Dual-adjuvant effect of pH-sensitive liposomes loaded with STING and TLR9 agonists regress tumor development by enhancing Th1 immune response

Nucleic acid-based pattern recognition receptor agonists are effective adjuvants and immunotherapeutic agents. Rather than single applications, ligand combinations could synergistically potentiate immune responses by elevating cytokine and chemokine production via triggering multiple signaling pathways. However, short half-lives of such labile ligands due to nuclease attack and limited cellular uptake due to their structure significantly hamper their in vivo performances. More importantly, simultaneous delivery and activity presentation of protein antigen and nucleic acid ligands critically limit the clinical development of these constructs. In this work, we approached this problem by co-encapsulating a model antigen ovalbumin along with TLR9 and STING ligands within liposomes, a well-established drug delivery system that enables payload stability and enhanced cellular activity upon internalization. Moreover, by loading dual ligands we postulated to achieve heightened Th-1 immune response that would yield pronounced protective vaccine efficacy. We show that, pH-sensitive liposomes co-encapsulating CpG ODN and cGAMP induced synergistic innate immune response by elevating type I and type II interferon levels. Most importantly, this vaccine formulation led to similar to 70% regression of established melanoma tumor. pH-sensitive liposomal vaccine administration elevated IgG2c/IgG1 antibody ratio, indicative of augmented OVA-specific Th1-biased immunity. Importantly, while the frequency of tumor-specific IFN-gamma producing CD8(+) T-cells was significantly increased, the M2-type anti-inflammatory macrophage levels were decreased in the tumor bed. In conclusion, our strategy induces reversal of immunosuppressive tumor microenvironment, while enhancing effective anti-tumor immune-response. We propose that this could be coupled with standard therapies during combating tumor eradication

PMA-Ionomycin stimulation induced similar IFNγ producing T cells from healthy and SCI PBMCs.

<p>PBMCs (10⁶/ml) were incubated with PMA-Ionomycin for 6h in the presence of Brefeldin A (10μl/ml). (A) % IFNγ producing T cell number by FACS analysis (Healthy = 3, SCI = 4), (B) Representative dot plots showing IFNγ⁺/CD4⁺ T cells. 50000 cells were acquired for the analysis. Mann-Whitney test was used to test significance between healthy and SCI responses. Two-way ANOVA with Bonferroni Correction was used to test the significance when compared to medium alone group. (NS, not significant; ****, p≤0.0001).</p

Impaired toll like receptor-7 and 9 induced immune activation in chronic spinal cord injured patients contributes to immune dysfunction

<div><p>Reduced immune activation or immunosuppression is seen in patients withneurological diseases. Urinary and respiratory infections mainly manifested as septicemia and pneumonia are the most frequent complications following spinal cord injuries and they account for the majority of deaths. The underlying reason of these losses is believed to arise due to impaired immune responses to pathogens. Here, we hypothesized that susceptibility to infections of chronic spinal cord injured (SCI) patients might be due to impairment in recognition of pathogen associated molecular patterns and subsequently declining innate and adaptive immune responses that lead to immune dysfunction. We tested our hypothesis on healthy and chronic SCI patients with a level of injury above T-6. Donor PBMCs were isolated and stimulated with different toll like receptor ligands and T-cell inducers aiming to investigate whether chronic SCI patients display differential immune activation to multiple innate and adaptive immune cell stimulants. We demonstrate that SCI patients' B-cell and plasmacytoid dendritic cells retain their functionality in response to TLR7 and TLR9 ligand stimulation as they secreted similar levels of IL6 and IFNα. The immune dysfunction is not probably due to impaired T-cell function, since neither CD4⁺ T-cell dependent IFNγ producing cell number nor IL10 producing regulatory T-cells resulted different outcomes in response to PMA-Ionomycin and PHA-LPS stimulation, respectively. We showed that TLR7 dependent IFNγ and IP10 levels and TLR9 mediated APC function reduced substantially in SCI patients compared to healthy subjects. More importantly, IP10 producing monocytes were significantly fewer compared to healthy subjects in response to TLR7 and TLR9 stimulation of SCI PBMCs. When taken together this work implicated that these defects could contribute to persistent complications due to increased susceptibility to infections of chronic SCI patients.</p></div

Healthy and SCI donor PBMCs respond similarly to different immune cell stimulants.

<p>PBMCs (2x10⁵/200μl/well) were incubated with different immune stimulants for 24h. (A) IL6 and (B) IFNα secretion in response to R848 (5μg/ml) and D35 (3μM), (<b>C)</b> IL10 production in response to PHA-LPS (5μg/ml-1μg/ml).</p

Co-stimulatory molecule and MHC-II expressions altered in response to TLR ligand stimulation of healthy and SCI PBMCs.

<p>PBMCs (10⁶/ml) were stimulated with R848 (5μg/ml) or D35 (3μM) for 24h. (A) Average levels of CD83/HLA-DR double positive cells (n = 4), (B) Representative dot plots demonstrating double positive cell percent from PBMCs. During analyses 30000 cells were acquired. Two-way ANOVA with Bonferroni Correction was used to test the significance when compared to medium alone group. (*, p≤0.05; **, p≤0.01).</p

Baseline characteristics of the patients and control group.

<p>Baseline characteristics of the patients and control group.</p

Hematological characteristics of the patients and the control group.

<p>Hematological characteristics of the patients and the control group.</p

SCI monocytes are defective in IP10 production in response to D35 stimulation.

<p>PBMCs (2x10⁵/200μl/well) were incubated with D35 (3μM) for 24h. (A) IP10 secretion levels of healthy (n = 5) and SCI (n = 10) PBMC supernatants. PBMCs (10⁶/ml) were stimulated with D35 (3μM) for 20h, at 12h Brefeldin A (10μl/ml) was added. (B) Percent IP10 producing cells following intracellular cytokine staining of healthy (n = 7) and SCI (n = 7) PBMCs, (C) Representative dot plots showing IP10 producing monocytes. 30000 cells were acquired during the analysis. Mann-Whitney test was used to test significance between healthy and SCI responses to D35. Two-way ANOVA with Bonferroni Correction was used to test the significance when compared to medium alone group. (*, p≤0.05; **, p≤0.01; ****, p≤0.0001).</p

Circulating LL37 targets plasma extracellular vesicles to immune cells and intensifies Behcet's disease severity

Behcet's disease (BD) activity is characterised by sustained, over-exuberant immune activation, yet the underlying mechanisms leading to active BD state are poorly defined. Herein, we show that the human cathelicidin derived antimicrobial peptide LL37 associates with and directs plasma extracellular vesicles (EV) to immune cells, thereby leading to enhanced immune activation aggravating BD pathology. Notably, disease activity was correlated with elevated levels of circulating LL37 and EV plasma concentration. Stimulation of healthy PBMC with active BD patient EVs induced heightened IL1 beta, IFN alpha, IL6 and IP10 secretion compared to healthy and inactive BD EVs. Remarkably, when mixed with LL37, healthy plasma-EVs triggered a robust immune activation replicating the pathology inducing properties of BD EVs. The findings of this study could be of clinical interest in the management of BD, implicating LL37/EV association as one of the major contributors of BD pathogenesis