First of all, certain distinctive properties of pathogenic anti-DNA autoantibodies were crucial for isolating and characterizing the Th cells that specifically help them. The pathogenic anti-dsDNA autoantibodies that are deposited in kidneys with lupus nephritis have distinct features, as they are complement-fixing IgG in isotypes, with cationic charge, and clonally restricted by isoelectric focusing, and are able to cause glomerulonephritis in vivo (35–41). Moreover, their antigen combining V regions share recurrent idiotype and fine-specificity patterns for autoantigens (39, 42). Sequence analysis of the pathogenic autoantibodies confirmed their clonal expansion, as they shared V_H region CDR3 sequences containing numerous cationic residues generated by somatic mutation (43–45), a signature of Th cell drive. Contemporary studies had shown that immune complexes with cationic charge preferentially bind to anionic residues in glomerular basement membrane proteoglycans and collagen (46–48). It was shown later that glomerular binding of these “anti-DNA” antibodies could also be mediated via histones in nucleosomes bound in situ (49–52).

As described above, pathogenic anti-dsDNA antibodies in lupus are class-switched (35, 36) and clonally expanded (43, 44) suggesting a T helper cell dependent response, but it was mysterious up to 1980s and early 1990s how the Th cells actually helped Pathogenic anti-dsDNA autoantibody-producing B cells, because conventional Th cells do not recognize DNA.

In the first step, it was established that special autoimmune T helper (Th) cell subsets expanded the select population of pathogenic anti-dsDNA autoantibody producing B cells in mice with lupus (41, 53). The production of these pathogenic autoantibodies is also driven by select Th cells that are detectable in patients with active lupus nephritis (54–56).

In the next step, to define their antigenic specificity, the autoimmune Th cells were cloned from lupus prone mice, and also from patients with lupus nephritis (~ 800 clones). Prior to these studies, isolation of the pathogenic T helper (Th) cells of lupus was not done, because their antigenic specificities were unknown. However, using their special functional property of inducing the pathogenic variety of anti-dsDNA autoantibodies as a selection marker, the lupus nephritis-inducing Th cells were isolated. Only 12-15% of activated T cells in lupus patients and mouse models, could induce the production of pathogenic IgG anti-DNA autoantibodies (53–57). When administered into young pre-clinical stage lupus-prone mice, pathogenic autoantibody-inducing Th clones could rapidly induce immune-deposit glomerulonephritis (57, 58). Sequences of antigen-binding CDR loops of the TCRs of these pathogenic Th clones of lupus show recurrent motifs of anionic residues, indicating their selection by autoantigens with cationic residues (56, 57, 59). Indeed, a majority of such pathogenic Th clones produced IL-2 and IFN-γ in response to nucleosomes that contain histone peptides bearing cationic determinants, and nucleosome-specific T cells are detectable in pre-clinical stage lupus-prone mice before pathogenic autoantibodies are detectable in their serum (56, 60, 61). In addition, immunization of pre-clinical lupus mice, but not normal mice, with whole nucleosome particles induces accelerated lupus nephritis indicating the need for pre-existing autoimmune T and B cells in a lupus-prone background (60).

