Despite the impressive rates of clinical response to programmed death 1 (PD-1) blockade in multiple cancers, the majority of patients still fail to respond to this therapy. The CT26 tumor in mice showed similar heterogeneity, with most tumors unaffected by anti-PD-1. As in humans, response of CT26 to anti-PD-1 correlated with increased T-and B-cell infiltration and IFN expression. We show that intratumoral injection of a highly interferogenic TLR9 agonist, SD-101, in anti-PD-1 nonresponders led to a complete, durable rejection of essentially all injected tumors and a majority of uninjected, distant-site tumors. Therapeutic efficacy of the combination was also observed with the TSA mammary adenocarcinoma and MCA38 colon carcinoma tumor models that show little response to PD-1 blockade alone. Intratumoral SD-101 substantially increased leukocyte infiltration and IFN-regulated gene expression, and its activity was dependent on CD8 + T cells and type I IFN signaling.Anti-PD-1 plus intratumoral SD-101 promoted infiltration of activated, proliferating CD8 + T cells and led to a synergistic increase in total and tumor antigen-specific CD8 + T cells expressing both IFN-γ and TNF-α.Additionally, PD-1 blockade could alter the CpG-mediated differentiation of tumor-specific CD8 + T cells into CD127 low KLRG1 high shortlived effector cells, preferentially expanding the CD127 high KLRG1 low long-lived memory precursors. Tumor control and intratumoral T-cell proliferation in response to the combined treatment is independent of T-cell trafficking from secondary lymphoid organs. These findings suggest that a CpG oligonucleotide given intratumorally may increase the response of cancer patients to PD-1 blockade, increasing the quantity and the quality of tumor-specific CD8 + T cells.PD-1 blockade | TLR9 agonist | multifunctional CD8+ T cells

We propose an experimental strategy for highly accurate selection of candidates for bacterial vaccines without using in vitro and/or in vivo protection assays. Starting from the observation that efficacious vaccines are constituted by conserved, surface-associated and/or secreted components, the strategy contemplates the parallel application of three high throughput technologies, i.e. mass spectrometry-based proteomics, protein array, and flow-cytometry analysis, to identify this category of proteins, and is based on the assumption that the antigens identified by all three technologies are the protective ones. When we tested this strategy for Group A Streptococcus, we selected a total of 40 proteins, of which only six identified by all three approaches. When the 40 proteins were tested in a mouse model, only six were found to be protective and five of these belonged to the group of antigens in common to the three technologies. Finally, a combination of three protective antigens conferred broad protection against a panel of four different Group A Streptococcus strains. This approach may find general application as an accelerated and highly accurate path to bacterial vaccine discovery.

Vaccines are the most effective agents to control infections. In addition to the pathogen antigens, vaccines contain adjuvants that are used to enhance protective immune responses. However, the molecular mechanism of action of most adjuvants is ill-known, and a better understanding of adjuvanticity is needed to develop improved adjuvants based on molecular targets that further enhance vaccine efficacy. This is particularly important for tuberculosis, malaria, AIDS, and other diseases for which protective vaccines do not exist. Release of endogenous danger signals has been linked to adjuvanticity; however, the role of extracellular ATP during vaccination has never been explored. Here, we tested whether ATP release is involved in the immune boosting effect of four common adjuvants: aluminum hydroxide, calcium phosphate, incomplete Freund's adjuvant, and the oil-in-water emulsion MF59. We found that intramuscular injection is always associated with a weak transient release of ATP, which was greatly enhanced by the presence of MF59 but not by all other adjuvants tested. Local injection of apyrase, an ATP-hydrolyzing enzyme, inhibited cell recruitment in the muscle induced by MF59 but not by alum or incomplete Freund's adjuvant. In addition, apyrase strongly inhibited influenza-specific T-cell responses and hemagglutination inhibition titers in response to an MF59-adjuvanted trivalent influenza vaccine. These data demonstrate that a transient ATP release is required for innate and adaptive immune responses induced by MF59 and link extracellular ATP with an enhanced response to vaccination.vaccine adjuvants | danger associated molecular pattern | DAMP | inflammation V accine adjuvants are used to enhance immune responses toward coadministered antigens, thereby improving vaccine potency, immunological memory, or cross-protection (1, 2). Experimental adjuvants range from simple molecules such as calcium phosphate (CaPi) to very complex mixtures such as incomplete Freund's adjuvant (IFA), made of a water-in-oil emulsion, or complete Freund's adjuvant, which also includes killed Mycobacteria (3). However, for human vaccines, adjuvants of highly defined properties that combine efficacy with complete safety are needed; to date, very few compounds have been licensed. Some of the safest and most efficient adjuvants licensed for human use, such as aluminum hydroxide (alum) and the oil-in-water squalene-based emulsion MF59, have been empirically identified, and their mechanism of action is still not fully understood (4, 5). A better understanding of their mechanism of action is needed to develop improved adjuvants that further enhance vaccine efficacy. This is particularly important for diseases for which protective vaccines do not exist (6).An examination of the chemical nature of four major vaccine adjuvants (alum, CaPi, IFA, and MF59) suggested they could interact with the phospholipid bilayer of cell membranes via hydrogen bonding or ionic interactions with the head groups of phospholipids/glycolipids and/or via hydrophobic...

