Hepatitis B virus (HBV) infection is a major public health concern. The clearance of HBV may involve cytotoxic T-lymphocyte (CTL) activity and T helper type 1 reactions. Exosomes generated from dendritic cells (DCs) can induce immunological responses capable of eradicating viruses. However, exosomes loaded with antigens have not yet demonstrated therapeutic potential in HBV infection. Therefore, the present study aimed to investigate the antiviral effects of DC-derived exosomes (Dexs) loaded with ubiquitinated HBV core antigen (Dexs-Ub-HBcAg). Murine bone marrow-derived DCs were loaded with a recombinant lentivector encoding the ubiquitinated form of HBcAg. High-purity Dexs were generated using differential velocity centrifugation. Splenic T-lymphocytes were stimulated with Dexs-Ub-HBcAg and the specific T-cell-mediated immune responses were examined. Cytokine expression was analyzed using enzyme-linked immunosorbent assays. T-lymphocyte proliferation was detected using a Cell Counting Kit-8 assay and HBcAg-specific CTL activity was determined using a lactate dehydrogenase release assay. The results revealed that Dexs-Ub-HBcAg effectively stimulated T-cell proliferation and induced the activation of antigen-specific CTLs to exhibit HBcAg-specific CTL immune responses in vitro. These results suggest the potential of Dexs-Ub-HBcAg for development as a future therapeutic option for the elimination of HBV.

The blockade of inhibitory receptors such as CTLA-4 (CD152) is being used as immune-checkpoint therapy, offering a powerful strategy to restore effective immune responses against tumors. To determine signal components that are induced under the control of CTLA-4 we analyzed activated murine CD8+ T cells by quantitative proteomics. Accurate mass spectrometry revealed that CTLA-4 engagement led to central changes in the phosphorylation of proteins involved in T-cell differentiation. Beside other targets, we discovered a CTLA-4-mediated induction of the translational inhibitor programmed cell death-4 (PDCD4) as a result of FoxO1 nuclear re-localization. PDCD4 further bound a distinct set of mRNAs including Glutaminase, which points out a critical role for CTLA-4 in CD8+ T-cell metabolism. Consequently, PDCD4-deficient cytotoxic T-lymphocytes (CTLs) expressed increased amounts of otherwise repressed effector molecules and ultimately led to superior control of tumor growth in vivo. These findings reveal a novel CTLA-4-mediated pathway to attenuate CTLs and indicate the importance of post-transcriptional mechanisms in the regulation of anti-tumor immune responses.

The aim of the present study was to determine whether the combination of two modalities of immunotherapy, targeting two different tumor antigens, may be feasible and non-toxic, yet enhance the killing of a human breast cancer cell line. The first modality was tumor growth factor α-Pseudomonas exotoxin 38 (TGFα-PE38), which specifically targets and kills tumor cells that express the epidermal growth factor receptor. The second modality was mucin-1 (MUC1)-specific cytotoxic T lymphocytes (CTLs), generated by MUC1 stimulation of peripheral blood mononuclear cells, to target the human breast cancer cell line, MCF7. TGFα-PE38 exhibited specific lysis of the MCF7 cells in a concentration- and time-dependent manner. TGFα-PE38 did not kill the normal hematopoietic stem cells or CTLs. Furthermore, TGFα-PE38 was not inhibitory for the growth or differentiation of the normal human hematopoietic stem cells into erythroid and myeloid colonies. In addition, TGFα-PE38 did not inhibit the killing function of CTLs, either when preincubated or co-incubated with CTLs. Finally, therapeutic enhancement was observed, in that TGFα-PE38 and CTLs were additive in the specific lysis of the MCF7 cells. These two modalities of immunotherapy may be beneficial for humans with breast cancer with or without other therapies, including autologous hematopoietic stem cell transplantation, specifically for purging cancer cells from hematopoietic stem cells prior to transplantation.

