Costimulation Endows Immunotherapeutic CD8 T Cells with IL-36 Responsiveness during Aerobic Glycolysis.

CD134- and CD137-primed CD8 T cells mount powerful effector responses upon recall, but even without recall these dual-costimulated T cells respond to signal 3 cytokines such as IL-12. We searched for alternative signal 3 receptor pathways and found the IL-1 family member IL-36R. Although IL-36 alone did not stimulate effector CD8 T cells, in combination with IL-12, or more surprisingly IL-2, it induced striking and rapid TCR-independent IFN-γ synthesis. To understand how signal 3 responses functioned in dual-costimulated T cells we showed that IL-2 induced IL-36R gene expression in a JAK/STAT-dependent manner. These data help delineate a sequential stimulation process where IL-2 conditioning must precede IL-36 for IFN-γ synthesis. Importantly, this responsive state was transient and functioned only in effector T cells capable of aerobic glycolysis. Specifically, as the effector T cells metabolized glucose and consumed O2, they also retained potential to respond through IL-36R. This suggests that T cells use innate receptor pathways such as the IL-36R/axis when programmed for aerobic glycolysis. To explore a function for IL-36R in vivo, we showed that dual costimulation therapy reduced B16 melanoma tumor growth while increasing IL-36R gene expression. In summary, cytokine therapy to eliminate tumors may target effector T cells, even outside of TCR specificity, as long as the effectors are in the correct metabolic state.

Algal chloroplast produced camelid VH H antitoxins are capable of neutralizing botulinum neurotoxin.

We have produced three antitoxins consisting of the variable domains of camelid heavy chain-only antibodies (VH H) by expressing the genes in the chloroplast of green algae. These antitoxins accumulate as soluble proteins capable of binding and neutralizing botulinum neurotoxin. Furthermore, they accumulate at up to 5% total soluble protein, sufficient expression to easily produce these antitoxins at scale from algae. The genes for the three different antitoxins were transformed into Chlamydomonas reinhardtii chloroplasts and their products purified from algae lysates and assayed for in vitro biological activity using toxin protection assays. The produced antibody domains bind to botulinum neurotoxin serotype A (BoNT/A) with similar affinities as camelid antibodies produced in Escherichia coli, and they are similarly able to protect primary rat neurons from intoxication by BoNT/A. Furthermore, the camelid antibodies were produced in algae without the use of solubilization tags commonly employed in E. coli. These camelid antibody domains are potent antigen-binding proteins and the heterodimer fusion protein containing two VH H domains was capable of neutralizing BoNT/A at near equimolar concentrations with the toxin. Intact antibody domains were detected in the gastrointestinal (GI) tract of mice treated orally with antitoxin-producing microalgae. These findings support the use of orally delivered antitoxins produced in green algae as a novel treatment for botulism.

Presensitizing with a Toll-like receptor 3 ligand impairs CD8 T-cell effector differentiation and IL-33 responsiveness.

The synthetic double-stranded RNA poly(I:C) is commonly used as an adjuvant to boost CD8 T-cell function; however, polyinosinic:polycytidylic acid [poly(I:C)] can also suppress autoimmune disease. The mechanism by which a single adjuvant achieves two distinct immunoregulatory roles is unknown. Although it is clear that coadministration of poly(I:C) with antigen elicits strong adjuvant effects in mice, we found that poly(I:C) injection before antigen substantially reduced antigen-dependent CD8 T-cell responses. Notably, CD8 T cells sensitized in poly(I:C)-pretreated mice failed to fully up-regulate IL-33R (ST2), which led to impaired T-cell receptor-independent responses to IL-33. In contrast, nonsensitized effector CD8 T cells responded robustly to IL-33 using a two-signal cytokine mechanism. During an acute lung response to Staphylococcus aureus enterotoxin, peripheral injection of poly(I:C) manifested a suppressive process by inhibiting the differentiation of both antigen- and IL-33-responsive CD8 effectors systemically. These findings highlight that early exposure to double-stranded RNA reverses its role as an adjuvant and, importantly, prevents IL-33R up-regulation on CD8 effector T cells to dampen inflammation.

The oxazolidinone derivative locostatin induces cytokine appeasement.

Damaging inflammation arising from autoimmune pathology and septic responses results in severe cases of disease. In both instances, anti-inflammatory compounds are used to limit the excessive or deregulated cytokine responses. We used a model of robust T cell stimulation to identify new proteins involved in triggering a cytokine storm. A comparative proteomic mining approach revealed the differential mapping of Raf kinase inhibitory protein after T cell recall in vivo. Treatment with locostatin, an Raf kinase inhibitory protein inhibitor, induced T cell anergy by blocking cytokine production after Ag recall. This was associated with a reduction in Erk phosphorylation. Importantly, in vivo treatment with locostatin profoundly inhibited TNF-alpha production upon triggering the Ag-specific T cells. This effect was not limited to a murine model because locostatin efficiently inhibited cytokine secretion by human lymphocytes. Therefore, locostatin should be a useful therapeutic to control inflammation, sepsis, and autoimmune diseases.

Distinct haplotype profiles and strong linkage disequilibrium at the MDR1 multidrug transporter gene locus in three ethnic Asian populations.

