Harnessing Tolerogenic Histone Peptide Epitopes From Nucleosomes for Selective Down-Regulation of Pathogenic Autoimmune Response in Lupus (Past, Present, and Future).

Autoantigen-directed tolerance can be induced by certain nucleosomal histone peptide epitope/s in nanomolar dosage leading to sustained remission of disease in mice with SLE. By contrast, lupus is by administration of intact (whole) histones, or whole nucleosomes in microparticles from apoptotic cells, or by post-translationally acetylated histone-peptides. Low-dose therapy with the histone-peptide epitopes simultaneously induces TGFβ and inhibits IL-6 production by DC , especially pDC, which then induce CD4+CD25+ Treg and CD8+ Treg cells that suppress pathogenic autoimmune response.

Five year follow-up after autologous peripheral blood hematopoietic stem cell transplantation for refractory, chronic, corticosteroid-dependent systemic lupus erythematosus: effect of conditioning regimen on outcome.

Some patients with systemic lupus erythematosus (SLE) are refractory to traditional therapies, dependent on chronic corticosteroids, have organ damage, and are at high risk of mortality. In this group of patients, we report outcome at a median of five years after autologous hematopoietic stem cell transplant (HSCT) using two different non-myeloablative regimens. Four patients received a conditioning regimen of cyclophosphamide (200 mg/kg) and alemtuzumab (60 mg), while 26 patients underwent conditioning with cyclophosphamide (200 mg/kg), rATG (Thymoglobulin) (5.

Hematopoietic stem and multipotent progenitor cells produce IL-17, IL-21 and other cytokines in response to TLR signals associated with late apoptotic products and augment memory Th17 and Tc17 cells in the bone marrow of normal and lupus mice.

We studied effects of early and late apoptotic (necroptotic) cell products, related damage associated alarmins and TLR agonists, on hematopoietic stem and progenitor cells (HSPC). Surprisingly, normal HSPC themselves produced IL-17 and IL-21 after 1½days of stimulation, and the best stimulators were TLR 7/8 agonist; HMGB1-DNA; TLR 9 agonist, and necroptotic B cells. The stimulated HSPC expressed additional cytokines/mediators, directly causing rapid expansion of IL-17(+) memory CD4 T (Th17), and CD8 T (Tc17) cells, and antigen-experienced IL-17(+) T cells with "naïve" phenotype.

CD11c-mediated deletion of Flip promotes autoreactivity and inflammatory arthritis.

Dendritic cells (DCs) are critical for immune homeostasis. To target DCs, we generated a mouse line with Flip deficiency in cells that express cre under the CD11c promoter (CD11c-Flip-KO). CD11c-Flip-KO mice spontaneously develop erosive, inflammatory arthritis, resembling rheumatoid arthritis, which is dramatically reduced when these mice are crossed with Rag(-/-) mice.

Human megakaryocyte progenitors derived from hematopoietic stem cells of normal individuals are MHC class II-expressing professional APC that enhance Th17 and Th1/Th17 responses.

Platelets, like stromal cells, present antigen only via MHC class I, but the immune potential of their progenitors has not been explored in humans. We derived CD34(+)CD117(+)CD41(+)CD151(+) megakaryocyte progenitors (MKp) in vitro from mobilized peripheral blood hematopoietic stem and progenitor cells (HSPC) of normal subjects using culture conditions akin to bone marrow niche, or organs that support extramedullary hematopoiesis. The MKp expressed MHC Class II in contrast to platelets and functioned as professional APC before they matured further.

Major pathogenic steps in human lupus can be effectively suppressed by nucleosomal histone peptide epitope-induced regulatory immunity.

Low-dose tolerance therapy with nucleosomal histone peptide epitopes blocks lupus disease in mouse models, but effect in humans is unknown. Herein, we found that CD4(+)CD25(high)FoxP3(+) or CD4(+)CD45RA(+)FoxP3(low) T-cells, and CD8(+)CD25(+)FoxP3(+) T-cells were all induced durably in PBMCs from inactive lupus patients and healthy subjects by the histone peptide/s themselves, but in active lupus, dexamethasone or hydroxychloroquine unmasked Treg-induction by the peptides. The peptide-induced Treg depended on TGFβ/ALK-5/pSmad 2/3 signaling, and they expressed TGF-β precursor LAP.

Megakaryocyte progenitors are the main APCs inducing Th17 response to lupus autoantigens and foreign antigens.

In search of autoantigen-presenting cells that prime the pathogenic autoantibody-inducing Th cells of lupus, we found that CD41(+)CD151(+) cells among Lineage(-) (Lin(-)) CD117(+) (c-Kit(+)) CX3CR1(-) splenocytes depleted of known APCs were most proficient in presenting nuclear autoantigens from apoptotic cells to induce selectively an autoimmune Th17 response in different lupus-prone mouse strains. The new APCs have properties resembling megakaryocyte and/or bipotent megakaryocyte/erythroid progenitors of bone marrow, hence they are referred to as MM cells in this study. The MM cells produce requisite cytokines, but they require contact for optimal Th17 induction upon nucleosome feeding, and can induce Th17 only before undergoing differentiation to become c-Kit(-)CD41(+) cells.

