Chronic inflammation via dysregulation of T cell immune responses is critically involved in the pathogenesis of atherosclerotic cardiovascular disease. Improving the balance between proinflammatory T cells and anti-inflammatory regulatory T cells (Tregs) may be an attractive approach for treating atherosclerosis. Although C-C chemokine receptor 4 (CCR4) has been shown to mediate the recruitment of T cells to inflamed tissues, its role in atherosclerosis is unclear. Here, we show that genetic deletion of CCR4 in hypercholesterolemic mice accelerates the development of early atherosclerotic lesions characterized by an inflammatory plaque phenotype. This was associated with the augmentation of proinflammatory T helper type 1 (Th1) cell responses in peripheral lymphoid tissues, para-aortic lymph nodes, and atherosclerotic aorta. Mechanistically, CCR4 deficiency in Tregs impaired their suppressive function and tended to inhibit their migration to the atherosclerotic aorta, and subsequently augmented Th1 cell-mediated immune responses through defective regulation of dendritic cell function, which accelerated aortic inflammation and atherosclerotic lesion development. Thus, we revealed a previously unrecognized role for CCR4 in controlling the early stage of atherosclerosis via Treg-dependent regulation of proinflammatory T cell responses. Our data suggest that CCR4 is an important negative regulator of atherosclerosis.

Conventional type 1 dendritic cells (cDC1s) play a crucial role in antitumor immunity through the induction and activation of tumor-specific CD8+ cytotoxic T cells (CTLs). The chemokine XCL1 is a major chemotactic factor for cDC1s and its receptor XCR1 is selectively expressed on cDC1s. Here, we investigated the effect of intratumoral delivery of a highly active form of murine XCL1 (mXCL1-V21C/A59C) on cDC1-mediated antitumor immunity using a hydrophilic gel patch. The hydrophilic gel patch containing mXCL1-V21C/A59C increased cDC1 accumulation in the tumor masses and promoted their migration to the regional lymph nodes, resulting in enhanced induction of tumor-specific CTLs. Tumor-infiltrating cDC1s not only expressed XCR1 but also produced CXCL9, a ligand for CXCR3 which is highly expressed on CTLs and NK cells. Consequently, CTLs and NK cells were increased in the tumor masses of mice treated with mXCL1-V21C/A59C, while immunosuppressive cells such as monocyte-derived suppressive cells and regulatory T cells were decreased. We also confirmed that anti-CXCL9 treatment decreased the tumor infiltration of CTLs. The intratumoral delivery of mXCL1-V21C/A59C significantly decreased tumor growth and prolonged survival in E.G7-OVA and B16-F10 tumor-bearing mice. Furthermore, the antitumor effect of mXCL1-V21CA59C was enhanced in combination with anti-programmed cell death protein 1 treatment. Finally, using The Cancer Genome Atlas database, we found that XCL1 expression was positively correlated with tumor-infiltrating cDC1s and a better prognosis in melanoma patients. Collectively, our findings provide a novel therapeutic approach to enhance tumor-specific CTL responses through the selective recruitment of CXCL9-expressing cDC1s into the tumor masses.

Cancer tissue generally possesses an immunosuppressive microenvironment. However, some cancers are associated with lymphoid stroma (i.e., a widely developed tertiary lymphoid structure). The T-cell zone (paracortex) of secondary lymphoid organs, particularly lymph nodes, is characterized by an abundance of T-cell zone fibroblastic reticular cells (TCZ-FRCs) that express C-C motif chemokine ligand 21 (CCL21) and smooth muscle actin (SMA). We analyzed the presence of TCZ-FRCs in 30 cases of carcinomas with lymphoid stroma of the breast, stomach, colon, tongue, and skin. Immunohistochemistry corroborated the abundance of CCL21+ SMA+ TCZ-FRCs in the normal lymph nodes. In sharp contrast, all 30 carcinomas with lymphoid stroma displayed no CCL21+ SMA+ TCZ-FRCs despite the affluence of T cells. Real-time reverse transcription polymerase chain reaction confirmed a marked decrease in the messenger ribonucleic acid expression of CCL21 and its receptor C-C motif chemokine receptor 7 in cancer lymphoid stroma compared to that in lymph nodes. Next, we analyzed the T cell phenotypes. The cancer lymphoid stroma demonstrated an abundance of CD3+ CD62L- memory-type T cells, in contrast to the presence of CD3+ CD62L+ naïve- and central memory T cells in the T cell zone of lymphoid tissues. Our data demonstrated the following: 1) Cancer lymphoid stroma lacked TCZ-FRCs with abundance of more activated T cells than in lymph nodes and 2) these were common phenomena in cancer lymphoid stroma irrespective of the histological types and organs involved.

The oral cavity serves as the primary path through which substances from the outside world enter our body. Therefore, it functions as a critical component of host defense. Saliva is essential for maintaining a stable oral environment by catching harmful agents, including pathogens, allergens, and chemicals, in the air or food. CCL28, highly expressed in mucosal tissues, such as the colon and salivary glands, is a chemokine that attracts CCR10/CCR3 expressing cells. However, the role of CCL28 in salivary gland formation remains unclear. In this study, we investigated the salivary gland structure in CCL28-deficient mice. Histological analysis showed decreased staining intensity of Alcian blue, which detects acidic mucous, reduced expression of MUC2, and higher infiltration of gram-positive bacteria in the salivary glands of CCL28-deficient mice. In addition, CCL28-deficient mice contained ectopically MUC2-expressed cells in the ducts and reduced the expression of cytokeratin 18, a marker for ductal cells, within the submandibular glands, resulting in decreased duct numbers. Additionally, the submandibular glands of CCL28-deficient mice showed reduced expression of several stem cell markers. These results suggest that CCL28 regulates saliva production via proper differentiation of salivary gland stem cells and could be a valuable biomarker of salivary gland function.

Increasing evidence indicates that immune abnormalities are associated with the pathogenesis of depression. CCR4 is a chemokine receptor that regulates regulatory T cell (Treg) and Th17 cell migration. Here, using a lipopolysaccharide (LPS)-induced depression mouse model, we demonstrated that CCR4 deficiency exacerbated depressive-like behavior. Tregs and M2 macrophages, but not Th17 cells, were decreased in the brain of CCR4-deficient mice. Consistently, treatment with a CCR4 inhibitor reduced Tregs and M2 macrophages in the brain and exacerbated depressive-like behavior. Thus, CCR4 may contribute to the reduction of depressive symptoms by promoting Treg recruitment to the brain and subsequent M2 macrophage polarization.

Four major mucosal-associated chemokines, CCL25, CCL28, CXCL14, and CXCL17, play an important role in protecting mucosal surfaces from infectious pathogens. However, their role in protection against genital herpes remains to be fully explored. The CCL28 is a chemoattractant for the CCR10 receptor-expressing immune cells and is produced homeostatically in the human vaginal mucosa (VM). In this study, we investigated the role of the CCL28/CCR10 chemokine axis in mobilizing protective antiviral B and T cell subsets into the VM site of herpes infection. We report a significant increase in the frequencies of HSV-specific memory CCR10+CD44+CD8+ T cells, expressing high levels of CCR10, in herpes-infected asymptomatic (ASYMP) women compared with symptomatic women. Similarly, a significant increase in the CCL28 chemokine (a ligand of CCR10), was detected in the VM of herpes-infected ASYMP C57BL/6 mice, associated with the mobilization of high frequencies of HSV-specific effector memory CCR10+CD44+CD62L-CD8+ TEM cells and memory CCR10+B220+CD27+ B cells in the VM of HSV-infected ASYMP mice. Inversely, compared with wild-type C57BL/6 mice, the CCL28 knockout (CCL28-/-) mice (1) appeared to be more susceptible to intravaginal infection and reinfection with HSV type 2, and (2) exhibited a significant decrease in the frequencies of HSV-specific effector memory CCR10+CD44+CD62L-CD8+ TEM cells and of memory CD27+B220+ B cells in the infected VM. These findings suggest a critical role of the CCL28/CCR10 chemokine axis in the mobilization of antiviral memory B and T cells within the VM to protect against genital herpes infection and disease.

