Nanovaccine-based strategies for lymph node targeted delivery and imaging in tumor immunotherapy.

Therapeutic tumor vaccines have attracted considerable attention in the past decade; they can induce tumor regression, eradicate minimal residual disease, establish lasting immune memory and avoid non-specific and adverse side effects. However, the challenge in the field of therapeutic tumor vaccines is ensuring the delivery of immune components to the lymph nodes (LNs) to activate immune cells. The clinical response rate of traditional therapeutic tumor vaccines falls short of expectations due to inadequate lymph node delivery. With the rapid development of nanotechnology, a large number of nanoplatform-based LN-targeting nanovaccines have been exploited for optimizing tumor immunotherapies. In addition, some nanovaccines possess non-invasive visualization performance, which is benefit for understanding the kinetics of nanovaccine exposure in LNs. Herein, we present the parameters of nanoplatforms, such as size, surface modification, shape, and deformability, which affect the LN-targeting functions of nanovaccines. The recent advances in nanoplatforms with different components promoting LN-targeting are also summarized. Furthermore, emerging LNs-targeting nanoplatform-mediated imaging strategies to both improve targeting performance and enhance the quality of LN imaging are discussed. Finally, we summarize the prospects and challenges of nanoplatform-based LN-targeting and /or imaging strategies, which optimize the clinical efficacy of nanovaccines in tumor immunotherapies.

Tumor cell-derived autophagosomes (DRibbles)-activated B cells induce specific naïve CD8+ T cell response and exhibit antitumor effect.

Dendritic cell (DC) vaccine has been proved to be an effective way in cancer immunotherapy in both preclinical and clinical studies. However, limitations in DC isolation and culture have hampered its practice and promoted the development of other antigen-presenting cells (APCs) sources to fulfill that role. Our previous studies have shown that B cells loaded by tumor cell-derived autophagosomes, which we named as DRibbles (defective ribosomal products-containing blebs), could reactivate DC-induced effector T cell response. In this study, the roles of DRibble-loaded B cells in priming naïve CD8+ T cell responses and controlling tumors were investigated. We found that high-mobility group box 1 protein (HMGB1) on DRibbles was involved in DRibble-induced B cell activation, and the DRibble-triggered B cell phagocytosis via the caveolae-mediated endocytosis pathway. By using OT-I mouse-derived T cells, we demonstrated that DRibble-loaded B cells could activate specific naïve CD8+ T cells in vitro and ex vivo. In a tumor-bearing mouse model, DRibble-loaded B cells elicited systemic antitumor immunity and significantly suppressed the tumor growth. Moreover, the antitumor efficacy of DRibble-loaded B cells was enhanced when they were combined with CpG and anti-CD40 stimulation. These results suggest that DRibble-loaded B cells represent a viable and practical therapeutic vaccination strategy that might have important clinical implications for tumor immunotherapy.

Current status and perspective of tumor immunotherapy for head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) have a high incidence and mortality rate, and investigating the pathogenesis and potential therapeutic strategies of HNSCC is required for further progress. Immunotherapy is a considerable therapeutic strategy for HNSCC due to its potential to produce a broad and long-lasting antitumor response. However, immune escape, which involves mechanisms including dyregulation of cytokines, perturbation of immune checkpoints, and recruitment of inhibitory cell populations, limit the efficacy of immunotherapy. Currently, multiple immunotherapy strategies for HNSCC have been exploited, including immune checkpoint inhibitors, costimulatory agonists, antigenic vaccines, oncolytic virus therapy, adoptive T cell transfer (ACT), and epidermal growth factor receptor (EGFR)-targeted therapy. Each of these strategies has unique advantages, and the appropriate application of these immunotherapies in HNSCC treatment has significant value for patients. Therefore, this review comprehensively summarizes the mechanisms of immune escape and the characteristics of different immunotherapy strategies in HNSCC to provide a foundation and consideration for the clinical treatment of HNSCC.

Thymosin α-1 Reverses M2 Polarization of Tumor-Associated Macrophages during Efferocytosis.

The immunologic effects of chemotherapy-induced tumor cell death are not completely understood. Accumulating evidence suggests that phagocytic clearance of apoptotic tumor cells, also known as efferocytosis, is an immunologically silent process, thus maintaining an immunosuppressive tumor microenvironment (TME). Here we report that, in the breast tumor microenvironment, thymosin α-1 (Tα-1) significantly reverses M2 polarization of IL10-producing tumor-associated macrophages (TAM) during efferocytosis induced by apoptotic cells. Mechanistically, Tα-1, which bound to phosphatidylserine on the surface of apoptotic tumor cells and was internalized by macrophages, triggered the activation of SH2-containing inositol 5'-phosphatase 1 (SHIP1) through the lysosomal Toll-like receptor 7 (TLR7)/MyD88 pathway, subsequently resulting in dephosphorylation of efferocytosis-activated TBK1 and reduction of efferocytosis-induced IL10. Tα-1 combined with epirubicin chemotherapy markedly suppressed tumor growth in an in vivo breast cancer model by reducing macrophage-derived IL10 and enhancing the number and function of tumor-infiltrating CD4+ and CD8+ T cells. In conclusion, Tα-1 improved the curative effect of chemotherapy by reversing M2 polarization of efferocytosis-activated macrophages, suggesting that Tα-1 injection immediately after chemotherapy may contribute to highly synergistic antitumor effects in patients with breast cancer. SIGNIFICANCE: Thymosin α-1 improves the curative effect of chemotherapy by reversing efferocytosis-induced M2 polarization of macrophages via activation of a TLR7/SHIP1 axis.

