Identifying and controlling tumor escape mechanisms is crucial for improving cancer treatment effectiveness. Experimental studies reveal tumor hypoxia and adenosine as significant contributors to such mechanisms. Hypoxia exacerbates adenosine levels in the tumor microenvironment. Combining inhibition of these factors with dendritic cell (DC)-based immunotherapy promises improved clinical outcomes. However, challenges include understanding dynamics, optimal vaccine dosages, and timing. Mathematical models, including agent-based, diffusion, and ordinary differential equations, address these challenges. Here, we employ these models for the first time to elucidate how hypoxia and adenosine facilitate tumor escape in DC-based immunotherapy. After parameter estimation using experimental data, we optimize vaccination protocols to minimize tumor growth. Sensitivity analysis highlights adenosine\'s significant impact on immunotherapy efficacy. Its suppressive role impedes treatment success, but inhibiting adenosine could enhance therapy, as suggested by the model. Our findings shed light on hypoxia and adenosine-mediated tumor escape mechanisms, informing future treatment strategies. Additionally, identifiability analysis confirms accurate parameter determination using experimental data.

Dendritic cells mediate innate and adaptive immune responses and are directly involved in the activation of cytotoxic T lymphocytes that kill tumor cells. Dendritic cell-based cancer immunotherapy has clinical benefits. Dendritic cell subsets are diverse, and tumors can be hot or cold, depending on their immunogenicity; this heterogeneity affects the success of dendritic cell-based immunotherapy. Here, we review the ontogeny of dendritic cells and dendritic cell subsets. We also review the characteristics of hot and cold tumors and briefly introduce therapeutic trials related to hot and cold tumors. Lastly, we discuss dendritic cell-based cancer immunotherapy in hot and cold tumors.

The combination of radiotherapy and immunotherapy improves the survival rate of patients with malignancies developed through escape from T-cell-mediated immune surveillance. Immune checkpoint inhibitors, such as anti-programmed cell death protein-ligand 1 (anti-PD-L1) antibody, are used to rescue exhausted T cells. Simultaneously, dendritic cells (DCs) which are antigen-presenting cells that can initiate T-cell activation, are used to induce a tumor-specific immune response. However, the synergistic antitumor efficacy of the aforementioned combinational immunotherapy with intratumoral injection of low-dose DCs has not been reported, and the underlying therapeutic mechanism requires further investigation. Herein, we present the special case of a psoriatic patient with cutaneous squamous cell carcinoma (cSCC) in the right inguinal region, these two diseases characterized by opposing contradiction, further complicating treatments and side-effect management efforts. To treat the intractable SCC without exaggerating psoriasis, we developed the triple-regimen therapy (TRT) with the intratumoral injection of low-dose autologous DCs and anti-PD-L1 combined with radiotherapy. The injected DCs were obtained simply through leukapheresis without prior G-CSF administration for mobilization nor tumor-antigen loading for expansion. The patient received three radiation doses (24, 18, and 18 Gy) combined with three intratumoral injections of anti-PD-L1 antibody (40, 60, and 120 mg) plus autologous DCs (80% of the DC subpopulation being CD16+ myeloid DC with approximate amounts of 7.3 × 104, 2.5 × 106, and 1.7 × 107) within 10 weeks. The efficacy of the TRT was encouraging in shrinking tumor mass with remarkable SUVmax reduction (approximately 42%) on FDG PET-Scan despite relatively low-dose DCs were available. The low-dose intratumoral immunotherapy induced mild cutaneous side effects as expected. The transcriptomes were compared between pre-TRT and post-TRT biopsies to analyze underlying mechanical pathways of the TRT protocol. Over 10 highly significantly enriched T-cell-related pathways (P <0.0001) were identified in post-TRT biopsies. In addition, the activation of both innate and adaptive immunity was significantly enriched in post-TRT peripheral blood samples. We develop the easily accessible TRT which produces both local anti-tumor T-cell responses and systemic antitumor immunity for treating cSCC patients, especially for those with autoimmune disease.

Clonal anergy and depletion of antigen-specific CD8+ T cells are characteristics of immunosuppressed patients such as cancer and post-transplant patients. This has promoted translational research on the adoptive transfer of T cells to restore the antigen-specific cellular immunity in these patients. In the present work, we compared the capability of PBMCs and two types of mature monocyte-derived DCs (moDCs) to prime and to expand ex-vivo antigen-specific CD8+ T cells using culture conditioned media supplemented with IL-7, IL-15, and IL-21. The data obtained suggest that protocols involving moDCs are as efficient as PBMCs-based cultures in expanding antigen-specific CD8+ T cell to ELA and CMV model epitopes. These three gamma common chain cytokines promote the expansion of naïve-like and central memory CD8+ T cells in PBMCs-based cultures and the expansion of effector memory T cells when moDCs were used. Our results provide new insights into the use of media supplemented with IL-7, IL-15, and IL-21 for the in-vitro expansion of early-differentiated antigen-specific CD8+ T cells for immunotherapy purposes.