The MDR1 multidrug transporter plays a key role in determining drug bioavailability, and differences in drug response exist amongst different ethnic groups. Numerous studies have identified an association between the MDR1 single nucleotide polymorphism (SNP) exon 26 3435C>T and differences in MDR1 function. We performed a haplotype analysis of the MDR1 gene in three major ethnic groups (Chinese, Malays and Indians) by examining 10 intragenic SNPs. Four were polymorphic in all three ethnic groups: one occurring in the non-coding region and three occurring in coding exons. All three coding SNPs (exon 12 1236C>T, exon 21 2677G>T/A and exon 26 3435C>T) were present in high frequency in each ethnic group, and the derived haplotype profiles exhibited distinct differences between the groups. Fewer haplotypes were observed in the Malays (n = 6) compared to the Chinese (n = 10) and Indians (n = 9). Three major haplotypes (> 10% frequency) were observed in the Malays and Chinese; of these, two were observed in the Indians. Strong linkage disequilibrium (LD) was detected between the three SNPs in all three ethnic groups. The strongest LD was present in the Chinese, followed by Indians and Malays, with the corresponding LD blocks estimated to be approximately 80 kb, 60 kb and 40 kb, respectively. These data strongly support the hypothesis that strong LD between the neutral SNP exon 26 3435C>T and a nearby unobserved causal SNP underlies the observed associations between the neutral SNP and MDR1 functional differences. Furthermore, strong LD between exon 26 3435T and different unobserved causal SNPs in different study populations may provide a plausible explanation for conflicting reports associating the same exon 26 3435T allele with different MDR1 functional changes.

The role of microbial byproducts in protection against immunological disorders and the hygiene hypothesis.

Over the past three decades the incidence of allergic disorders and autoimmune diseases has risen and this trend is particularly prominent in developed nations. The hygiene hypothesis suggests that as a living environment becomes more sanitized, children are not exposed to microbial and parasitic stimulations that were once commonly acquired since early in life, leading to a lack of immune sensitization tending towards T helper 2 (Th2) dominance. This postulation is sufficient to explain allergic disorders, which mostly result from hyper Th2 responses, but inadequate to explain the increase in Th1 or Th17-based autoimmunity. Recent advances in experimental mouse models revealed that stimulation of Toll-like receptors (TLRs) by pathogen-associated molecular patterns could reduce symptoms of allergic airway disease and prevent the onset of autoimmunity. The underlying mechanism for the protective effects of TLR ligands is currently under intense investigation and there are indications that IL-10-producing B cells, regulatory T cells, and innate immune cells play an important role during this process. The finding that early exposure to microbial byproducts contributes to the modulation of immunological disorders may once again modify our interpretation of the hygiene hypothesis.

The systemic and pulmonary immune response to staphylococcal enterotoxins.

In response to environmental cues the human pathogen Staphylococcus aureus synthesizes and releases proteinaceous enterotoxins. These enterotoxins are natural etiologic entities of severe food poisoning, toxic shock syndrome, and acute diseases. Staphylococcal enterotoxins are currently listed as Category B Bioterrorism Agents by the Center for Disease Control and Prevention. They are associated with respiratory illnesses, and may contribute to exacerbation of pulmonary disease. This likely stems from the ability of Staphylococcal enterotoxins to elicit powerful episodes of T cell stimulation resulting in release of pro-inflammatory cytokines. Here, we discuss the role of the immune system and potential mechanisms of disease initiation and progression.

Potent intestinal Th17 priming through peripheral lipopolysaccharide-based immunization.

Lipopolysaccharide (LPS) is a potent natural adjuvant, commonly used to amplify Th1 responses. Here, we report that systemic immunization using LPS generates large numbers of specific Th17 cells in murine small intestinal lamina propria. The priming of these Th17 cells required IL-23p19 production by bone marrow-derived cells. In contrast, IL-23 had no impact on Th1 differentiation or overall numbers of Ag-specific regulatory T cells. Experiments using T-cell adoptive transfers revealed a previously unappreciated mechanism for how Th17 responses are amplified in vivo: stimulation through LPS expanded precommitted Th17 cells rather than causing Th17 differentiation. Second, LPS drove Th17 cell expansion independently of IL-23, demonstrating that this cytokine is not necessary for expansion and possibly functions at an earlier stage in Th17 priming. Our data provide an impetus for using LPS-based peripheral vaccination to augment specific T-cell-mediated immunity in the gut mucosa.

TLR4 hyperresponsiveness via cell surface expression of heat shock protein gp96 potentiates suppressive function of regulatory T cells.

As one of the main mediators of the endoplasmic reticulum unfolded protein response, heat shock protein gp96 is also an obligate chaperone for multiple TLRs including TLR4. We demonstrated recently that enforced cell surface expression of gp96 in a transgenic (Tg) mouse (96tm-Tg) conferred hyperresponsiveness to LPS and induced TLR4-dependent lupus-like autoimmune diseases. In this study, we investigated the function of CD4(+)CD25(+) Foxp3(+) regulatory T cells (T(reg)) in these mice in light of the important roles of T(reg) in the maintenance of peripheral tolerance against self-Ag as well as the increasing appreciation of TLR signaling on the regulation of T(reg). We found that the development of T(reg) was not impaired in 96tm-Tg mice. Contrary to the prediction of dampened T(reg) activity, we discovered that the suppressive functions of T(reg) were increased in 96tm-Tg mice. Inactivation of T(reg) during the neonatal stage of life exacerbated not only organ-specific diseases but also systemic autoimmune diseases. By crossing 96tm-Tg mice into the TLR4 null background, we demonstrated the critical roles of TLR4 in the amplification of T(reg) suppressive function. These findings illustrate that gp96 plays dual roles in regulating immune responses by augmenting proinflammatory responses and inducing T(reg) function, both of which are dependent on its ability to chaperone TLR4. Our study provides strong support to the notion of compensatory T(reg) activation by TLR ligation to dampen inflammation and autoimmune diseases.