Abstract CCR4 is a major trafficking receptor for Th2 cells and Th17 cells and is considered as a potential therapeutic target for atopic dermatitis (AD). The CCR4 ligands CCL17 and CCL22 have been reported to be upregulated in the skin lesions of AD patients. Of note, Thymic stromal lymphopoietin (TSLP), a master regulator of the Th2 immune response, promotes the expression of CCL17 and CCL22 in AD skin lesions. Here, we investigated the role of CCR4 in an AD mouse model induced by MC903, a TSLP inducer. Topical application of MC903 to ear skin increased the expression of not only TSLP but also CCL17, CCL22, the Th2 cytokine IL-4, and the Th17 cytokine IL-17A. Consistently, MC903 induced AD-like skin lesions as shown by increased epidermal thickness; increased infiltration of eosinophils, mast cells, type 2 innate lymphoid cells, Th2 cells, and Th17 cells; and elevated serum levels of total IgE. We also found increased expansion of Th2 cells and Th17 cells in the regional lymph nodes (LNs) of AD mice. Compound 22, a CCR4 inhibitor, ameliorated AD-like skin lesions with reduction of Th2 cells and Th17 cells in the skin lesions and regional LNs. We further confirmed that compound 22 diminished the expansion of Th2 cells and Th17 cells in the coculture of CD11c + dendritic cells and CD4 + T cells derived from the regional LNs of AD mice. Collectively, CCR4 antagonists may exhibit anti-allergic effects by inhibiting both the recruitment and expansion of Th2 cells and Th17 cells in AD.

Vaginal mucosa-resident anti-viral effector memory B- and T cells appeared to play a crucial role in protection against genital herpes. However, how to mobilize such protective immune cells into the vaginal tissue close to infected epithelial cells remains to be determined. In the present study, we investigate whether and how, CCL28, a major mucosal-associated chemokine, mobilizes effector memory B- and T cells in leading to protecting mucosal surfaces from herpes infection and disease. The CCL28 is a chemoattractant for the CCR10 receptor-expressing immune cells and is produced homeostatically in the human vaginal mucosa (VM). We found the presence of significant frequencies of HSV-specific memory CCR10+CD44+CD8+ T cells, expressing high levels of CCR10 receptor, in herpes-infected asymptomatic (ASYMP) women compared to symptomatic (SYMP) women. A significant amount of the CCL28 chemokine (a ligand of CCR10), was detected in the VM of herpes-infected ASYMP B6 mice, associated with the mobilization of high frequencies of HSV-specific effector memory CCR10+CD44+ CD62L- CD8+ TEM cells and memory CCR10+B220+CD27+ B cells in the VM of HSV-infected asymptomatic mice. In contrast, compared to wild-type (WT) B6 mice, the CCL28 knockout (CCL28(-/-)) mice: (i) Appeared more susceptible to intravaginal infection and re-infection with HSV-2; (ii) Exhibited a significant decrease in the frequencies of HSV-specific effector memory CCR10+CD44+ CD62L- CD8+ TEM cells and of memory CD27+B220+ B cells in the infected VM. The results imply a critical role of the CCL28/CCR10 chemokine axis in the mobilization of anti-viral memory B and T cells within the VM to protect against genital herpes infection and disease.

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma associated with chronic inflammation (DLBCL-CI) develops in the setting of long-standing inflammation. This type of lymphoma may have specific expression profiles of chemokines involved in the pathogenesis of DLBCL-CI. EBV-positive pyothorax-associated lymphoma (PAL) is a prototype of DLBCL-CI and represents a valuable model for the study of this disease category. Using a panel of PAL cell lines, we found that PAL cells expressed and secreted C-X-C motif chemokine ligands 9 and 10 (CXCL9 and CXCL10), the ligands of CXCR3, in contrast to EBV-negative DLBCL cell lines, which did not. Culture supernatants from PAL cell lines attracted CXCR3-expressing CD4+ T cells, CD8+ T cells, and CD56+ natural killer cells from human peripheral blood mononuclear cells. PAL cells injected into mice also attracted CXCR3-positive cytotoxic lymphocytes that expressed interferon-γ. The expression of CXCL9 and CXCL10 was detected in PAL tumor biopsy samples from patients, and CXCR3-positive lymphocytes were abundant in the tissue samples. Collectively, these findings suggest that CXCL9 and CXCL10 are produced by PAL cells and can elicit cytotoxic responses via CXCR3. This chemokine system is also likely to contribute to tissue necrosis, which is a signature histological feature of DLBCL-CI. Further studies are warranted to determine whether the CXCL9-CXCL10/CXCR3 axis exerts antitumor effects in DLBCL-CI.

Single-cell transcriptomics has allowed unprecedented resolution of cell types/states in the human lung, but their spatial context is less well defined. To (re)define tissue architecture of lung and airways, we profiled five proximal-to-distal locations of healthy human lungs in depth using multi-omic single cell/nuclei and spatial transcriptomics (queryable at lungcellatlas.org ). Using computational data integration and analysis, we extend beyond the suspension cell paradigm and discover macro and micro-anatomical tissue compartments including previously unannotated cell types in the epithelial, vascular, stromal and nerve bundle micro-environments. We identify and implicate peribronchial fibroblasts in lung disease. Importantly, we discover and validate a survival niche for IgA plasma cells in the airway submucosal glands (SMG). We show that gland epithelial cells recruit B cells and IgA plasma cells, and promote longevity and antibody secretion locally through expression of CCL28, APRIL and IL-6. This new 'gland-associated immune niche' has implications for respiratory health.

Th17 cells express CCR4 and secrete cytokines such as IL-17A and GM-CSF, while dendritic cells (DCs) produce CCL22, a CCR4 ligand, upon stimulation with GM-CSF. Th17 cells are known to play a critical role in the pathogenesis of rheumatoid arthritis (RA). CCL22 has also been shown to be upregulated in the synovial tissues of RA patients. Here, we investigated the role of CCR4 in collagen-induced arthritis (CIA), a mouse model of RA. DBA/1J mice efficiently developed CIA as shown by erythema, paw swelling, joint rigidity, and joint destruction. Th17 cells were increased in the arthritic joints and regional lymph nodes (LNs) of CIA mice. A fraction of Th17 cells were also shown to produce GM-CSF. On the other hand, we observed no significant increases of Th2 cells or Treg cells, the T cell subsets also known to express CCR4, in these tissues. We further observed clusters of CCR4-expressing memory Th17 cells and CCL22-producing DCs in the regional LNs of CIA mice, supporting the role of the CCR4-CCL22 axis in the expansion of Th17 cells in the regional LNs. Compound 22, a CCR4 inhibitor, ameliorated the disease severity with reduction of Th17 cells in the arthritic joints and regional LNs and Th17-DC clusters in the regional LNs. We further confirmed that CCR4-deficient mice in the C57BL/6J background were highly resistant to CIA induction compared with wild-type mice. Collectively, CCR4 contributes to the pathogenesis of CIA and may thus represent a new therapeutic target for RA.

Atopic dermatitis (AD) is the most common inflammatory skin disease, which is characterized by excessive Th2 immune responses. In AD patients, the expression of the chemokines CCL17 and CCL22 is increased in skin lesions, leading to the infiltration of Th2 cells. In addition, typical pro-inflammatory cytokines, including TNF-α, IL-1β and IL-6, have also been shown to be associated with the pathogenesis of AD. Recently, DDH-1, an ascorbic acid derivative, has been synthesized and demonstrated to have a more stabilized structure and better skin penetrability. Furthermore, DDH-1 has been shown to suppress pro-inflammatory cytokine expression in vitro and in vivo. Therefore, using an AD mouse model, we evaluated the effect of DDH-1 to reduce allergic skin inflammation. We found that cutaneous administration of DDH-1 significantly reduced the expression levels of TNF-α, IL-1β and IL-6 in the skin lesions of AD-like mice. Additionally, DDH-1 administration also significantly reduced the expression levels of CCL17 and CCL22, resulting in decreased skin infiltration of Th2 cells. Consequently, DDH-1 reduced ear and epidermal thickness, the serum IgE levels and the number of infiltrating inflammatory cells and mast cells into the AD-like skin lesions. Combination treatment with DDH-1 and corticosteroid more efficiently improved the skin lesions compared with corticosteroid alone. Collectively, our results suggest that DDH-1 has an anti-allergic effect in an AD mouse model by reducing not only the pro-inflammatory cytokine expression but also the Th2-associated chemokine expression. Thus, DDH-1 may be beneficial for AD treatment and prevention as a monotherapy or in combination with corticosteroids.

