ATP-dependent transporters: emerging players at the crossroads of immunity and metabolism

Nearly 50 ATP-binding cassette (ABC) transporters are encoded by mammalian genomes. These transporters are characterized by conserved nucleotide-binding and hydrolysis (i.e., ATPase) domains, and power directional transport of diverse substrate classes – ions, small molecule metabolites, xenobiotics, hydrophobic drugs, and even polypeptides – into or out of cells or subcellular organelles. Although immunological functions of ABC transporters are only beginning to be unraveled, emerging literature suggests these proteins have under-appreciated roles in the development and function of T lymphocytes, including many of the key effector, memory and regulatory subsets that arise during responses to infection, inflammation or cancers. One transporter in particular, MDR1 (Multidrug resistance-1; encoded by the ABCB1 locus in humans), has taken center stage as a novel player in immune regulation. Although MDR1 remains widely viewed as a simple drug efflux pump in tumor cells, recent evidence suggests that this transporter fills key endogenous roles in enforcing metabolic fitness of activated CD4 and CD8 T cells. Here, we summarize current understanding of the physiological functions of ABC transporters in immune regulation, with a focus on the anti-oxidant functions of MDR1 that may shape both the magnitude and repertoires of antigen-specific effector and memory T cell compartments. While much remains to be learned about the functions of ABC transporters in immunobiology, it is already clear that they represent fertile new ground, both for the definition of novel immunometabolic pathways, and for the discovery of new drug targets that could be leveraged to optimize immune responses to vaccines and cancer immunotherapies

Domain Requirements and Sequence Specificity of DNA Binding for the Forkhead Transcription Factor FOXP3

The forkhead, winged-helix transcription factor FOXP3 is preferentially expressed in T regulatory (Treg) cells and is critical for their immunosuppressive function. Mutations that abolish FOXP3 function lead to systemic autoimmunity in mice and humans. However, the manner by which FOXP3 recognizes cognate DNA elements is unclear. Here we identify an in vitro optimized DNA sequence to assess FOXP3 DNA binding by electrophoretic mobility shift assay (EMSA). The optimized sequence contains two tandem copies of a core DNA element resembling, but not identical to, the canonical forkhead (FKH) binding element. The tandem nature of this optimized FOXP3-binding oligonucleotide suggests a requirement for multimerization, and EMSA experiments confirm that both the DNA-binding FKH domain and an intact leucine-zipper domain, which mediates homo-multimerization of FOXP3, are required for DNA binding. These results establish a practical framework for understanding the molecular basis by which FOXP3 regulates gene transcription and programs Treg suppressive function

HIV Infection of Naturally Occurring and Genetically Reprogrammed Human Regulatory T-cells

A T-cell subset, defined as CD4(+)CD25(hi) (regulatory T-cells [Treg cells]), was recently shown to suppress T-cell activation. We demonstrate that human Treg cells isolated from healthy donors express the HIV-coreceptor CCR5 and are highly susceptible to HIV infection and replication. Because Treg cells are present in very few numbers and are difficult to expand in vitro, we genetically modified conventional human T-cells to generate Treg cells in vitro by ectopic expression of FoxP3, a transcription factor associated with reprogramming T-cells into a Treg subset. Overexpression of FoxP3 in naïve human CD4(+) T-cells recapitulated the hyporesponsiveness and suppressive function of naturally occurring Treg cells. However, FoxP3 was less efficient in reprogramming memory T-cell subset into regulatory cells. In addition, FoxP3-transduced T-cells also became more susceptible to HIV infection. Remarkably, a portion of HIV-positive individuals with a low percentage of CD4(+) and higher levels of activated T-cells have greatly reduced levels of FoxP3(+)CD4(+)CD25(hi) T-cells, suggesting disruption of the Treg cells during HIV infection. Targeting and disruption of the T-cell regulatory system by HIV may contribute to hyperactivation of conventional T-cells, a characteristic of HIV disease progression. Moreover, the ability to reprogram human T-cells into Treg cells in vitro will greatly aid in decoding their mechanism of suppression, their enhanced susceptibility to HIV infection, and the unique markers expressed by this subset

Small-Molecule RORγt Antagonists Inhibit T Helper 17 Cell Transcriptional Network by Divergent Mechanisms

SummaryWe identified three retinoid-related orphan receptor gamma t (RORγt)-specific inhibitors that suppress T helper 17 (Th17) cell responses, including Th17-cell-mediated autoimmune disease. We systemically characterized RORγt binding in the presence and absence of drugs with corresponding whole-genome transcriptome sequencing. RORγt acts as a direct activator of Th17 cell signature genes and a direct repressor of signature genes from other T cell lineages; its strongest transcriptional effects are on cis-regulatory sites containing the RORα binding motif. RORγt is central in a densely interconnected regulatory network that shapes the balance of T cell differentiation. Here, the three inhibitors modulated the RORγt-dependent transcriptional network to varying extents and through distinct mechanisms. Whereas one inhibitor displaced RORγt from its target loci, the other two inhibitors affected transcription predominantly without removing DNA binding. Our work illustrates the power of a system-scale analysis of transcriptional regulation to characterize potential therapeutic compounds that inhibit pathogenic Th17 cells and suppress autoimmunity