The histone peptide H4 71-94 alone is more effective than a cocktail of peptide epitopes in controlling lupus: immunoregulatory mechanisms.

Tolerance therapy with nucleosomal histone peptides H4(71-94), H4(16-39), or H1'(22-42) controls disease in lupus-prone SNF1 mice. It would be clinically important to determine whether a cocktail of the above epitopes would be superior. Herein, we found that compared with cocktail peptides, H4(71-94) monotherapy more effectively delayed nephritis onset, prolonged lifespan, diminished immunoglobulin G autoantibody levels, reduced autoantigen-specific Th1 and Th17 responses and frequency of T(FH) cells in spleen and the helper ability of autoimmune T cells to B cells, by inducing potent CD8 Treg cells.

Regulatory T cell (Treg) subsets return in patients with refractory lupus following stem cell transplantation, and TGF-beta-producing CD8+ Treg cells are associated with immunological remission of lupus.

Compared with conventional drug therapy, autologous hemopoietic stem cell transplantation (HSCT) can induce very-long-term remission in refractory lupus patients. Herein, we show that in posttransplant patients, both CD4(+)CD25(high)FoxP3(+) and an unusual CD8(+)FoxP3(+) Treg subset return to levels seen in normal subjects; accompanied by almost complete inhibition of pathogenic T cell response to critical peptide autoepitopes from histones in nucleosomes, the major lupus autoantigen from apoptotic cells. In addition to a stably sustained elevation of FoxP3, posttransplant CD8 T cells also maintained markedly higher expression levels of latency-associated peptide (LAP), CD103, PD-1, PD-L1, and CTLA-4, as compared with pretransplant CD8 T cells that were identically treated by a one-time activation and rest in short-term culture.

Apigenin, a non-mutagenic dietary flavonoid, suppresses lupus by inhibiting autoantigen presentation for expansion of autoreactive Th1 and Th17 cells.

Introduction: Lupus patients need alternatives to steroids and cytotoxic drugs. We recently found that apigenin, a non-mutagenic dietary flavonoid, can sensitize recurrently activated, normal human T cells to apoptosis by inhibiting nuclear factor-kappa-B (NF-kappaB)-regulated Bcl-xL, cyclooxygenase 2 (COX-2), and cellular FLICE-like inhibitory protein (c-FLIP) expression. Because sustained immune activation and hyperexpression of COX-2 and c-FLIP contribute to lupus, we treated SNF1 mice that spontaneously develop human lupus-like disease with apigenin.

Apigenin, a dietary flavonoid, sensitizes human T cells for activation-induced cell death by inhibiting PKB/Akt and NF-kappaB activation pathway.

Resistance of T cells to activation-induced cell death (AICD) is associated with autoimmunity and lymphoproliferation. We found that apigenin (4',5,7-trihydroxyflavone), a non-mutagenic dietary flavonoid, augmented both extrinsic and intrinsic pathways of apoptosis in recurrently activated, but not in primarily stimulated, human blood CD4+ T cells. Apigenin potentiated AICD by inhibiting NF-kappaB activation and suppressing NF-kappaB-regulated anti-apoptotic molecules, cFLIP, Bcl-x(L), Mcl-1, XIAP and IAP, but not Bcl-2.

Hyperexpression of cyclooxygenase 2 in the lupus immune system and effect of cyclooxygenase 2 inhibitor diet therapy in a murine model of systemic lupus erythematosus.

Objective: To investigate the role of cyclooxygenase 2 (COX-2) in the functioning of different cell types involved in the lupus autoimmune response, and to examine the therapeutic effect of COX-2 inhibitors in mice prone to spontaneously develop systemic lupus erythematosus (SLE).

Methods: Lupus-prone (SWR x NZB)F(1) mice were fed with a diet containing different doses of the COX-2-specific inhibitor celecoxib or the nonspecific inhibitor aspirin, or a combination of both, and the effects of the therapy on autoantibody production, development of lupus nephritis, and mortality were determined. Expression of COX-2 by different cells of the lupus immune system and the effect of COX-2 inhibitors on the function of these cells in vitro and in vivo were assessed.

Low-dose peptide tolerance therapy of lupus generates plasmacytoid dendritic cells that cause expansion of autoantigen-specific regulatory T cells and contraction of inflammatory Th17 cells.

Subnanomolar doses of an unaltered, naturally occurring nucleosomal histone peptide epitope, H4(71-94), when injected s.c. into lupus-prone mice, markedly prolong lifespan by generating CD4+25+ and CD8+ regulatory T cells (Treg) producing TGF-beta.