Lung function deterioration is significantly associated with poor prognosis in patients with chronic obstructive pulmonary disease (COPD). We previously reported that CC chemokine ligand 17/thymus and activation-regulated chemokine (CCL17/TARC) could be a predictive factor of lung function decline in patients with COPD. However, the role of CCL17 in the pathogenesis of COPD is unclear. Here we examined the role of CCL17 in lung inflammation using mouse COPD models. Exposure to cigarette smoking induced CCL17 production in bronchial epithelial cells and accumulation of alveolar macrophages in the lungs. Intranasal administration of recombinant CCL17 further enhanced cigarette smoke-induced macrophage accumulation and also aggravated elastase-induced pulmonary emphysema. We confirmed that cigarette smoke (CS) extract as well as hydrogen peroxide upregulated CCL17 in BAES-2B cells. Of note, macrophages of both M1 and M2 surface markers were accumulated by cigarette smoke. Both alveolar macrophage accumulation via exposure to cigarette smoking and emphysematous changes induced by elastase administration were significantly reduced in CCL17-deficient mice. We further demonstrated that CCL17 strongly induced the expression of CC chemokine ligand 2 (CCL2), a chemoattractant for macrophages, in RAW264.7 cells, and its production was inhibited by knockdown of CCR4, the receptor of CCL17. Collectively, the present results demonstrate that CCL17 is produced by lung epithelial cells upon CS exposure. Furthermore, CCL17 is involved in CS-induced accumulation of alveolar macrophages and development of elastase-induced pulmonary emphysema, possibly through CCL17-induced production of CCL2 by macrophages. Our findings may provide a new insight into the pathogenesis of COPD.

Various immune cells are involved in host tumor immune responses. In particular, there are many T cell subsets with different roles in tumor immunity. T-helper (Th) 1 cells are involved in cellular immunity and thus play the major role in host anti-tumor immunity by inducing and activating cytotoxic T lymphocytes (CTLs). On the other hand, Th2 cells are involved in humoral immunity and suppressive to Th1 responses. Regulatory T (Treg) cells negatively regulate immune responses and contribute to immune evasion of tumor cells. Th17 cells are involved in inflammatory responses and may play a role in tumor progression. However, recent studies have also shown that Th17 cells are capable of directly inducting CTLs and thus may promote anti-tumor immunity. Besides these T cell subsets, there are many other innate immune cells such as dendritic cells (DCs), natural killer (NK) cells, and myeloid-derived suppressor cells (MDSCs) that are involved in host immune responses to cancer. The migratory properties of various immune cells are critical for their functions and largely regulated by the chemokine superfamily. Thus, chemokines and chemokine receptors play vital roles in the orchestration of host immune responses to cancer. In this review, we overview the various immune cells involved in host responses to cancer and their migratory properties regulated by the chemokine superfamily. Understanding the roles of chemokines and chemokine receptors in host immune responses to cancer may provide new therapeutic opportunities for cancer immunotherapy.

Psoriasis is a chronic skin disease associated with T helper (Th)17-mediated inflammation. Because CCR4 is a major chemokine receptor expressed on Th17 cells, we investigated the role of CCR4 in a modified imiquimod-induced psoriasis model that showed enhanced skin infiltration of Th17 cells. CCR4-deficient mice had less severe skin disease than wild-type mice. Th17 cells were decreased in the skin lesions and regional lymph nodes of CCR4-deficient mice. In the regional lymph nodes of wild-type mice, CD44+ memory Th17 cells expressing CCR4 were found to be clustered with dendritic cells expressing CCL22, a ligand for CCR4. Such dendritic cell‒Th17 cell clusters were significantly decreased in CCR4-deficient mice. Similar results were obtained using the IL-23‒induced psoriasis model. In vitro, compound 22, a CCR4 antagonist, significantly reduced the expansion of Th17 cells in the coculture of CD11c+ dendritic cells and CD4+ T cells separately prepared from the regional lymph nodes of wild-type mice with psoriasis. In vivo, compound 22 ameliorated the psoriasis-like skin disease in wild-type mice with significant decreases of Th17 cells in the regional lymph nodes and skin lesions. Collectively, CCR4 is likely to play a role in the pathogenesis of psoriasis through the expansion of Th17 cells.

Cancer immunotherapy aims to treat cancer by enhancing cancer-specific host immune responses. Recently, cancer immunotherapy has been attracting much attention because of the successful clinical application of immune checkpoint inhibitors targeting the CTLA-4 and PD-1/PD-L1 pathways. However, although highly effective in some patients, immune checkpoint inhibitors are beneficial only in a limited fraction of patients, possibly because of the lack of enough cancer-specific immune cells, especially CD8+ cytotoxic T-lymphocytes (CTLs), in the host. On the other hand, studies on cancer vaccines, especially DC-based ones, have made significant progress in recent years. In particular, the identification and characterization of cross-presenting DCs have greatly advanced the strategy for the development of effective DC-based vaccines. In this review, we first summarize the surface markers and functional properties of the five major DC subsets. We then describe new approaches to induce antigen-specific CTLs by targeted delivery of antigens to cross-presenting DCs. In this context, the chemokine receptor XCR1 and its ligand XCL1, being selectively expressed by cross-presenting DCs and mainly produced by activated CD8+ T cells, respectively, provide highly promising molecular tools for this purpose. In the near future, CTL-inducing DC-based cancer vaccines may provide a new breakthrough in cancer immunotherapy alone or in combination with immune checkpoint inhibitors.

FoxP3+ regulatory T cells (Tregs) play crucial roles in peripheral immune tolerance. In addition, Tregs that reside or accumulate in nonlymphoid tissues, called tissue Tregs, exhibit tissue-specific functions and contribute to the maintenance of tissue homeostasis and repair. In an experimental mouse model of crescentic glomerulonephritis induced by an anti-glomerular basement membrane antibody, Tregs started to accumulate in the kidney on day 10 of disease onset and remained at high levels (~30-35% of CD4+ T cells) during the late stage (days 21-90), which correlated with stable disease control. Treg depletion on day 21 resulted in the relapse of renal dysfunction and an increase in Th1 cells, suggesting that Tregs are essential for disease control during the convalescence stage. The Tregs that accumulated in the kidney showed tissue Treg phenotypes, including high expression of GATA3, ST2 (the IL33 receptor subunit), amphiregulin (Areg), and PPARγ. Although T-bet+ Tregs and RORγt+ Tregs were observed in the kidney, GATA3+ Tregs were predominant during the convalescence stage, and a PPARγ agonist enhanced the accumulation of GATA3+ Tregs in the kidney. To understand the function of specific genes in kidney Tregs, we developed a novel T cell transfer system to T cell-deficient mice. This experiment demonstrates that ST2, Areg, and CCR4 in Tregs play important roles in the accumulation of GATA3+ Tregs in the kidney and in the amelioration of renal injury. Our data suggest that GATA3 is important for the recruitment of Tregs into the kidney, which is necessary for convalescence after renal tissue destruction.

Exposure to moderate doses of ionizing radiation (IR), which is sufficient for causing skin injury, can occur during radiation therapy as well as in radiation accidents. Radiation-induced skin injury occasionally recovers, although its underlying mechanism remains unclear. Moderate-dose IR is frequently utilized for bone marrow transplantation in mice; therefore, this mouse model can help understand the mechanism. We had previously reported that bone marrow-derived cells (BMDCs) migrate to the epidermis-dermis junction in response to IR, although their role remains unknown. Here, we investigated the role of BMDCs in radiation-induced skin injury in BMT mice and observed that BMDCs contributed to skin recovery after IR-induced barrier dysfunction. One of the important mechanisms involved the action of CCL17 secreted by BMDCs on irradiated basal cells, leading to accelerated proliferation and recovery of apoptosis caused by IR. Our findings suggest that BMDCs are key players in IR-induced skin injury recovery.