Cellular immunotherapy is a promising new therapeutic approach for hepatocellular carcinoma (HCC), which has a high recurrence rate, irrespective of the treatment administered. In this study, we attempted to improve the cytolytic activity of effector T-lymphocytes against HCC. T-lymphocytes were activated by monocyte-derived dendritic cells (DCs) pulsed with cell lysate or RNA prepared from HCC cell lines. Monocytes were activated for differentiation into DCs by treatment with the IL4 and GM-CSF. DCs were pulsed with cell lysate or RNA prepared from a single cell line or combinations of two or three HCC cell lines, and then co-cultured with autologous T-lymphocytes with the intent of creating specific cytotoxicity. We discovered that DCs pulsed with total RNA effectuated greater T-lymphocyte function than DCs pulsed with total cell lysate, as evidenced by greater cytolytic activities against HCC target cells. The percentage of Huh7, HepG2, and SNU449 cell apoptosis at effector:target ratio of 10:1 was 42.6 ± 4.5% (p = 0.01), 33.6 ± 3.1% (p = 0.007), and 21.4 ± 1.4% (p < 0.001), respectively. DCs pulsed with pools of antigens prepared from three cell lines improved the cytolytic function of effector T-lymphocytes by approximately two-fold (p < 0.001), which suggests that this approach be further developed and applied for adoptive transfer treatment of HCC.

Use of prebiotics is a growing topic in healthcare. A lightweight molecule and water-soluble fiber ingredient, longish glucomannan hydrolysates (LGH), has been developed to improve the intestinal mucosal barrier and confer gut health benefits. This study aims to investigate the implications of continuous LGH intervening in intestinal epithelium integrity and protective immunity against chemical dextran sodium sulfate (DSS)-induced colitis. Twelve male BALB/c mice were randomly arranged into four groups. The LGH/DSS group had results in bodyweight variance, epithelial cell density, and aberrancy score as good as the LGH group, and both were equivalent to the control group. LGH consumption effectively protects the distal intestinal epithelium by activating innate T lymphocytes. Meanwhile, T-cell subsets in subepithelial interspersion take a bystander role in these microenvironmental alterations. Under this stress, the cluster of differentiation 3 (CD3)+ T cells infiltrate the epithelium, while CD4+ T cells inversely appear in submucosal large lymphoid aggregates/isolated lymphoid follicles (ILFs) in which significant CD3+, CD4+, and CD8+ T-cell populations agglomerate. Moreover, forkhead box P3 (Foxp3) and interleukin 17 (IL-17) are observed in these ILFs. Agglomerated CD4+ T-cell lineages may have roles with proinflammatory T helper 17 cells and anti-inflammatory regulatory T cells in balancing responses to intraluminal antigens. Collectively, LGH administration may function in immune modulation to protect against DSS-induced inflammation.

We have previously shown that small B16 melanomas can be successfully treated using a combination of anti-cytotoxic T lymphocyte antigen (CTLA)-4 monoclonal antibody with a granulocyte/macrophage colony-stimulating factor (GM-CSF) producing irradiated tumor cell vaccine. Regression of tumors results in long-lasting immunity and is frequently accompanied by autoimmune depigmentation. Here we examine the cellular and molecular mechanisms of this combined treatment. Histological examination of depigmented lesions revealed infiltration of polymorphonuclear cells and deposition of antibody. The combination therapy also induced tumor rejection and skin depigmentation in B cell-deficient and in CD4(+) T cell-depleted mice. Both effects of the treatment absolutely required CD8(+) T cells. Analysis of the response in successfully treated mice revealed elevated levels of CD8(+) T cells specific for a nonameric peptide consisting of residues 180-188 of the melanocyte differentiation antigen tyrosinase-related protein (TRP)2. There was no evidence of reactivity to the melanocyte antigens gp100, tyrosinase, Mart1/MelanA, or TRP1. Fas-FasL interactions and perforin played a role in mounting the effector response, whereas the tumor necrosis factor pathway was not required. The cellular requirements for tumor rejection in this therapeutic setting were strikingly different from those in a prophylactic setting. In particular, if mice received a prophylactic vaccine consisting of anti-CTLA-4 and B16-GM-CSF before tumor challenge, full protection was obtained even in the absence of CD8(+) T cells. Our data demonstrate that therapeutic autoreactive CD8(+) T cell responses can effectively be generated in tumor-bearing mice and stresses the value of studying tumor immunity in a therapeutic rather than a prophylactic setting.

