Local immunomodulation Fas ligand-engineered biomaterials achieves allogeneic islet graft acceptance.

Islet transplantation is a promising therapy for type 1 diabetes. However, chronic immunosuppression to control rejection of allogeneic islets induces morbidities and impairs islet function. T effector cells are responsible for islet allograft rejection and express Fas death receptors following activation, becoming sensitive to Fas-mediated apoptosis. Here, we report that localized immunomodulation using microgels presenting an apoptotic form of the Fas ligand with streptavidin (SA-FasL) results in prolonged survival of allogeneic islet grafts in diabetic mice. A short course of rapamycin treatment boosted the immunomodulatory efficacy of SA-FasL microgels, resulting in acceptance and function of allografts over 200 days. Survivors generated normal systemic responses to donor antigens, implying immune privilege of the graft, and had increased CD4+CD25+FoxP3+ T regulatory cells in the graft and draining lymph nodes. Deletion of T regulatory cells resulted in acute rejection of established islet allografts. This localized immunomodulatory biomaterial-enabled approach may provide an alternative to chronic immunosuppression for clinical islet transplantation.

Characterization of TRIF selectivity in the AGP class of lipid A mimetics: role of secondary lipid chains.

TLR4 agonists that favor TRIF-dependent signaling and the induction of type 1 interferons may have potential as vaccine adjuvants with reduced toxicity. CRX-547 (4), a member of the aminoalkyl glucosaminide 4-phosphate (AGP) class of lipid A mimetics possessing three (R)-3-decanoyloxytetradecanoyl groups and d-relative configuration in the aglycon, selectively reduces MyD88-dependent signaling resulting in TRIF-selective signaling, whereas the corresponding secondary ether lipid 6a containing (R)-3-decyloxytetradecanoyl groups does not. In order to determine which secondary acyl groups are important for the reduction in MyD88-dependent signaling activity of 4, the six possible ester/ether hybrid derivatives of 4 and 6a were synthesized and evaluated for their ability to induce NF-κB in a HEK293 cell reporter assay. An (R)-3-decanoyloxytetradecanoyl group on the 3-position of the d-glucosamine unit was found to be indispensable for maintaining low NF-κB activity irrespective of the substitutions (decyl or decanoyl) on the other two secondary positions. These results suggest that the carbonyl group of the 3-secondary lipid chain may impede homodimerization and/or conformational changes in the TLR4-MD2 complex necessary for MyD88 binding and pro-inflammatory cytokine induction.

A spatially anchored transcriptomic atlas of the human kidney papilla identifies significant immune injury in patients with stone disease.

Kidney stone disease causes significant morbidity and increases health care utilization. In this work, we decipher the cellular and molecular niche of the human renal papilla in patients with calcium oxalate (CaOx) stone disease and healthy subjects. In addition to identifying cell types important in papillary physiology, we characterize collecting duct cell subtypes and an undifferentiated epithelial cell type that was more prevalent in stone patients. Despite the focal nature of mineral deposition in nephrolithiasis, we uncover a global injury signature characterized by immune activation, oxidative stress and extracellular matrix remodeling. We also identify the association of MMP7 and MMP9 expression with stone disease and mineral deposition, respectively. MMP7 and MMP9 are significantly increased in the urine of patients with CaOx stone disease, and their levels correlate with disease activity. Our results define the spatial molecular landscape and specific pathways contributing to stone-mediated injury in the human papilla and identify associated urinary biomarkers.

A Novel Form of 4-1BBL Prevents Cancer Development via Nonspecific Activation of CD4+ T and Natural Killer Cells.

Costimulation through 4-1BB (CD137) receptor generates robust CD8+ T-effector and memory responses. The only known ligand, 4-1BBL, is a trimeric transmembrane protein that has no costimulatory activity as a soluble molecule. Thus, agonistic antibodies to the receptor have been used for cancer immunotherapy in preclinical models and are currently being evaluated in the clinic. Here, we report that treatment with an oligomeric form of the ligand, SA-4-1BBL, as a single agent is able to protect mice against subsequent tumor challenge irrespective of the tumor type. Protection was long-lasting (>8 weeks) and a bona fide property of SA-4-1BBL, as treatment with an agonistic antibody to the 4-1BB receptor was ineffective in generating immune protection against tumor challenge. Mechanistically, SA-4-1BBL significantly expanded IFNγ-expressing, preexisting memory-like CD44+CD4+ T cells and NK cells in naïve mice as compared with the agonistic antibody. In vivo blockade of IFNγ or depletion of CD4+ T or NK cells, but not CD8+ T or B cells, abrogated the immunopreventive effects of SA-4-1BBL against cancer. SA-4-1BBL as a single agent also exhibited robust efficacy in controlling postsurgical recurrences. This work highlights unexpected features of SA-4-1BBL as a novel immunomodulator with implications for cancer immunoprevention and therapy. SIGNIFICANCE: This study demonstrates the unique and unexpected immunomodulatory features of SA-4-1BBL that bridge innate and adaptive immune responses with both preventive and therapeutic efficacy against cancer.

The 'Ethereal' nature of TLR4 agonism and antagonism in the AGP class of lipid A mimetics.

