Turning anecdotal irradiation-induced anticancer immune responses into reproducible in situ cancer vaccines via disulfiram/copper-mediated enhanced immunogenic cell death of breast cancer cells.

Irradiation (IR) induces immunogenic cell death (ICD) in tumors, but it rarely leads to the abscopal effect (AE); even combining IR with immune checkpoint inhibitors has shown only anecdotal success in inducing AEs. In this study, we aimed to enhance the IR-induced immune response and generate reproducible AEs using the anti-alcoholism drug, disulfiram (DSF), complexed with copper (DSF/Cu) to induce tumor ICD. We measured ICD in vitro and in vivo. In mouse tumor models, DSF/Cu was injected intratumorally followed by localized tumor IR, creating an in situ cancer vaccine. We determined the anticancer response by primary tumor rejection and assessed systemic immune responses by tumor rechallenge and the occurrence of AEs relative to spontaneous lung metastasis. In addition, we analyzed immune cell subsets and quantified proinflammatory and immunosuppressive chemokines/cytokines in the tumor microenvironment (TME) and blood of the vaccinated mice. Immune cell depletion was investigated for its effects on the vaccine-induced anticancer response. The results showed that DSF/Cu and IR induced more potent ICD under hypoxia than normoxia in vitro. Low-dose intratumoral (i.t.) injection of DSF/Cu and IR(12Gy) demonstrated strong anti-primary and -rechallenged tumor effects and robust AEs in mouse models. These vaccinations also increased CD8+ and CD4+ cell numbers while decreasing Tregs and myeloid-derived suppressor cells in the 4T1 model, and increased CD8+, dendritic cells (DC), and decreased Treg cell numbers in the MCa-M3C model. Depleting both CD8+ and CD4+ cells abolished the vaccine's anticancer response. Moreover, vaccinated tumor-bearing mice exhibited increased TNFα levels and reduced levels of immunosuppressive chemokines/cytokines. In conclusion, our novel approach generated an anticancer immune response that results in a lack of or low tumor incidence post-rechallenge and robust AEs, i.e., absence of or decreased spontaneous lung metastasis in tumor-bearing mice. This approach is readily translatable to clinical settings and may increase IR-induced AEs in cancer patients.

Modulation of immune-responses by DSF/Cu enhances the anti-tumor effects of DTX for metastasis breast cancer.

Metastasis has been one of the most important causes of death from breast cancer, and chemotherapy remains the major option for metastatic breast cancer. However, drug resistance and higher toxicity from chemotherapy have been an obstacle for clinical practice, and the combination of chemotherapy with immunotherapy has emerged as a promising treatment strategy. Here, we describe a therapy based on the combination of disulfiram (DSF) and Cu2+ with widely used cytotoxic docetaxel (DTX). DSF/Cu-induced immunogenic cell death promoted the release of type I interferon and human monocyte-induced dendritic cell maturation, which established a foundation for the combination with chemotherapy. Consequently, the combination of DSF/Cu and DTX resulted in significantly more potent anti-tumor effects in 4T1-bearing mice than in single therapy. The present study has shed new light on combining DSF/Cu-induced immune responses with traditional chemotherapeutic agents to achieve greater benefits for patients with metastasis.

Turning anecdotal irradiation-induced anti-cancer immune responses into reproducible in situ cancer vaccines via disulfiram/copper-mediated enhanced immunogenic cell death of breast cancer cells.

Irradiation (IR) induces immunogenic cell death (ICD) in tumors, but it rarely leads to the abscopal effect (AE). However, combining IR with immune checkpoint inhibitors has shown anecdotal success in inducing AEs. In this study, we aimed to enhance the IR-induced immune response and generate reproducible AEs using the anti-alcoholism drug disulfiram (DSF) and copper complex (DSF/Cu) via induction of tumor ICD. We measured ICD in vitro and in vivo. In mouse tumor models, DSF/Cu was injected intratumorally followed by localized tumor IR, creating an in situ cancer vaccine. We determined the anti-cancer response by primary tumor rejection and assessed systemic immune responses by tumor rechallenge and the occurrence of AEs, i.e., spontaneous lung metastasis. Additionally, we analyzed immune cell subsets and quantified proinflammatory and immunosuppressive chemokines/cytokines in the tumor microenvironment (TME) and blood of the vaccinated mice. Immune cell depletion was investigated for its effects on the vaccine-induced anti-cancer response. The results showed that DSF/Cu and IR induced more potent ICD under hypoxia than normoxia in vitro. Low-dose intratumoral injection of DSF/Cu and IR demonstrated strong anti-primary and -rechallenged tumor effects and robust AEs in mouse models. These vaccinations also increased CD8 + and CD4 + cell numbers while decreasing Tregs and myeloid-derived suppressor cells in the 4T1 model, and increased CD8+, DC, and decreased Treg cell numbers in the MCa-M3C model. Depleting both CD8 + and CD4 + cells abolished the vaccine's anticancer response. Moreover, vaccinated tumor-bearing mice exhibited increased TNFα levels and reduced levels of immunosuppressive chemokines/cytokines. In conclusion, our novel approach generated an anti-cancer immune response, resulting in a lack of or low tumor incidence post-rechallenge and robust AEs, i.e., the absence of or decreased spontaneous lung metastasis in tumor-bearing mice. This approach is readily translatable to clinical settings and may increase IR-induced AEs in cancer patients.