Autologous chondrocyte implantation is the current gold standard cell therapy for cartilage lesions. However, in some instances, the heavily compromised health of the patient can either impair or limit the recovery of the autologous chondrocytes and a satisfactory outcome of the implant. Allogeneic human articular chondrocytes (hAC) could be a good alternative, but the possible immunological incompatibility between recipient and hAC donor should be considered. Herein, we report that allogeneic hAC inhibited T lymphocyte response to antigen-dependent and -independent proliferative stimuli. This effect was maximal when T cells and hAC were in contact and it was not relieved by the addition of exogenous lymphocyte growth factor interleukin (IL)-2. More important, hAC impaired the differentiation of peripheral blood monocytes induced with granulocyte monocyte colony-stimulating factor and IL-4 (Mo) to professional antigen-presenting cells, such as dendritic cells (DC). Indeed, a marked inhibition of the onset of the CD1a expression and an ineffective downregulation of CD14 antigens was observed in Mo-hAC co-cultures. Furthermore, compared to immature or mature DC, Mo from Mo-hAC co-cultures did not trigger an efficacious allo-response. The prostaglandin (PG) E2 present in the Mo-hAC co-culture conditioned media is a putative candidate of the hAC-mediated inhibition of Mo maturation. Altogether, these findings indicate that allogeneic hAC inhibit, rather than trigger, immune response and strongly suggest that an efficient chondrocyte implantation could be possible also in an allogeneic setting.

Dendritic cells (DCs) play a key role in initiating and regulating the immune responses to pathogens, self-antigens, and cancers. Human blood DCs comprise a family of different subsets: plasmacytoid DCs (pDCs) and CD16+, CD1c/BDCA1+, and BDCA3+ (CD141+) myeloid DCs and possess different phenotypes and functional characteristics. Lung cancer is the most common cancer, with the highest morbidity and mortality in the world. However, which DC subset plays a leading role in the lung cancer immune responses is unclear. We reanalyzed C-type lectin domain family 9 member A (CLEC9A) and CD141 (THBD) gene expression profiles from the Cancer Genome Atlas (TCGA) database and performed the Kaplan-Meier survival analysis of overall survival for several cancers according to their expression levels. Next, we investigated the capacities of five human blood DC subsets to stimulate T cell proliferation and capture, process and (cross-) present tumor antigen. Human BDCA3+ (CD141+) DCs have a superior capacity to stimulate allogeneic CD4+T cells proliferation and induce superior Th1 response compared with other DC subsets. Interestingly, toll-like receptor (TLR) agonists have little effect on DCs to induce the proliferation of naïve CD4+ T cells, but contribute to their differentiation. Importantly, BDCA3+ (CD141+) DCs possess the most potent ability to cross-present human tumor antigen after their uptake of necrotic lung cancer cells despite their lower antigen uptake. These findings suggest that human BDCA3+ (CD141+) DCs are critical mediators of cytotoxic T lymphocyte responses against EGFR-positive lung cancer. Therefore, our findings may provide theoretical basis for the development of DC-based antitumor vaccines.

