BackgroundImmune checkpoint blockade (ICB) promotes adaptive immunity and tumor regression in some cancer patients. However, in patients with immunologically “cold” tumors, tumor-resident innate immune cell activation may be required to prime an adaptive immune response and so exploit the full potential of ICB. Whilst Toll-like receptor (TLR) agonists have been used topically to successfully treat some superficial skin tumors, systemic TLR agonists have not been well-tolerated.MethodsThe response of human immune cells to TLR7 and 8 agonism was measured in primary human immune cell assays. MEDI9197 (3M-052) was designed as a novel lipophilic TLR7/8 agonist that is retained at the injection site, limiting systemic exposure. Retention of the TLR7/8 agonist at the site of injection was demonstrated using quantitative whole-body autoradiography, HPLC-UV, and MALDI mass spectrometry imaging. Pharmacodynamic changes on T cells from TLR7/8 agonist treated B16-OVA tumors was assessed by histology, quantitative real time PCR, and flow cytometry. Combination activity of TLR7/8 agonism with immunotherapies was assessed in vitro by human DC-T cell MLR assay, and in vivo using multiple syngeneic mouse tumor models.ResultsTargeting both TLR7 and 8 triggers an innate and adaptive immune response in primary human immune cells, exemplified by secretion of IFNα, IL-12 and IFNγ. In contrast, a STING or a TLR9 agonist primarily induces release of IFNα. We demonstrate that the TLR7/8 agonist, MEDI9197, is retained at the sight of injection with limited systemic exposure. This localized TLR7/8 agonism leads to Th1 polarization, enrichment and activation of natural killer (NK) and CD8+ T cells, and inhibition of tumor growth in multiple syngeneic models. The anti-tumor activity of this TLR7/8 agonist is enhanced when combined with T cell-targeted immunotherapies in pre-clinical models.ConclusionLocalized TLR7/8 agonism can enhance recruitment and activation of immune cells in tumors and polarize anti-tumor immunity towards a Th1 response. Moreover, we demonstrate that the anti-tumor effects of this TLR7/8 agonist can be enhanced through combination with checkpoint inhibitors and co-stimulatory agonists.Electronic supplementary materialThe online version of this article (10.1186/s40425-019-0724-8) contains supplementary material, which is available to authorized users.

Human and animal studies strongly suggest that dietary gluten could play a causal role in the etiopathogenesis of type 1 diabetes (T1D). However, the mechanisms have not been elucidated. Recent reports indicate that the intestinal microbiome has a major influence on the incidence of T1D. Since diet is known to shape the composition of the intestinal microbiome, we investigated using non-obese diabetic (NOD) mice whether changes in the intestinal microbiome could be attributed to the pro- and anti-diabetogenic effects of gluten-containing and gluten-free diets, respectively. NOD mice were raised on gluten-containing chows (GCC) or gluten-free chows (GFC). The incidence of diabetes was determined by monitoring blood glucose levels biweekly using a glucometer. Intestinal microbiome composition was analyzed by sequencing 16S rRNA amplicons derived from fecal samples. First of all, GCC-fed NOD mice had the expected high incidence of hyperglycemia whereas NOD mice fed with a GFC had significantly reduced incidence of hyperglycemia. Secondly, when the fecal microbiomes were compared, Bifidobacterium, Tannerella, and Barnesiella species were increased (p = 0.03, 0.02, and 0.02, respectively) in the microbiome of GCC mice, where as Akkermansia species was increased (p = 0.02) in the intestinal microbiomes of NOD mice fed GFC. Thirdly, both of the gluten-free chows that were evaluated, either egg white based (EW-GFC) or casein based (C-GFC), significantly reduced the incidence of hyperglycemia. Interestingly, the gut microbiome from EW-GFC mice was similar to C-GFC mice. Finally, adding back gluten to the gluten-free diet reversed its anti-diabetogenic effect, reduced Akkermansia species and increased Bifidobacterium, Tannerella, and Barnesiella suggesting that the presence of gluten is directly responsible for the pro-diabetogenic effects of diets and it determines the gut microflora. Our novel study thus suggests that dietary gluten could modulate the incidence of T1D by changing the gut microbiome.