The regulation of Tfh cell differentiation by ß-hydroxybutyrylation modification of transcription factor Bcl6.

Transcriptional repressor B cell lymphoma 6 (Bcl6) is a major transcription factor involved in Tfh cell differentiation and germinal center response, which is regulated by a variety of biological processes. However, the functional impact of post-translational modifications, particularly lysine ß-hydroxybutyrylation (Kbhb), on Bcl6 remains elusive. In this study, we revealed that Bcl6 is modified by Kbhb to affect Tfh cell differentiation, resulting in the decrease of cell population and cytokine IL-21. Furthermore, the modification sites are identified from enzymatic reactions to be lysine residues at positions 376, 377, and 379 by mass spectrometry, which is confirmed by site-directed mutagenesis and functional analyses. Collectively, our present study provides evidence on the Kbhb modification of Bcl6 and also generates new insights into the regulation of Tfh cell differentiation, which is a starting point for a thorough understanding of the functional involvement of Kbhb modification in the differentiations of Tfh and other T cells.

Regulation of RDN on Th1/ILC1 cell imbalance in HFMD patients caused by EV71 infection.

Enterovirus 71 (EV71) infection is more likely to cause hand, foot and mouth disease (HFMD) in children, which can lead to neurogenic complications and higher mortality. As a commonly used clinical medicine, Reduning injection (RDN) helps to shorten the symptoms of patients with HFMD and facilitate the early recovery of children. However, the regulatory mechanism of RDN on the HFMD immune system disorder caused by EV71 remains to be discussed. This study collected detailed treatment data of 56 children with HFMD who entered the affiliated Children's Hospital of Nanjing Medical University during 2019. Retrospective analysis of clinical data showed that the symptoms of the RDN treatment group were improved compared with the untreated group. To explore its mechanism, the relevant detection indicators were detected by flow cytometry, enzyme-linked immunosorbent assay and real-time quantitative PCR. It was found that the number and function of innate immune (ILCs) and adaptive immunity (Th1, Th2 and secreted cytokines) were reduced, suggesting that RDN plays a role by regulating cellular immunity. The in vitro differentiation inhibition test further confirmed that RDN affected Th1 differentiation by inhibiting the expression of transcription factors on the basis of Th1 cell differentiation in vitro.

Immunomodulatory effects of platelets on the severity of hand, foot, and mouth disease infected with enterovirus 71.

BACKGROUND: Enterovirus 71 (EV71) infection contributes to hand, foot, and mouth disease (HFMD) with severe neurogenic complications, leading to higher morbidity. In addition to their typical roles in coagulation, platelets could serve as essential immune regulatory cells to play a key role in the pathogenesis of this viral infection. METHODS: Platelet parameters were measured using an automatic hematology analyzer. T-helper type 1 (Th1) and Th2 cells were analyzed by flow cytometry. The levels of cytokines and key transcription factors were determined. RESULTS: The levels of platelet count and plateletcrit were positively associated with the severity of HFMD. Th1 and Th2 cells as well as their corresponding cytokines were increased in the severe group compared to the healthy volunteers. Moreover, the levels of platelets were negatively correlated with the level of interferon-γ (IFN-γ), but positively correlated with the frequency of Th1 cells. Coculture of platelets and naive CD4+ T cells showed that platelets from mild patients promote Th1 cell differentiation and IFN-γ secretion. CONCLUSIONS: Our study has shown for the first time that the distinct roles of platelets are responsible for the regulation of pathogenic CD4+ T cell differentiation and function in the pathogenesis of HFMD caused by EV71. IMPACT: Our study has shown for the first time that the distinct roles of platelets are responsible for the regulation of pathogenic CD4+ T cell differentiation and function in the pathogenesis of HFMD caused by EV71. For the first time, we have discovered the role of platelets in children's HFMD caused by EV71 infection, which may provide a better treatment for HFMD in the future. This article describes new discoveries in platelet immunity.

