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.

Efficacy of bath-psoralen and ultraviolet A therapy for mycosis fungoides - retrospective analysis of 62 cases.

Photochemotherapy with psoralen and ultraviolet A (PUVA) is widely used for refractory skin diseases. Bathwater delivery of 8-methoxypsoralen (8-MOPS) with subsequent UVA irradiation (bath-PUVA) or oral administration of 8-MOPS with UVA is used to treat mycosis fungoides. We retrospectively analyzed 62 patients with mycosis fungoides (8 stage IA, 30 stage IB, 5 stage IIB, 18 stage IIIA, and 1 stage IVA2) treated with bath-PUVA at the Dermatology Clinic of Nagoya City University Hospital from November 2004 to December 2013. A complete response was achieved in 37 (59.7%) patients, a partial response was achieved in 16 (25.8%), and stable disease was achieved in 6 (9.7%). Progressive disease was observed in 3 (4.8%) patients. Almost all patients in stage IA/IB achieved a complete response. Of the 5 stage IIB patients, 2 achieved a partial response, 1 achieved stable disease, and 2 had progressive disease. The serum concentrations of soluble interleukin-2 receptor and lactate dehydrogenase decreased significantly following treatment with bath-PUVA (p < 0.001). We examined the risk factors of patients whose stage progressed despite PUVA treatment. A multivariate Cox regression analysis of risk factors associated with stage progression yielded a hazard ratio of 28.5 for stage IIb. Treatment with bath-PUVA is highly effective in the early stages of mycosis fungoides, and partially effective in advanced stages.

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.

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.

Regulatory T cells expressing abundant CTLA-4 on the cell surface with a proliferative gene profile are key features of human head and neck cancer.

FOXP3+ regulatory T (Treg) cells suppress anti-tumor immunity. The suppression of Treg cells is regulated by cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), whose expression on the cell surface is tightly regulated. Here we found that Treg cells expressing abundant CTLA-4 on the cell surface (surface-CTLA-4+ Treg) were expanded in human head and neck cancer tissues. RNA sequencing of surface-CTLA-4+ and surface-CTLA-4- Treg cells infiltrating human head and neck cancer tissues revealed that surface-CTLA-4+ Treg cells have a previously undescribed gene expression profile correlating to cell cycle, cell proliferation, and DNA replication. Moreover, surface-CTLA-4+ Treg cells were PD-1+ , actively proliferated and associated with CD45RA- FOXP3high Treg cells with strong suppressive function. Thus, surface-CTLA-4+ Treg cells with a proliferative gene expression signature and phenotype are key features of head and neck cancer. Targeting surface-CTLA-4+ Treg cells might be new strategies to evoke effective immune responses to head and neck cancer.

Ezh2 Controls Skin Tolerance through Distinct Mechanisms in Different Subsets of Skin Dendritic Cells.

Ezh2, a well-established epigenetic repressor, can down-regulate leukocyte inflammatory responses, but its role in cutaneous health remains elusive. Here we demonstrate that Ezh2 controls cutaneous tolerance by regulating Langerhans cell (LC) transmigration across the epidermal basement membrane directly via Talin1 methylation. Ezh2 deficiency impaired disassembly of adhesion structures in LCs, leading to their defective integrin-dependent emigration from the epidermis and failure in tolerance induction. Moreover, mobilization of Ezh2-deficient Langerin- dermal dendritic cells (dDCs) via high-dose treatment with a weak allergen restored tolerance, which is associated with an increased tolerogenic potential of Langerin- dDCs likely due to epigenetic de-repression of Aldh in the absence of Ezh2. Our data reveal novel roles for Ezh2 in governing LC- and dDC-mediated host protection against cutaneous allergen via distinct mechanisms.

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.

Bath-PUVA therapy improves impaired resting regulatory T cells and increases activated regulatory T cells in psoriasis.