The mechanisms whereby autoreactive T cells escape peripheral tolerance establishing thus autoimmune diseases in humans remain an unresolved question. Here, we demonstrate that autoreactive polyfunctional CD8+ T cells recognizing self-antigens (i.e., vimentin, actin cytoplasmic 1, or non-muscle myosin heavy chain 9 epitopes) with high avidity, counter-regulate Tregs by killing them, in a consistent percentage of rheumatoid arthritis (RA) patients. Indeed, these CD8+ T cells express a phenotype and gene profile of effector (eff) cells and, upon antigen-specific activation, kill Tregs indirectly in an NKG2D-dependent bystander fashion in vitro. This data provides a mechanistic basis for the finding showing that AE-specific (CD107a+) CD8+ T killer cells correlate, directly with the disease activity score, and inversely with the percentage of activated Tregs, in both steady state and follow-up studies in vivo. In addition, multiplex immunofluorescence imaging analyses of inflamed synovial tissues in vivo show that a remarkable number of CD8+ T cells express granzyme-B and selectively contact FOXP3+ Tregs, some of which are in an apoptotic state, validating hence the possibility that CD8+ Teff cells can counteract neighboring Tregs within inflamed tissues, by killing them. Alternatively, the disease activity score of a different subset of patients is correlated with the expansion of a peculiar subpopulation of autoreactive low avidity, partially-activated (pa)CD8+ T cells that, despite they conserve the conventional naïve (N) phenotype, produce high levels of tumor necrosis factor (TNF)-α and exhibit a gene expression signature of a progressive activation state. Tregs directly correlate with the expansion of this autoreactive (low avidity) paCD8+ TN cell subset in vivo, and efficiently control their differentiation rather their proliferation in vitro. Interestingly, autoreactive high avidity CD8+ Teff cells or low avidity paCD8+ TN cells are significantly expanded in RA patients who would become non-responders or patients who would become responders to TNF-α inhibitor therapy, respectively. These data provide evidence of a previously undescribed role of such mechanisms in the progression and therapy of RA.

CD4+ T cell help is required for the generation of CD8+ cytotoxic T lymphocyte (CTL) memory. Here, we use genome-wide analyses to show how CD4+ T cell help delivered during priming promotes memory differentiation of CTLs. Help signals enhance IL-15-dependent maintenance of central memory T (TCM) cells. More importantly, help signals regulate the size and function of the effector memory T (TEM) cell pool. Helped TEM cells produce Granzyme B and IFNγ upon antigen-independent, innate-like recall by IL-12 and IL-18. In addition, helped memory CTLs express the effector program characteristic of helped primary CTLs upon recall with MHC class I-restricted antigens, likely due to epigenetic imprinting and sustained mRNA expression of effector genes. Our data thus indicate that during priming, CD4+ T cell help optimizes CTL memory by creating TEM cells with innate and help-independent antigen-specific recall capacities.

The maintenance of T-cell homeostasis must be tightly regulated. Here, we have identified a coordinated role of Poly(ADP-ribose) polymerase-1 (PARP-1) and PARP-2 in maintaining T-lymphocyte number and function. Mice bearing a T-cell specific deficiency of PARP-2 in a PARP-1-deficient background showed defective thymocyte maturation and diminished numbers of peripheral CD4+ and CD8+ T-cells. Meanwhile, peripheral T-cell number was not affected in single PARP-1 or PARP-2-deficient mice. T-cell lymphopenia was associated with dampened in vivo immune responses to synthetic T-dependent antigens and virus, increased DNA damage and T-cell death. Moreover, double-deficiency in PARP-1/PARP-2 in T-cells led to highly aggressive T-cell lymphomas with long latency. Our findings establish a coordinated role of PARP-1 and PARP-2 in T-cell homeostasis that might impact on the development of PARP-centred therapies.