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Currently approved inhibitors of the PD-1/PD-L1 pathway represent a major advance for the treatment of lung cancers, yet they are ineffective in a majority of patients due to lack of preexisting T-cell reactivity. Here, we show that a TLR9 agonist delivered by inhalation is able to prime T-cell responses against poorly immunogenic lung tumors and to complement the effects of PD-1 blockade. Inhaled TLR9 agonist causes profound remodeling in tumor-bearing lungs, leading to the formation of tertiary lymphoid structures adjacent to the tumors, CD8 T-cell infiltration into the tumors, dendritic cell expansion, and antibody production. Inhalation of TLR9 agonist also increased the pool of functional PD-1T-bet effector CD8 T cells in tumor-bearing lungs. Effector CD8 T cells generated by inhaled TLR9 agonist treatment were licensed by PD-1 blockade to become highly functional CTLs, leading to a durable rejection of both lung tumors and tumor lesions outside the lungs. CD4 T cells activated in response to inhaled TLR9 play a critical role in this process by controlling the proliferation, preventing exhaustion, and guiding the differentiation of optimally functional CTLs. This study characterizes a strategy to apply localized TLR9 stimulation to a tumor type not accessible for direct injection, a strategy that may expand the therapeutic potential of PD-1 blockade in non-small cell lung cancer. These findings demonstrate that local delivery of a toll-like receptor 9 agonist can change the immune content of an entire organ and enhance the efficacy of immune checkpoint inhibition. http://cancerres.aacrjournals.org/content/canres/78/17/4943/F1.large.jpg .

Pneumococcal pili have been shown to influence pneumococcal colonization, disease development, and the inflammatory response in mice. The role of the pilus-associated RrgA adhesin in pneumococcal interactions with murine and human macrophages was investigated. Expression of pili with RrgA enhanced the uptake of pneumococci by murine and human macrophages that was abolished by antibodies to complement receptor 3 (CR3) and not seen in CR3-deficient macrophages. Recombinant RrgA, but not pilus subunit RrgC, promoted CR3-mediated phagocytosis of coated beads by murine and human macrophages. Flow cytometry showed that purified CR3 binds pneumococcal cells expressing RrgA, and purified RrgA was shown to interact with CR3 and its I domain. In vivo, RrgA facilitated spread of pneumococci from the upper airways and peritoneal cavity to the bloodstream. Earlier onset of septicemia and more rapidly progressing disease was observed in wild-type mice compared to CR3-deficient mice challenged intranasally or intraperitoneally with pneumococci. Motility assays and time-lapse video microscopy showed that pneumococcal stimulation of macrophage motility required RrgA and CR3. These findings, together with the observed RrgA-dependent increase of intracellular survivors up to 10 h following macrophage infection, suggest that RrgA-CR3-mediated phagocytosis promotes systemic pneumococcal spread from local sites.

Background. Streptococcus pneumoniae serotype 1 has a high likelihood of causing invasive disease. Serotype 1 isolates belonging to CC228 are associated with low mortality, while CC217 isolates exhibit high mortality in patients.Methods. Clinical pneumococcal isolates and mutants were evaluated in wild-type C57BL/6 mice, macrophage-depleted mice, neutrophil-depleted mice, and SIGN-R1 knockout mice. In vitro models included binding and phagocytosis by THP-1 cells, capsule measurements, hydrogen peroxide production, and viability assays.Results. During early systemic infection in mice with serotype 1, large-colony variants appeared in blood. Similar large colonies were found in blood specimens from patients with invasive disease. Large morphotypes contained higher numbers of viable bacteria, grew faster, produced no or little hydrogen peroxide, and contained mutations in the spxB gene. spxB mutants were considerably more virulent in wild-type mice, less susceptible to early host clearance than wild-type strains after intravenous infection, but impaired in colonization. spxB mutants were less efficiently phagocytosed by macrophages than wild-type bacteria, which, in contrast to spxB mutants, caused more-severe disease when macrophages or SIGN-R1 were depleted.Conclusions. Hypervirulent spxB mutants are selected in both mice and patients and are resistant to early macrophage-mediated clearance.

The synthetic oligonucleotide SD-101 is a potent and specific agonist for tolllike receptor 9. Intratumoral injection of SD-101 induces significant anti-tumor immunity in preclinical and clinical studies, especially when combined with PD-1 blockade. To build upon this strategy, we studied the enhancement of SD-101 activities by combination with low-dose cyclophosphamide, a well-characterized agent with potentially complementary activities. In multiple mouse tumor models, we demonstrate substantial anti-tumor activity of the combination, compared to each single agent. Combination therapy generated CD8+ T cell dependent immunity leading to rejection of both non-injected and injected tumors and long-term survival, even in very large tumors. Mechanistic studies encompassing global gene expression changes and characterization of immune cell infiltrates show the rapid, sequential induction of innate and adaptive responses and identify discrete contributions of SD-101 and cyclophosphamide. Importantly, these changes were prominent in tumors not injected directly with SD-101. Combination treatment resulted in creation of a permissive environment for a systemic anti-tumor immune response, including a reduction of intratumoral regulatory T cells (Tregs) and an increase in "M1" versus "M2" tumorassociated macrophage (TAM) phenotypes. Additionally, we observed increased immunogenic cell death as well as antigen processing in response to combination treatment.