Among the exotoxins produced by Staphylococcus aureus and Streptococcus pyogenes, the superantigens (SAgs) are the most potent T-cell activators known to date. SAgs are implicated in several serious diseases including toxic shock syndrome (TSS), Kawasaki disease, and sepsis. However, the immunopathogenesis of TSS and other diseases involving SAgs are still not completely understood. The commonly used conventional laboratory mouse strains do not respond robustly to SAgs in vivo. Therefore, they must be artificially rendered susceptible to TSS by using sensitizing agents such as D-galactosamine (D-galN), which skews the disease exclusively to the liver and, hence, is not representative of the disease in humans. SAg-induced TSS was characterized using transgenic mice expressing HLA class II molecules that are extremely susceptible to TSS without D-galN. HLA-DR3 transgenic mice recapitulated TSS in humans with extensive multiple-organ inflammation affecting the lung, liver, kidneys, heart, and small intestines. Heavy infiltration with T lymphocytes (both CD4 ؉ and CD8؉), neutrophils, and macrophages was noted. In particular, the pathologic changes in the small intestines were extensive and accompanied by significantly altered absorptive functions of the enterocytes. In contrast to massive liver failure alone in the D-galN sensitization model of TSS, findings of the present study suggest that gut dysfunction might be a key pathogenic event that leads to high morbidity and mortality in humans with

Toxic shock syndrome (TSS) caused by the superantigen exotoxins of Staphylococcus aureus and Streptococcus pyogenes is characterized by robust T cell activation, profound elevation in systemic levels of multiple cytokines, including interferon-γ (IFN-γ), followed by multiple organ dysfunction and often death. As IFN-γ possesses pro- as well as anti-inflammatory properties, we delineated its role in the pathogenesis of TSS. Antibody-mediated in vivo neutralization of IFN-γ or targeted disruption of IFN-γ gene conferred significant protection from lethal TSS in HLA-DR3 transgenic mice. Following systemic high dose SEB challenge, whereas the HLA-DR3.IFN-γ+/+ mice became sick and succumbed to TSS, HLA-DR3.IFN-γ−/− mice appeared healthy and were significantly protected from SEB-induced lethality. SEB-induced systemic cytokine storm was significantly blunted in HLA-DR3.IFN-γ−/− transgenic mice. Serum concentrations of several cytokines (IL-4, IL-10, IL-12p40 and IL-17) and chemokines (KC, rantes, eotaxin and MCP-1) were significantly lower in HLA-DR3.IFN-γ−/− transgenic mice. However, SEB-induced T cell expansion in the spleens was unaffected and expansion of SEB-reactive TCR Vβ8+ CD4+ and CD8+ T cells was even more pronounced in HLA-DR3.IFN-γ−/− transgenic mice when compared to HLA-DR3.IFN-γ+/+ mice. A systematic histopathological examination of several vital organs revealed that both HLA-DR3.IFN-γ+/+ and HLA-DR3.IFN-γ−/− transgenic mice displayed comparable severe inflammatory changes in lungs, and liver during TSS. Remarkably, whereas the small intestines from HLA-DR3.IFN-γ+/+ transgenic mice displayed significant pathological changes during TSS, the architecture of small intestines in HLA-DR3.IFN-γ−/− transgenic mice was preserved. In concordance with these histopathological changes, the gut permeability to macromolecules was dramatically increased in HLA-DR3.IFN-γ+/+ but not HLA-DR3.IFN-γ−/− mice during TSS. Overall, IFN-γ seemed to play a lethal role in the immunopathogenesis of TSS by inflicting fatal small bowel pathology. Our study thus identifies the important role for IFN-γ in TSS.

Chronic nasal and skin colonization with superantigen (SAg)-producing Staphylococcus aureus is well documented in humans. Given that trans-mucosal and trans-cutaneous absorption of SAgs can occur, we determined whether chronic exposure to small amounts of SAg per se could activate autoreactive CD4+ and CD8+ T cells and precipitate any autoimmune disease without further external autoantigenic stimulation. Because HLA class II molecules present SAg more efficiently than do mouse MHC class II molecules, HLA-DQ8 transgenic mice were implanted s.c. with mini-osmotic pumps capable of continuously delivering the SAg, staphylococcal enterotoxin B (total of 10 μg/mouse), or PBS over 4 wk. Chronic exposure to staphylococcal enterotoxin B resulted in a multisystem autoimmune inflammatory disease with features similar to systemic lupus erythematosus. The disease was characterized by mononuclear cell infiltration of lungs, liver, and kidneys, accompanied by the production of anti-nuclear Abs and deposition of immune complexes in the renal glomeruli. The inflammatory infiltrates in various organs predominantly consisted of CD4+ T cells bearing TCR Vβ8. The extent of immunopathology was markedly reduced in mice lacking CD4+ T cells and CD28, indicating that the disease is CD4+ T cell mediated and CD28 dependent. The absence of disease in STAT4-deficient, as well as IFN-γ–deficient, HLA-DQ8 mice suggested the pathogenic role of Th1-type cytokines, IL-12 and IFN-γ. In conclusion, our study suggests that chronic exposure to extremely small amounts of bacterial SAg could be an etiological factor for systemic lupus erythematosus.