Mature dendritic cells enriched in regulatory molecules may control regulatory T cells and the prognosis of head and neck cancer.

We previously reported that regulatory T (Treg) cells expressing CTLA-4 on the cell surface are abundant in head and neck squamous cell carcinoma (HNSCC). The role of expanded Treg cells in the tumor microenvironment of HNSCC remains unclear. In this study, we reveal that the tumor microenvironment of HNSCC is characterized by the high expression of genes related to Treg cells, dendritic cells (DCs), and interleukin (IL)-17-related molecules. Increased expression of IL17A, IL17F, or IL23A contributes to a favorable prognosis of HNSCC. In the tumor microenvironment of HNSCC, IL23A and IL12B are expressed in mature dendritic cells enriched in regulatory molecules (mregDCs). The mregDCs in HNSCC are a migratory and mature phenotype; their signature genes strongly correlate with Treg signature genes in HNSCC. We also observed that IL17A was highly expressed in Th17 cells and exhausted CD8+ T cells in HNSCC. These data suggest that mregDCs in HNSCC may contribute to the prognosis by balancing Treg cells and effector T cells that produce IL-17. Targeting mregDCs may be a novel strategy for developing new immune therapies against HNSCC.

Foxp3+ CD4+ regulatory T cells control dendritic cells in inducing antigen-specific immunity to emerging SARS-CoV-2 antigens.

Regulatory T (Treg) cells, which constitute about 5-10% of CD4+T cells expressing Foxp3 transcription factor and CD25(IL-2 receptor α chain), are key regulators in controlling immunological self-tolerance and various immune responses. However, how Treg cells control antigen-specific immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains unclear. In this study, we examined the effect of transient breakdown of the immunological tolerance induced by Treg-cell depletion on adaptive immune responses against administered SARS-CoV-2 antigen, spike protein 1 (S1). Notably, without the use of adjuvants, transient Treg-cell depletion in mice induced anti-S1 antibodies that neutralized authentic SARS-CoV-2, follicular helper T cell formation and S1-binding germinal center B cell responses, but prevented the onset of developing autoimmune diseases. To further clarify the mechanisms, we investigated maturation of dendritic cells (DCs), which is essential to initiate antigen-specific immunity. We found that the transient Treg-cell depletion resulted in maturation of both migratory and resident DCs in draining lymph nodes that captured S1-antigen. Moreover, we observed S1-specific CD4+ T cells and CD8+ T cells with interferon-γ production. Thus, captured S1 was successfully presented by DCs, including cross-presentation to CD8+ T cells. These data indicate that transient Treg-cell depletion in the absence of adjuvants induces maturation of antigen-presenting DCs and succeeds in generating antigen-specific humoral and cellular immunity against emerging SARS-CoV-2 antigens. Finally, we showed that SARS-CoV-2 antigen-specific immune responses induced by transient Treg-cell depletion in the absence of adjuvants were compatible with those induced with an effective adjuvant, polyriboinosinic:polyribocytidyl acid (poly IC) and that the combination of transient Treg-cell depletion with poly IC induced potent responses. These findings highlight the capacity for manipulating Treg cells to induce protective adaptive immunity to SARS-CoV-2 with activating antigen-presenting DCs, which may improve the efficacy of ongoing vaccine therapies and help enhance responses to emerging SARS-CoV-2 variants.

Proenkephalin+ regulatory T cells expanded by ultraviolet B exposure maintain skin homeostasis with a healing function.

Regulatory T (Treg) cells, expressing CD25 (interleukin-2 receptor α chain) and Foxp3 transcription factor, maintain immunological self-tolerance and suppress various immune responses. Here we report a feature of skin Treg cells expanded by ultraviolet B (UVB) exposure. We found that skin Treg cells possessing a healing function are expanded by UVB exposure with the expression of an endogenous opioid precursor, proenkephalin (PENK). Upon UVB exposure, skin Treg cells were expanded with a unique TCR repertoire. Also, they highly expressed a distinctive set of genes enriched in "wound healing involved in inflammatory responses" and the "neuropeptide signaling pathway," as indicated by the high expression of Penk. We found that not only was PENK expression at the protein level detected in the UVB-expanded skin Treg (UVB-skin Treg) cells, but that a PENK-derived neuropeptide, methionine enkephalin (Met-ENK), from Treg cells promoted the outgrowth of epidermal keratinocytes in an ex vivo skin explant assay. Notably, UVB-skin Treg cells also promoted wound healing in an in vivo wound closure assay. In addition, UVB-skin Treg cells produced amphiregulin (AREG), which plays a key role in Treg-mediated tissue repair. Identification of a unique function of PENK+ UVB-skin Treg cells provides a mechanism for maintaining skin homeostasis.