Thus the relevant autoantigens for pathogenic anti-dsDNA autoantibody inducing Th cells of lupus were discovered by an unbiased experimental approach, using pathogenic autoantibody inducing Th cells as sensors to detect the relevant autoantigen epitopes. This property provided the lupus Th cells the ability of “linked recognition” (62) for interaction with pathogenic anti-DNA autoantibody producing B cells (Figure 1). In this way, for the first time a true autoantigen for spontaneous SLE; namely endogenous nucleosomes from host’s apoptotic cells, and not some speculative component in microbes (63, 64), was found to be the real text book-like hapten- carrier link between the pathogenic Th and B cell in lupus for cognate interaction (57, 60),—and from that critical experimental step further studies led to identification of the histone peptide epitopes in nucleosomes recognized by those Th cells, and showed how to harness those particular epitopes for regulatory T cell induction for lupus therapy, as described below. All this was possible in 1980s and early 1990s by cloning the select population of pathogenic anti-dsDNA autoantibody-producing B cells, and then the special autoimmune T helper cells that drive such B cells in lupus. To emphasize again, despite the obstacle that the antigenic specificities of lupus T cells were then unknown, using the experimental steps described above, the pathogenic anti-dsDNA autoantibody-inducing T helper cells were cloned to define the structure and specificity of their receptor genes in human and murine lupus. To achieve the ultimate goal of understanding the cause, and designing a cure for spontaneous autoimmune diseases like lupus, it was essential at that time to identify the major autoantigen/s that drives the pathogenic autoimmune response. In lupus, DNA is a target antigen for autoantibodies but paradoxically immunization of mice with DNA does not cause lupus. The studies with the pathogenic autoantibody-inducing T helper clones in early 1990s led to the initial identification of one of the major immunogens that drives the pathogenic T helper cells of lupus (57, 60).

Many studies soon followed that demonstrated or suggested mechanisms that could initiate or amplify the response of pathogenic Th cells to nucleosomal peptides in lupus. Briefly, in lupus, products from apoptotic cells accumulate and become immunogenic because, scavenging molecules in phagocytic cells, such as Marco and other scavenger receptors are functionally deficient in lupus, and so are complement components such as C1q, which facilitate phagocytosis of apoptotic cells without causing an immune response (65–68). Nucleosomes, HMGB1, DNA or RNA from apoptotic cell components not being disposed of properly, act as endogenous TLR ligands, stimulating cells of the innate and adaptive immune system (19, 23, 60, 69–74). For example, HMGB1 chromosomal protein from apoptotic cells that have not been removed properly, forms inflammatory complexes with other accumulating debris like DNA or nucleosomes particles, stimulating immune cells via TLR 2, TLR 4, and RAGE on the cell-surface, or TLR9 in the endosome/lysosome (74–76). Similarly, accumulating extra-cellular nucleosomes in micro particles containing DNA, or ribonucleoproteins containing RNA can stimulate cells of the innate immune system respectively by TLR9 or by TLR 7/8 (29, 31, 77, 78), thus augmenting autoantigen presentation to pathogenic Th cells by those activated APC. The case for TLR 9 is actually more complex, because in early stages, TLR9 actually protects against lupus (79), possibly by promoting tolerance in APC, B cells and helping Treg generation (80, 81). The other possible autoantigen-derived epitope with cationic charge, which could be recognized in a lupus B cell-linked fashion by the pathogenic autoantibody inducing Th cells possessing reciprocally charged anionic residues in their CDR3 region, would be derived from CDR3 region peptides of somatically mutated anti-DNA autoantibodies, as suggested (57, 60); and this possibility was independently demonstrated to be true by several laboratories (82–85). This issue is dealt in other contributions to this research topic.

Th1, T_FH, T_PH, Gamma Delta Th, CD8 Th, CD4^-CD8^- Th cells and more, participate in contributing to the pathogenic response in lupus. However, is it mainly Th1 → T_FH cells initiating/sustaining pathogenic autoantibody production; whereas the others evolve as amplifiers at extrafollicular inflammatory sites? First of all as mentioned above, only particular subclasses of IgG anti-DNA antibodies are more closely associated with a pathogenic potential in lupus patients and mice, and these pathogenic IgG antibodies belong to Th1-induced isotype classes. In lupus patients, Th1-induced anti-DNA IgG1antibodies are always elevated before the occurrence of renal relapse, and IgG1 plus IgG2 anti-DNA antibodies are found in patient’s renal eluates, whereas in lupus prone mice, murine Th1-induced IgG2a, IgG2b, and IgG3 anti-DNA are more frequently eluted from kidneys with active nephritis (39, 42, 86–88). In contrast to T_FH cells which conventionally produce IL-21; Th1 cytokine IFNγ not only mediates class switch for the nephritogenic isotypes, but Th1 derived IFNγ signal is also critical for autoantibody production by germinal center B cells (89, 90). Furthermore, many non-autoantigen specific, bystander T_FH cells expand as a secondary event with progression of disease, which could amplify (but not initiate) anti-DNA autoantibody production (91). Indeed, Ig class-switch recombination (CSR), during T and B cell cognate interaction, which is Th1 IFNγ cytokine dependent in lupus, may occur before the T_FH IL-21 driven expansion of autoantibody producing B cells in germinal center, which comes later (92). Moreover, Th1-biased GC T_FH cells have been reported (93), and another group reported that the differentiation and function of a Th1-derived T_FH1-like cell population is driven by IL-12 signaling, which is important for differentiation of Th1 cells in the first place (94–96).

