Palash Bhattacharya

GM-CSF-induced, bone-marrow-derived dendritic cells can expand natural Tregs and induce adaptive Tregs by different mechanisms

In our earlier work, we had shown that GM‐CSF treatment of CBA/J mice can suppress ongoing thyroiditis by inducing tolerogenic CD8α− DCs, which helped expand and/or induce CD4+Foxp3+ Tregs. To identify the primary cell type that was affected by the GM‐CSF treatment and understand the mechanism by which Tregs were induced, we compared the effect of GM‐CSF on matured spDCs and BMDC precursors in vitro. Matured spDCs exposed to GM‐CSF ex vivo induced only a modest increase in the percentage of Foxp3‐expressing T cells in cocultures. In contrast, BM cells, when cultured in the presence of GM‐CSF, gave rise to a population of CD11c+CD11bHiCD8α− DCs (BMDCs), which were able to expand Foxp3+ Tregs upon coculture with CD4+ T cells. This contact‐dependent expansion occurred in the absence of TCR stimulation and was abrogated by OX40L blockage. Additionally, the BMDCs secreted high levels of TGF‐β, which was required and sufficient for adaptive differentiation of T cells to Foxp3+ Tregs, only upon TCR stimulation. These results strongly suggest that the BMDCs differentiated by GM‐CSF can expand nTregs and induce adaptive Tregs through different mechanisms.

GM-CSF: An immune modulatory cytokine that can suppress autoimmunity.

GM-CSF was originally identified as a colony stimulating factor (CSF) because of its ability to induce granulocyte and macrophage populations from precursor cells. Multiple studies have demonstrated that GM-CSF is also an immune-modulatory cytokine, capable of affecting not only the phenotype of myeloid lineage cells, but also T-cell activation through various myeloid intermediaries. This property has been implicated in the sustenance of several autoimmune diseases like arthritis and multiple sclerosis. In contrast, several studies using animal models have shown that GM-CSF is also capable of suppressing many autoimmune diseases such as Crohns disease, Type-1 diabetes, Myasthenia gravis and experimental autoimmune thyroiditis. Knockout mouse studies have suggested that the role of GM-CSF in maintaining granulocyte and macrophage populations in the physiological steady state is largely redundant. Instead, its immune-modulatory role plays a significant role in the development or resolution of autoimmune diseases. This is mediated either through the differentiation of precursor cells into specialized non-steady state granulocytes, macrophages and dendritic cells, or through the modulation of the phenotype of mature myeloid cells. Thus, outside of myelopoiesis, GM-CSF has a profound role in regulating the immune response and maintaining immunological tolerance.

Intravaginal Zinc Oxide Tetrapod Nanoparticles as Novel Immunoprotective Agents against Genital Herpes

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.

OX40L/Jagged1 Cosignaling by GM-CSF–Induced Bone Marrow-Derived Dendritic Cells Is Required for the Expansion of Functional Regulatory T Cells

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.

Nanodisc-Incorporated Hemagglutinin Provides Protective Immunity against Influenza Virus Infection

ABSTRACT Every year, influenza virus infection causes significant mortality and morbidity in human populations. Although egg-based inactivated viral vaccines are available, their effectiveness depends on the correct prediction of the circulating viral strains and is limited by the time constraint of the manufacturing process. Recombinant subunit vaccines are easier to manufacture with a relatively short lead time but are limited in their efficacy partly because the purified recombinant membrane proteins in the soluble form most likely do not retain their native membrane-bound structure. Nanodisc (ND) particles are soluble, stable, and reproducibly prepared discoid shaped nanoscale structures that contain a discrete lipid bilayer bound by two amphipathic scaffold proteins. Because ND particles permit the functional reconstitution of membrane/envelope proteins, we incorporated recombinant hemagglutinin (HA) from influenza virus strain A/New Caledonia/20/99 (H1N1) into NDs and investigated their potential to elicit an immune response to HA and confer immunity to influenza virus challenge relative to the commercial vaccines Fluzone and FluMist. HA-ND vaccination induced a robust anti-HA antibody response consisting of predominantly the immunoglobulin G1 (IgG1) subclass and a high hemagglutination inhibition titer. Intranasal immunization with HA-ND induced an anti-HA IgA response in nasal passages. HA-ND vaccination conferred protection that was comparable to that of Fluzone and FluMist against challenge with influenza virus strain A/Puerto Rico/8/1934 (H1N1).