Anti-oxidative Amino Acid L-ergothioneine Modulates the Tumor Microenvironment to Facilitate Adjuvant Vaccine Immunotherapy.

Cancer vaccines consist of a tumor-associated antigen (TAA) and adjuvant. These vaccines induce and activate proliferation of TAA-specific cytotoxic T lymphocytes (CTLs), suppressing tumor growth. The therapeutic efficacy of TAA-specific CTLs depends on the properties of tumor microenvironment. The environments make immunosuppressive by function of regulatory T cells and tumor-associated myeloid cells; thus, regulation of these cells is important for successful cancer immunotherapy. We report here that L-ergothioneine (EGT) with the adjuvant Toll-like receptor 2 (TLR2) ligand modulated suppressive microenvironments to be immune-enhancing. EGT did not augment DC-mediated CTL priming or affect CTL activation in draining lymph node and spleen. However, EGT decreased the immuno-suppressive function of tumor-associated macrophages (TAMs). TLR2 stimulation accompanied with EGT administration downregulated expression of PD-L1, CSF-1R, arginase-1, FAS ligand, and TRAIL in TAMs, reflecting reduction of CTL suppression. An anti-oxidative thiol-thione residue of EGT was essential to dampening CTL suppression. The effect was specific to the thiol-thione residue of EGT because no effect was observed with another anti-oxidant N-acetyl-L-cysteine (NAC). A CTL-suppressive environment made by TLR2 is relieved to be improved by the addition of EGT, which may ameliorate the efficacy of vaccine immunotherapy.

Hyperglycemia Is Associated with Psoriatic Inflammation in Both Humans and Mice.

Chronic low-grade inflammation can cause several metabolic syndromes. Patients with psoriasis, a chronic immunological skin inflammation, often develop diabetes. However, it is not clear to date how psoriasis leads to, or is correlated with, glucose intolerance. Here, we investigate whether psoriasis itself is correlated with hyperglycemia in humans and mice. In patients, the severity of psoriasis was correlated with high blood glucose levels, and treatment of psoriasis by phototherapy improved insulin secretion. Imiquimod-induced systemic and cutaneous inflammation in mice, with features of human psoriasis, also resulted in hyperglycemia. Although it should be determined if psoriasis-like cutaneous inflammation alone can induce hyperglycemia, imiquimod-treated mice showed impairment of insulin secretion without significant islet inflammation. Administration of anti-IL-17A monoclonal antibody improved hyperglycemia in patients with psoriasis and imiquimod-treated mice with psoriasiform features. These results suggest that hyperglycemia is highly associated with psoriasis, mainly through IL-17.

Vaccine immunotherapy with ARNAX induces tumor-specific memory T cells and durable anti-tumor immunity in mouse models.

Immunological checkpoint blockade therapies benefit a limited population of cancer patients. We have previously shown that vaccine immunotherapy with Toll-like receptor (TLR)3-adjuvant and tumor antigen overcomes anti-programmed death ligand-1 (PD-L1) resistance in mouse tumor models. In the present study, 4 different ovalbumin (OVA)-expressing tumor cell lines were implanted into syngeneic mice and subjected to anti-tumor immunotherapy using ARNAX and whole OVA protein. ARNAX is a TLR3-specific agonist that does not activate the mitochondrial antiviral-signaling protein (MAVS) pathway, and thus does not induce systemic inflammation. Dendritic cell priming and proliferative CTL were induced by ARNAX + OVA, but complete remission was achieved only in a PD-L1-low cell line of EG7. Addition of anti-PD-L1 antibody to the ARNAX + OVA therapy brought complete remission to another PD-L1-high subline of EG7. Tumor shrinkage but not remission was observed in MO5 in that regimen. We analyzed tumor cells and tumor-infiltrating immune cells to identify factors associated with successful ARNAX vaccine therapy. Tumors that responded to ARNAX therapy expressed high levels of MHC class I and low levels of PD-L1. The tumor-infiltrating immune cells in ARNAX-susceptible tumors contained fewer immunosuppressive myeloid cells with low PD-L1 expression. Combination with anti-PD-L1 antibody functioned not only within tumor sites but also within lymphoid tissues, augmenting the therapeutic efficacy of the ARNAX vaccine. Notably, ARNAX therapy induced memory CD8+ T cells and rejection of reimplanted tumors. Thus, ARNAX vaccine + anti-PD-L1 therapy enabled permanent remission against some tumors that stably present antigens.