Regulatory T cells in lupus.

Naturally occurring, CD4+ CD25+ regulatory T cells that are exported from the thymus early in life play an important role in controlling organ-specific autoimmune diseases, but they may not be critical for suppressing systemic autoimmunity in lupus. On the other hand, lupus-prone subjects appear to be deficient in generation of adaptive T-regulatory cells that can be induced by various means. We review autoantigen-specific therapeutic approaches that induce such regulatory T cells.

A defect in deletion of nucleosome-specific autoimmune T cells in lupus-prone thymus: role of thymic dendritic cells.

To study central tolerance to the major product of ongoing apoptosis in the thymus, we made new lines of transgenic (Tg) mice expressing TCR of a pathogenic autoantibody-inducing Th cell that was specific for nucleosomes and its histone peptide H4(71-94). In the lupus-prone (SWR x NZB)F1 (SNF1) thymus, introduction of the lupus TCR transgene caused no deletion, but marked down-regulation of the Tg TCR and up-regulation of endogenous TCRs. Paradoxically, autoimmune disease was suppressed in the alphabetaTCR Tg SNF1 mice with induction of highly potent regulatory T cells in the periphery.

Very low-dose tolerance with nucleosomal peptides controls lupus and induces potent regulatory T cell subsets.

We induced very low-dose tolerance by injecting lupus prone (SWR x NZB)F1 (SNF1) mice with 1 mug nucleosomal histone peptide autoepitopes s.c. every 2 wk.

Genetic origin of lupus in NZB/SWR hybrids: lessons from an intercross study.

Objective: (SWR x NZB)F(1) (or SNF(1)) hybrid mice succumb to lupus nephritis. A previous analysis of SNF(1) x NZB backcross mice revealed the existence of 4 SWR loci (H2 on chromosome 17, Swrl-1 on chromosome 1, Swrl-2 on chromosome 14, and Swrl-3 on chromosome 18) and 2 NZB loci (Nba1 and Lbw2/Sbw2, both on chromosome 4). A second study focusing on SNF(1) x SWR backcross offspring uncovered 5 suggestive loci for antinuclear antibody formation, consisting of 3 dominant NZB contributions (Nba4 on chromosome 5, Lbw4 on chromosome 6, and Nba5 on chromosome 7) and 2 recessive SWR contributions (Swrl-1 on chromosome 1 and Swrl-4 on chromosome 10).

T-helper cell intrinsic defects in lupus that break peripheral tolerance to nuclear autoantigens.

Special populations of T helper cells drive B cells to produce IgG class switched, pathogenic autoantibodies in lupus. The major source of antigenic determinants (epitopes) that trigger interactions between lupus T and B cells is nucleosomes of apoptotic cells. These epitopes can be used for antigen-specific therapy of lupus.

Human lupus T cells resist inactivation and escape death by upregulating COX-2.

Autoimmune T-helper cells drive pathogenic autoantibody production in systemic lupus erythematosus (SLE), but the mechanisms maintaining those T cells are unknown. Autoreactive T cells are normally eliminated by functional inactivation (anergy) and activation-induced cell death (AICD) or apoptosis through death receptor (Fas) signaling. However, mutations in the genes encoding Fas and its ligand (FasL) are rare in classical SLE.

Major peptide autoepitopes for nucleosome-centered T and B cell interaction in human and murine lupus.

The potential cross-reactivity of normal T and B cells to nuclear antigens is vast, probably due to their "education" by apoptotic cell antigens in generative lymphoid organs. Despite this "nucleocentric repertoire," as we call it, the peripheral immune system normally remains tolerant or ignorant of the products of apoptosis. However, the T helper (Th) cells, and also B cells of lupus, have a regulatory defect in the expression of CD40 ligand (CD40L).

B cell stimulatory effects of alpha-enolase that is differentially expressed in NZB mouse B cells.

Intrinsic polyclonal B cell activation is characteristic of NZB mice and it contributes to the development of lupus nephritis in NZB crosses. Although multiple autosomal genes appear to be involved, the major loci for B cell hyperactivity have been mapped on chromosome 4. To identify various genes determining B cell hyperactivity, differential mRNA display was done comparing B cells of NZB and BALB/c mice.

Naturally processed chromatin peptides reveal a major autoepitope that primes pathogenic T and B cells of lupus.

Major autoepitopes for pathogenic Th cells of lupus were previously found in core histones of nucleosomes by testing overlapping synthetic peptides. To detect other dominant epitopes, we eluted peptides from MHC class II molecules of a murine lupus APC line that was fed with crude chromatin. The eluted peptides were purified by reverse-phase HPLC and tested for their ability to stimulate autoimmune Th clones, and then analyzed by mass spectrometry.