Abnormalities of ILC1 in children with hand, foot and mouth disease during enterovirus 71 infection.

Children with HFMD due to EV71 infection are more likely to suffer from neurogenic complications, leading to higher morbidity and mortality. ILCs play crucial roles in the initiation of host immunity. However, the contribution of ILCs to the occurrence and development of HFMD due to EV71 infection remains to be explored. The results of our study showed that the levels of peripheral ILC1s and Th1 cells were increased in children with severe HFMD compared to healthy children, as were ILC1- and Th1-related cytokines and transcription factors. Furthermore, HFMD children with a higher frequency of circulating ILC1s exhibited a 2.9-fold greater risk of severity when HFMD was accompanied by VEM. Our study is the first to show that ILC1 abnormalities contribute to the pathogenesis of the severity of HFMD, in which ILC1s are aberrant increased and affect the cellular and humoral immunity. ILC1s could be used in the diagnosis of HFMD.

Gm15575 functions as a ceRNA to up-regulate CCL7 expression through sponging miR-686 in Th17 cells.

Compelling evidence has demonstrated that Th17 cells play an essential role in the pathogenesis of multiple sclerosis (MS). Long noncoding RNAs (lncRNAs) have been confirmed as vital regulators of immune cell differentiation and other functions. However, whether and how lncRNAs influence Th17 cell differentiation and functional behaviors remain largely unclear. Here, we identified that a lncRNA, namely Gm15575, is specifically enriched in Th17 cells and spleen tissues of EAE mice. Functionally, knockdown of Gm15575 in Th17 cells suppressed the secretion of IL17A. Mechanistically, Gm15575 served as a competing endogenous RNA (ceRNA) to block the function of miR-686, positively regulating the expression of CCL7, a pro-inflammatory chemokine with high expression in Th17 cells, and Th17 differentiation. Taken together, our study revealed that Gm15575-miR-686 axis promoted the progression of EAE by regulating Th17 differentiation and expression of CCL7 which elucidated the pathogenesis of autoimmune diseases at genetic level. Gm15575 can be involved in the course of Th17-related autoimmune diseases.

Anti-IL-12/23 p40 antibody attenuates chronic graft-versus-host disease with lupus nephritis via inhibiting Tfh cell in mice.

Systemic lupus erythematosus (SLE) is an autoimmune disease that is mainly caused by excessive accumulation of autoantibodies that target autoantibodies such as nucleic acids. T helper (Th) cell have been associated with the development of SLE. Typically, different subsets of Th cells secrete various cytokines to regulate the disease progression. IL-12 and IL-23 participate in the differentiation and activation of multiple Th cell subsets, including Th1, Th2, Th9, Th17, regulatory T (Treg) and follicular helper T (Tfh) cells. Because of the signature p40 subunit shared by IL-12 and IL-23, blocking IL-12/IL-23 signaling may interfere the differentiation of Th cell and directly inhibit the secretion of proinflammatory cytokines. In this study, we examined the effects of anti-IL-12/23 p40 antibody on chronic graft-versus-host disease with lupus nephritis, and found that the therapeutic effectiveness was mediated through the inhibition of Tfh cell in mice. Moreover, anti-IL-12/23 p40 antibody inhibited human Tfh cell differentiation in vitro. These results strongly suggest that Tfh cell contribute to the pathogenesis of SLE, and the neutralization of IL-12/IL-23 signaling during Tfh cell differentiation may be critical for the treatment of SLE.

Human IL-23R Cytokine-Binding Homology Region-Fc Fusion Protein Ameliorates Psoriasis via the Decrease of Systemic Th17 and ILC3 Cell Responses.

Interleukin (IL)-23 is considered an effective therapeutic target for the treatment of psoriasis because of the crucial role of the IL-23/IL-17 axis in the pathogenesis of psoriasis, and it has recently been reported to be involved in ILC3 cell differentiation. In this study, we report that eukaryotically expressed rhIL23R-CHR/Fc, as an endogenous extracellular receptor analogue, could be a natural antagonist in an imiquimod (IMQ)-induced psoriasis-like mouse model, including the antagonizing effect of suppressed inflammation in the skin lesion, decreased production of pro-inflammatory cells, and reduced the expression of pro-inflammatory factors. The rhIL23R-CHR/Fc fusion protein inhibits both innate immune and adaptive immune-mediated inflammatory responses. These findings shed light on rhIL23R-CHR/Fc as a promising candidate therapy for the treatment of psoriasis.