Type I Interferon-Independent Dendritic Cell Priming and Antitumor T Cell Activation Induced by a Mycoplasma fermentans Lipopeptide.

Mycoplasma fermentans-derived diacylated lipoprotein M161Ag (MALP404) is recognized by human/mouse toll-like receptor (TLR) 2/TLR6. Short proteolytic products including macrophage-activating lipopeptide 2 (MALP2) have been utilized as antitumor immune-enhancing adjuvants. We have chemically synthesized a short form of MALP2 named MALP2s (S-[2,3-bis(palmitoyloxy)propyl]-CGNNDE). MALP2 and MALP2s provoke natural killer (NK) cell activation in vitro but only poorly induce tumor regression using in vivo mouse models loading NK-sensitive tumors. Here, we identified the functional mechanism of MALP2s on dendritic cell (DC)-priming and cytotoxic T lymphocyte (CTL)-dependent tumor eradication using CTL-sensitive tumor-implant models EG7 and B16-OVA. Programmed death ligand-1 (PD-L1) blockade therapy in combination with MALP2s + ovalbumin (OVA) showed a significant additive effect on tumor growth suppression. MALP2s increased co-stimulators CD80/86 and CD40, which were totally MyD88-dependent, with no participation of toll-IL-1R homology domain-containing adaptor molecule-1 or type I interferon signaling in DC priming. MALP2s + OVA consequently augmented proliferation of OVA-specific CTLs in the spleen and at tumor sites. Chemokines and cytolytic factors were upregulated in the tumor. Strikingly, longer duration and reinvigoration of CTLs in spleen and tumors were accomplished by the addition of MALP2s + OVA to α-PD-L1 antibody (Ab) therapy compared to α-PD-L1 Ab monotherapy. Then, tumors regressed better in the MALP2s/OVA combination than in the α-PD-L1 Ab monotherapy. Hence, MALP2s/tumor-associated antigens combined with α-PD-L1 Ab is a good therapeutic strategy in some mouse models. Unfortunately, numerous patients are still resistant to PD-1/PD-L1 blockade, and good DC-priming adjuvants are desired. Cytokine toxicity by MALP2s remains to be settled, which should be improved by chemical modification in future studies.

Toll-like receptor 3 signal augments radiation-induced tumor growth retardation in a murine model.

Radiotherapy induces anti-tumor immunity by induction of tumor antigens and damage-associated molecular patterns (DAMP). DNA, a representative DAMP in radiotherapy, activates the stimulator of interferon genes (STING) pathway which enhances the immune response. However, the immune response does not always parallel the inflammation associated with radiotherapy. This lack of correspondence may, in part, explain the radiation-resistance of tumors. Additive immunotherapy is expected to revive tumor-specific CTL facilitating radiation-resistant tumor shrinkage. Herein pre-administration of the double-stranded RNA, polyinosinic-polycytidylic acid (polyI:C), in conjunction with radiotherapy, was shown to foster tumor suppression in mice bearing radioresistant, ovalbumin-expressing Lewis lung carcinoma (LLC). Extrinsic injection of tumor antigen was not required for tumor suppression. No STING- and CTL-response was induced by radiation in the implant tumor. PolyI:C was more effective for induction of tumor growth retardation at 1 day before radiation than at post-treatment. PolyI:C targeted Toll-like receptor 3 with minimal effect on the mitochondrial antiviral-signaling protein pathway. Likewise, the STING pathway barely contributed to LLC tumor suppression. PolyI:C primed antigen-presenting dendritic cells in draining lymph nodes to induce proliferation of antigen-specific CTL. By combination therapy, CTL efficiently infiltrated into tumors with upregulation of relevant chemokine transcripts. Batf3-positive DC and CD8+ T cells were essential for therapeutic efficacy. Furthermore, polyI:C was shown to stimulate tumor-associated macrophages and release tumor necrosis factor alpha, which acted on tumor cells and increased sensitivity to radiation. Hence, polyI:C treatment prior to radiotherapy potentially induces tumor suppression by boosting CTL-dependent and macrophage-mediated anti-tumor responses. Eventually, polyI:C and radiotherapy in combination would be a promising therapeutic strategy for radiation-resistant tumors.