Extracellular ATP is known to promote Th17 cell differentiation in the intestinal lamina propria by stimulating CD70+CD11clow dendritic cells (DCs) via P2X receptors (P2XRs). Recent studies have also shown that Th17 cells enhance antitumor immunity by directly promoting proliferation of cytotoxic T lymphocytes (CTLs). These finding led us to test a P2XR agonist, αβ-methylene ATP (αβ-ATP), as a mucosal vaccine adjuvant to promote CTL responses through Th17 induction. We demonstrated that (i) CD70+CD11clow DCs were present in the nasal lamina propria and expressed P2X1R, P2X2R and P2X4R; (ii) CD70+CD11clow DCs isolated from the nasal lamina propria enhanced Th17 cell differentiation of cocultured splenic CD4+ T cells upon stimulation with αβ-ATP; (iii) mice intranasally immunized with ovalbumin (OVA) and αβ-ATP had increased OVA-specific Th17 cells and CTLs in the nasal lamina propria and regional lymph nodes; (iv) mice intranasally immunized with OVA and αβ-ATP also had elevated resistance to E.G7-OVA tumor growth compared with those intranasally immunized with OVA alone; (v) suramin, a broad-range inhibitor of P2 receptors, suppressed the increases of OVA-specific Th17 cells and CTLs in mice intranasally immunized with OVA and αβ-ATP; and (vi) suramin also abrogated the enhanced antitumor immunity of mice intranasally immunized with OVA and αβ-ATP against E.G7-OVA. Collectively, αβ-ATP may be a promising mucosal adjuvant that promotes antigen-specific CTL responses via CD70+CD11clow DC-mediated Th17 induction.

Memory CD8+ cytotoxic T-lymphocytes (CTLs) play a key role in protective immunity against infection and cancer. However, the induction of memory CTLs with currently available vaccines remains difficult. The chemokine receptor XCR1 is predominantly expressed on CD103+ cross-presenting dendritic cells (DCs). Recently, we have demonstrated that a high activity form of murine lymphotactin/XCL1 (mXCL1-V21C/A59C), a ligand of XCR1, can induce antigen-specific memory CTLs by increasing the accumulation of CD103+ DCs in the vaccination site and the regional lymph nodes. Here, we combined a hydrophilic gel patch as a transcutaneous delivery device and mXCL1-V21C/A59C as an adjuvant to further enhance memory CTL responses. The transcutaneous delivery of ovalbumin (OVA) and mXCL1-V21C/A59C by the hydrophilic gel patch increased CD103+ DCs in the vaccination site and the regional lymph nodes for a prolonged period of time compared with the intradermal injection of OVA and mXCL1-V21C/A59C. Furthermore, the hydrophilic gel patch containing OVA and mXCL1-V21C/A59C strongly induced OVA-specific memory CTLs and efficiently inhibited the growth of OVA-expressing tumors more than the intradermal injection of OVA and mXCL1-V21C/A59C. Collectively, this type of hydrophilic gel patch and a high activity form of XCL1 may provide a useful tool for the induction of memory CTL responses.

免疫細胞は，生体内を循環することで異物侵入に対して全身をパトロールしているが，いざ異物が侵入してくると，その侵入部位へ即座に動員される．このような免疫細胞の細胞遊走には，方向性を持たないランダムな運動(chemokinesis)と細胞遊走因子の濃度勾配にしたがった方向性を持った運動(chemotaxis)に分けられる．Chemotaxis反応では，標的部位におけるケモカインの発現と免疫細胞におけるケモカイン受容体の選択的発現によって，免疫細胞の体内動態は緻密に制御されている．また細胞が，細胞外マトリックスに結合した細胞遊走因子と接触することで引き起こされる運動(haptotaxis)もある．

免疫系の主役となるリンパ球は，血管系とリンパ系を絶えず循環し，外界からの異物侵入に対してパトロール役として機能する．骨髄や胸腺などの一次リンパ組織で産生されたリンパ球は，二次リンパ組織に流入し，さらにその場を離れて全身を巡り，再び特定の二次リンパ組織に戻ってくる．この現象をリンパ球ホーミングという．この特定の組織への流入を制御する分子メカニズムには，リンパ球上に発現する接着分子やケモカイン受容体と標的組織の血管に発現するケモカインの組み合わせが重要である．例えば，リンパ節ホーミングには，接着分子L-セレクチンおよびケモカイン受容体CCR7が重要な役割を果たすことが明らかにされている．

Populations of CD8+ lung-resident memory T (TRM) cells persist in the interstitium and epithelium (airways) following recovery from respiratory virus infections. While it is clear that CD8+ TRM cells in the airways are dynamically maintained via the continuous recruitment of new cells, there is a vigorous debate about whether tissue-circulating effector memory T (TEM) cells are the source of these newly recruited cells. Here we definitively demonstrate that CD8+ TRM cells in the lung airways are not derived from TEM cells in the circulation, but are seeded continuously by TRM cells from the lung interstitium. This process is driven by CXCR6 that is expressed uniquely on TRM cells but not TEM cells. We further demonstrate that the lung interstitium CD8+ TRM cell population is also maintained independently of TEM cells via a homeostatic proliferation mechanism. Taken together, these data show that lung memory CD8+ TRM cells in the lung interstitium and airways are compartmentally separated from TEM cells and clarify the mechanisms underlying their maintenance.

Chemokines interact with their G protein-coupled receptors (GPCRs) through a two-step, two-site mechanism and, through this interaction, mediate various homeostatic and immune response mechanisms. Upon initial recognition of the chemokine by the receptor, the amino terminus of the chemokine inserts into the orthosteric pocket of the GPCR, causing conformational changes that trigger intracellular signaling. There is considerable structural and functional evidence to suggest that the amino acid composition and length of the chemokine amino terminus is critical for GPCR activation, complementing the size and amino acid composition of the orthosteric pocket. However, very few structures of a native chemokine-receptor complex have been solved. Here, we used a hybrid approach that combines structure-function data with Rosetta modeling to describe key contacts within a chemokine-GPCR interface. We found that the extreme amino-terminal residues of the chemokine XCL1 (Val1, Gly2, Ser3, and Glu4) contribute a large fraction of the binding energy to its receptor XCR1, whereas residues near the disulfide bond-forming residue Cys11 modulate XCR1 activation. Alterations in the XCL1 amino terminus changed XCR1 activation, as determined by assessing inositol triphosphate accumulation, intracellular calcium release, and directed cell migration. Computational analysis of XCL1-XCR1 interactions revealed functional contacts involving Glu4 of XCL1 and Tyr117 and Arg273 of XCR1. Subsequent mutation of Tyr117 and Arg273 led to diminished binding and activation of XCR1 by XCL1. These findings demonstrate the utility of a hybrid approach, using biological data and homology modeling, to study chemokine-GPCR interactions.

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphomas associated with chronic inflammation (DLBCL-CI) develop in patients with chronic inflammation but without any predisposing immunodeficiency. Given the expression of the EBV latent genes, DLBCL-CI should have mechanisms for evasion of host antitumor immunity. EBV-positive pyothorax-associated lymphoma (PAL) is a prototype of DLBCL-CI and may provide a valuable model for the study of immune evasion by DLBCL-CI. This study demonstrates that PAL cell lines express and secrete CCL17 and/or CCL22 chemokines, the ligands of C-C motif chemokine receptor 4 (CCR4), in contrast to EBV-negative DLBCL cell lines. Accordingly, culture supernatants of PAL cell lines efficiently attracted CCR4-positive regulatory T (Treg) cells in human peripheral blood mononuclear cells. PAL cells injected into mice also attracted CCR4-expressing Treg cells. Furthermore, this study confirmed that CCR4-expressing Treg cells were abundantly present in primary PAL tissues. Collectively, these findings provide new insight into the mechanisms of immune evasion by PAL, and further studies are warranted on whether such mechanisms eventually lead to the development of DLBCL-CI.

CCR4 is a chemokine receptor highly expressed by Th2 cells, and regarded as a potential therapeutic target for atopic dermatitis (AD). CCL17 and CCL22 are the CCR4 ligands, and thymic stromal lymphopoietin (TSLP) is shown to promote the expression of CCL17 and CCL22 by dendritic cells. Here, by using dibutyl phthalate (DBP), a TSLP inducer, and a hydrogel patch as a transcutaneous delivery device for ovalbumin, we developed a novel murine AD model and investigated the effect of Compound 22, a CCR4 antagonist. We first found that the mRNA expression of TSLP together with CCL17 and CCL22 was increased in the skins treated with DBP. Furthermore, the topical application of ovalbumin and DBP efficiently and rapidly induced AD-like skin lesions in BALB/c mice, which were characterized by ear swelling accompanied by infiltration of eosinophils, mast cells, and CCR4-expressing Th2 cells in the skin lesions, and elevated total IgE levels in the sera. Using this AD model, we demonstrated that cutaneous administration of Compound 22 inhibited Th2 cell infiltration and ameliorated the AD-like skin lesions. These results suggest that our AD model could be useful for studying new therapeutic strategies. Collectively, CCR4 antagonists may be a promising approach for treating AD.

