TRIF is required for TLR4 mediated adjuvant effects on T cell clonal expansion.

Toll like receptor 4 (TLR4) is an important pattern recognition receptor with the ability to drive potent innate immune responses and also to modulate adaptive immune responses needed for long term protection. Activation of TLR4 by its ligands is mediated by engagement of the adapter proteins MyD88 (myeloid differentiation factor 88) and TRIF (Toll-interleukin 1 receptor domain-containing adapter inducing interferon-beta). Previously, we showed that TRIF, but not MyD88, plays an important role in allowing TLR4 agonists to adjuvant early T cell responses. In this study, we investigated the T cell priming events that are regulated specifically by the TRIF signaling branch of TLR4. We found that TRIF deficiency prevented the TLR4 agonist lipid A from enhancing T cell proliferation and survival in an adoptive transfer model of T cell priming. TRIF deficient DC showed defective maturation as evidenced by their failure to upregulate co-stimulatory molecules in response to lipid A stimulation. Importantly, TRIF alone caused CD86 and CD40 upregulation on splenic DC, but both TRIF and MyD88 were required for CD80 upregulation. The impairment of T cell adjuvant effects and defective DC maturation in TRIF (lps/lps) mice after TLR4 stimulation was mainly due to loss of type I IFN production, indicating that type I interferons are central to TLR4's adjuvant effects. These results are useful for the continued development of TLR4 based vaccine adjuvants that avoid inflammatory risks while retaining beneficial immune response

Lipid A adjuvanted T cells in TRIF^lps/lps recipients have impaired T cell proliferation, and survival ex vivo under growth factor deprived conditions.

<p>CFSE labeled OTI and OTII cells were activated in WT and TRIF^lps/lps recipients with antigen alone (Ag), antigen plus lipid A (Ag+Adj), or saline (No Ag) and harvested for flow cytometric analysis 96 h later. A) Histograms show the CFSE profiles of representative replicates after each treatment (black lines, WT; gray lines, TRIF^lps/lps; solid histogram, unstimulated cells obtained at the time of immunization).B) The average number of OTII cell divisions after each treatment were calculated as described in Methods. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056855#s3" target="_blank">Results</a> shown are the combined average ± SEM of 3 replicates from three independent experiments (n = 9). C–D) TLR4-induced survival effects on the proliferating T cells were estimated by placing the harvested splenocytes in ex vivo culture under growth factor restricted conditions and testing for viable cells after 9 h (CD8⁺ OTI cells (C)) or 18 h (CD4⁺OTII cells (D)). Data represented are the averages from all 3 replicates from 3 independent experiments. Statistical significance between the treatments was by two-way ANOVA. **; p-value≤0.01 and *; p-value≤0.05.</p

Type I IFN plays an important role in TLR4 mediated adjuvant effects on T cells.

<p>OTI and OTII cells were activated in WT or TRIF^lps/lps recipients with subcutaneous hock injections containing antigen alone (Ag), with antigen and lipid A (Ag+Adj), or with saline (No Ag). Effects of recombinant IFN-β on T cell adjuvant effects in TRIF^lps/lps recipients (A) were analyzed by estimating the lipid A induced fold change in OTI and OT II cell numbers in mice that were infused with 30,000 IU of IFN-β 6,12, and 18 h after immunization over non-adjuvanted treatment. Total OTI or OT II cell numbers observed in spleens of each treated mouse are shown at bottom. Effects of type I IFNα/β receptor blockade (mAb MAR1-5A3)(B) on T cell adjuvant effects in WT recipients was analyzed by estimating the lipid A induced fold change in OTI and OT II cell numbers over non adjuvanted treatment in mice that had been given MAR1-5A3 mAb or control IgG₁ 1 h before immunization. Total OTI or OT II cell numbers in recipient spleens of each treated mouse at the harvest are shown at bottom. Data shown are pooled from two independent experiments with 3 replicates in each. **; p-value≤0.01 and *; p-value≤0.05. ND; not determined.</p

Type I IFN receptor signal is required for TLR4-TRIF mediated upregulation of co-stimulatory molecules in vivo.

<p>C57BL/6 and TRIF^lps/lps mice were pre-treated with MAR1-5A3 mAb or isotype control and then injected with lipid A (10 µg/mouse) or saline. After 12 h, splenocytes were labeled with fluorochrome conjugated Abs to measure CD80, CD86, and CD40 expression by DC subsets. The data are representative of 2 independent experiments with 3 replicates in each treatment; nd, not determined. Statistical significance between the treatments at each time point was calculated by two-way ANOVA; **; p-value≤0.01 and *; p-value≤0.05 confidence interval.</p

Impaired Lipid A induced adjuvant effects on T cells in CXCLl10^−/− recipients.