BACKGROUND: Bath-psoralen plus ultraviolet light A (PUVA) therapy is an effective, safe, and inexpensive treatment for psoriasis. Psoriasis might be due to an unbalanced ratio of Th17 cells and regulatory T cells (Treg). The Treg functional ratio is significantly lower in patients with psoriasis compared with controls and is inversely correlated with the Psoriasis Area and Severity Index score. We previously reported that bath-PUVA therapy significantly increases the number of Treg and restores Treg function to almost normal in most patients with psoriasis. OBJECTIVES: We examined the effects of bath-PUVA therapy on three distinct Foxp3+ subsets: activated Treg (aTreg), resting Treg (rTreg), and cytokine-secreting non-suppressive T cells. METHODS: We enrolled 15 patients with psoriasis and 11 healthy controls. We examined aTreg, rTreg, and cytokine-secreting non-suppressive T cells in peripheral blood obtained from the psoriasis patients before and after every fifth bath-PUVA therapy session. RESULTS: Levels of aTreg, which are considered to have the strongest suppressive activity in patients with psoriasis, were significantly increased in the early bath-PUVA therapy sessions, and then diminished. Levels of rTreg were lower in psoriasis patients than in healthy controls, and increased during bath-PUVA therapy. CONCLUSIONS: Bath-PUVA therapy induced aTreg and rTreg concomitantly with an improvement in the psoriatic lesions, suggesting a mechanism for the effectiveness of bath-PUVA therapy for psoriasis patients.

The methyltransferase Ezh2 controls cell adhesion and migration through direct methylation of the extranuclear regulatory protein talin.

A cytosolic role for the histone methyltransferase Ezh2 in regulating lymphocyte activation has been suggested, but the molecular mechanisms underpinning this extranuclear function have remained unclear. Here we found that Ezh2 regulated the integrin signaling and adhesion dynamics of neutrophils and dendritic cells (DCs). Ezh2 deficiency impaired the integrin-dependent transendothelial migration of innate leukocytes and restricted disease progression in an animal model of multiple sclerosis. Direct methylation of talin, a key regulatory molecule in cell migration, by Ezh2 disrupted the binding of talin to F-actin and thereby promoted the turnover of adhesion structures. This regulatory effect was abolished by targeted disruption of the interactions of Ezh2 with the cytoskeletal-reorganization effector Vav1. Our studies reveal an unforeseen extranuclear function for Ezh2 in regulating adhesion dynamics, with implications for leukocyte migration, immune responses and potentially pathogenic processes.

Detection of T cell responses to a ubiquitous cellular protein in autoimmune disease.

T cells that mediate autoimmune diseases such as rheumatoid arthritis (RA) are difficult to characterize because they are likely to be deleted or inactivated in the thymus if the self antigens they recognize are ubiquitously expressed. One way to obtain and analyze these autoimmune T cells is to alter T cell receptor (TCR) signaling in developing T cells to change their sensitivity to thymic negative selection, thereby allowing their thymic production. From mice thus engineered to generate T cells mediating autoimmune arthritis, we isolated arthritogenic TCRs and characterized the self antigens they recognized. One of them was the ubiquitously expressed 60S ribosomal protein L23a (RPL23A), with which T cells and autoantibodies from RA patients reacted. This strategy may improve our understanding of the underlying drivers of autoimmunity.

Homeostasis of thymus-derived Foxp3+ regulatory T cells is controlled by ultraviolet B exposure in the skin.

Accumulating evidence shows that immunological tolerance induced by Ag administration together with UVB irradiation is dependent on Foxp3(+) CD4(+) regulatory T (Treg) cells. However, the mechanisms by which UVB controls Treg cells in the skin are currently unclear. In this study, we have shown that exposure to UVB induced expansion of Treg cells up to 50-60% of the CD4(+) T cells in the irradiated skin. The Treg cell expansion in the skin lasted for 2 wk after exposure, which contributed to homeostasis of Treg cells in the periphery later. UVB-expanded Treg cells formed clusters with dendritic cells and proliferated in situ. Furthermore, the expanded Treg cells appeared to derive from neuropilin 1(+) thymus-derived Treg (tTreg) cells in the periphery because UVB-expanded Treg cells possessed Treg cell-specific CpG hypomethylation pattern, as seen in tTreg cells. These results collectively indicate that homeostasis of tTreg cells is controlled by UVB exposure in the skin. UVB therapy may be useful for not only inflammatory skin disorders, but also autoimmunity, transplantation, and allergy.

Antigen delivery to CD11c+CD8- dendritic cells induces protective immune responses against experimental melanoma in mice in vivo.