Therefore, Th1 → T_FH1 evolution/transition is a possibility in pathogenic anti-DNA autoantibody production in lupus.

And then there are the potent T_PH cells with T_FH like phenotype but are CXCR5^-; they help lupus B cells also by producing IL-21; and IL-10–producing CCR6⁺T cells populate lymph nodes of SLE patients. These Th cells probably evolve after receiving cytokine and other signals from activated B cells and other APC at extra-follicular sites, as the disease progresses (27, 97).

In addition, helper activity of CD8⁺ and CD4^-/CD8^- αβ and γδ TCR⁺ Th cells, in pathogenic autoantibody production in human SLE has been reported (54, 55). The subset of T cells in humans that are CD4^-/CD8^- and αβ TCR⁺ with pathogenic anti-DNA autoantibody-inducing ability in SLE is interesting, because such Th cells were considered to be unique to MRL-lpr mice with lupus. However, similar pathogenic autoantibody-inducing T cells with double negative phenotype that express “forbidden”, autoreactive T cell receptors were described in non-lpr lupus prone mice (41, 53, 98) and then in human lupus (54, 55). Although these double negative T cells might be secondary events in lupus compared to the CD4+ Th cells, they make an important contribution to pathogenesis of the disease. The CD4^-/CD8^- and αβ TCR⁺ T cells also have important role in target organ inflammation (99, 100).

In the next step, the critical peptide autoepitopes recognized by lupus nephritis-inducing Th cells were localized initially to be in the core histones of nucleosomes, at amino acid (aa) positions: 10-33 of H-2B, 16-39 and 71-94 of H4, and 85-102 of H3 (61). Altogether 154 overlapping 15-mer peptides spanning the entire length of all four core histones were tested to find the buried epitopes in nucleosomal histones that were recognized specifically autoimmune Th cells that cause lupus in mouse models. In addition, another dominant epitope was identified in position 22-42 of H1’, by mass spectrometry analysis of naturally processed peptides eluted from class II molecules of lupus B cell (APC) lines fed with chromatin (101). Remarkably, all these epitopes are located in regions of histones that contact with DNA in the nucleosome, and they are also targeted by autoantibodies from lupus B cells (B-cell epitopes), indicating that the epitopes could be protected from degradation during autoantigen processing and thus preferentially presented to the Th cells (61, 101–103). Surprisingly, the nucleosomal peptides have the features of “universal epitopes” (104), for instance, the peptide epitopes are promiscuously recognized by pathogenic Th cells derived from lupus-prone SNF₁ mice (MHC I-A^d/q) even when presented by APC bearing I-A molecules of all other mouse haplotypes, and human HLA-DR as well! Due to reciprocal charge interaction, the lupus TCRs probably contact the nucleosomal peptide-complexed with MHC promiscuously to sustain TCR signaling (105, 106). The promiscuity of lupus TCRs influences their selection in the thymus of lupus-prone mice and ability to generate Treg cells for tolerance spreading in the periphery, as described below (80, 107–109).