In addition to maintaining immune tolerance, FOXP3+ regulatory T (Treg) cells perform specialized functions in tissue homeostasis and remodelling1,2. However, the characteristics and functions of brain Treg cells are not well understood because there is a low number of Treg cells in the brain under normal conditions. Here we show that there is massive accumulation of Treg cells in the mouse brain after ischaemic stroke, and this potentiates neurological recovery during the chronic phase of ischaemic brain injury. Although brain Treg cells are similar to Treg cells in other tissues such as visceral adipose tissue and muscle3-5, they are apparently distinct and express unique genes related to the nervous system including Htr7, which encodes the serotonin receptor 5-HT7. The amplification of brain Treg cells is dependent on interleukin (IL)-2, IL-33, serotonin and T cell receptor recognition, and infiltration into the brain is driven by the chemokines CCL1 and CCL20. Brain Treg cells suppress neurotoxic astrogliosis by producing amphiregulin, a low-affinity epidermal growth factor receptor (EGFR) ligand. Stroke is a leading cause of neurological disability, and there are currently few effective recovery methods other than rehabilitation during the chronic phase. Our findings suggest that Treg cells and their products may provide therapeutic opportunities for neuronal protection against stroke and neuroinflammatory diseases.

Psoriasis is a chronic inflammatory skin disease in which inflammatory cytokines play a major role in its pathogenesis. Because DDH-1, a novel amphipathic ascorbic acid derivative, has been recently shown to reduce inflammatory cytokine expression in human keratinocytes in vitro, we investigated its effect on imiquimod-induced psoriasis-like skin lesions in C57BL/6 mice. We first found that IL-1β and TNF-α mRNA expression was significantly decreased in the skin lesions treated with DDH-1. Furthermore, cutaneous administration of DDH-1 ameliorated psoriasis-like skin lesions. These results suggest that DDH-1 may be effective in the prevention and supplemental treatment of psoriasis.

Atopic dermatitis is a chronic inflammatory skin disease involving T-helper (Th) 2 cells, eosinophils, and mast cells. Although CCR4 is a major chemokine receptor expressed on Th2 cells and regarded as a potential therapeutic target for allergic diseases, its role in atopic dermatitis remains unclear. Here, by using a hydrogel patch as a transcutaneous delivery device for ovalbumin (an antigen) and Staphylococcus aureus δ-toxin (a mast cell activator), we efficiently induced acute atopic dermatitis-like skin lesions in BALB/c mice, a strain prone to Th2 responses, which were characterized by increased numbers of eosinophils, mast cells, and CCR4-expressing Th2 cells in the skin lesions; elevated levels of total and ovalbumin-specific IgE in the sera; and increased expression of IL-4, IL-17A, IL-22, CCL17, CCL22, and CCR4 in the skin lesions. Of note, the same model was less efficient in C57BL/6 mice, a strain prone to Th1 responses. Using this atopic dermatitis model in BALB/c mice, we demonstrated that CCR4-deficiency or a CCR4 antagonist ameliorated the allergic responses. Collectively, these results demonstrate that CCR4 plays a pivotal role in skin allergic inflammation of BALB/c mice by recruiting CCR4-expressing Th2 cells and Th17 cells.

CCR4 is a major chemokine receptor expressed by Treg cells that downregulate immune responses. Here, we investigated the role of CCR4-mediated Treg cell recruitment in antigen-specific immune responses. CCR4-deficient mice immunized intramuscularly with ovalbumin (OVA) showed enhanced OVA-specific IgG responses. Furthermore, intramuscular administration of OVA induced the expression of MDC/CCL22, a ligand for CCR4, in macrophages of the muscle tissues, and enhanced the recruitment of CCR4+ Treg cells in wild-type mice, whereas this recruitment of Treg cells was severely impaired in CCR4-deficient mice. Furthermore, OVA-loaded dendritic cells (DCs) derived from the muscle injection site of CCR4-deficient mice had an upregulated expression of the DC activation marker CD40 and 86, and the lymphoid organ homing receptor CCR7 resulting in an increased number of migratory DCs in the regional lymph node. Compound 22, a CCR4 antagonist, also inhibited the recruitment of Treg cells to the muscle tissue, and further enhanced DC activation and homing to the regional lymph node. Consequently, Compound 22 enhanced OVA-specific IgG responses, and the expression levels of IL-4 and IFN-γ in CD4+ T cells and the levels of IFN-γ in CD8+ T cells. Finally, intramuscular administration of OVA and Compound 22 significantly inhibited the growth of OVA-expressing tumors. Collectively, CCR4 plays a pivotal role in Treg cell recruitment to the muscle tissue, and intramuscular administration of CCR4 antagonists may be a promising approach for enhancing vaccine efficacy.

CCL28 induces the migration of IgA Ab-secreting cells (ASCs) via CCR10 and also displays a potent antimicrobial activity in vitro. To explore the role of CCL28 in vivo, we generated CCL28-deficient mice. The mice exhibited a significant reduction and abnormal distribution of IgA ASCs in the lamina propria of the colon. The concentrations of total and Ag-specific IgA in the fecal extracts of CCL28-deficient mice were also drastically reduced. The average amount of IgA secreted by a single IgA ASC derived from the colon was also substantially reduced in CCL28-deficient mice. Furthermore, CCL28 was found to significantly increase the average amount of IgA secreted by a single IgA ASC derived from the colon in vitro. In contrast, the generation of IgA ASCs in Peyer's and cecal patches was not significantly impaired in CCL28-deficient mice. We also found a relative increase in the Class Bacilli in the fecal extracts of CCL28-deficient mice and demonstrated a potent antimicrobial activity of CCL28 against Bacillus cereus and Enterococcus faecalis, both of which belong to Class Bacilli. Thus, CCL28 may also suppress the outgrowth of some bacterial species by its direct antimicrobial activity. Finally, CCL28-deficient mice exhibited a highly aggravated dextran sodium sulfate-induced colitis that was ameliorated by pretreatment with antibiotics. Collectively, CCL28 plays a pivotal role in the homing, distribution, and function of IgA ASCs in the colon and may also affect the intestinal microbiota through its direct antimicrobial activity.

The chemokine receptor XCR1 is known to be selectively expressed by cross-presenting dendritic cells (DCs), while its ligand XCL1/lymphotactin is mainly produced by activated CD8+ T cells and natural killer cells. Recent studies have shown that XCL1-antigen fusion proteins efficiently induce CD8+ T cell responses by preferentially delivering antigens to XCR1+ DCs. However, XCL1 per se was found to be a poor adjuvant for induction of CD8+ T cell responses. XCL1 is unique because of its lack of one of the two disulfide bonds commonly conserved in all other chemokines and thus has an unstable structure with a relatively weak chemokine activity. In the present study, we generated a variant form of murine XCL1 termed mXCL1-V21C/A59C that contained a second disulfide bond to stabilize its chemokine structure. We confirmed that mXCL1-V21C/A59C had much more potent chemotactic and calcium mobilization activities than the wild type XCL1 (mXCL1-WT). Intradermal injection of mXCL1-V21C/A59C, but not that of mXCL1-WT, significantly increased the accumulation of XCR1+CD103+ DCs in the injection site, and most of the accumulated XCR1+CD103+ DCs were found to take up co-injected ovalbumin (OVA). Furthermore, recruited XCR1+CD103+ DCs efficiently migrated to the draining lymph nodes and stayed for a prolonged period of time. Consequently, mXCL1-V21C/A59C strongly induced OVA-specific CD8+ T cells. The combination of OVA and mXCL1-V21C/A59C well protected mice from E.G7-OVA tumor growth in both prophylactic and therapeutic protocols. Finally, memory CTL responses were efficiently induced in mice immunized with OVA and mXCL1-V21C/A59C. Although intradermal injection of OVA and polyinosinic-polycytidylic acid (poly(I:C)) as an adjuvant also induced CD8+ T cell responses to OVA, poly (I:C) poorly recruited XCR1+CD103+ DCs in the injection site and failed to induce significant memory CTL responses to OVA. Collectively, our findings demonstrate that a highly active form of XCL1 is a promising vaccine adjuvant for cross-presenting DCs to induce antigen-specific effector and memory CD8+ T cells.

