Combination of TLR8 and TLR4 agonists reduces the degrading effects of nicotine on DC-NK mediated effector T cell generation.

The magnitude of immune responses to vaccination is a critical factor in determining protection from disease. It is known that cigarette smoke dampens the immune system and increases the risk of vaccine-preventable diseases. We reported that nicotine, the immunosuppressive component of cigarette smoke, disrupts the differentiation and functional properties of DC, which are pivotal in the initiation of immune response to vaccines. We also reported that TLR agonists act in synergy and boost DC maturation, DC-NK crosstalk and ultimately naïve T cell polarization into effector Th1 and Tc1 cells. Here, we investigated whether the combination of TLR agonists could diminish the degrading effects of nicotine on DC-NK mediated effector T cell generation. We found that none of TLR agonists, single or combined, were able to diminish completely the adverse effects of nicotine on DC. However, TLR3, TLR4, and TLR8 agonists acted as the most effective adjuvants to increase the expression levels of antigen-presenting, costimulatory molecules and production of cytokines by nicotine-exposed DC (nicDC). When combined, TLR3 + 8 and TLR4 + 8 synergistically optimized nicDC maturation and IFN-γ secretion from nicotine-exposed NK (nicNK) during co-cultures. Interestingly, in contrast to DC-NK-T, co-cultures of nicDC-nicNK-T treated with TLR3 + 8 or TLR4 + 8 agonists produced a similar frequency of effector memory Th1 and Tc1 cells. However, the effector cells from TLR4 + 8 followed by TLR3 + 8 treated nicDC-nicNK-T co-cultures produced significantly more IFN-γ when compared with aluminum salt treated co-culture. Our data suggest that addition of appropriate TLR agonists to vaccine formulation could potentially augment the immune response to vaccination in smokers.

TLR8 combined withTLR3 or TLR4 agonists enhances DC-NK driven effector Tc1 cells.

BACKGROUND: Most current prophylactic vaccines confer protection primarily through humoral immunity. Indeed, aluminum salts which have been widely used as adjuvants in vaccines primarily enhance Th2-driven antibody responses. Therefore, new vaccines formulation is moving toward a careful selection of adjuvants that also elicit significant Th1 or Tc1 responses. Several TLR agonists have been tested as potential new adjuvants in clinical and preclinical studies with some efficacy. These studies suggest that combining more than one of TLR ligands enhances the magnitude of immune responses to cancer and infectious disease. OBJECTIVES: In order to evaluate the synergistic effect of TLR agonists for effective induction of cellular immunity, we investigated the effects of single and/or combined TLR agonists on monocyte-derived DC maturation, DC-NK crosstalk and ultimately naïve T cells polarization into effector T cells. RESULTS: Among the adjuvants tested, we found that TLR3, TLR4, TLR7/8 and TLR8 agonists were the most effective adjuvants to increase the expression levels of antigen-presenting, co-stimulatory molecules and production of cytokines by maturing DCs. When combined, TLR3+8 and TLR4+8 synergistically optimized DC maturation and IFN-γ secretion from NK cells co-cultured with DCs. Interestingly, co-culture of DC-NK-T treated with aluminum salt produced the highest percentage of effector memory CFSE-CCR7- Th1 cells whereas TLR3+8 and TLR4+8 treated co-cultures produced the highest percentage of effector memory CFSE-CCR7- Tc1 cells producing IFN-γ. Finally, while both TLR3+8 or TLR4+8 treated co-cultures generated similar frequency of Th1 and Tc1 effector cells, the effector cells from the latter co-culture produced quantitatively more IFN-γ in the supernatant. CONCLUSION: Our data indicate that if in need of an enhanced DC-NK mediated cellular immunity one may select TLR agonists with defined synergistic effects.

Phenotyping and comparing the immune cell populations of free-ranging Atlantic bottlenose dolphins (Tursiops truncatus) and dolphins under human care.

BACKGROUND: Studies suggest that free-ranging bottlenose dolphins exhibit a suppressed immune system because of exposure to contaminants or microorganisms. However, due to a lack of commercially available antibodies specific to marine mammal immune cell surface markers, the research has been indecisive. The purpose of this study was to identify cross-reactive terrestrial-specific antibodies in order to assess the changes in the immune cell populations of dolphins under human care and free-ranging dolphins. The blood and PBMC fraction of blood samples from human care and free-ranging dolphins were characterized by H&E staining of cytospin slides and flow cytometry using a panel of terrestrial-specific antibodies. RESULTS: In this study, we show that out of 65 terrestrial-specific antibodies tested, 11 were cross-reactive and identified dolphin immune cell populations within their peripheral blood. Using these antibodies, we found significant differences in the absolute number of cells expressing specific markers within their lymphocyte and monocyte fractions. Interestingly, the peripheral blood mononuclear cell profile of free-ranging dolphins retained an additional population of cells that divided them into two groups showing a low (<27%) or high (>56%) percentage of smaller cells resembling granulocytes. CONCLUSIONS: We found that the cross-reactive antibodies not only identified specific changes in the immune cells of free-ranging dolphins, but also opened the possibility to investigate the causal relationship between immunosuppression and mortality seen in free-ranging dolphins.