Priming and activation of CD8+ T cell responses is crucial to achieve anti-viral and anti-tumor immunity. Live attenuated measles vaccine strains have been used successfully for immunization for decades and are currently investigated in trials of oncolytic virotherapy. The available reverse genetics systems allow for insertion of additional genes, including heterologous antigens. Here, we designed recombinant measles vaccine vectors for priming and activation of antigen-specific CD8+ T cells. For proof-of-concept, we used cytotoxic T lymphocyte (CTL) lines specific for the melanoma-associated differentiation antigen tyrosinase-related protein-2 (TRP-2), or the model antigen chicken ovalbumin (OVA), respectively. We generated recombinant measles vaccine vectors with TRP-2 and OVA epitope cassette variants for expression of the full-length antigen or the respective immunodominant CD8+ epitope, with additional variants mediating secretion or proteasomal degradation of the epitope. We show that these recombinant measles virus vectors mediate varying levels of MHC class I (MHC-I)-restricted epitope presentation, leading to activation of cognate CTLs, as indicated by secretion of interferon-gamma (IFNγ) in vitro. Importantly, the recombinant OVA vaccines also mediate priming of naïve OT-I CD8+ T cells by dendritic cells. While all vaccine variants can prime and activate cognate T cells, IFNγ release was enhanced using a secreted epitope variant and a variant with epitope strings targeted to the proteasome. The principles presented in this study will facilitate the design of recombinant vaccines to elicit CD8+ responses against pathogens and tumor antigens.

Recently, oncolytic vaccinia viruses (VACVs) have shown their potential to provide for clinically effective cancer treatments. The reason for this clinical usefulness is not only the direct destruction of infected cancer cells but also activation of immune responses directed against tumor antigens. For eliciting a robust antitumor immunity, a dominant T helper 1 (Th1) cell differentiation of the response is preferred, and such polarization can be achieved by activating the Toll-like receptor 3 (TLR3)-interferon regulatory factor 3 (IRF3) signaling pathway. However, current VACVs used as oncolytic viruses to date still encode several immune evasion proteins involved in the inhibition of this signaling pathway. By inactivating genes of selected regulatory virus proteins, we aimed for a candidate virus with increased potency to activate cellular antitumor immunity but at the same time with a fully maintained replicative capacity in cancer cells. The removal of up to three key genes (C10L, N2L, and C6L) from VACV did not reduce the strength of viral replication, both in vitro and in vivo, but resulted in the rescue of IRF3 phosphorylation upon infection of cancer cells. In syngeneic mouse tumor models, this activation translated to enhanced cytotoxic T lymphocyte (CTL) responses directed against tumor-associated antigens and neo-epitopes and improved antitumor activity.

RNA vaccines based on lipid nanoparticles (LNPs) with in vitro transcribed mRNA (IVT-mRNA) encapsulated are now a currently successful but still evolving modality of vaccines. One of the advantages of RNA vaccines is their ability to induce CD8+ T-cell-mediated cellular immunity that is indispensable for excluding pathogen-infected cells or cancer cells from the body. In this study, we report on the development of LNPs with an enhanced capability for inducing cellular immunity by using an ionizable lipid with a vitamin E scaffold. An RNA vaccine that contained this ionizable lipid and an IVT-mRNA encoding a model antigen ovalbumin (OVA) induced OVA-specific cytotoxic T cell responses and showed an antitumor effect against an E.G7-OVA tumor model. Vaccination with the LNPs conferred protection against lethal infection by Toxoplasma gondii using its antigen TgPF. The vitamin E scaffold-dependent type I interferon response was important for effector CD8+ T cell differentiation induced by the mRNA-LNPs. Our findings also revealed that conventional dendritic cells (cDCs) were essential for achieving CD8+ T cell responses induced by the mRNA-LNPs, while the XCR1-positive subset of cDCs, cDC1 specialized for antigen cross-presentation, was not required. Consistently, the mRNA-LNPs were found to selectively transfect another subset of cDCs, cDC2 that had migrated from the skin to lymph nodes, where they could make vaccine-antigen-dependent contacts with CD8+ T cells. The findings indicate that the activation of innate immune signaling by the adjuvant activity of the vitamin E scaffold and the expression of antigens in cDC2 are important for subsequent antigen presentation and the establishment of antigen-specific immune responses.