Ultraviolet B-Induced Maturation of CD11b-Type Langerin- Dendritic Cells Controls the Expansion of Foxp3+ Regulatory T Cells in the Skin.

Skin dendritic cells (DCs) are divided into several subsets with distinctive functions. This study shows a previously unappreciated role of dermal CD11b-type Langerin- DCs in maintaining immunological self-tolerance after UVB exposure. After UVB exposure, dermal CD11b-type Langerin- DCs upregulated surface CD86 expression, induced proliferation of Foxp3+ regulatory T (Treg) cells without exogenous Ags, and upregulated a set of genes associated with immunological tolerance. This Treg-expansion activity was significantly hampered by CD80/CD86 blockade in vivo. These results indicate that CD11b-type Langerin- DCs from the UVB-exposed skin are specialized to expand Treg cells in the skin, which suppress autoimmunity.

Tumor cell death by pattern-sensing of exogenous RNA: Tumor cell TLR3 directly induces necroptosis by poly(I:C) in vivo, independent of immune effector-mediated tumor shrinkage.

Poly(I:C) acts on dendritic cells to induce potent antitumor effects through the production of cytokines/interferons, activation of natural killer cells and proliferation of cytotoxic T lymphocytes. In some tumor or myeloid lineages, poly(I:C) seemed to induce necroptosis in concert with a pan-caspase inhibitor by directly acting on toll-like receptor (TLR) 3 in both in vivo and in vitro models.

The TLR3/TICAM-1 signal constitutively controls spontaneous polyposis through suppression of c-Myc in Apc Min/+ mice.

BACKGROUND: Intestinal tumorigenesis is promoted by myeloid differentiation primary response gene 88 (MyD88) activation in response to the components of microbiota in Apc Min/+ mice. Microbiota also contains double-stranded RNA (dsRNA), a ligand for TLR3, which activates the toll-like receptor adaptor molecule 1 (TICAM-1, also known as TRIF) pathway. METHODS: We established Apc Min/+ Ticam1 -/- mice and their survival was compared to survival of Apc Min/+ Myd88 -/- and wild-type (WT) mice. The properties of polyps were investigated using immunofluorescence staining and RT-PCR analysis. RESULTS: We demonstrate that TICAM-1 is essential for suppression of polyp formation in Apc Min/+ mice. TICAM-1 knockout resulted in shorter survival of mice compared to WT mice or mice with knockout of MyD88 in the Apc Min/+ background. Polyps were more frequently formed in the distal intestine of Apc Min/+ Ticam1 -/- mice than in Apc Min/+ mice. Infiltration of immune cells such as CD11b+ and CD8α+ cells into the polyps was detected histologically. CD11b and CD8α mRNAs were increased in polyps of Apc Min/+ Ticam1 -/- mice compared to Apc Min/+ mice. Gene expression of inducible nitric oxide synthase (iNOS), interferon (IFN)-γ, CXCL9 and IL-12p40 was increased in polyps of Apc Min/+ Ticam1 -/- mice. mRNA and protein expression of c-Myc, a critical transcription factor for inflammation-associated polyposis, were increased in polyps of Apc Min/+ Ticam1 -/- mice. A Lactobacillus strain producing dsRNA was detected in feces of Apc Min/+ mice. CONCLUSION: These results imply that the TLR3/TICAM-1 pathway inhibits polyposis through suppression of c-Myc expression and supports long survival in Apc Min/+ mice.

