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Human γ9δ2 T cells potently inhibit pathogenic microbes, including intracellular mycobacteria, but the key inhibitory mechanism(s) involved have not been identified. We report a novel mechanism involving the inhibition of intracellular mycobacteria by soluble granzyme A. γ9δ2 T cells produced soluble factors that could pass through 0.45 mm membranes and inhibit intracellular mycobacteria in human monocytes cultured below transwell inserts. Neutralization of TNF-α in cocultures of infected monocytes and γ9δ2 T cells prevented inhibition, suggesting that TNF-α was the critical inhibitory factor produced by γ9δ2 T cells. However, only siRNA- mediated knockdown of TNF-α in infected monocytes, but not in γ9δ2 T cells, prevented mycobacterial growth inhibition. Investigations of other soluble factors produced by γ9δ2 T cells identified a highly significant correlation between the levels of granzyme A produced and intracellular mycobacterial growth inhibition. Furthermore, purified granzyme A alone induced inhibition of intracellular mycobacteria, while knockdown of granzyme A in γ9δ2 T cell clones blocked their inhibitory effects. The inhibitory mechanism was independent of autophagy, apoptosis, nitric oxide production, type I interferons, Fas/FasL and perforin. These results demonstrate a novel microbial defense mechanism involving granzyme A-mediated triggering of TNF-α production by monocytes leading to intracellular mycobacterial growth suppression. This pathway may provide a protective mechanism relevant for the development of new vaccines and/or immunotherapies for macrophage-resident chronic microbial infections. [ABSTRACT FROM AUTHOR]

Mouse models of orthopoxvirus disease provide great promise for probing basic questions regarding host responses to this group of pathogens, which includes the causative agents of monkeypox and smallpox. However, some essential tools for their study that are taken for granted with other mouse models are not available for these viruses. Here we map and characterize the initial CD8+ T-cell determinants for poxviruses in H-2d-haplotype mice. CD8+ T cells recognizing these three determinants make up around 40% of the total responses to vaccinia virus during and after resolution of infection. We then use these determinants to test if predicted conservation across orthopoxvirus species matches experimental observation and find an unexpectedly cross-reactive variant peptide encoded by ectromelia (mousepox) virus. [ABSTRACT FROM AUTHOR]

TLR-7/8 agonists are a well-known class of vaccine adjuvants, with a leading example now included in Covaxin, a licensed human COVID-19 vaccine. This thereby provides the opportunity to develop newer, more potent adjuvants based on structure-function studies of these classes of compounds. Imidazoquinoline-based TLR7/8 agonists are the most potent, but when used as a vaccine adjuvant side effects can arise due to diffusion from the injection site into a systemic circulation. In this work, we sought to address this issue through structural modifications in the agonists to enhance their adsorption capacity to the classic adjuvant alum. We selected a potent TLR7-selective agonist, BBIQ (EC 50 = 0.85 μM), and synthesized polyphenolic derivatives to assess their TLR7 agonistic activity and adjuvant potential alone or in combination with alum. Most of the phenolic derivatives were more active than BBIQ and, except for 12b , all were TLR7 specific. Although the synthesized compounds were less active than resiquimod, the immunization data on combination with alum, specifically the IgG1, IgG2b and IgG2c responses, were superior in comparison to BBIQ as well as the reference standard resiquimod. Compound 12b was 5-fold more potent (EC 50 = 0.15 μM in TLR7) than BBIQ and induced double the IgG response to SARS-CoV-2 and hepatitis antigens. Similarly, compound 12c (EC 50 = 0.31 μM in TLR7) was about 3-fold more potent than BBIQ and doubled the IgG levels. Even though compound 12d exhibited low TLR7 activity (EC 50 = 5.13 μM in TLR7), it demonstrated superior adjuvant results, which may be attributed to its enhanced alum adsorption capability as compared with BBIQ and resiquimod. Alum-adsorbed polyphenolic TLR7 agonists thereby represent promising combination adjuvants resulting in a balanced Th1/Th2 immune response., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