<p>OTI and OTII T cells were activated in WT vs. CXCL10^−/− recipients with subcutaneous hock injections containing antigen alone (Ag), antigen plus lipid A (Ag+Adj) or saline (No Ag). Spleens from recipient mice were harvested 96 h after immunization and OTI and OTII T cells were enumerated by flow cytometry. The ratios of OT and OTII cells recovered from the adjuvant treated mice vs. antigen treated mice are plotted as a fold change (A) and in absolute numbers (B). Data are from three independent experiments combining with 3 replicates in each experiment (n = 9). **;P value<0.001.</p

R-DOTAP Cationic Lipid Nanoparticles Outperform Squalene-Based Adjuvant Systems in Elicitation of CD4 T Cells after Recombinant Influenza Hemagglutinin Vaccination

It is clear that new approaches are needed to promote broadly protective immunity to viral pathogens, particularly those that are prone to mutation and escape from antibody-mediated immunity. Prototypic pathogens of this type are influenza and SARS-CoV-2, where the receptor-binding protein exhibits extremely high variability in its receptor-binding regions. T cells, known to target many viral proteins, and within these, highly conserved peptide epitopes, can contribute greatly to protective immunity through multiple mechanisms but are often poorly recruited by current vaccine strategies. Here, we have studied a promising novel pure enantio-specific cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (R-DOTAP), which was previously recognized for its ability to generate anti-tumor immunity through the induction of potent cytotoxic CD8 T cells. Using a preclinical mouse model, we have assessed an R-DOTAP nanoparticle adjuvant system for its ability to promote CD4 T cell responses to vaccination with recombinant influenza protein. Our studies revealed that R-DOTAP consistently outperformed a squalene-based adjuvant emulsion, even when it was introduced with a potent TLR agonist CpG, in the ability to elicit peptide epitope-specific CD4 T cells when quantified by IFN-γ and IL-2 ELISpot assays. Clinical testing of R-DOTAP containing vaccines in earlier work by others has demonstrated an acceptable safety profile. Hence, R-DOTAP can offer exciting opportunities as an immune stimulant for next-generation prophylactic recombinant protein-based vaccines

Flublok Quadrivalent Vaccine Adjuvanted with R-DOTAP Elicits a Robust and Multifunctional CD4 T Cell Response That Is of Greater Magnitude and Functional Diversity Than Conventional Adjuvant Systems

It is clear that new approaches are needed to promote broadly protective immunity to viral pathogens, particularly those that are prone to mutation and escape from antibody-mediated immunity. CD4+ T cells, known to target many viral proteins and highly conserved peptide epitopes, can contribute greatly to protective immunity through multiple mechanisms. Despite this potential, CD4+ T cells are often poorly recruited by current vaccine strategies. Here, we have analyzed a promising new adjuvant (R-DOTAP), as well as conventional adjuvant systems AddaVax with or without an added TLR9 agonist CpG, to promote CD4+ T cell responses to the licensed vaccine Flublok containing H1, H3, and HA-B proteins. Our studies, using a preclinical mouse model of vaccination, revealed that the addition of R-DOTAP to Flublok dramatically enhances the magnitude and functionality of CD4+ T cells specific for HA-derived CD4+ T cell epitopes, far outperforming conventional adjuvant systems based on cytokine EliSpot assays and multiparameter flow cytometry. The elicited CD4+ T cells specific for HA-derived epitopes produce IL-2, IFN-γ, IL-4/5, and granzyme B and have multifunctional potential. Hence, R-DOTAP, which has been verified safe by human studies, can offer exciting opportunities as an immune stimulant for next-generation prophylactic recombinant protein-based vaccines

Altered populations of natural killer cells, cytotoxic T lymphocytes, and regulatory T cells in major depressive disorder: Association with sleep disturbance

A subset of individuals with major depressive disorder (MDD) have impaired adaptive immunity characterized by a greater vulnerability to viral infection and a deficient response to vaccination along with a decrease in the number and/or activity of T cells and natural killer cells (NKC). Nevertheless, it remains unclear which specific subsets of lymphocytes are altered in MDD, a shortcoming we address here by utilizing an advanced fluorescence-activated cell sorting (FACS) method that allows for the differentiation of important functionally-distinct lymphocyte sub-populations. Furthermore, despite evidence that sleep disturbance, which is a core symptom of MDD, is itself associated with alterations in lymphocyte distributions, there is a paucity of studies examining the contribution of sleep disturbance on lymphocyte populations in MDD populations. Here, we measured differences in the percentages of 13 different lymphocytes and 6 different leukocytes in 54 unmedicated MDD patients (partially remitted to moderate) and 56 age and sex-matched healthy controls (HC). The relationship between self-reported sleep disturbance and cell counts was evaluated in the MDD group using the Pittsburgh Sleep Quality Index (PSQI). The MDD group showed a significantly increased percentage of CD127low/CCR4+ Treg cells, and memory Treg cells, as well as a reduction in CD56+CD16- (putative immunoregulatory) NKC counts, the latter, prior to correction for body mass index. There also was a trend for higher effector memory CD8+ cell counts in the MDD group versus the HC group. Further, within the MDD group, self-reported sleep disturbance was associated with an increased percentage of effector memory CD8+ cells but with a lower percentage of CD56+CD16- NKC. These results provide important new insights into the immune pathways involved in MDD, and provide novel evidence that MDD and associated sleep disturbance increase effector memory CD8+ and Treg pathways. Targeting sleep disturbance may have implications as a therapeutic strategy to normalize NKC and memory CD8+ cells in MDD