Dendritic cells (DCs) are central modulators of immune responses and, therefore, interesting target cells for the induction of antitumor immune responses. Ag delivery to select DC subpopulations via targeting Abs to DC inhibitory receptor 2 (DCIR2, clone 33D1) or to DEC205 was shown to direct Ags specifically to CD11c(+)CD8(-) or CD11c(+)CD8(+) DCs, respectively, in vivo. In contrast to the increasing knowledge about the induction of immune responses by efficiently cross-presenting CD11c(+)CD8(+) DCs, little is known about the functional role of Ag-presenting CD11c(+)CD8(-) DCs with regard to the initiation of protective immune responses. In this study, we demonstrate that Ag targeting to the CD11c(+)CD8(-) DC subpopulation in the presence of stimulating anti-CD40 Ab and TLR3 ligand polyinosinic-polycytidylic acid induces protective responses against rapidly growing tumor cells in naive animals under preventive and therapeutic treatment regimens in vivo. Of note, this immunization protocol induced a mixed Th1/Th2-driven immune response, irrespective of which DC subpopulation initially presented the Ag. Our results provide important information about the role of CD11c(+)CD8(-) DCs, which have been considered to be less efficient at cross-presenting Ags, in the induction of protective antitumor immune responses.

Dendritic cells in the periphery control antigen-specific natural and induced regulatory T cells.

Dendritic cells (DCs) are specialized antigen-presenting cells that regulate both immunity and tolerance. DCs in the periphery play a key role in expanding naturally occurring Foxp3(+) CD25(+) CD4(+) regulatory T cells (Natural T-regs) and inducing Foxp3 expression (Induced T-regs) in Foxp3(-) CD4(+) T cells. DCs are phenotypically and functionally heterogeneous, and further classified into several subsets depending on distinct marker expression and their location. Recent findings indicate the presence of specialized DC subsets that act to expand Natural T-regs or induce Foxp3(+) T-regs from Foxp3(-) CD4(+) T cells. For example, two major subsets of DCs in lymphoid organs act differentially in inducing Foxp3(+) T-regs from Foxp3(-) cells or expanding Natural T-regs with model-antigen delivery by anti-DC subset monoclonal antibodies in vivo. Furthermore, DCs expressing CD103 in the intestine induce Foxp3(+) T-regs from Foxp3(-) CD4(+) T cells with endogenous TGF-ß and retinoic acid. In addition, antigen-presenting DCs have a capacity to generate Foxp3(+) T-regs in the oral cavity where many antigens and commensals exist, similar to intestine and skin. In skin and skin-draining lymph nodes, at least six DC subsets have been identified, suggesting a complex DC-T-reg network. Here, we will review the specific activity of DCs in expanding Natural T-regs and inducing Foxp3(+) T-regs from Foxp3(-) precursors, and further discuss the critical function of DCs in maintaining tolerance at various locations including skin and oral cavity.

Photo(chemo)therapy reduces circulating Th17 cells and restores circulating regulatory T cells in psoriasis.

BACKGROUND: Photo(chemo)therapy is widely used to treat psoriasis, the pathogenesis of which might be caused by an imbalance of Th17 cells/regulatory T cells (Treg). In the present study, we evaluated the effects of photo(chemo)therapy on the Th17/Treg balance and Treg function. METHODS: Peripheral blood was obtained from psoriasis patients treated with bath-psoralen ultraviolet A (UVA, n = 50) or narrowband ultraviolet B (UVB, n = 18), and age-matched healthy volunteers (n = 20). CD3(+)CD4(+)IL-17A(+) or CD4(+)CD25(+)Foxp3(+)cells were analyzed to estimate Th17 or Treg number by fluorescence-activated cell sorting. Moreover, CD4(+) CD25(-) T cells from patients treated with PUVA(n = 14) were incubated in CFSE and activated with or without CD4(+) CD25(+)T cells, and the suppressive function of CD4(+) CD25(+)T cells were analyzed. RESULTS: Photo(chemo)therapy significantly reduced Th17 levels from 5.66 ± 3.15% to 2.96 ± 2.89% in patients with increased Th17 (Th17/CD4>3.01% [mean+SD of controls]). In contrast, photo(chemo)therapy significantly increased Treg levels from 2.77 ± 0.75 to 3.40 ± 1.88% in patients with less than 4.07% Treg level, defined as the mean of controls. Furthermore, while Treg suppressed the CD4(+)CD25(-) T cell proliferation to a greater extent in controls (Treg Functional Ratio 94.4 ± 4.28%) than in patients (70.3±25.1%), PUVA significantly increased Treg Functional Ratio to 88.1 ± 6.47%. Th17 levels in severe patients (>30 PASI) were significantly higher as compared to controls. Th17 levels that were left after treatment in the patients not achieving PASI 50 (3.78 ± 4.18%) were significantly higher than those in the patients achieving PASI 75 (1.83±1.87%). Treg levels in patients achieving PASI 90 (4.89 ± 1.70%) were significantly higher than those in the patients not achieving PASI 90 (3.90 ± 1.66%). Treg levels prior to treatment with Th17 high decreased group (5.16 ± 2.20%) was significantly higher than that with Th17 high increased group (3.33 ± 1.39%). CONCLUSION: These findings indicate that Treg is dysfunctional in psoriasis patients, and photochemotherapy restores those dysfunctional Treg. Photo(chemo)therapy resolved the Th17/Treg imbalance in patients with psoriasis.