Inhibition of serine palmitoyltransferase delays the onset of radiation-induced pulmonary fibrosis through the negative regulation of sphingosine kinase-1 expression

The enforcement of sphingosine-1-phosphate (S1P) signaling network protects from radiation-induced pneumonitis. We now demonstrate that, in contrast to early postirradiation period, late postirradiation sphingosine kinase-1 (SphK1) and sphingoid base-1-phosphates are associated with radiation-induced pulmonary fibrosis (RIF). Using the mouse model, we demonstrate that RIF is characterized by a marked upregulation of S1P and dihydrosphingosine-1-phosphate (DHS1P) levels in the lung tissue and in circulation accompanied by increased lung SphK1 expression and activity. Inhibition of sphingolipid de novo biosynthesis by targeting serine palmitoyltransferase (SPT) with myriocin reduced radiation-induced pulmonary inflammation and delayed the onset of RIF as evidenced by increased animal lifespan and decreased expression of markers of fibrogenesis, such as collagen and α-smooth muscle actin (α-SMA), in the lung. Long-term inhibition of SPT also decreased radiation-induced SphK activity in the lung and the levels of S1P-DHS1P in the lung tissue and in circulation. In vitro, inhibition or silencing of serine palmitoyltransferase attenuated transforming growth factor-β1 (TGF-β)-induced upregulation of α-SMA through the negative regulation of SphK1 expression in normal human lung fibroblasts. These data demonstrate a novel role for SPT in regulating TGF-β signaling and fibrogenesis that is linked to the regulation of SphK1 expression and S1P-DHS1P formation.

Age-dependent divergent effects of OX40L treatment on the development of diabetes in NOD mice

Abstract Earlier, we have shown that GM-CSF derived bone marrow (BM) dendritic cells (G-BMDCs) can expand Foxp3+ regulatory T-cells (Tregs) through a TCR-independent, but IL-2 dependent mechanism that required OX40L/OX40 interaction. While some reports have shown suppression of autoimmunity upon treatment with an OX40 agonist, others have shown exacerbation of autoimmune disease instead. To better understand the basis for these differing outcomes, we compared the effects of OX40L treatment in 6-week-old pre-diabetic and 12-week-old near diabetic NOD mice. Upon treatment with OX40L, 6-week-old NOD mice remained normoglycemic and showed a significant increase in Tregs in their spleen and lymph nodes, while 12-week-old NOD mice very rapidly developed hyperglycemia and failed to show Treg increase in spleen or LN. Interestingly, OX40L treatment increased Tregs in the thymus of both age groups. However, it induced Foxp3+CD103+CD38− stable-phenotype Tregs in the thymus and reduced the frequency of autoreactive Teff cells in 6-week-old mice; while it induced Foxp3+CD103−CD38+ labile-phenotype Tregs in the thymus and increased autoreactive CD4+ T cells in the periphery of 12-week-old mice. This increase in autoreactive CD4+ T cells was likely due to either a poor suppressive function or conversion of labile Tregs into Teff cells. Using ex vivo cultures, we found that the reduction in Treg numbers in 12-week-old mice was likely due to IL-2 deficit, and their numbers could be increased upon addition of exogenous IL-2. The observed divergent effects of OX40L treatment were likely due to differences in the ability of 6- and 12-week-old NOD mice to produce IL-2.