Peritumoral administration of DRibbles-pulsed antigen-presenting cells enhances the antitumor efficacy of anti-GITR and anti-PD-1 antibodies via an antigen presenting independent mechanism.

BACKGROUND: TNF receptor family agonists and checkpoint blockade combination therapies lead to minimal tumor clearance of poorly immunogenic tumors. Therefore, a need to enhance the efficacy of this combination therapy arises. Antigen-presenting cells (APCs) present antigen to T cells and steer the immune response through chemokine and cytokine secretion. DRibbles (DR) are tumor-derived autophagosomes containing tumor antigens and innate inflammatory adjuvants. METHODS: Using preclinical murine lung and pancreatic cancer models, we assessed the triple combination therapy of GITR agonist and PD-1 blocking antibodies with peritumoral injections of DRibbles-pulsed-bone marrow cells (BMCs), which consisted mainly of APCs, or CD103+ cross-presenting dendritic cells (DCs). Immune responses were assessed by flow cytometry. FTY720 was used to prevent T-cell egress from lymph nodes to assess lymph node involvement, and MHC-mismatched-BMCs were used to assess the necessity of antigen presentation by the peritumorally-injected DR-APCs. RESULTS: Tritherapy increased survival and cures in tumor-bearing mice compared to combined antibody therapy or peritumoral DR-BMCs alone. Peritumorally-injected BMCs remained within the tumor for at least 14 days and tritherapy efficacy was dependent on both CD4+ and CD8+ T cells. Although the overall percent of tumor-infiltrating T cells remained similar, tritherapy increased the ratio of effector CD4+ T cells-to-regulatory T cells, CD4+ T-cell cytokine production and proliferation, and CD8+ T-cell cytolytic activity in the tumor. Despite tritherapy-induced T-cell activation and cytolytic activity in lymph nodes, this T-cell activation was not required for tumor regression and enhanced survival. Replacement of DR-BMCs with DR-pulsed-DCs in the tritherapy led to similar antitumor effects, whereas replacement with DRibbles was less effective but delayed tumor growth. Interestingly, peritumoral administration of DR-pulsed MHC-mismatched-APCs in the tritherapy led to similar antitumor effects as MHC-matched-APCs, indicating that the observed enhanced antitumor effect was mediated independently of antigen presentation by the administered APCs. CONCLUSIONS: Overall, these results demonstrate that peritumoral DR-pulsed-BMC/DC administration synergizes with GITR agonist and PD-1 blockade to locally modulate and sustain tumor effector T-cell responses independently of T cell priming and perhaps through innate inflammatory modulations mediated by the DRibbles adjuvant. We offer a unique approach to modify the tumor microenvironment to benefit T-cell-targeted immunotherapies.

Tumor-released autophagosomes induces CD4+ T cell-mediated immunosuppression via a TLR2-IL-6 cascade.

BACKGROUND: CD4+ T cells are critical effectors of anti-tumor immunity, but how tumor cells influence CD4+ T cell effector function is not fully understood. Tumor cell-released autophagosomes (TRAPs) are being recognized as critical modulators of host anti-tumor immunity during tumor progression. Here, we explored the mechanistic aspects of TRAPs in the modulation of CD4+ T cells in the tumor microenvironment. METHODS: TRAPs isolated from tumor cell lines and pleural effusions or ascites of cancer patients were incubated with CD4+ T cells to examine the function and mechanism of TRAPs in CD4+ T cell differentiation and function. TRAPs-elicited CD4+ T cells were tested for their suppression of effector T cell function, induction of regulatory B cells, and promotion of tumorigenesis and metastasis in a mouse model. RESULTS: Heat shock protein 90α (HSP90α) on the surface of TRAPs from malignant effusions of cancer patients and tumor cell lines stimulated CD4+ T cell production of IL-6 via a TLR2-MyD88-NF-κB signal cascade. TRAPs-induced autocrine IL-6 further promoted CD4+ T cells secretion of IL-10 and IL-21 via STAT3. Notably, TRAPs-elicited CD4+ T cells inhibited CD4+ and CD8+ effector T cell function in an IL-6- and IL-10-dependent manner and induced IL-10-producing regulatory B cells (Bregs) via IL-6, IL-10 and IL-21, thereby promoting tumor growth and metastasis. Consistently, inhibition of tumor autophagosome formation or IL-6 secretion by CD4+ T cells markedly retarded tumor growth. Furthermore, B cell or CD4+ T cell depletion impeded tumor growth by increasing effector T cell function. CONCLUSIONS: HSP90α on the surface of TRAPs programs the immunosuppressive functions of CD4+ T cells to promote tumor growth and metastasis. TRAPs or their membrane-bound HSP90α represent important therapeutic targets to reverse cancer-associated immunosuppression and improve immunotherapy.