Resistance to the breakpoint cluster region-abelson 1 (BCR-ABL1) tyrosine kinase inhibitor (TKI) imatinib poses a major problem when treating chronic myeloid leukemia (CML). Imatinib resistance often results from a secondary mutation in BCR-ABL1. However, in the absence of a mutation in BCR-ABL1, the basis of BCR-ABL1-independent resistance must be elucidated. To gain insight into the mechanisms of BCR-ABL1-independent imatinib resistance, we performed an array-based comparative genomic hybridization. We identified various resistance-related genes, and focused on MET. Treatment with a MET inhibitor resensitized K562/IR cells to BCR-ABL1 TKIs. Combined treatment of K562/IR cells with imatinib and a MET inhibitor suppressed extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) activation, but did not affect AKT activation. Our findings implicate the MET/ERK and MET/JNK pathways in conferring resistance to imatinib, providing new insights into the mechanisms of BCR-ABL1 TKI resistance in CML.

CCR4 is a major chemokine receptor expressed by Treg cells and Th17 cells. While Treg cells are known to suppress antitumor immunity, Th17 cells have recently been shown to enhance the induction of antitumor cytotoxic T lymphocytes. Here, CCR4-deficient mice displayed enhanced tumor growth upon intradermal inoculation of B16-F10 melanoma cells. In CCR4-deficient mice, while IFN-γ+CD8+ effector T cells were decreased in tumor sites, IFN-γ+CD8+ T cells and Th17 cells were decreased in regional lymph nodes. In wild-type mice, CD4+IL-17A+ cells, which were identified as CCR4+CD44+ memory Th17, were found to be clustered around dendritic cells expressing MDC/CCL22, a ligand for CCR4, in regional lymph nodes. Compound 22, a CCR4 antagonist, also enhanced tumor growth and decreased Th17 cells in regional lymph nodes in tumor-bearing mice treated with Dacarbazine. In contrast, CCR6 deficiency did not affect the tumor growth and the numbers of Th17 cells in regional lymph nodes. These findings indicate that CCR4 is critically involved in regional lymph node DC-Th17 cell interactions that are necessary for Th17 cell-mediated induction of antitumor CD8+ effector T cells in mice bearing B16 melanoma.

Chemokine receptors CCR3 and CCR4 are preferentially expressed by TH2 cells, mast cells, and/or eosinophils, all of which are involved in the pathogenesis of allergic diseases. Therefore, CCR3 and CCR4 have long been highlighted as potent therapeutic targets for allergic diseases. Japanese traditional herbal medicine Kampo consists of multiple crude drugs/herbs, which further consist of numerous chemical substances. Recent studies have demonstrated that such chemical substances appear to promising sources in the development of novel therapeutic agents. Based on these findings, we hypothesize that Kampo-related crude drugs/herbs would contain chemical substances that inhibit the cell migration mediated by CCR3 and/or CCR4. To test this hypothesis, we screened 80 crude drugs/herbs to identify candidate substances using chemotaxis assay. Among those tested, Ephedra Herb inhibited the chemotaxis mediated by both CCR3 and CCR4, Cornus Fruit inhibited that mediated by CCR3, and Rhubarb inhibited that mediated by CCR4. Furthermore, Ephedra Herb specifically inhibited the chemotaxis mediated by not only CCR3 and CCR4 but CCR8, all of which are selectively expressed by TH2 cells. This result led us to speculate that ephedrine, a major component of Ephedra Herb, would play a central role in the inhibitory effects on the chemotaxis mediated by CCR3, CCR4, and CCR8. However, ephedrine exhibited little effects on the chemotaxis. Therefore, we fractionated Ephedra Herb into four subfractions and examined the inhibitory effects of each subfraction. As the results, ethyl acetate-insoluble fraction exhibited the inhibitory effects on chemotaxis and calcium mobilization mediated by CCR3 and CCR4 most significantly. In contrast, chloroform-soluble fraction exhibited a weak inhibitory effect on the chemotaxis mediated by CCR8. Furthermore, maoto, one of the Kampo formulations containing Ephedra Herb, exhibited the inhibitory effects on the chemotaxis mediated by CCR3, CCR4, and CCR8. Taken together, our data suggest that these crude drugs/herbs might be useful sources to develop new drugs targeting TH2-mediated allergic diseases.

我々は最近、Taxを発現しているHTLV-1感染細胞株においてTax siRNAにより発現が変動する遺伝子をマイクロアレイにより解析した結果、Tax依存性発現の可能性のある多数の遺伝子を同定した。その中に最近注目を浴びている翻訳伸長因子のeukaryotic elongation factor 1 alpha 2 (eEF1A2)が含まれていた。eEF1A2はEF-1αサブユニットのイソ型で、eEF1A1がハウスキーピング遺伝子として筋肉以外の多くの組織で発現しているのに対し、eEF1A2は主に筋肉と神経で発現している。両者は異なる染色体上にマップされ、92％のアミノ酸相同性を持つものの互いに排他的な発現調節を受けている。さらに興味深いことに、異所性に発現したeEF1A2はがん化との関連が報告されている。したがって、eEF1A2は発がん遺伝子としての性格も有しているが、その作用メカニズムについてはまだ十分明らかにされていない。 我々がeEF1A2に特に注目する理由は、最近明らかになってきているeEF1A2の翻訳伸長因子以外の作用である。eEF

成人T細胞性白血病において、Fra-2の過剰発現がCCR4と細胞増殖を引き起こすことを報告した。

【目的と意義】HTLV-1が感染してから数十年の潜伏感染の後、一部の感染者に成人T細胞白血病（ATL）が発症する。ATL腫瘍細胞の特徴は、本来は末梢血CD4＋T細胞の20%程度しか発現していないケモカイン受容体CCR4の高頻度発現（＞90%）である。前回我々は、HTLV-1感染細胞がTax依存性にCCL22を産生し、末梢血CCR4＋CD4＋T細胞を選択的に遊走させることを報告した。今回、HTLV-1初期感染におけるCCL22/CCR4の役割をさらに追究し、CD4＋T細胞への感染におけるCCL22/CCR4の役割を検討した｡ 【材料と方法】HTLV-1の感染様式を理解するために、①感染開始となる細胞間接着時､②感染早期(1週間前後)、③感染後期（2～3ヶ月）の各時期に分けて検討した｡HTLV-1感染細胞株と末梢血CCR4＋CD4＋T細胞を共培養し、CCR4特異的阻害剤あるいは百日咳毒素（PTX）処理が細胞間結合に与える影響を検討した｡また、マイトマイシンC処理したHTLV-1感染細胞とPHA処理した末梢血単核球細胞（PBMC）を共培養してHTLV-1感染実験

We examined the effect of Tax on expression of CCL22, a ligand of CCR4. Most Tax-expressing HTLV-1+ T cell lines produced a large amount of CCL22 in their culture supernatants. Transient expression of Tax also induced CCL22. Knockdown of Tax gene by siRNA resulted in a reduction of CCL22 mRNA. These results indicate CCL22 is one of the target genes of Tax. Culture supernatants from the HTLV-1+ T cell lines selectively attracted primary CCR4+CD4+ T cells in chemotaxis assays. The migration was blocked by pretreatment of the supernatants with anti-CCL22 Ab or pretreatment of PBMCs with a CCR4-specific antagonist. Further, HTLV-1+ T cell lines showed significant CCR4-dependent cell-to-cell interactions with sorted primary CCR4+CD4+ T cells, and anti-CCL22 Ab inhibited HTLV-1 primary infection. These results indicate that HTLV-1-infected T cells produce CCL22 upon expression of Tax and selectively recruit CCR4+CD4+ T cells, resulting in preferential transmission of HTLV-1 to these cells.