To overcome the chemical and metabolic instability of the secondary fatty acyl residues in the AGP class of lipid A mimetics, the secondary ether lipid analogs of the potent TLR4 agonist CRX-527 (2) and TLR4 antagonist CRX-526 (3) were synthesized and evaluated along with their ester counterparts for agonist/antagonist activity in both in vitro and in vivo models. Like CRX-527, the secondary ether lipid 4 showed potent agonist activity in both murine and human models. Ether lipid 5, on the other hand, showed potent TLR4 antagonist activity similar to CRX-526 in human cell assays, but did not display any antagonist activity in murine models and, in fact, was weakly agonistic. Glycolipids 2, 4, and 5 were synthesized via a new highly convergent method utilizing a common advanced intermediate strategy. A new method for preparing (R)-3-alkyloxytetradecanoic acids, a key component of ether lipids 4 and 5, is also described.

Current challenges for cancer vaccine adjuvant development.

INTRODUCTION: Although much progress has been made in the last decade(s) toward development of effective cancer vaccines, there are still important obstacles to therapeutic successes. New generations of cancer vaccines will benefit from a combination adjuvant approach that targets multiple branches of the immune response. AREAS COVERED: Herein we describe how combinatorial adjuvant strategies can help overcome important obstacles to cancer vaccine development, including antigen immunogenicity and tumor immune suppression. Tumor antigens may be both tolerogenic and may utilize active mechanisms to suppress host immunity, including downregulation of MHC molecules to evade recognition and upregulation of immune inhibitory receptors, to subvert an effective immune response. The current cancer vaccine literature was surveyed to identify advancements in the understanding of the biological mechanisms underlying poor antigen immunogenicity and tumor immune evasion, as well as adjuvant strategies designed to overcome them. EXPERT COMMENTARY: Poor immunogenicity of tumor antigens and tumor immune evasion mechanisms make the design of cancer vaccines challenging. Growing understanding of the tumor microenvironment and associated immune responses indicate the importance of augmenting not only the effector response, but also overcoming the endogenous regulatory response and tumor evasion mechanisms. Therefore, new vaccines will benefit from multi-adjuvanted approaches that simultaneously stimulate immunity while preventing inhibition.

Immunopharmacology of lipid A mimetics.

The structural core of bacterial lipopolysaccharide, lipid A, has played a role in medicine since the 1890s when William Coley sought to harness its immunostimulatory properties in the form of a crude bacterial extract. Recent decades have brought remarkable clarity to the structure of lipid A and the multicomponent endotoxin receptor system that evolved to detect it. A range of therapeutically useful versions of lipid A now exists, including preparations of detoxified lipid A, synthetic copies of naturally occurring biological intermediates such as lipid IVa, and synthetic mimetics. These agents are finding use as vaccine adjuvants, antagonists and immunostimulants whose structural features have been refined to potentiate efficacy while decreasing the risk of inflammatory side effects.

Selective TRIF-dependent signaling by a synthetic toll-like receptor 4 agonist.

In response to ligand binding to the Toll-like receptor 4 (TLR4) and myeloid differentiation-2 (MD-2) receptor complex, two major signaling pathways are activated that involve different adaptor proteins. One pathway depends on myeloid differentiation marker 88 (MyD88), which elicits proinflammatory responses, whereas the other depends on Toll-IL-1 receptor (TIR) domain-containing adaptor inducing interferon-ß (TRIF), which elicits type I interferon production. Here, we showed that the TLR4 agonist and vaccine adjuvant CRX-547, a member of the aminoalkyl glucosaminide 4-phosphate (AGP) class of synthetic lipid A mimetics, displayed TRIF-selective signaling in human cells, which was dependent on a minor structural modification to the carboxyl bioisostere corresponding to the 1-phosphate group on most lipid A types. CRX-547 stimulated little or no activation of MyD88-dependent signaling molecules or cytokines, whereas its ability to activate the TRIF-dependent pathway was similar to that of a structurally related inflammatory AGP and of lipopolysaccharide from Salmonella minnesota. This TRIF-selective signaling response resulted in the production of substantially less of the proinflammatory mediators that are associated with MyD88 signaling, thereby potentially reducing toxicity and improving the therapeutic index of this synthetic TLR4 agonist and vaccine adjuvant.

Interaction of thiostrepton and elongation factor-G with the ribosomal protein L11-binding domain.

Ribosomal protein L11 and the L11 binding region of ribosomal RNA constitute an important domain involved in active functions of the ribosome during translation. We studied the effects of L11 knock-out and truncation mutations on the structure of the rRNA in this region and on its interactions with a translation elongation factor and the antibiotic thiostrepton. The results indicated that the structure of the L11-binding rRNA becomes conformationally flexible when ribosomes lack the entire L11 protein, but not when the C-terminal domain is present on ribosomes. Probing wild type and mutant ribosomes in the presence of the antibiotic thiostrepton and elongation factor-G (EF-G) rigorously localized the binding cleft of thiostrepton and suggested a role for the rRNA in the L11-binding domain in modulating factor binding. Our results also provide evidence that the structure of the rRNA stabilized by the C-terminal domain of L11 is necessary to stabilize EF-G binding in the post-translocation state, and thiostrepton may modulate this structure in a manner that interferes with the ribosome-EF-G interaction. The implications for recent models of thiostrepton activity and factor interactions are discussed.