Nested Epitopes for CD8 T Cells. The tolerogenic nucleosomal peptide autoepitopes bind to MHC class II as described above, but CD8⁺ Treg cells were also induced by injection of the epitopes. Indeed algorithms showed, MHC class I-binding motifs were nested in their sequences, as described (108, 110). The rationale being that the relatively long chain peptides epitopes would be processed further by APC for cross-presentation to CD8 T cells (111). For an example, H4_71–94 nucleosomal epitope has the nested CD8 sequence shown in bold letters TYTEHAKRKTVTAMDVVYALKRQG, and similarly individually distinct nested CD8 epitopes were detected in each of the longer peptide epitopes from the nucleosomes with CD4 Treg inducing ability, as detailed (108).

(Experimental Steps of More Recent Publications in Mouse Models and Then in Human Lupus Are Described in Brief Below):

It is noteworthy that the immune response against nuclear autoantigens is inter-connected by cross-reactive recognition at the B cell level (38, 39, 42), and importantly at the Th cell level (61, 101, 102, 105, 109). Thus the same lupus Th clone can help either a B cell specific for nucleosomes, or a B cell specific for dsDNA, or for ssDNA, or histone, or HMG, because each B cell can take up and process the whole chromatin particle by recognizing its own specific epitope in the chromatin, and then present the Th clone’s relevant histone peptide epitope derived from chromatin processing, and that results in linked intermolecular help (56, 60, 120). Therefore, suppressing the Th cells of lupus could block spreading of response to multiple epitopes in chromatin (Figure 4). This hypothesis of “Tolerance Spreading” was experimentally supported as described above showing that progression of established lupus nephritis in the lupus-prone mouse models can be delayed, diminishing proteinuria and prolonging life by administering the nucleosomal peptide epitopes singly in high dose IV or low dose SC in tolerogenic regimens (80, 108, 109).

Indeed, the production of a variety of pathogenic autoantibodies to nuclear autoantigens was inhibited by tolerance therapy with any one of the epitopes. Due to promiscuous recognition described above, multiple autoimmune T cells with different TCRs can respond to the same peptide from a nucleosomal histone, and on the other hand, an individual autoimmune T cell can recognize multiple nucleosome-derived peptides that are distinct in sequence (61, 105). Therefore, when injected in a soluble tolerance-inducing form, in the absence of adjuvants, even one peptide epitope can tolerize autoimmune Th cells of diverse specificity for chromatin-derived autoantigens and conversely suppressing Th cells with specificty for one nuclear autoantigenic epitope deprives help for multiple autoimmune B cells of lupus. Thus, tolerance induced by any one of the dominant peptide epitopes can suppress autoimmune response to other nucleosome-derived pathogenic epitopes (“Tolerance Spreading”). Indeed, such “cross-reactive” suppression directed at the broad spectrum of pathogenic autoimmune response is more desirable in lupus therapy rather than pinpoint precision for antigen-specificity, which is the goal of some studies using modern techniques (121). Furthermore, the peptide epitopes are very effective in tolerance induction because, they are simultaneously recognized by autoimmune T and B cells, and they may inhibit autoimmune B cells and DC directly in lupus (80, 101, 108, 109).

Despite “Tolerance Spreading to other lupus autoepitopes”, the histone peptide therapy resulted in autoantigen-specific regulation because, pathogenic autoimmune responses in lupus-prone subjects were preferentially and selectively downregulated, without any suppression of immune responses to foreign antigens, as detailed in these References (80, 108, 109). Moreover, ability of the treated animals to survive environmental microbes/pathogens appeared to be intact and robust as compared to untreated controls and non-autoimmune “normal” mice housed in the same “dirty” mouse facilities (80, 108, 109). Finally, experiments showed that the Treg cells generated by the peptide epitope therapy suppressed T cell response and T cell helper activity specifically directed to the peptide autoepitopes, but not for a foreign antigen, such as Hen Egg Lysozyme (80, 108).