Most Tax+ HTLV-1+ T cell lines produced large amounts of CCL22 in their culture supernatants. Transient Tax expression also expressed CCL22. siRNA-induced knockdown of Tax resulted in a significant reduction of CCL22 mRNA in Tax+ cells. These results indicated CCL22 is one of the target genes of Tax. By using PBMCs from healthy donors in chemotaxis assays, it was demonstrated that culture supernatants of Tax+ cells strongly mobilized CCR4+CD4+ T cells. The migration was blocked by pretreatment of the supernatants with anti-CCL22 neutralizing antibody or pretreatment of the PBMCs with a CCR4-specific inhibitor. Further, Tax+ cells showed significant CCR4-dependent cell-cell interactions with the sorted CCR4+CD4+ T cells. These results imply that HTLV-1+ T cells, which were vertically transmitted from the breast milk of a carrier mother, produced CCL22 upon expression of Tax and preferentially recruited CCR4+CD4+ T cells in the child.

（目的）ケモカインは白血球の主要な遊走制御因子であるが、近年、一部のケモカインにおいて抗菌活性を有することが報告された。抗菌ペプチドの一部はエンベロープウイルスに対しても抗ウイルス活性を持つことから、今回、ケモカインによる抗ウイルス活性について検討を行った。 （方法）単純ヘルペスウイルス1型（HSV-1）のプラーク解析を指標に、ケモカインによる抗ウイルス活性を探索した。結合解析によりケモカインとHSV-1との直接の結合を確認した。ケモカインのHSV-1粒子構造への影響を電子顕微鏡により検討した。さらに、ヘルペス性角膜実質炎（HSK）モデルマウスを用いて、ケモカインによる治療効果についても検討を行った。 （結果）22のケモカインのうちRANTES、MIP-1aおよびMIP-1bを含む8つのケモカインはHSV-1のVero細胞への感染を優位に抑制した。これらのケモカインは直接HSV-1と結合し、その結合はエンベロープタンパクgBに対する中和抗体で特異的に阻害された

目的と意義】HTLV-1が感染してから数十年の潜伏感染の後、一部の感染者に成人T細胞白血病（ATL）が発症する。我々は以前、ATL腫瘍細胞の特徴として、本来末梢血CD4+T細胞で高々10%程度しか発現していないケモカイン受容体CCR4が高頻度に発現していることを報告した（Blood, 2002）｡今回、ATL細胞がCCR4+CD4＋T細胞に集束されるメカニズムの1つとして、HTLV-1感染初期に発現すると考えられるTax蛋白との関係に注目した。Taxは、細胞遺伝子の発現制御や発がん関連遺伝子の修飾を通して腫瘍化の初期に重要な役割を果たすと考えられている。我々はCCR4発現細胞を選択的に細胞遊走するケモカインMDCとTaxとの関係を検討した｡ 【材料と方法】各種HTLV-1感染細胞株、Tax発現誘導系細胞株JPX-9、Jurkat T細胞株などを用いて、RT-PCR、ELISA、siRNA、プロモーター解析、Noshift転写因子解析などにより検討した｡ 【結果】HTLV-1感染細胞株の多くがTaxを強発現しており、解析したTax発現細胞のほとんどでMDCの遺

角膜上皮細胞と角膜実質細胞は炎症性サイトカインやHSV-1感染により、様々なケモカインの発現を誘導した。マウスヘルペス性角膜実質炎では、その急性期には細胞性免疫を担うTh1、一方治癒に向かう後期には液性免疫を担うTh2が優勢な環境が形成されることが示唆された。

角膜上皮細胞と角膜実質細胞は炎症性サイトカインやHSV-1感染により、それぞれ独自のパターンで、未熟樹状細胞、単球やT細胞などを遊走するケモカインを産生することが明らかとなった。マウスヘルペス性角膜実質炎では、その急性期には細胞性免疫を担うTh1、一方治癒に向かう後期には液性免疫を担うTh2が優勢な環境が形成されることが示唆された。

炎症性サイトカインやHSV-1感染により角膜上皮細胞や角膜実質細胞からCCL20が産生されることが分かった。また、ヘルペス性角膜実質炎において、CCL20はその受容体であるCCR6を介した樹状細胞浸潤を誘導することにより、ヘルペス性角膜実質炎の病態形成に重要な役割を果たすと考えられた。

乾癬の発症には、Th17細胞が重要な役割を果たすことが知られている。ケモカイン受容体CCR4は、Th2細胞に発現し、Th2細胞の遊走を介してアトピー性皮膚炎等のアレルギー性疾患の発症に寄与している。近年、このCCR4がTh17細胞にも発現していることが明らかとなった。しかしながら、乾癬におけるCCR4の関与については不明である。本申請課題では、乾癬におけるCCR4を介したTh17細胞の遊走制御機構の解析を通じて、CCR4の乾癬の発症における役割を追究する。 前年度、イミキモド長期塗布により乾癬様病態を誘発したところ、野生型マウスと比べ、CCR4欠損マウスでは病態の軽減および病変部へのTh17細胞の浸潤の減少が認められた。 本年度は、CCR4の選択的阻害薬の投与による乾癬病態の変化について解析した。CCR4阻害薬compound22の2日に1回の投与により、臨床スコア（皮膚肥厚、紅斑、鱗屑）の軽減、病変皮膚および所属リンパ節でのTh17細胞の減少、乾癬関連遺伝子（IL-17A、IL-22、IL-23、STAT3、Keratin 14）の減少がみられた。以上より、CCR4の阻害が乾癬の治療戦略となる可能性が示された。

本研究の目的は、P2受容体が粘膜特有のCD70陽性樹状細胞に発現すること、また、メモリーCTLの生存維持に関与することに着目し、P2受容体を介したエフェクター/メモリーCTL誘導およびその維持機構を解明し、P2受容体のアゴニストである加水分解性抵抗性APTのワクチンアジュバントとしての有用性を検証することである。 本年度は、加水分解抵抗性ATPによって誘導されたエフェクターあるいはメモリーCTLが抗腫瘍活性を示すかどうかについてin vivo担癌モデルマウスを用いて検討した。モデル抗原OVAと加水分解抵抗性ATPを経鼻投与し、エフェクター期およびメモリー期にOVA発現腫瘍細胞 (E.G7-OVA)を皮内に接種した。経日的に腫瘍体積をモニタリングした結果、加水分解抵抗性ATPを併用した場合において、有意な腫瘍増殖抑制効果を認められた。さらに、この効果はP2受容体の阻害剤であるスラミンの投与によって消失した。これらの結果から、加水分解性抵抗性ATPによって誘導されたCTLによって有意な抗腫瘍効果を示すことが明らかとなり、加水分解抵抗性ATPは機能的なCTLを誘導できるアジュバントとしての活性を有することを示した。

本研究の目的は、ケモカインELC/CCL19とその新規受容体の相互作用解析を通じて、ELC/CCL19の新規受容体を介した生理的および病的役割を解明することにある。 研究代表者は、これまでにELC/CCL19はGPCR#13に対して既知受容体であるCCR7と同様に強い細胞遊走活性を示すことを見出した。そして、ヒト末梢血正常細胞において、GPCR#13は既知受容体CCR7の発現していないエフェクター細胞に広く発現することを明らかにした。 平成30年度は、ヒト乾癬患者の皮膚臨床検体のreal-time PCR解析により、ELC/CCL19および新規受容体GPCR#13の発現が認められ、特に重症度の高い患者の検体で高い値を示すことを明らかにした。 これらの結果は、ELC/CCL19はGPCR#13に対する機能的な新規アゴニストであり、エフェクター細胞の遊走を介して乾癬の病態形成において重要な役割を果たすことが示唆された。

光変換蛋白質KikGRを発現するマウスによって同定可能な炎症皮膚に「来た」「留まった」制御性T細胞(Treg)を対象に、シングルセル遺伝子・蛋白質多因子発現解析を行い、異なるTreg機能分子を発現する複数のサブセットを同定した。サブセット間を比較すると、発現する遊走関連分子も、皮膚に「留まる」性質も異なった。機能分子と遊走関連分子の多因子発現解析により、KikGR発現データを用いずに皮膚に「来た」「留まった」Tregを区別した。Tregサブセットの分化責任因子の同定及び炎症皮膚への集積法の樹立も試みた。本研究により、異なる機能分子を発現するTregは異なる炎症組織間遊走を行うことが示唆された。

