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Foxp3+T regulatory cells (Tregs) control autoimmune response by suppressing proliferation and effector functions of self-reactive Foxp3-CD4+/CD8+ T cells and thereby maintain the critical balance between self-tolerance and autoimmunity. Earlier, we had shown that OX40L-JAG1 cosignaling mediated through their cognate receptors OX40 and Notch3 preferentially expressed on murine Tregs can selectively induce their proliferation in the absence of TCR stimulation. However, the differential molecular mechanisms regulating TCR-independent versus TCR-dependent Treg proliferation and lineage stability of the expanded Tregs remained unknown. In this study, we show that OX40L-JAG1 treatment induced TCR-independent proliferation of Tregs in the thymus and periphery. The use of Src kinase inhibitor permitted us to demonstrate selective inhibition of TCR-dependent T cell proliferation with little to no effect on OX40L-JAG1--induced TCR-independent Treg expansion in vitro, which was critically dependent on noncanonical NF-κB signaling. OX40L-JAG1--expanded Tregs showed sustained lineage stability as indicated by stable demethylation marks in Treg signature genes such as Foxp3, Il2ra, Ctla4, Ikzf2, and Ikzf4. Furthermore, OX40L-JAG1 treatment significantly increased CTLA4+ and TIGIT+ Tregs and alleviated experimental autoimmune thyroiditis in mice. Relevance of our findings to humans became apparent when human OX40L and JAG1 induced TCR-independent selective expansion of human Tregs in thymocyte cultures and increased human Tregs in the liver tissue of humanized NSG mice. Our findings suggest that OX40L-JAG1--induced TCR-independent Treg proliferation is a conserved mechanism that can be used to expand lineage-stable Tregs to treat autoimmune diseases. [ABSTRACT FROM AUTHOR]

Virtually all efforts to generate an effective protection against the life-long, recurrent genital infections caused by HSV-2 have failed. Apart from sexual transmission, the virus can also be transmitted from mothers to neonates, and it is a key facilitator of HIV coacquisition. In this article, we uncover a nanoimmunotherapy using specially designed zinc oxide tetrapod nanoparticles (ZOTEN) with engineered oxygen vacancies. We demonstrate that ZOTEN, when used intravaginally as a microbicide, is an effective suppressor of HSV-2 genital infection in female BALB/c mice. The strong HSV-2 trapping ability of ZOTEN significantly reduced the clinical signs of vaginal infection and effectively decreased animal mortality. In parallel, ZOTEN promoted the presentation of bound HSV-2 virions to mucosal APCs, enhancing T cell-mediated and Ab-mediated responses to the infection, and thereby suppressing a reinfection. We also found that ZOTEN exhibits strong adjuvant-like properties, which is highly comparable with alum, a commonly used adjuvant. Overall, to our knowledge, our study provides the very first evidence for the protective efficacy of an intravaginal microbicide/vaccine or microbivac platform against primary and secondary female genital herpes infections. [ABSTRACT FROM AUTHOR]

Alternative NF-κB signaling is crucial for B cell activation and Ig production, and it is mainly regulated by the inhibitor of κ B kinase (IKK) regulatory complex. Dysregulation of alternative NF-κB signaling in B cells could therefore lead to hyperactive B cells and Ig overproduction. In our previous, study we found that deleted in breast cancer 1 (DBC1) is a suppressor of the alternative NF-κB pathway to attenuate B cell activation. In this study, we report that loss of DBC1 results in spontaneous overproduction of Ig in mice after 10 mo of age. Using a double mutant genetic model, we confirm that DBC1 suppresses B cell activation through RelB inhibition. At the molecular level, we show that DBC1 interacts with alternative NF-κB members RelB and p52 through its leucine zipper domain. In addition, phosphorylation of DBC1 at its C terminus by IKKa facilitates its interaction with RelB and IKKa, indicating that DBCl-mediated suppression of alternative NF-κB is regulated by IKKa. Our results define the molecular mechanism of DBC1 inhibition of alternative NF-κB activation in suppressing B cell activation. [ABSTRACT FROM AUTHOR]