Identification of murine B cell lines that undergo somatic hypermutation focused to A:T and G:C residues

Activation‐induced deaminase (AID) is the master regulator of class switch recombination (CSR) and somatic hypermutation (SHM), but the mechanisms regulating AID function are obscure. The differential pattern of switch plasmid activity in three IgM+/AID+ and two IgG+/AID+ B cell lines prompted an analysis of global gene expression to discover the origin of these cells. Gene profiling suggested that the IgG+/AID+ B cell lines derived from germinal center B cells. Analysis of SHM potential demonstrates that the IgVκ domains are inducibly diversified at high rate during in vitro culture. The mutation spectra focused to A:T base pairs, revealing a component of the hypermutation program that occurs preferentially during phase 2 of SHM. The A:T error spectra were analyzed and were not characteristic of polymerase η activity. A differential pattern of three consensus motifs used for A:T base substitutions was observed in WT and Polη‐, Msh2‐ and Msh6‐deficient B cells. Strikingly, mutations in our B cell lines recapitulated the mutable motif profile for Polη and Msh2 deficiency, respectively, and suggest that an additional pathway for the generation of A:T mutations in SHM is conserved in mouse and human.

IL-1β Promotes TGF-β1 and IL-2 Dependent Foxp3 Expression in Regulatory T Cells

Earlier, we have shown that GM-CSF-exposed CD8α− DCs that express low levels of pro-inflammatory cytokines IL-12 and IL-1β can induce Foxp3+ Tregs leading to suppression of autoimmunity. Here, we examined the differential effects of IL-12 and IL-1β on Foxp3 expression in T cells when activated in the presence and absence of DCs. Exogenous IL-12 abolished, but IL-1β enhanced, the ability of GM-CSF-exposed tolerogenic DCs to promote Foxp3 expression. Pre-exposure of DCs to IL-1β and IL-12 had only a modest effect on Foxp3− expressing T cells; however, T cells activated in the absence of DCs but in the presence of IL-1β or IL-12 showed highly significant increase and decrease in Foxp3+ T cell frequencies respectively suggesting direct effects of these cytokines on T cells and a role for IL-1β in promoting Foxp3 expression. Importantly, purified CD4+CD25+ cells showed a significantly higher ability to maintain Foxp3 expression when activated in the presence of IL-1β. Further analyses showed that the ability of IL-1β to maintain Foxp3 expression in CD25+ T cells was dependent on TGF-β1 and IL-2 expression in Foxp3+Tregs and CD25− effectors T cells respectively. Exposure of CD4+CD25+ T cells to IL-1β enhanced their ability to suppress effector T cell response in vitro and ongoing experimental autoimmune thyroidits in vivo. These results show that IL-1β can help enhance/maintain Tregs, which may play an important role in maintaining peripheral tolerance during inflammation to prevent and/or suppress autoimmunity.

Switch Region Identity Plays an Important Role in Ig Class Switch Recombination

Ig class switch recombination (CSR) is regulated through long-range intrachromosomal interactions between germline transcript promoters and enhancers to initiate transcription and create chromatin accessible to activation-induced deaminase attack. CSR occurs between switch (S) regions that flank Cμ and downstream CH regions and functions via an intrachromosomal deletional event between the donor Sμ region and a downstream S region. It is unclear to what extent S region primary sequence influences differential targeting of CSR to specific isotypes. We address this issue in this study by generating mutant mice in which the endogenous Sγ3 region was replaced with size-matched Sγ1 sequence. B cell activation conditions are established that support robust γ3 and γ1 germline transcript expression and stimulate IgG1 switching but suppress IgG3 CSR. We found that the Sγ1 replacement allele engages in μ→γ3 CSR, whereas the intact allele is repressed. We conclude that S region identity makes a significant contribution to CSR. We propose that the Sγ1 region is selectively targeted for CSR following the induction of an isotype-specific factor that targets the S region and recruits CSR machinery.