As described above, the nucleosomal histone peptide epitopes when administered in nanomolar doses (1µg) subcutaneously (S.C.) every 2 weeks or even every month to lupus-prone mice, are effective in delaying or even preventing nephritis. This dose is lower by almost 1000 fold compared to some other peptides being tried as therapeutic agents. Furthermore, a major histone peptide epitope, administered singly, can suppress lupus disease also via nasal tolerance (122, 123). The peptide epitopes are a constituent of nucleosome, a highly conserved, ubiquitous self-antigen produced during ongoing apoptosis in generative lymphoid organs and recognized by developing cells of the immune system bearing appropriate receptors. Therefore, anaphylactic reactions were not observed with these self-peptides when administered in close to 1000 lupus-prone mice for various studies. The histone peptide epitopes, administered S.C. in a very low doses, generate Treg cells that suppress by producing minute amounts of TGFβ that act in close range cell to cell interaction, rather than causing Th2 deviation with consequent allergic reactions seen in the case of therapy of other autoimmune diseases, such as, EAE/MS and diabetes in NOD mice using other peptides. The histone peptide epitopes induce stable Treg that are autoepitope-specific and cross-reactive autoantigen-directed Treg cells by simultaneously decreasing IL-6 and increasing TGF-β production by DCs, which consequently caused Smad-3 phosphorylation in the peptide epitope-specific auto-immune CD4+ Th cells, and the peptide tolerance therapy is effective even at an age when they manifest clinically active disease. A single dominant epitope such as H4_71-94, is capable of inhibiting the diverse autoimmune process in lupus, because, potent and durable regulatory T cells (Treg) are generated by low-dose tolerance therapy mediating “tolerance spreading”. Both sets of regulatory T cells act via TGFβ in close range, and suppress autoimmune Th and B cells and other autoantigen presenting cells.

Although the phenomenon of low-dose tolerance was well known (124), most work since then have dealt with tolerance induction to foreign antigens in non-autoimmune subjects. However, the histone peptide induced low-dose tolerance was achieved in subjects with spontaneous SLE, whose immune system is already primed for autoimmune response against ubiquitous nucleosomal self-epitopes. The Autoantigen-specific and cross-reactive autoantigen-directed Treg cells generated by the peptide epitope therapy was effective even in the presence of complex lupus abnormalities, such as hyperactivity of lupus T and B cells and DC; particularly IFN-α producing pDC.

Unlike the peptide epitopes in low doses, whole (intact) histones worsen lupus (60) probably by binding to DNA or reciprocally charged molecules in vivo to make complex nucleosome like particle structures. Moreover, processing by APC of intact histones generates altered epitopes by post-translational modifications, such as acetylation or citrulination (125). Therefore, intact whole (complete) histones should not be used for tolerance induction.

Another group induced Treg cells by continuous infusion of a model laboratory antigen using hemagglutinin (HA)-specific TCR−Tg mouse system, and they also targeted the peptide to a surface receptor DEC-205 on DC, and administered considerable quantities of TGFβ in vivo (126). However, those approaches for therapy of diabetes were found to be deleterious (127). Moreover, in vivo administration of TGF−β as a drug in the presence of high IL−6 levels in lupus could induce pathogenic Th17 cells and T_FH cells, instead of generating Treg cells (128, 129).

Unexpectedly, prolonged remission achieved by patients with refractory lupus after autologous hematopoietic stem cell transplantation (HSCT) have a different mechanistic basis than “clinical remission” in conventional drug-treated patients, who do not achieve true immunologic remission, although they have a Systemic Lupus Disease Activity Index (SLEDAI) of 0–2 (80% of the drug induced remission patients were at zero level). In patients with stem cell transplant induced remission, CD4+CD25^highFoxP3+ Treg, and CD8+FoxP3+ Treg cells are generated, accompanied by almost total suppression of pathogenic T cells that respond to the histone peptide autoepitopes (135).