CD40 and BAFFR signaling play important roles in B cell proliferation and Ig production. In this study, we found that B cells from mice with deletion of Dbc1 gene (Dbc1-/-) show elevated proliferation, and IgG1 and IgA production upon in vitro CD40 and BAFF, but not BCR and LPS stimulation, indicating that DBC1 inhibits CD40/BAFF-mediated B cell activation in a cell-intrinsic manner. Microarray analysis and chromatin immunoprecipitation experiments reveal that DBC1 inhibits B cell function by selectively suppressing the transcriptional activity of alternative NF-κB members RelB and p52 upon CD40 stimulation. As a result, when immunized with nitrophenylated-keyhole limpet hemocyanin, Dbc1-/- mice produce significantly increased levels of germinal center B cells, plasma cells, and Ag-specific Ig. Finally, loss of DBC1 in mice leads to higher susceptibility to experimental autoimmune myasthenia gravis. Our study identifies DBC1 as a novel regulator of B cell activation by suppressing the alternative NF-κB pathway. [ABSTRACT FROM AUTHOR]

Earlier, we had demonstrated that treatment with low dose of GM-CSF can prevent the development of experimental autoimmune thyroiditis (EAT), experimental autoimmune myasthenia gravis, and type 1 diabetes, and could also reverse ongoing EAT and experimental autoimmune myasthenia gravis. The protective effect was mediated through the induction of tolerogenic CD11C+CD8α- dendritic cells (DCs) and consequent expansion of Foxp3+ regulatory T cells (Tregs). Subsequently, we showed that GM-CSF acted specifically on bone marrow precursors and facilitated their differentiation into tolerogenic dendritic cells (DCs; GM-CSF-induced bone marrow-derived DCs [GM-BMDCs]), which directed Treg expansion in a contact-dependent manner. This novel mechanism of Treg expansion was independent of TCR-mediated signaling but required exogenous IL-2 and cosignaling from DC-bound OX40L. In this study, we observed that OX40L-mediated signaling by GM-BMDCs, although necessary, was not sufficient for Treg expansion and required signaling by Jagged1. Concurrent signaling induced by OX40L and Jagged1 via OX40 and Notch3 receptors expressed on Tregs was essential for the Treg expansion with sustained FoxP3 expression. Adoptive transfer of only OX40L+Jagged1+ BMDCs led to Treg expansion, increased production of IL-4 and IL-10, and suppression of EAT in the recipient mice. These results showed a critical role for OX40L- and Jagged1-induced cosignaling in GM-BMDC-induced Treg expansion. [ABSTRACT FROM AUTHOR]

Although the role of IL-7 and IL-7R has been implicated in the pathogenesis of rheumatoid arthritis (RA), the majority of the studies have focused on the effect of IL-7/IL-7R in T cell development and function. Our novel data, however, document that patients with RA and greater disease activity have higher levels of IL-7, IL-7R, and TNF-α in RA monocytes, suggesting a feedback regulation between IL-7/IL-7R and TNF-α cascades in myeloid cells that is linked to chronic disease progression. Investigations into the involved mechanism showed that IL-7 is a novel and potent chemoattractant that attracts IL-7R+ monocytes through activation of the PI3K/AKT1 and ERK pathways at similar concentrations of IL-7 detected in RA synovial fluid. To determine whether ligation of IL-7 to IL-7R is a potential target for RA treatment and to identify their mechanism of action, collagen-induced arthritis (CIA) was therapeutically treated with anti-IL-7 Ab or IgG control. Anti-IL-7 Ab treatment significantly reduces CIA monocyte recruitment and osteoclast differentiation as well as potent joint monocyte chemoattractants and bone erosion markers, suggesting that both direct and indirect pathways might contribute to the observed effect. We also demonstrate that reduction in joint MIP-2 levels is responsible for suppressed vascularization detected in mice treated with anti-IL-7 Ab compared with the control group. To our knowledge, we show for the first time that expression of IL-7/IL-7R in myeloid cells is strongly correlated with RA disease activity and that ligation of IL-7 to IL-7R contributes to monocyte homing, differentiation of osteoclasts, and vascularization in the CIA effector phase. [ABSTRACT FROM AUTHOR]