The Anti-Oxidant Ergothioneine Augments the Immunomodulatory Function of TLR Agonists by Direct Action on Macrophages.

L-Ergothioneine (EGT) is a naturally-occurring amino acid which is characterized by its antioxidant property; yet, the physiological role of EGT has yet to be established. We investigated the immune-enhancing properties of EGT, and found that it acts as a potentiator of toll-like receptor (TLR) signaling. When mouse bone marrow-derived macrophages (BMDMs) were pretreated with EGT, TLR signal-mediated cytokine production was augmented in BMDMs. The results were reproducible with TLR2, 3, 4 and 7 agonists. In particular, IL-6 and IL-12p40 were elevated further by pretreatment with EGT in BMDMs, suggesting the induction of M1 polarization. In co-culture assay with OT-II CD4+ T cells and splenic F4/80+ macrophages, EGT significantly induced Th17 skewing in CD4+ T cells. Thus, EGT is an immune modifier as well as a redox controller under TLR stimulation that induces M1 macrophages and a Th17 shift in inflammation.

Double-stranded RNA promotes CTL-independent tumor cytolysis mediated by CD11b+Ly6G+ intratumor myeloid cells through the TICAM-1 signaling pathway.

PolyI:C, a synthetic double-stranded RNA analog, acts as an immune-enhancing adjuvant that regresses tumors in cytotoxic T lymphocyte (CTL)-dependent and CTL-independent manner, the latter of which remains largely unknown. Tumors contain CD11b+Ly6G+ cells, known as granulocytic myeloid-derived suppressor cells (G-MDSCs) or tumor-associated neutrophils (TANs) that play a critical role in tumor progression and development. Here, we demonstrate that CD11b+Ly6G+ cells respond to polyI:C and exhibit tumoricidal activity in an EL4 tumor implant model. PolyI:C-induced inhibition of tumor growth was attributed to caspase-8/3 cascade activation in tumor cells that occurred independently of CD8α+/CD103+ dendritic cells (DCs) and CTLs. CD11b+Ly6G+ cells was essential for the antitumor effect because depletion of CD11b+Ly6G+ cells totally abrogated tumor regression and caspase activation after polyI:C treatment. CD11b+Ly6G+ cells that had been activated with polyI:C showed cytotoxicity and inhibited tumor growth through the production of reactive oxygen species (ROS)/reactive nitrogen species (RNS). These responses were abolished in either Toll/interleukin-1 receptor domain-containing adaptor molecule-1 (TICAM-1)-/- or interferon (IFN)-αß receptor 1 (IFNAR1)-/- mice. Thus, our results suggest that polyI:C activates the TLR3/TICAM-1 and IFNAR signaling pathways in CD11b+Ly6G+ cells in tumors, thereby eliciting their antitumor activity, independent of those in CD8α+/CD103+ DCs that prime CTLs.

Toll-like receptor 2 ligand and interferon-γ suppress anti-tumor T cell responses by enhancing the immunosuppressive activity of monocytic myeloid-derived suppressor cells.

CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs) suppress activation/proliferation of cytotoxic T cells, thereby hindering cancer immunotherapy. MDSCs are increased after adjuvant therapy with toll-like receptor (TLR) 2 ligands, such as Pam2CSK4, in tumor-bearing mice. However, it remains unknown if the activation of TLR2 in MDSCs affects their function and the therapeutic efficacy of TLR2 ligand. Here, we show that TLR2 signaling in CD11b+Ly6G-Ly6Chigh monocytic MDSCs (M-MDSCs), but not CD11b+Ly6G+Ly6Clow granulocytic MDSCs (G-MDSCs), enhances their immunosuppressive activity, thereby limiting anti-tumor T cell responses induced by TLR2-activated dendritic cells (DCs). iNOS induction was critical for Pam2CSK4-enhanced T cell suppression by M-MDSCs. iNOS was expressed in M-MDSC-derived macrophages, but not undifferentiated M-MDSCs, in cocultures with CD8+ T cells, CD11c+ DCs, antigen peptide and Pam2CSK4. Pam2CSK4 increased the differentiation frequency of M-MDSCs to macrophages, and iNOS expression required interferon-γ (IFN-γ) production by CD8+ T cells that had been transiently stimulated by M-MDSC-derived macrophages in an antigen/TLR2-dependent manner. Although Pam2CSK4 triggered DC maturation and tumor regression via induction of tumor antigen-specific cytotoxic T lymphocyte (CTL) responses in tumor-bearing mice, Pam2CSK4 plus antigen increased the frequency of iNOS+ macrophages in the tumor. Treatment with iNOS inhibitor enhanced the therapeutic efficacy of Pam2CSK4. Hence, the results suggest that TLR2 ligand and T cell-derived IFN-γ enhance M-MDSC-mediated immunosuppression, which may negatively regulate anti-tumor CTL response.