Aluminum-containing adjuvants have been used for nearly 100 years to enhance immune responses in billions of doses of vaccines. To date, only a few adjuvants have been approved for use in humans, among which aluminum-containing adjuvants are the only ones widely used. However, the medical need for potent and safe adjuvants is currently continuously increasing, especially those triggering cellular immune responses for cytotoxic T lymphocyte activation, which are urgently needed for the development of efficient virus and cancer vaccines. Manganese is an essential micronutrient required for diverse biological activities, but its functions in immunity remain undefined. We previously reported that Mn2+ is important in the host defense against cytosolic dsDNA by facilitating cGAS-STING activation and that Mn2+ alone directly activates cGAS independent of dsDNA, leading to an unconventional catalytic synthesis of 2'3'-cGAMP. Herein, we found that Mn2+ strongly promoted immune responses by facilitating antigen uptake, presentation, and germinal center formation via both cGAS-STING and NLRP3 activation. Accordingly, a colloidal manganese salt (Mn jelly, MnJ) was formulated to act not only as an immune potentiator but also as a delivery system to stimulate humoral and cellular immune responses, inducing antibody production and CD4+/CD8+ T-cell proliferation and activation by either intramuscular or intranasal immunization. When administered intranasally, MnJ also worked as a mucosal adjuvant, inducing high levels of secretory IgA. MnJ showed good adjuvant effects for all tested antigens, including T cell-dependent and T cell-independent antigens, such as bacterial capsular polysaccharides, thus indicating that it is a promising adjuvant candidate.

Cytokines play pleiotropic, antagonistic, and collaborative in viral disease. The high morbidity and mortality of coronavirus disease 2019 (COVID-19) make it a significant threat to global public health. Elucidating its pathogenesis is essential to finding effective therapy. A retrospective study was conducted on 71 patients hospitalized with COVID-19. Data on cytokines, T lymphocytes, and other clinical and laboratory characteristics were collected from patients with variable disease severity. The effects of cytokines on the overall survival (OS) and event-free survival (EFS) of patients were analyzed. The critically severe and severe patients had higher infection indexes and significant multiple organ function abnormalities than the mild patients (P < 0.05). IL-6 and IL-10 were significantly higher in the critically severe patients than in the severe and mild patients (P < 0.05). IL-6 and IL-10 were closely associated with white blood cells, neutrophils, T lymphocyte subsets, D-D dimer, blood urea nitrogen, complement C1q, procalcitonin C-reactive protein. Moreover, the IL-6 and IL-10 levels were closely correlated to dyspnea and dizziness (P < 0.05). The patients with higher IL-10 levels had shorter OS than the group with lower levels (P < 0.05). The older patients with higher levels of single IL-6 or IL-10 tended to have shorter EFS (P < 0.05), while the patients who had more elevated IL-6 and IL-10 had shorter OS (P < 0.05). The Cox proportional hazard model revealed that IL-6 was the independent factor affecting EFS. IL-6 and IL-10 play crucial roles in COVID-19 prognosis.

On the basis of immunological results, it is not in doubt that the immune system is able to recognize and eliminate transformed cells. A plethora of studies have investigated the immune system of patients with cancer and how it is prone to immunosuppression, due in part to the decrease in lymphocyte proliferation and cytotoxic activity. The series of experiments published following the demonstration by Dr Allison's group of the potential effect of anti-cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) paved the way for a new perception in cancer immunotherapy: Immune checkpoints. Several T cell‑co-stimulatory molecules including cluster of differentiation (CD)28, inducible T cell co-stimulatory, 4-1BB, OX40, glucocorticoid-induced tumor necrosis factor receptor-related gene and CD27, and inhibitory molecules including T cell immunoglobulin and mucin domain-containing-3, programmed cell death-1 (PD-1), programmed cell death ligand-1 (PD-L1), V-domain immunoglobulin suppressor of T cells activation, T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain, and B and T lymphocyte attenuator have been described in regulating T cell functions, and have been demonstrated to be essential targets in immunotherapy. In preclinical studies, glioblastoma multiforme, a high-grade glioma, the monotherapy targeting PD-1/PD-L1 and CTLA-4 resulted in increased survival times. An improved understanding of the pharmacodynamics and immune monitoring on glioma cancers, particularly in diffuse intrinsic pontine glioma (DIPG), an orphan type of cancer, is expected to have a major contribution to the development of novel therapeutic approaches. On the basis of the recent preclinical and clinical studies of glioma, but not of DIPG, the present review makes a claim for the importance of investigating the tumor microenvironment, the immune response and the use of immune checkpoints (agonists or antagonists) in preclinical/clinical DIPG samples by immune monitoring approaches and high-dimensional analysis. Evaluating the potential predictive and correlative biomarkers in preclinical and clinical studies may assist in answering certain crucial questions that may be useful to improve the clinical response in patients with DIPG.