Class-switched antinuclear autoantibodies produced by T follicular helper (TFH) cell-dependent germinal center (GC) B cell response play an essential pathogenic role in lupus nephritis (LN). The role of T follicular regulatory (TFR) cells, an effector subset of CD4 + Foxp3 + T regulatory cells (Tregs), which are specialized in suppressing TFH-GC response and Ab production, remains elusive in LN. Contrasting reports have shown increased/reduced circulating TFR cells in human lupus that might not accurately reflect their presence in the GCs of relevant lymphoid organs. In this study, we report a progressive reduction in TFR cells and decreased TFR/TFH ratio despite increased Tregs in the renal lymph nodes of NZBWF1/j mice, which correlated with increased GC-B cells and proteinuria onset. Cotreatment with soluble OX40L and Jagged-1 (JAG1) proteins increased Tregs, TFR cells, and TFR/TFH ratio, with a concomitant reduction in TFH cells, GC B cells, and anti-dsDNA IgG Ab levels, and suppressed LN onset. Mechanistic studies showed attenuated TFH functions and diminished GC events such as somatic hypermutation and isotype class-switching in OX40L-JAG1-treated mice. RNA sequencing studies revealed inhibition of hypoxia-inducible factor 1-α (HIF-1a) and STAT3 signaling in T conventional cells from OX40L-JAG1-treated mice, which are critical for the glycolytic flux and differentiation into TFH cell lineage. Therefore, the increased TFR/TFH ratio seen in OX40L-JAG1-treated mice could involve both impaired differentiation of TFH cells from T conventional cells and expansion of TFR cells. We show a key role for GC-TFR/TFH imbalance in LN pathogenesis and how restoring homeostatic balance can suppress LN., (Copyright © 2022 by The American Association of Immunologists, Inc.)

The levels of expression of alternatively spliced variants of CTLA-4 and insufficient CTLA-4 signaling have been implicated in type 1 diabetes. Hence, we hypothesized that increasing CTLA-4-specific ligand strength on autoantigen-presenting dendritic cells (DCs) can enhance ligation of CTLA-4 on T cells and lead to modulation of autoreactive T cell response. In this study, we show that DC-directed enhanced CTLA-4 engagement upon pancreatic beta cell Ag presentation results in the suppression of autoreactive T cell response in NOD mice. The T cells from prediabetic NOD mice treated with an agonistic anti-CTLA-4 Ab-coated DC (anti-CTLA-4-Ab DC) showed significantly less proliferative response and enhanced IL-10 and TGF-beta1 production upon exposure to beta cell Ags. Furthermore, these mice showed increased frequency of Foxp3+ and IL-10+ T cells, less severe insulitis, and a significant delay in the onset of hyperglycemia compared with mice treated with control Ab-coated DCs. Further analyses showed that diabetogenic T cell function was modulated primarily through the induction of Foxp3 and IL-10 expression upon Ag presentation by anti-CTLA-4-Ab DCs. The induction of Foxp3 and IL-10 expression appeared to be a consequence of increased TGF-beta1 production by T cells activated using anti-CTLA-4-Ab DCs, and this effect could be enhanced by the addition of exogenous IL-2 or TGF-beta1. Collectively, this study demonstrates the potential of a DC-directed CTLA-4 engagement approach not only in treating autoimmunity in type 1 diabetes, but also in altering diabetogenic T cell function ex vivo for therapy.