Detailed experiments in the above ref (135)., demonstrated that the post-transplant CD8 Treg cells suppressive activity was nucleosomal histone peptide-specific, as well as nonspecific, but directed to cross-reactive autoreactive and activated T cells. Both types of Treg cell’s suppressive activity was mainly TGF-β-dependent, but independent of cell-cell contact. The post-transplant patients’ CD8 Treg cells were stably FoxP3+ and they expressed markedly increased levels of CTLA-4, CD103, PD-1, PD-L1 and LAP, when compared to CD8 T cells from the same patients before undergoing transplantation. By contrast, the pre-transplant lupus patient’s CD8 T cells have cell-contact dependent helper activity for autoantibody production. The CD8 Treg found only in post-transplant patients are considerably more potent in suppressive activity compared to the CD4+CD25^high Treg cells that appear during clinical “remission” in lupus patients treated by conventional drugs, in whom autoimmune response of CD4 T cells to nucleosome-derived autoepitopes persists even during “clinical remission” (SLEDAI of zero). Therefore, autologous HSCT leads to the generation of a newly differentiated population of LAP^highCD103^high CD8^TGF-β Treg cells that maintain the lupus patients in “true immunological remission”, unlike patients with conventional drug therapy. Remarkably, very similar, highly potent CD8 Treg cells are also generated by low-dose nucleosomal peptide tolerance therapy that can prevent or treat lupus disease in mouse models of spontaneous SLE, as described above.

As stated, autoantibodies in lupus that belong to IFN-γ (Th1) dependent IgG subclasses fix complement and bind to activating FcγR on inflammatory cells to mediate pathogenicity. A CD4 T cell population in untreated lupus patients PBMC produces IFN-γ in response to histone peptide autoepitopes, and this autoimmune IFN-γ production response was almost completely suppressed in fresh PBMC from lupus patients in remission post-transplant. Removal of CD8 T cells (total) from the PBMC of post-transplant patients in remission, restored the IFN-γ response of CD4 T cells to nucleosomes and histone epitopes, much more strongly than removal of CD4+CD25^high cell subset enriched for Treg. Therefore, the latter subset probably cannot restore immunologic remission in conventionally treated lupus patients although they are increased in such patients after “clinical remission” (SLEDAI of 0–2).

The Post-transplant CD8 T cells suppressed by secreting mainly TGF-β and they expressed high levels of TGF-β latency-associated peptide (LAP), but they produced IL-10 to a much lesser extent; which is desirable because IL-10, by causing expansion of autoimmune B cells, is deleterious in lupus (136).

As low-dose tolerance induced by the histone peptide epitopes effectively inhibited lupus disease in mouse models, the effect of the epitopes on lupus patients’ PBMC cultures was tested in vitro. As discussed above, the major Peptide Autoepitopes for nucleosome-specific T Cells of human lupus were identical in sequence to the peptide autoepitopes for pathogenic T cells of lupus-prone mice (61, 102), and they shared similar properties of promiscuous MHC class II binding and being B cell autoantibody epitopes as well. Thus the peptide epitopes could be effective tolerogens for inhibiting both autoimmune T and B cell populations in lupus patients with diverse HLA alleles (61, 102, 105, 147).

