs and/ or to bioengineer T regs into transformational medicines that could benefit patients with various inflammatory and autoimmune diseases including SLE.
While a decrease in the number and/or function of CD4 + T regs has been extensively studied in SLE, [11][12][13][14][15][16][17][18][19] the role and characterization of CD8 + T regs in the disease is less clear. Our group identified and characterized a CD8 + T cell subset that prevented the generation of pathogenic autoantibody production and maintained immune self-tolerance in murine lupus. [6,8] The investigation of the regulatory networks, genes, and signaling pathways involved in the regulation of the functional activity and survival of CD8 + T regs can be important for the development of therapies of restoration of immune homeostasis in SLE and other autoimmune diseases. The critical questions toward a clinical translational use of the findings are: (1) What is/are the precise surface phenotype(s) of the CD8 + T regs which suppress autoantibody production? (2) What are the critical molecular elements in the CD8 + T regs that are required for their survival, expansion, and suppression of helper T cell activity and suppression of autoantibody production by B cells? (3) What are the roles of transforming growth factor (TGF)-β, Bcl2, regulator of G-protein signaling (RGS) proteins, and interferons (IFNs) expression in the suppressive mechanisms of the CD8 + T regs ? (4) Can peptides that target Major Histocompatibility Complex (MHC) I/II T-cell domains augment the CD8 + T reg activity in SLE patients?
This review will discuss the aspects of T reg -mediated immune regulation, current knowledge in the field and approaches of T reg -based immunotherapy for improved management of SLE.

Cellular and molecular phenotypes of CD8 + T regulatory Cells (T regs )
Although several cellular and molecular markers have been described for the identification of CD8 + T regs (see Table 1, Figure 1, 2 and [20] ), there is no single surface marker that is specific for CD8 + T regs .
Also CD4 + FoxP3 − type-1 regulatory (Tr1) cells that express IL-10 are involved in the maintenance of tolerance and display strong immunosuppressive functions. [52][53][54] Duhen et al. identified CD4 + T regs subsets based on the expression of chemokine receptors, with differentially expressed lineage-specific transcription factors that responded differently to Th1, Th2, and Th17. [55,56] Pesenacker et al. and Afzali et al. defined a new subset of T regs in human cord blood with a CD4 + CD161 + phenotype that, although proinflammatory in nature, had a similar suppressive potential as conventional T regs, [57,58] while Chung et al., and Linterman et al. identified a subset of CD4 + T regs expressing CXCR5 and Bcl6 that localized in the germinal centers of both mice and humans. [59,60] Other tissue-resident T regs can be mostly activated cells with memory suppression. [61,62]

Transcription factors and mechanisms of action of T regs
FoxP3 is a critical transcription factor in the regulatory activity of both CD4 + and CD8 + T regs . [109] A decreased expression of FoxP3 results in loss of tolerance to self-antigens in SLE patients, [110] and SLE patients have a decreased expression of FoxP3 as compared to healthy matched controls. [77] Recent studies have shown that both CD4 + and CD8 + T regs express another transcription factor, Helios, which appears as essential for the maintenance of a stable phenotype and suppressive activity during inflammation and autoimmunity. [111] Helios is a member of the Ikaros gene transcription factor family expressed by FoxP3 + T regs (both in mice and humans). It is thought that Helios + cell subsets arise from thymus while  Helios − subsets are induced from FoxP3 − T cells. Helios + T cells are highly suppressive and express more highly demethylated T reg -specific demethylated region (TSDR) that facilitate FoxP3 transcription and therefore expression. [112] Helios + human memory T regs appear to co-express (T cell immunoreceptor with Ig and ITIM domains (TIGIT) and Fc receptor-like protein 3 (FCRL3), [113] and suppressive Helios + FoxP3 + T regs with migratory potential are expanded in inflamed tissues of SLE patients with active disease. [114] It seems that transcription factor, BTB Domain And CNC Homolog 2 (Bach2), is also important for T regs , since a loss of it results in Th2-mediated inflammatory lung disease while its expression is required for TGF-β-induced FoxP3 expression and the suppression of T effector cells. [115][116][117] CD4 + CD25 + LAG + T regs are instead regulated by Early growth response 2 (Egr2), a zinc-finger transcription factor required for the induction of T cell anergy, and produce TGF-β3 in an Egr2-and Fas-dependent manner. [118] Another transcription factor, nuclear factor erythroid 2-related factor 2 (NRF2), is a transcriptional activator which regulates oxidative stress. [119] Although specific functions of NRF2 in T regs are not fully understood, a recent study has shown that NRF2 is a negative regulator of T reg function and that FoxP3 specific activation of NRF2 results in the loss of immune tolerance and the accumulation of IFN-γ-producing T effector cells and inflammation. [120] In SLE, several lines of evidence suggest that NRF2 plays a central role in the pathogenesis of the disease by exerting anti-inflammatory effects-although others show pro-inflammatory effects. One study showed that aged female NRF2-deficient mice were prone to develop a condition closely resembling human SLE, [121] and another study in B6/lpr mice associated NRF2 deficiency with lupus nephritis and Th17 cells. [122] Mechanistically, NRF2 binds together with small Maf proteins to the antioxidant response element (ARE) in the regulatory regions of target genes and with KEAP1 (Kelch ECH associating protein 1), a repressor protein that binds to NRF2 and promotes its degradation by the ubiquitin-proteasome pathway. Genetic deletion of Keap1 resulted in higher percentages of T regs, [123] and the absence of NRF2 in donor T cells enhanced the persistence of T regs and reduced systemic inflammation in murine GVHD. [124] Notwithstanding the above consideration, the general mechanisms of actions of the T regs include: (1) suppression of T and B cells through inhibitory cytokines; (2) induction of cytolysis in target cells; (3) targeting antigen presenting cells (APC) such as dendritic cells, and (4) metabolic disruption in target cells.
T regs secrete inhibitory cytokines such as IL-10, TGF-β, and IL-35 that can suppress target cells including APCs and CD4 + CD25 − T effector cells. [68,69,125,126] For example, pConsinduced T regs secreted TGF-β and IL-10, [6,8,18,21,76] is also observed in other studies. [70][71][72] The cytolysis of target cells by T regs involved perforin and granzyme B. [18,90] T regs can also target directly APCs to suppress their function or render them tolerogenic through an upregulation of inhibitory receptors such as Ig-like transcript (ILT)-3 and ILT-4. [37,38] Bezie et al. showed that CD8 + FoxP3 + T regs depend on the expression of CTLA-4 to suppress T effector cells in vitro, [73] and other studies found that T regs can downregulate costimulatory molecules such as CD80 and CD86 on the APCs. [36,74,79] Finally, the "metabolic disruption" in target cells by T regs causes suppression of T effector cells by utilizing/sequestering IL-2 and/or IL-15, thus depriving the target cells of critical growth factors. [75,127]

Concluding remarks
Studies and findings on T regs are ready to be translated into approaches for the restoration of immune tolerance in SLE and advancement toward clinical settings. In particular, the bioengineering of T regs and the use of polyclonal and antigenspecific T reg cell therapies based on CD4 + and CD8 + chimericantigen-receptor (CAR) T regs in ongoing investigations by many biotechnology and pharmaceutical companies are providing encouraging results that appear to rapidly translate into the clinical practices. [128][129][130][131][132][133][134] More research will allow to fine-tuning and avoid off-target effects in different T regs -based immunotherapies, optimizing the immunotherapeutic benefits for SLE patients.