Allogeneic cancer cell lines serve as universal source of tumor-associated antigens in cancer vaccines. Immunogenic high hydrostatic pressure-killed cancer cells derived from cell lines can be used for the generation of dendritic cell (DC)-based active cellular immunotherapy of non-small cell lung cancer (NSCLC). We investigated the expression of 12 known NSCLC tumor-associated antigens (TAA) (CEA, MAGE-A1, MAGE-A3, MAGE-A4, PRAME, hTERT, HER2, MUC1, Survivin, STEAP1, SOX2 and NY-ESO-1) in 6 NSCLC cell lines as candidates for the generation of DC-based lung cancer vaccine. We showed that the selected antigenic profile of these cell lines overlaps to various degrees with that of primary NSCLC tumors (n = 52), indicating that 4 out of 6 NSCLC cell lines would be suitable for DC-based vaccine generation. We further investigated the presence of TAA-specific T cells in blood of NSCLC patients (n = 32) using commercially available peptide mixes in an in vitro stimulation assay. IFN-γ+CD8+ and IFN-γ+CD4+ T cell responses to all antigens were detected in NSCLC patients. Interestingly, despite higher TAA expression in squamous cell carcinoma (SCC) the responsiveness of patients\' T cells to stimulation was significantly lower in SCC patients than in adenocarcinoma (AC) patients. This suggests qualitative differences in T cell functionality between NSCLC subtypes. Based on this study, and in order to maximize the amount of treatable patients, we selected a mix of H520 and H522 NSCLC cell lines for DC-based vaccine preparation. We also established a minimal panel of antigenic peptide mixes (CEA, hTERT, PRAME, HER2) for immunomonitoring of T cell responses during the DC-based lung cancer immunotherapy in Phase I lung cancer clinical trial (NCT02470468).

Dendritic cells (DCs) are the professional antigen-presenting cells of the immune system. Proper function of DCs is crucial to elicit an effective immune response against pathogens and to induce antitumor immunity. Different members of the nuclear receptor (NR) family of transcription factors have been reported to affect proper function of immune cells. Nur77 is a member of the NR4A subfamily of orphan NRs that is expressed and has a function within the immune system. We now show that Nur77 is expressed in different murine DCs subsets in vitro and ex vivo, in human monocyte-derived DCs (moDCs) and in freshly isolated human BDCA1+ DCs, but its expression is dispensable for DC development in the spleen and lymph nodes. We show, by siRNA-mediated knockdown of Nur77 in human moDCs and by using Nur77-/- murine DCs, that Nur77-deficient DCs have enhanced inflammatory responses leading to increased T cell proliferation. Treatment of human moDCs with 6-mercaptopurine, an activator of Nur77, leads to diminished DC activation resulting in an impaired capacity to induce IFNγ production by allogeneic T cells. Altogether, our data show a yet unexplored role for Nur77 in modifying the activation status of murine and human DCs. Ultimately, targeting Nur77 may prove to be efficacious in boosting or diminishing the activation status of DCs and may lead to the development of improved DC-based immunotherapies in, respectively, cancer treatment or treatment of autoimmune diseases.

The aim of this study is to optimize the dendritic cell (DC)-mediated T-cell activation using reporter gene imaging and flow cytometric analysis in living mice.
A murine dendritic cell line (DC2.4) co-expressing effluc and Thy1.1 genes were established by transfection with retroviral vectors. Thy1.1 positive cells were sorted by magnetic bead separation system (DC2.4/effluc). Cell proliferation assay and phenotype analysis to determine the effects of gene transduction on the function of dendritic cells between parental DC2.4 and DC2.4/effluc were performed. To optimize the DC-mediated immune response by cell number or frequency, different cell numbers (5 × 105, 1 × 106, and 2 × 106 DC2.4/effluc) or different frequencies of DC2.4/effluc (first, second, and third injections) were injected in the right footpad of mice. The migration of the DC2.4/effluc into the draining popliteal lymph node of mice was monitored by bioluminescence imaging (BLI). Flow cytometric analysis was performed with splenocytes to determine the cytotoxic T-cell population after injection of DC2.4/effluc.
Parental DC2.4 and DC2.4/effluc exhibit no significant differences in their proliferation and phenotype. BLI signals were observed in the draining popliteal lymph node at day 1 after injection of DC2.4/effluc in 1 × 106 and 2 × 106 cells-injected groups. The highest BLI signal intensity was detected in 2 × 106 cells-injected mice. On day 11, the BLI signal was detected in only 2 × 106 cell-injected group but not in other groups. Optimized cell numbers (2 × 106) were injected in three animal groups with a different frequency (first, second, and third injection groups). The BLI signal was detected at day 1 and maintained until day 7 in the first injection group, but there is low signal intensity in the second and the third injection groups. Although the expression levels of Thy1.1 gene in the first injection group were very high, there reveals no expression of Thy1.1 gene in the second and the third injection groups. The number of tumor-specific CD8+ T-cells in the spleen significantly increased, as the number of DC injections increases.
Successful optimization of DC-mediated cytotoxic T-cell activation in living mice using reporter gene imaging and flow cytometric analysis was achieved. The optimization of DC-mediated cytotoxic T-cell activation could be applied for the future DC-based immunotherapy.

Considerable evidence shows that the tumor microenvironment is an active participant in preventing immunosurveillance and limiting the efficacy of anticancer therapies. Hypoxia is a prominent characteristic of the solid tumor microenvironment. The transcription factor hypoxia-inducible factor-1α (HIF-1α) is an important mediator of hypoxic response of tumor cells that modulates the expression of specific genes involved in tumor immunosuppression. Using a 4T1 breast cancer model, we show that in vivo administration of PX-478, an inhibitor of oxygen-sensitive HIF-1α, led to reduced expression of Foxp3 and VEGF transcript and/or protein, molecules that are directly controlled by HIF-1. When combined with dendritic cell (DC)-based vaccination, HIF-1α inhibition resulted in an augmented cytotoxic T lymphocyte effector function, improved proliferation status of T cells, increased production of inflammatory cytokine IFN-γ, as well as reduced regulatory function of T cells in association with slower tumor growth. Taken together, our findings indicate that the use of HIF-1α inhibition provides an immune adjuvant activity, thereby improves the efficacy of tumor antigen-based DC vaccine.