STING in tumor and host cells cooperatively work for NK cell-mediated tumor growth retardation.

An interferon-inducing DNA sensor STING participates in tumor rejection in mouse models. Here we examined what mechanisms contribute to STING-dependent growth retardation of B16 melanoma sublines by NK cells in vivo. The studies were designed using WT and STING KO black mice, and B16D8 (an NK-sensitive melanoma line having STING) and STING KO B16D8 sublines established for this study. The results from tumor-implant studies suggested that STING in host immune cells and tumor cells induced distinct profiles of chemokines including CXCL10, CCL5 and IL-33, and both participated in NK cell infiltration and activation in B16D8 tumor. Spontaneous activation of STING occurs in host-immune and tumor cells of this NK-sensitive tumor, thereby B16D8 tumor growth being suppressed in this model. Our data show that STING induces tumor cytotoxicity by NK cells through tumor and host immune cell network to contribute to innate surveillance and suppression of tumors in vivo.

Double-stranded RNA analog and type I interferon regulate expression of Trem paired receptors in murine myeloid cells.

BACKGROUND: Triggering receptors expressed on myeloid cells (Trem) proteins are a family of cell surface receptors used to control innate immune responses such as proinflammatory cytokine production in mice. Trem genes belong to a rapidly expanding family of receptors that include activating and inhibitory paired-isoforms. RESULTS: By comparative genomic analysis, we found that Trem4, Trem5 and Trem-like transcript-6 (Treml6) genes typically paired receptors. These paired Trem genes were murine-specific and originated from an immunoreceptor tyrosine-based inhibition motif (ITIM)-containing gene. Treml6 encoded ITIM, whereas Trem4 and Trem5 lacked the ITIM but possessed positively-charged residues to associate with DNAX activating protein of 12 kDa (DAP12). DAP12 was directly associated with Trem4 and Trem5, and DAP12 coupling was mandatory for their expression on the cell surface. In bone marrow-derived dendritic cells (BMDCs) and macrophages (BMDMs), and splenic DC subsets, polyinosinic-polycytidylic acid (polyI:C) followed by type I interferon (IFN) production induced Trem4 and Treml6 whereas polyI:C or other TLR agonists failed to induce the expression of Trem5. PolyI:C induced Treml6 and Trem4 more efficiently in BMDMs than BMDCs. Treml6 was more potentially up-regulated in conventional DC (cDCs) and plasmacytoid DC (pDCs) than Trem4 in mice upon in vivo stimulation with polyI:C. DISCUSSION: Treml6-dependent inhibitory signal would be dominant in viral infection compared to resting state. Though no direct ligands of these Trem receptors have been determined, the results infer that a set of Trem receptors are up-regulated in response to viral RNA to regulate myeloid cell activation through modulation of DAP12-associated Trem4 and ITIM-containing Treml6.

Live imaging of transforming growth factor-ß activated kinase 1 activation in Lewis lung carcinoma 3LL cells implanted into syngeneic mice and treated with polyinosinic:polycytidylic acid.