Although allogeneic bone marrow transplantation has been shown to prevent autoimmune diabetes in heavily irradiated nonobese diabetic (NOD) mice, a similar procedure is not suitable for the treatment of patients with type 1 diabetes because of associated severe side effects. Therefore, we evaluated whether mouse newborn blood (NBB), equivalent to human umbilical cord blood, could be used for diabetes prevention without recipient preconditioning. To test this hypothesis, unconditioned, prediabetic female NOD mice were given a single injection of whole NBB derived from the allogeneic diabetes-resistant mouse strain C57BL/6. Transfusion of allogeneic NBB but not adult blood prevented diabetes incidence in a majority of treated mice for a prolonged period of time. This was accompanied by the release of insulin in response to a challenge with glucose. Invasive cellular infiltration of islets was also substantially reduced in these mice. Although NBB transfusion induced a low level of hematopoietic microchimerism, it did not strictly correlate with amelioration of diabetes. Induction of genes implicated in diabetes, such as Il18, Tnfa, and Inos but not Il4, Il17 or Ifng, was repressed in splenocytes derived from protected mice. Notably, expression of the transcription factor Tbet/Tbx21 but not Gata3 or Rorgt was upregulated in protected mice. These data indicate that allogeneic NBB transfusion can prevent diabetes in NOD mice associated with modulation of selected cytokine genes implicated in diabetes manifestation. The data presented in this study provide the proof of principle for the utility of allogeneic umbilical cord blood transfusion to treat patients with autoimmune diabetes.

Costimulatory ligands CD80 and CD86 have different binding preferences and affinities to their receptors, CD28 and CTLA-4. Earlier, we demonstrated that CD80 binds to CTLA-4 with higher affinity and has a role in suppressing T cell response. The current study demonstrates that not only did blockade of CD86 upon Ag presentation by bone marrow-derived dendritic cells (DC) to OVA-specific T cells result in induction of hyporesponsive T cells but also that these T cells could suppress the proliferative response of effector T cells. These T cells showed TGF-beta1 on their surface and secreted TGF-beta1 and IL-10 upon restimulation. Although blockade of CTLA-4 and neutralization of IL-10 profoundly inhibited the induction of these TGF-beta1(+) T cells, their ability to suppress the effector T cell proliferation was abrogated by neutralization of TGF-beta1 alone. Induction of TGF-beta1(+) and IL-10(+) T cells was found to be independent of natural CD4(+)CD25(+) regulatory T cells, demonstrating that preferential ligation of CTLA-4 by CD80 induced IL-10 production by effector T cells, which in turn promoted the secretion of TGF-beta1. Treatment of prediabetic NOD mice with islet beta cell Ag-pulsed CD86(-/-) DCs, but not CD80(-/-) DCs, resulted in the induction of TGF-beta1- and IL-10-producing cells, significant suppression of insulitis, and delay of the onset of hyperglycemia. These observations demonstrate not only that CD80 preferentially binds to CTLA-4 but also that interaction during Ag presentation can result in IL-10-dependent TGF-beta1(+) regulatory T cell induction, reinstating the potential of approaches to preferentially engage CTLA-4 through CD80 during self-Ag presentation in suppressing autoimmunity.

CTLA-4 is a critical negative regulator of T cell response and is instrumental in maintaining immunological tolerance. In this article, we report that enhanced selective engagement of CTLA-4 on T cells by Ag-presenting dendritic cells resulted in the induction of Ag-specific CD4(+)CD25(+)Foxp3(+) and CD4(+)CD25(-)TGF-beta1(+) adaptive Tregs. These cells were CD62L(low) and hyporesponsive to stimulation with cognate Ag but demonstrated a superior ability to suppress Ag-specific effector T cell response compared with their CD62L(high) counterparts. Importantly, treatment of mice with autoimmune thyroiditis using mouse thyroglobulin (mTg)-pulsed anti-CTLA-4 agonistic Ab-coated DCs, which results in a dominant engagement of CTLA-4 upon self-Ag presentation, not only suppressed thyroiditis but also prevented reemergence of the disease upon rechallenge with mTg. Further, the disease suppression was associated with significantly reduced mTg-specific T cell and Ab responses. Collectively, our results showed an important role for selective CTLA-4 signaling in the induction of adaptive Tregs and suggested that approaches that allow dominant CTLA-4 engagement concomitant with Ag-specific TCR ligation can be used for targeted therapy.