Indeed, in PBMC cultures from inactive lupus patients and healthy subjects, addition of the histone peptide epitopes induced CD4⁺CD25^highFoxP3⁺ or CD4⁺CD45RA⁺FoxP3^low Treg cells, as well as CD8⁺CD25⁺FoxP3⁺ Treg cells with stable FoxP3 expression and suppressive activity (147). In the case of PBMC from patients with active lupus, dexamethasone or hydroxychloroquine were additionally needed for Treg-induction by the peptide epitopes in cultures. The peptide-induced regulatory T cells in lupus PBMC depended on TGFβ/ALK-5/pSmad 2/3 signaling, and TGF-β precursor LAP was expressed by those Treg cells, indicating that TGFβ production was responsible for their suppressive activity, and a positive feedback mechanism as well. The peptide epitope-induced Treg cells also inhibited type I IFN related gene expression in lupus PBMC. Expression of major members of Type I IFN genes themselves, as well as type I IFN induced genes (ISG) were markedly reduced by histone peptide epitopes in TLR9-stimulated PBMC of lupus patients. As stated above, pDCs in lupus are the main producers of Type I IFN upon stimulation by nuclear autoantigens complexed with anti-nuclear autoantibodies (7, 153, 154), and in lupus-prone mice, histone peptide epitopes act on pDC rendering them to become tolerogenic (80). Secondly, expression of 13 ISG genes, which have been reported to be upregulated in patients with active lupus (153, 155, 156) were also inhibited by the peptide epitopes. Moreover, the histone peptide Th cell epitopes, which were also shared by autoantibody producing B cell epitopes in lupus, could inhibit production of pathogenic autoantibodies by PBMC from active lupus patients as potently as an anti-IL6 antibody. Experimental details are in reference (147). Importantly, a mixture of the peptide epitopes (cocktail) was more effective in uniformly suppressing pathogenic activities in Human lupus PBMC cultures, as compared to single epitopes, because patients are heterogeneous in contrast to inbred lupus-prone mice. For example, suppression by a histone peptide cocktail #1 (C1), which is a mixture of H1^22-42, H3^115-135 and H4^16-39 at a concentration of 1.5 μM for each peptide or histone peptide epitope cocktail #2 (C2), which is a mixture of H1^22-42, H3^115-135 and H4^16-39 at a concentration of 4 μM of each peptide were very efficient in suppressing pathogenic autoantibody production and type I IFN related gene expression in lupus PBMC (147). Thus, low-dose histone peptide epitopes could durably inhibit pathogenic autoimmune response in human lupus by diverse pathways.

Multiple laboratories have shown that histone peptide epitope/s or other peptide epitopes administered without IL-2 injection, can induce generation of effective Treg in vivo, which inhibit disease in various lupus-prone mice (80, 82–85, 108, 122, 168). Although lupus T cells are deficient in IL-2 production (169, 170), that situation is relative not absolute, as lupus patients do not succumb to recurrent infections found in IL-2 knockout Immunodeficiency. A possibility in the case of these peptide epitopes is that in low doses, they could transiently activate autoreactive T cells, which could then provide small amounts of IL-2 for generating the regulatory T cells. Indeed this proposed mechanism (171), has actually been demonstrated by a similar situation occurring in the thymus where IL-2 is produced by a small population of self-reactive CD4 single positive (CD4SP) thymocytes, which then stimulates Treg precursor cells to differentiate (172). Those regulatory T cells induced in vivo may then be sustained also by other signals such as from ICOS, or TNFR2 (Tseng WY et al. Proc Natnl Acad Sci USA 2019, 116:21666-21672) (173). Still in view of the benefits of low dose IL-2 therapy in all autoimmune diseases, whether deficient in IL-2 or not (129, 169, 170, 174–177); adjunct therapy with low-dose IL-2 will be beneficial in the peptide-epitope therapy of lupus, as stated in the theme of this Topic. Moreover, Low dose IL-2 and corticosteroids in maintenance dose, actually were necessary for the peptide epitopes’ induction of Treg cells in ACTIVE lupus patients’ PBMC in vitro (147). Indeed, corticosteroids themselves induce Treg cells by various mechanisms to some extent (Tsai YG et al. Plos One 2014, 9:e81344) (178, 179), and thus could potentiate the autoantigen-specific and cross-reactive autoantigen-directed Treg response by the peptide epitopes (147).