Cytokine signals through PI-3 kinase pathway modulate Th17 cytokine production by CCR6+ human memory T cells

PI-3K–mediated repression of FOXO1 and KLF2 promotes proinflammatory cytokine expression by lineage-committed human CCR6+ Th17/Th22 memory cells

Human Natural Killer T Cells Are Heterogeneous in Their Capacity to Reprogram Their Effector Functions

BACKGROUND: Natural killer T (NKT) cells are a subset of T cells that help potentiate and regulate immune responses. Although human NKT cell subsets with distinct effector functions have been identified, it is unclear whether the effector functions of these subsets are imprinted during development or can be selectively reprogrammed in the periphery. RESULTS: We found that neonatal NKT cells are predominantly CD4+ and express higher levels of CCR7 and CD62L and lower levels of CD94 and CD161 than adult CD4+ or CD4− NKT cell subsets. Accordingly, neonatal NKT cells were more flexible than adult CD4+ NKT cells in their capacity to acquire Th1- or Th2-like functions upon either cytokine-mediated polarization or ectopic expression of the Th1 or Th2 transcription factors T-bet and GATA-3, respectively. Consistent with their more differentiated phenotype, CD4- NKT cells were predominantly resistant to functional reprogramming and displayed higher cytotoxic function. In contrast to conventional T cells, neither the expression of CXCR3 nor the cytotoxic capacity of neonatal NKT cells could be reprogrammed. CONCLUSIONS AND SIGNIFICANCE: Together, these results suggest that neonatal CD4+, adult CD4+, and adult CD4− NKT may represent unique states of maturation and that some functions of human NKT cells may be developmentally imprinted, while others are acquired similar to conventional T cell subsets during peripheral maturation and differentiation. Given the potent immuno-regulatory functions of NKT cells, these findings have important implications for the development of novel NKT cell-based therapeutics and vaccines

Th17 Cells and IL-17 in Protective Immunity to Vaginal Candidiasis

Background: Th17 cells play a major role in coordinating the host defence in oropharyngeal candidiasis. In this study we investigated the involvement of the Th17 response in an animal model of vulvovaginal candidiasis (VVC). Methods: To monitor the course of infection we exploited a new in vivo imaging technique. Results: i) The progression of VVC leads to a strong influx of neutrophils in the vagina soon after the challenge which persisted despite the resolution of infection; ii) IL-17, produced by vaginal cells, particularly CD4 T cells, was detected in the vaginal wash during the infection, reaching a maximum 14 days after the challenge; iii) The amount and kinetics of IL-23 in vaginal fluids were comparable to those in vaginal cells; iv) The inhibition of Th17 differentiation led to significant inhibition of IL-17 production with consequent exacerbation of infection; v) An increased production of bdefensin 2 was manifested in cells of infected mice. This production was strongly reduced when Th17 differentiation was inhibited and was increased by rIL-17 treatment. Conclusions: These results imply that IL-17 and Th17, along with innate antimicrobial factors, have a role in the immune response to vaginal candidiasis

FOXP3:DNA binding is regulated by spacing between tandem elements.

<p>(A) Sequences of the oligonucleotides used as probes in EMSA assays. Each FOXP3 binding site is underlined with the first two nucleotides of each site highlighted in red text. The space (in number of nucleotides) separating the 5′ ends of the two binding sites is indicated below each sequence. The total number of base pairs in each sequence is listed next to the probe name in parentheses. (B) <i>In vitro</i>-translated firefly luciferase (Luc), or FOXP3-ΔN were incubated with each labeled probe as indicated. Protein:DNA complexes and free probe are indicated. Quantification values of bound probe were indicated below the lanes. All data shown are representative of at least 2 separate experiments.</p

DNA binding specificities of Foxp1 and FOXP3.

<p>(A) Sequences of the A probe <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008109#pone.0008109-Wang1" target="_blank">[34]</a>, A′ and A″ oligonucleotides used for EMSA experiments. The 5′ putative FOXP3-binding site (A1) is similar to a predicted Foxp3 binding site <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008109#pone.0008109-Zheng2" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008109#pone.0008109-Marson1" target="_blank">[30]</a> (see insert adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008109#pone.0008109-Marson1" target="_blank">[30]</a> below sequence text). The 3′ binding site (A2) represents the Foxp1 consensus site (blue text) as determined previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008109#pone.0008109-Wang1" target="_blank">[34]</a>. A′ has two putative FOXP3-binding sites (A1-A1). A″ has two putative Foxp1 sites (A2-A2). (B) <i>In vitro</i>-translated firefly luciferase (Luc), Foxp1 (full-length or ΔN) or FOXP3 (full-length or ΔN) were incubated with each labeled probe as indicated. Protein:DNA complexes and free probe are indicated at left (by arrowhead in one case and a square bracket in the other). NS – non-specific. Quantification values of bound probe (shown as % bound of total detected probe in each lane) were indicated below the lanes. (D) Expression of Foxp1 or FOXP3 constructs was evaluated by western blotting using anti-Foxp1 monoclonal or anti-FOXP3 polyclonal antisera respectively. Loading amounts of <i>in vitro</i>-translated lysates were 4 µl for Luc and 1 µl, 2 µl and 4 µl for the Foxp1 and FOXP3 constructs as in (C). Data are representative of at least two independent experiments.</p