Dendritic cells (DCs) have the potential to activate or tolerize T cells in an Ag-specific manner. Although the precise mechanism that determines whether DCs exhibit tolerogenic or immunogenic functions has not been precisely elucidated, growing evidence suggests that DC function is largely dependent on differentiation status, which can be manipulated using various growth factors. In this study, we investigated the effects of mobilization of specific DC subsets-using GM-CSF and fms-like tyrosine kinase receptor 3-ligand (Flt3-L)-on the susceptibility to induction of experimental autoimmune myasthenia gravis (EAMG). We administered GM-CSF or Flt3-L to C57BL/6 mice before immunization with acetylcholine receptor (AChR) and observed the effect on the frequency and severity of EAMG development. Compared with AChR-immunized controls, mice treated with Flt3-L before immunization developed EAMG at an accelerated pace initially, but disease frequency and severity was comparable at the end of the observation period. In contrast, GM-CSF administered before immunization exerted a sustained suppressive effect against the induction of EAMG. This suppression was associated with lowered serum autoantibody levels, reduced T cell proliferative responses to AChR, and an expansion in the population of FoxP3+ regulatory T cells. These results highlight the potential of manipulating DCs to expand regulatory T cells for the control of autoimmune diseases such as MG.

Our earlier study showed that GM-CSF has the potential not only to prevent, but also to suppress, experimental autoimmune thyroiditis (EAT). GM-CSF-induced EAT suppression in mice was accompanied by an increase in the frequency of CD4(+)CD25(+) regulatory T cells that could suppress mouse thyroglobulin (mTg)-specific T cell responses in vitro, but the underlying mechanism of this suppression was not elucidated. In this study we show that GM-CSF can induce dendritic cells (DCs) with a semimature phenotype, an important characteristic of DCs, which are known to play a critical role in the induction and maintenance of regulatory T cells. Adoptive transfer of CD4(+)CD25(+) T cells from GM-CSF-treated and mTg-primed donors into untreated, but mTg-primed, recipients resulted in decreased mTg-specific T cell responses. Furthermore, lymphocytes obtained from these donors and recipients after adoptive transfer produced significantly higher levels of IL-10 compared with mTg-primed, untreated, control mice. Administration of anti-IL-10R Ab into GM-CSF-treated mice abrogated GM-CSF-induced suppression of EAT, as indicated by increased mTg-specific T cell responses, thyroid lymphocyte infiltration, and follicular destruction. Interestingly, in vivo blockade of IL-10R did not affect GM-CSF-induced expansion of CD4(+)CD25(+) T cells. However, IL-10-induced immunosuppression was due to its direct effects on mTg-specific effector T cells. Taken together, these results indicated that IL-10, produced by CD4(+)CD25(+) T cells that were probably induced by semimature DCs, is essential for disease suppression in GM-CSF-treated mice.