Dendritic cells from oral cavity induce Foxp3(+) regulatory T cells upon antigen stimulation.

Evidence is accumulating that dendritic cells (DCs) from the intestines have the capacity to induce Foxp3(+)CD4(+) regulatory T cells (T-regs) and regulate immunity versus tolerance in the intestines. However, the contribution of DCs to controlling immunity versus tolerance in the oral cavity has not been addressed. Here, we report that DCs from the oral cavity induce Foxp3(+) T-regs as well as DCs from intestine. We found that oral-cavity-draining cervical lymph nodes contained higher frequencies of Foxp3(+) T-regs and ROR-γt(+) CD4(+)T cells than other lymph nodes. The high frequency of Foxp3(+) T-regs in the oral-cavity-draining cervical lymph nodes was not dependent on the Toll like receptor (TLR) adaptor molecules, Myd88 and TICAM-1 (TRIF). In contrast, the high frequency of ROR-γt(+) CD4(+)T cells relies on Myd88 and TICAM-1. In vitro data showed that CD11c(+) DCs from oral-cavity-draining cervical lymph nodes have the capacity to induce Foxp3(+) T-regs in the presence of antigen. These data suggest that, as well as in the intestinal environment, antigen-presenting DCs may play a vital role in maintaining tolerance by inducing Foxp3(+) T-regs in the oral cavity.

TLR2-dependent induction of IL-10 and Foxp3+ CD25+ CD4+ regulatory T cells prevents effective anti-tumor immunity induced by Pam2 lipopeptides in vivo.

16 S-[2,3-bis(palmitoyl)propyl]cysteine (Pam2) lipopeptides act as toll-like receptor (TLR)2/6 ligands and activate natural killer (NK) cells and dendritic cells (DCs) to produce inflammatory cytokines and cytotoxic NK activity in vitro. However, in this study, we found that systemic injection of Pam2 lipopeptides was not effective for the suppression of NK-sensitive B16 melanomas in vivo. When we investigated the immune suppressive mechanisms, systemic injection of Pam2 lipopeptides induced IL-10 in a TLR2-dependent manner. The Pam2 lipopeptides increased the frequencies of Foxp3(+)CD4(+) regulatory T (T reg) cells in a TLR2- and IL-10- dependent manner. The T reg cells from Pam2-lipopeptide injected mice maintained suppressor activity. Pam2 lipopeptides, plus the depletion of T reg with an anti-CD25 monoclonal antibody, improved tumor growth compared with Pam2 lipopeptides alone. In conclusion, our data suggested that systemic treatment of Pam2 lipopeptides promoted IL-10 production and T reg function, which suppressed the effective induction of anti-tumor immunity in vivo. It is necessary to develop an adjuvant that does not promote IL-10 and T reg function in vivo for the future establishment of an anti-cancer vaccine.

Failure of mycoplasma lipoprotein MALP-2 to induce NK cell activation through dendritic cell TLR2.

Macrophage-activating lipopeptide 2 (MALP-2), a mycoplasmal diacylated lipopeptide with palmitic acid moiety (Pam2), activates Toll-like receptor (TLR) 2 to induce inflammatory cytokines. TLR2 is known to mature myeloid dendritic cells (mDC) to drive mDC contact-mediated natural killer (NK) cell activation. Here we tested if MALP-2 activates NK cells through stimulation of TLR2 on mDC. Although synthetic MALP-2 with 6 or 14 amino acids (a.a.) stretch (designated as s and f) matured mDC to induce IL-6, IL-12p40 and TNF-α to a similar extent, they far less activated NK cells than Pam2CSK4, a positive control of 6 a.a.-containing diacyl lipopeptide. MALP-2s and f were TLR2/6 agonists and activate the MyD88 pathway similar to Pam2CSK4, but MALP-2s having the CGNNDE sequence acted on mDC TLR2 to barely induce external NK activation. Even the s form, with slightly high induction of IL-6 compared to the f form, barely induced in vivo growth retardation of NK-sensitive implant tumor. Pam2CSK4 and MALP-2 have the common lipid moiety but different peptides, which are crucial for NK cell activation. The results infer that MALP-2 is applicable to a cytokine inducer but not to an adjuvant for antitumor NK immunotherapy.