Polyethyleneimine modification of aluminum hydroxide nanoparticle enhances antigen transportation and cross-presentation of dendritic cells.

BACKGROUND: The aim of this study was to explore the feasibility of delivering tumor antigens and enhancing the antigen cross-presentation of dendritic cells (DCs) by aluminum hydroxide nanoparticle with polyethyleneimine (PEI) modification (LV@HPA/PEI). MATERIALS AND METHODS: The LV@HPA nanoparticles were modified by PEI first, then the influence of LV@HPA/PEI on DCs was examined. The distinct expression of ovalbumin (OVA) protein transported into DCs by LV@HPA/PEI was observed by flow cytometry and Western blot. The biocompatibility of LV@HPA/PEI, maturity and antigen cross-presentation of DCs was observed in vitro. Tumor derived autophagosomes (DRibbles) combined with LV@HPA/PEI were loaded into DCs, and DC vaccines were used to immunize mice. The percentage of CD3+CD8+IFN-γ+ T cells in immunized mice was determined by flow cytometry. Additionally, the functional properties of the LV@HPA/PEI-DRibble-DCs vaccine were examined in vivo in PancO2 tumor-bearing mice. RESULTS: In our study, we described how LV@HPA/PEI can be a functionalized antigen delivery system with notable antigen transport effect and negligible cytotoxicity. It was found that LV@HPA/PEI could be easily internalized into DCs to assist antigen release into the cytoplasm. In addition, DCs matured gradually after loading with LV@HPA/PEI-OVA, which increased significantly the cytokine IL-12 secretion and expression of surface molecules CD80 and CD86. Interestingly, DCs loaded with LV@HPA/PEI-DRibbles could promote the activation of tumor-specific T cells both in murine and in human T cells. In the following in vivo experiments, the vaccine of LV@HPA/PEI-DRibble-DCs significantly inhibited tumor growth and improved the survival rate of the PancO2 tumor-bearing mice. CONCLUSION: We established a high-performance anti-tumor vaccine of DCs loaded with LV@ HPA/PEI nanoparticles and tumor-associated antigens in autophagosomes (DRibbles), which could serve as a therapeutic strategy in cancer immunotherapy.

Tumor cell-released autophagosomes (TRAPs) promote immunosuppression through induction of M2-like macrophages with increased expression of PD-L1.

BACKGROUND: Tumor-associated macrophages (TAMs) facilitate tumor progression via establishment of an immunosuppressive tumor microenvironment (TME). However, it is poorly understood how tumor cells could functionally modulate TAMs. Our previous work indicated that tumor cell-released autophagosomes (TRAPs), a type of LC3-II+ double-membrane extracellular vesicles (EVs) was sufficient to suppress anti-tumor immune responses by inducing IL-10-producing B cells and immune suppressive neutrophils. Here, we hypothesized that TRAPs may participate in regulating macrophage polarization. METHODS: TRAPs isolated from multiple murine tumor cell lines and pleural effusions or ascites of cancer patients were incubated with bone marrow-derived macrophages (BMDMs) and monocytes, respectively. Cellular phenotypes were examined by flow cytometry, ELISA and quantitative PCR. TRAPs treated BMDMs were tested for the ability to suppress T-cell proliferation in vitro, and for promotion of tumor growth in vivo. Transwell chamber and neutralization antibodies were added to ascertain the inhibitory molecules expressed on BMDMs exposed to TRAPs. Knockout mice were used to identify the receptors responsible for TRAPs-induced BMDMs polarization and the signaling mechanism was examined by western blot. Autophagy-deficient tumors were profiled for phenotypic changes of TAMs and IFN-γ secretion of T cells by flow cytometry. The phenotype of monocytes from pleural effusions or ascites of cancer patients was assessed by flow cytometry. RESULTS: TRAPs converted macrophages into an immunosuppressive M2-like phenotype characterized by the expression of PD-L1 and IL-10. These macrophages inhibited the proliferation of both CD4+ and CD8+ T cells in vitro, and promoted tumor growth mainly through PD-L1 in vivo. TRAPs-induced macrophage polarization was dependent on TLR4-mediated MyD88-p38-STAT3 signaling. In vivo studies indicated that disruption of autophagosome formation in B16F10 cells by silencing the autophagy gene Beclin1 resulted in a remarkable delay in tumor growth, which was associated with reduced autophagosome secretion, TAMs reprogramming and enhanced T cell activation. Moreover, the levels of LC3B+ EVs appeared to correlate significantly with up-regulation of PD-L1 and IL-10 in matched monocytes from effusions or ascites of cancer patients, and TRAPs isolated from these samples could also polarize monocytes to an M2-like phenotype with increased expression of PD-L1, CD163 and IL-10, decreased expression of HLA-DR, and T cell-suppressive function. CONCLUSIONS: These findings suggest the TRAPs-PD-L1 axis as a major driver of immunosuppression in the TME by eliciting macrophage polarization towards an M2-like phenotype, and highlight the potential novel therapeutic approach of simultaneously targeting autophagy and PD-L1.