CTLA-4 (CD152) is actively involved in down-regulating T cell activation and maintaining lymphocyte homeostasis. Our earlier studies showed that targeted engagement of CTLA-4 can down-modulate T cell response and suppress allo- and autoimmune responses. In this study, we report that targeted CTLA-4 engagement can induce immune tolerance to a specific target through selective induction of an Ag-specific CD4(+)CD25(+)CTLA-4(high) regulatory T cell (Treg cell) population. Allogeneic cells coated with anti-CTLA-4 Ab induced immune hyporesponsiveness through suppression of proinflammatory cytokines IFN-gamma and IL-2, and up-regulation of the regulatory cytokines IL-10, TGF-beta1, and IL-4, presumably through the engagement of CTLA-4 on activated T cells. Although rechallenge with alloantigen failed to break the unresponsiveness, a transient recovery from tolerance was observed in the presence of high concentrations of exogenous IL-2, saturating concentrations of neutralizing anti-TGF-beta1 and anti-IL-10 Abs, and blocking anti-CTLA-4 Ab, and upon depletion of CD4(+)CD25(+) Treg cells. The CD4(+)CD25(+)CTLA-4(high) Treg cells from tolerant mice suppressed the effector function of CD25(-) T cells from Ag-primed mice. Adoptive transfer of these Treg cells into Ag-primed mice resulted in a significantly reduced alloantigen-specific response. Further characterization demonstrated that the Treg cells with memory phenotype (CD62L(-)) were more potent in suppressing the alloantigen-specific T cell response. These results strongly support that the targeted engagement of CTLA-4 has therapeutic potential for the prevention of transplant rejection.

Fms-like tyrosine kinase receptor 3-ligand (Flt3-L) and GM-CSF cause expansion of different subsets of dendritic cells and skew the immune response toward predominantly Th1 and Th2 type, respectively. In the present study, we investigated their effects on experimental autoimmune thyroiditis in CBA/J mice. Relative to mouse thyroglobulin (mTg) immunized controls, mTg-immunized mice treated with Flt3-L showed more severe thyroiditis characterized by enhanced lymphocytic infiltration of the thyroid, and IFN-gamma and IL-2 production. In contrast, mice treated with GM-CSF, either before or after immunization with mTg, showed suppressed T cell response to mTg and failed to develop thyroiditis. Lymphocytes from these mice, upon activation with mTg in vitro, produced higher levels of IL-4 and IL-10. Additionally, GM-CSF-treated mice showed an increase in the frequency of CD4(+)/CD25(+) T cells, which suppressed the mTg-specific T cell response. Neutralization of IL-10, but not IL-4, or depletion of CD4(+)/CD25(+) cells resulted in increased mTg-specific in vitro T cell proliferation suggesting that IL-10 produced by the Ag-specific CD4(+)/CD25(+) regulatory T cells might be critical for disease suppression. These results indicate that skewing immune response toward Th2, through selective activation of dendritic cells using GM-CSF, may have therapeutic potential in Th1 dominant autoimmune diseases including Hashimoto's thyroiditis.

In autoimmune Graves' disease (GD), autoantibodies bind to the thyrotropin receptor (TSHR) and cause hyperthyroidism. We studied the effects of fms-like tyrosine kinase receptor 3 ligand (Flt3-L) or GM-CSF treatment on the development of experimental autoimmune GD (EAGD) in mice, a slowly progressing Ab-mediated organ-specific autoimmune disease of the thyroid induced by immunization with syngeneic cells expressing TSHR. Flt3-L and GM-CSF treatment resulted in up-regulation of CD8a(+) and CD8a(-) dendritic cells, and skewing of cytokine and immune responses to TSHR in favor of Th1 and Th2, respectively. However, this skewing did not persist until the later stages, and thus failed to affect the course or severity of the disease. To determine whether the total absence of either IL-4 or IFN-gamma could affect the development of EAGD, we immunized wild-type, IFN-gamma(-/-) and IL-4(-/-) BALB/c mice with TSHR. Nearly 100% of the wild-type and IFN-gamma(-/-) mice developed EAGD with optimal TSHR-specific immune responses, while IL-4(-/-) mice completely resisted disease and showed delayed and suboptimal pathogenic Ab response. These data demonstrated that skewing immune responses to TSHR, using either Flt3-L or GM-CSF, in favor of Th1 or Th2, respectively, may not be sufficient to alter the course of the disease, while the complete absence of IL-4, but not IFN-gamma, can prevent the development of EAGD.