Since the first appearance of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS- CoV-2) in China on December 2019, the world has now witnessed the emergence of the SARS- CoV-2 outbreak. Therefore, due to the high transmissibility rate of virus, there is an urgent need to design and develop vaccines against SARS-CoV-2 to prevent more cases affected by the virus. In this study, a computational approach is proposed for vaccine design against the envelope (E) protein of SARS-CoV-2, which contains a conserved sequence feature. First, we sought to gain potential B-cell and T-cell epitopes for vaccine designing against SARS-CoV-2. Second, we attempted to develop a multi-epitope vaccine. Immune targeting of such epitopes could theoretically provide defense against SARS-CoV-2. Finally, we evaluated the affinity of the vaccine to major histocompatibility complex (MHC) molecules to stimulate the immune system response to this vaccine. We also identified a collection of B-cell and T-cell epitopes derived from E proteins that correspond identically to SARS-CoV-2 E proteins. The in-silico design of our potential vaccine against E protein of SARS-CoV-2 demonstrated a high affinity to MHC molecules, and it can be a candidate to make a protection against this pandemic event.

Dendritic cells (DC) are powerful cells that play critical roles in anti-tumor immunity, and their use in cancer immunotherapy unlocks hidden capabilities as an effective therapeutic. In order to maximize the full potential of DC, we developed a DC vaccine named CellgramDC-WT1 (CDW). CDW was pulsed with WT1, an antigen commonly expressed in solid tumors, and induced with zoledronate to aid DC maturation. Although our previous study focused on using Rg3 as an inducer of DC maturation, problems with quality control and access led us to choose zoledronate as a better alternative. Furthermore, CDW secreted IL-12 and IFN-γ, which induced the differentiation of naïve T cells to active CD8+ T cells and elicited cytotoxic T lymphocyte (CTL) response against cancer cells with WT1 antigens. By confirming the identity and function of CDW, we believe CDW is an improved DC vaccine and holds promising potential in the field of cancer immunotherapy.

Tumour cells are characterized by having lost their differentiation state. They constitutively secrete small extracellular vesicles (sEV) called exosomes when they come from late endosomes. Dendrogenin A (DDA) is an endogenous tumour suppressor cholesterol-derived metabolite. It is a new class of ligand of the nuclear Liver X receptors (LXR) which regulate cholesterol homeostasis and immunity. We hypothesized that DDA, which induces tumour cell differentiation, inhibition of tumour growth and immune cell infiltration into tumours, could functionally modify sEV secreted by tumour cells. Here, we have shown that DDA differentiates tumour cells by acting on the LXRβ. This results in an increased production of sEV (DDA-sEV) which includes exosomes. The DDA-sEV secreted from DDA-treated cells were characterized for their content and activity in comparison to sEV secreted from control cells (C-sEV). DDA-sEV were enriched, relatively to C-sEV, in several proteins and lipids such as differentiation antigens, "eat-me" signals, lipidated LC3 and the endosomal phospholipid bis(monoacylglycero)phosphate, which stimulates dendritic cell maturation and a Th1 T lymphocyte polarization. Moreover, DDA-sEV inhibited the growth of tumours implanted into immunocompetent mice compared to control conditions. This study reveals a pharmacological control through a nuclear receptor of exosome-enriched tumour sEV secretion, composition and immune function. Targeting the LXR may be a novel way to reprogram tumour cells and sEV to stimulate immunity against cancer.