How can durable immunoregulatory mechanisms be established in the inflammatory environment of lupus? In lupus patients, tolerance therapy with histone peptide epitope would be optimal after inflammatory burden is reduced by drugs. Nevertheless, in animal models, the peptides alone are effective in ameliorating established lupus nephritis (80, 108, 109). The regulatory T cells are more stable in inflammatory environment because they were induced by the peptide epitopes in vivo, in contrast to Treg cells induced/expanded in vitro. Furthermore, dexamethasone or hydroxychloroquine in maintenance doses actually supported Treg-induction by the peptides in lupus patients’ PBMC cultures, indicating that drugs that counter the increased activity of IRF5 and TLR pathways in lupus APC would be of added benefit (147). The histone peptide epitopes also can directly regulate autoimmune B cells and DC in lupus, in addition to generating Treg cells (80, 101, 109); and indeed the peptides could suppress autoantibody production to baseline levels in lupus patient’s PBMC even before significantly increasing Treg cell numbers in culture (147). The select histone peptide epitopes, which are tolerogenic, can directly reduce IL-6 and increase TGFβ production by DC (80), a situation which renders the DC not only be able to induce Treg, but also become susceptible to suppression by Treg (180). This property of inducing TGFβ production and simultaneously decreasing IL-6 production by DC, especially pDC, in turn induces TGF-β signal (Smad-3 phosphorylation) in target auto-immune CD4+ T cells converting them to stable Treg cells; a property highly beneficial for lupus therapy (80, 147), also because T_FH cell differentiation is inhibited in germinal centers under such conditions (128).

Since apoptotic cells have immunosuppressive properties (17, 181); the unaltered histone peptide epitopes derived from apoptotic platform may have intrinsic tolerogenic property (80, 147). In very low doses, without immunostimulatory adjuvants, the histone peptide epitopes could possibly activate latent TGFβ, by inducing expression of the integrin αvβ8 in resting pDC, as shown in other systems (182).

Treg cells require continued antigen-specific stimulation from DC to maintain lineage stability and high affinity regulatory cell function (183, 184). In lupus-prone mice, regulatory T cells induced by the peptide epitopes are detectable up to six weeks after S.C. injection. Subcutaneous injection route works for the highly soluble and charged histone peptide epitopes which are very rapidly absorbed systemically (80). In humans many protein drugs, and mAb biologics, insulin, IVIg etc., are administered S.C. without causing local/systemic inflammatory response.

However, despite the promising beneficial effects in animal models of established lupus disease, there is always the possibility of adverse autoreactive response to the peptide epitopes in patients with lupus, although they might be selected at the earliest, pre-clinical stage of disease. Another issue is that peptide epitope cocktails in low doses were more effective than a single peptide epitope in suppressing lupus manifestations in human lupus PBMC, but cocktails may be more immunogenic when injected in the skin (147).

Therefore, peptide delivery should be considered in fail-safe tolerogenic vehicles, such as Nanoparticles (NP), which are described in detail by experts in this field in other articles as part of this research topic. Just as a brief synopsis, the peptide epitopes may be delivered within the nanoparticles, or administered around the same time, but separately from tolerogenic nanoparticles (185). There are many issues in choosing the right nanoparticles for such therapy, specifically for lupus; for instance, liposome derived NP can activate complement, and rapamycin containing NP may interfere with initial Treg generation (186, 187), although rapamycin is effective in maintaining Treg, once they are induced (188). It is noteworthy that injected nanoparticles might be nonspecifically immunosuppressive, like silica particles, by overloading the immune system’s APCs, which phagocytose and engorge themselves with any foreign particles (189, 190). Therefore, targeted nanoparticles designed to be directed against potentially autoreactive T cells are much more promising (140, 191), as addressed by articles from experts in this research topic.

Finally, emerging studies on epigenetic or metabolic mechanisms for Treg cell stability (192–194), and correcting other abnormalities in lupus T cells, such as, metabolic (12, 13), could be potentiated by utilizing the benefits of peptide epitope therapy, in the near future.