Lipopeptides including diacylated Pam 2 CSK 4 as well as triacylated Pam 3 CSK 4 act as ligands of toll-like receptor (TLR)-2, a promising target for the development of vaccine adjuvants. The highly investigated Pam 2 CSK 4 and Pam 3 CSK 4 , despite their aqueous solubility have not performed well as vaccine adjuvants which may be attributable to potential denaturation of protein antigens by these cationic surfactant-like lipopeptides. In the present investigation, we synthesized ( R ), ( S ) and racemic Pam 2 CS(OMe) analogs and their N -acetyl derivatives without the tetralysine component to systematically investigate the effect of stereochemistry at the thio-glycerol lipopeptide core of these lipopeptide based TLR2 agonists. The resulting compounds were compared using TLR2 reporter cell-based assays and the ability of the synthesized lipopeptides to stimulate cytokine production (IL-6, IL-10 and TNF-α) by freshly collected human PBMCs and CD40 and CD86 expressions by mouse spleen cells was also investigated. Notably, few synthesized lipopeptides were found to be potent TLR2/6 agonists, inducing cytokine production and upregulating CD40 and CD86 expressions. The TLR2/6 agonistic lipopeptides were further assessed for vaccine adjuvant effects in mice. The results confirmed that the R -stereochemistry at the thio-glycerol lipopeptide core was preferred for maximal TLR2/6 activity, as reflected in Th1 immune deviation, higher antibody levels and enhanced vaccine protection against a lethal influenza challenge., Competing Interests: There is no conflict of interest to declare., (This journal is © The Royal Society of Chemistry.)

γ9δ2 T cells provide a natural bridge between innate and adaptive immunity, rapidly and potently respond to pathogen infection in mucosal tissues, and are prominently induced by both tuberculosis (TB) infection and bacillus Calmette Guérin (BCG) vaccination. Mycobacterium-expanded γ9δ2 T cells represent only a subset of the phosphoantigen {isopentenyl pyrophosphate [IPP] and (E)-4-hydroxy-3-methyl-but-2-enylpyrophosphate [HMBPP]}-responsive γ9δ2 T cells, expressing an oligoclonal set of T cell receptor (TCR) sequences which more efficiently recognize and inhibit intracellular Mycobacterium tuberculosis infection. Based on this premise, we have been searching for M. tuberculosis antigens specifically capable of inducing a unique subset of mycobacterium-protective γ9δ2 T cells. Our screening strategy includes the identification of M. tuberculosis fractions that expand γ9δ2 T cells with biological functions capable of inhibiting intracellular mycobacterial replication. Chemical treatments of M. tuberculosis whole-cell lysates (MtbWL) ruled out protein, nucleic acid, and nonpolar lipids as the M. tuberculosis antigens inducing protective γ9δ2 T cells. Mild acid hydrolysis, which transforms complex carbohydrate to monomeric residues, abrogated the specific activity of M. tuberculosis whole-cell lysates, suggesting that a polysaccharide was required for biological activity. Extraction of MtbWL with chloroform-methanol-water (10:10:3) resulted in a polar lipid fraction with highly enriched specific activity; this activity was further enriched by silica gel chromatography. A combination of mass spectrometry and nuclear magnetic resonance analysis of bioactive fractions indicated that 6-O-methylglucose-containing lipopolysaccharides (mGLP) are predominant components present in this active fraction. These results have important implications for the development of new immunotherapeutic approaches for prevention and treatment of TB., (Copyright © 2016 Xia et al.)

There is increased interest in understanding protective immunity to smallpox for two principal reasons. First, it is the only disease that has been successfully eradicated using a live virus vaccine and, second, there exists a potential threat of intentional or unintentional release of variola virus, the causative agent of smallpox. Although mortality rates associated with smallpox were as high as 40%, a significant subset of those infected recovered. The basis of susceptibility or resistance, and the immune parameters associated with recovery, are still unknown. Animal models of poxvirus infections are being employed to understand what constitutes an effective host response. Ectromelia virus is closely related to variola virus and it causes a disease similar to smallpox in mice. This model is well established, resistant and susceptible strains of mice are defined and four genetic loci associated with resistance have been identified. Susceptibility to infec tion and disease severity is also influenced by virus immune evasion strategies. The outcome of infection is clearly dictated by several factors including host and viral genes, both of which influence the immune response. Here we present data on one virus-encoded immune modifier and its effect on the functions of two host genetic loci associ ated with resistance.

Mouse models of orthopoxvirus disease provide great promise for probing basic questions regarding host responses to this group of pathogens, which includes the causative agents of monkeypox and smallpox. However, some essential tools for their study that are taken for granted with other mouse models are not available for these viruses. Here we map and characterize the initial CD8+ T-cell determinants for poxviruses in H-2d-haplotype mice. CD8+ T cells recognizing these three determinants make up around 40% of the total responses to vaccinia virus during and after resolution of infection. We then use these determinants to test if predicted conservation across orthopoxvirus species matches experimental observation and find an unexpectedly cross-reactive variant peptide encoded by ectromelia (mousepox) virus.