Transforming growth factor-ß activated kinase 1 (TAK1) has been shown to play a crucial role in cell death, differentiation, and inflammation. Here, we live-imaged robust TAK1 activation in Lewis lung carcinoma 3LL cells implanted into the s.c. tissue of syngeneic C57BL/6 mice and treated with polyinosinic:polycytidylic acid (PolyI:C). First, we developed and characterized a Förster resonance energy transfer-based biosensor for TAK1 activity. The TAK1 biosensor, named Eevee-TAK1, responded to stress-inducing reagents such as anisomycin, tumor necrosis factor-α, and interleukin1-ß. The anisomycin-induced increase in Förster resonance energy transfer was abolished by the TAK1 inhibitor (5z)-7-oxozeaenol. Activity of TAK1 in 3LL cells was markedly increased by PolyI:C in the presence of macrophages. 3LL cells expressing Eevee-TAK1 were implanted into mice and observed through imaging window by two-photon excitation microscopy. During the growth of tumor, the 3LL cells at the periphery of the tumor showed higher TAK1 activity than the 3LL cells located at the center of the tumor, suggesting that cells at the periphery of the tumor mass were under stronger stress. Injection of PolyI:C, which is known to induce regression of the implanted tumors, induced marked and homogenous TAK1 activation within the tumor tissues. The effect of PolyI:C faded within 4 days. These observations suggest that Eevee-TAK1 is a versatile tool to monitor cellular stress in cancer tissues.

Adjuvant for vaccine immunotherapy of cancer--focusing on Toll-like receptor 2 and 3 agonists for safely enhancing antitumor immunity.

Immune-enhancing adjuvants usually targets antigen (Ag)-presenting cells to tune up cellular and humoral immunity. CD141(+) dendritic cells (DC) represent the professional Ag-presenting cells in humans. In response to microbial pattern molecules, these DCs upgrade the maturation stage sufficient to improve cross-presentation of exogenous Ag, and upregulation of MHC and costimulators, allowing CD4/CD8 T cells to proliferate and liberating cytokines/chemokines that support lymphocyte attraction and survival. These DCs also facilitate natural killer-mediated cell damage. Toll-like receptors (TLRs) and their signaling pathways in DCs play a pivotal role in DC maturation. Therefore, providing adjuvants in addition to Ag is indispensable for successful vaccine immunotherapy for cancer, which has been approved in comparison with antimicrobial vaccines. Mouse CD8α(+) DCs express TLR7 and TLR9 in addition to the TLR2 family (TLR1, 2, and 6) and TLR3, whereas human CD141(+) DCs exclusively express the TLR2 family and TLR3. Although human and mouse plasmacytoid DCs commonly express TLR7/9 to respond to their agonists, the results on mouse adjuvant studies using TLR7/9 agonists cannot be simply extrapolated to human adjuvant immunotherapy. In contrast, TLR2 and TLR3 are similarly expressed in both human and mouse Ag-presenting DCs. Bacillus Calmette-Guerin peptidoglycan and polyinosinic-polycytidylic acid are representative agonists for TLR2 and TLR3, respectively, although they additionally stimulate cytoplasmic sensors: their functional specificities may not be limited to the relevant TLRs. These adjuvants have been posted up to a certain achievement in immunotherapy in some cancers. We herein summarize the history and perspectives of TLR2 and TLR3 agonists in vaccine-adjuvant immunotherapy for cancer.

Functional Alteration of Tumor-infiltrating Myeloid Cells in RNA Adjuvant Therapy.

Macrophages, as well as dendritic cells (DCs), are derived from myeloid progenitor cells. Recent evidence suggests that tumor-infiltrating macrophages differ in many aspects from conventional tissue macrophages, including nature, function and markers. Tumors usually contain various myeloid lineage cells in their non-parenchymal environment. In immunotherapy for cancer, tumor cells and non-parenchymal cells are exposed to tumor-associated antigens (TAA) and tumor-cell-derived nucleic acids. In addition, a dsRNA mimic, polyinosinic:polycytidylic acid (polyI:C), exhibits strong adjuvant activity, which acts both on the immune system and tumor constituents. Herein we discuss the RNA recognition system and unique cellular output in tumor-associated myeloid cells in response to immunotherapy. We especially focus on the mechanism by which RNA adjuvant alters the tumor-supportive nature of tumor-infiltrated myeloid cells to those with tumoricidal activity. We discuss how RNA administration makes tumor cells collapse and its significance of evoking cell death signals in tumor cells and macrophages. This knowledge will be applicable to the development of an alternative immunotherapy for cancer.