CCL28遺伝子欠損マウスでは、大腸粘膜でのIgA産生細胞の減少と分布異常、糞便中のIgA量の著明な減少、および個々のIgA産生細胞のIgA産生量の低下が見いだされ、CCL28は大腸粘膜でのIgA産生に重要な役割を担っていることが確認された。さらにCCL28遺伝子欠損マウスの糞便ではバシラス綱が相対的に増加していた。大腸でのIgA産生量の低下と細菌叢の変化と関連して、CCL28遺伝子欠損マウスではデキストラン硫酸誘発腸炎の増悪が見られ、それは抗菌薬投与で軽減された。

これまでに研究代表者は和漢薬ライブラリーを用いて細胞遊走阻害活性を指標としたケモカインアンタゴニストの探索を行ってきた。本研究において、麻黄エキスがケモカイン受容体CCR3、CCR4、およびCCR8特異的なアンタゴニスト活性を示し、そのアンタゴニスト活性が麻黄エキスの酢酸エチル非可溶性分画に存在することを明らかとした。また、このアンタゴニスト成分による阻害活性にリガンド特異性はなく、受容体を直接阻害することが示唆された。 CCR3、CCR4、およびCCR8は、いずれもTh2細胞あるいは好酸球や好塩基球に選択的に発現することから、アレルギー性疾患において重要な役割を果たすと考えられる。

近年、特発性肺線維症（IPF）の9割にEBウイルス（EBV）を始めとしたヘルペスウイルス感染が報告され、ヘルペスウイルスのIPF発症・増悪化への関与が示唆されている。IPFは肺上皮細胞の障害に伴う慢性炎症を基盤とする難治性疾患であり、筋線維芽細胞が産生する細胞外基質の過剰沈着を主体とする。しかしその病態発症機序については未だ不明な点が多い。 ガレクチン9（Gal-9）は上記肺線維症の発症要因とされる炎症促進作用や細胞外基質産生誘導能を有するβ-ガラクトシド認識結合タンパクである。本研究ではこのGal-9がヘルペスウイルス感染肺上皮細胞に強く発現したことから、EBVに相同性の高いマウスγヘルペスウイルス（MHV68）による肺線維症モデルを構築し、Gal-9の肺線維化における役割を解析してきた。 本年度までに肺線維症モデルマウスを構築し、Gal-9欠損マウスではMHV68感染による炎症反応および肺線維化が著しく軽減されることを明らかにした。さらにMHV68感染後の野生型マウス肺で検出されたIL-27の発現がGal-9欠損マウスで顕著に低下していたため、IL-27に対する中和抗体をMHV68感染後の野生型マウスに連続投与したところ、感染後の肺に認められた一連のケモカイン発現上昇および炎症細胞亜集団の浸潤が劇的に抑制された。また組織学的解析およびハイドロキシプロリン量測定によりMHV68誘導性の肺線維化もIL-27中和抗体投与により有意に抑制された。以上のことから、Gal-9に制御されるIL27が肺線維化を抑制するための治療標的分子となる可能性を示した。

我々は、成人T細胞白血病/リンパ腫（ATL）でのCCR4発現に関わる転写因子としてAP-1ファミリーのFRA-2を同定し、FRA-2はJUNDと共同してATLの増殖を促進するとともにc-Myb、SOX4などの原癌遺伝子の発現を誘導することを明らかにした。本研究では、ATLおよび皮膚T細胞リンパ腫（CTCL）でのCCR7発現に関与する転写因子を検討し、新規DNA結合配列GAGGAGを同定し、その部位にはZFYVE19が結合することを明らかにした。本研究から、ATLやCTCLでの新たな発がん遺伝子カスケードの存在が示唆され、これらの成熟型T細胞性腫瘍の診断や治療に有用な情報が提供された。

これまでに我々は、成人T細胞白血病/リンパ腫（ATL）や皮膚T細胞リンパ腫（CTCL)でFra-2が一貫して発現し、CCR4発現誘導や細胞増殖に関与することを明らかにしてきた。本研究では、ATLとCTCL発癌におけるSOX4発現とその役割について検討した結果、ATLとCTCLにおいて、Fra-2-JunDが直接SOX4の転写活性を促進し、SOX4下流標的遺伝子として見出したGCKR、NAP1、HDAC8を発現誘導することでこれらの腫瘍の細胞増殖能を高めることを明らかにした。したがって、これらの知見からFra-2-SOX4経路はATLとCTCL発癌において重要な役割を有することが示唆された。

本研究において研究代表者は、成熟T細胞リンパ腫での新規c-Myb標的遺伝子として薬物トランスポーターABCG2/BCRPを同定した。c-MybとABCG2/BCRPは、成熟T細胞リンパ腫において共発現することが確認された。さらにABCG2/BCRP特異的阻害剤であるKo143は、ドキソルビシンのATLL細胞株に対する抗腫瘍活性を優位に促進した。これらの結果より、c-Myb-ABCG2/BCRP経路は成熟T細胞リンパ腫の抗がん剤耐性機構において重要な役割を果たすことが示唆された。

本研究において研究代表者は、Fra-2とc-Mybが皮膚T細胞リンパ腫に普遍的に発現し、細胞増殖に関与することを見出した。また、c-MybはFra-2の直接的な標的遺伝子であることが示された。さらに細胞増殖に関わるc-Mybの下流標的遺伝子としてRas活性化因子であるRASGRP2を同定した。Fra-2-c-Myb経路は発癌遺伝子カスケードとして機能し、細胞増殖に寄与することが示唆された。

CCR4はTh2細胞、制御性T細胞、皮膚指向性T細胞などに選択的に発現するケモカイン受容体である。我々は成人T細胞白血病(ATL)でのCCR4の高頻度発現を初めて報告した(Yoshie et al., Blood 2002)。そしてATLでのCCR4発現の転写制御機構を調べることにより、ATLではAP-1ファミリーのFra-2が構成的に異常発現しており、Fra-2はJunDとヘテロダイマーを形成することにより、ATLでのCCR4の発現を誘導し、さらにc-Myb、MDM2、Bcl-6などの原がん遺伝子の発現も誘導していることを明らかにした(Nakayama et al., Oncogene 2008)。ATLでFra-2やこれらの原がん遺伝子の構成的発現が示されたのは初めてである。そこで本年度は、Fra-2/JunDによるc-Mybの発現誘導を転写レベルで証明した。また皮膚T細胞リンパ腫(CTCL)は菌状息肉腫やCD30陽性未分化大細胞型リンパ腫などの多様な成熟型T細胞リンパ腫よりなるが、ATLと同様にCCR4を高頻度で発現する。そこでCTCLでのFra-2の発現と役割を検討した。そ.の結果、CTCLでもCCR4の発現とともにFra-2とJunDの構成的な発現が確認され、ATLと同様にFra-2とJunDはCTCLの増殖に関与すること、さらにFra-2とJunDはCCR4の発現だけでなく、c-Myb、MDM2、Bcl-6の発現も誘導していることを明らかにした。これらの結果は、Fra-2の構成的発現と発がんにおける役割はATLに限られるものではなく、CTCLも含めてCCR4を発現する成熟型T細胞由来の白血病/リンパ腫に普遍的に当てはまるものであることを示す。そしてFra-2で発現が誘導されるCCR4はこれらの悪性腫瘍の有用な腫瘍マーカーであるとともに有望な治療標的でもあると言える。

CCR4はTh2細胞、制御性T細胞、皮膚指向性T細胞などに選択的に発現するケモカイン受容体である。我々はTh2型のマウス気道炎症で低分子CCR4阻害剤の治療効果を示した。またCCR4とそのリガンドMDC/CCL22 は腸管膜リンパ節での樹状細胞と制御性T 細胞の相互作用を誘導して免疫寛容を誘導することを示した。さらにEB ウイルスやHTLV-1といったヒトの腫瘍ウイルスは共通してCCR4のリガンド、特にMDC/CCL22の発現を感染細胞に誘導し、それによって感染細胞の周囲にCCR4陽性のTh2細胞や制御性T細胞を動員して免疫回避やウイルス伝播に役立てていることを明らかにした。

ケモカインeotaxin-3/CCL26は、好酸球に高発現するCCR3のリガンドであり、炎症局所への好酸球浸潤に重要な役割を果たすことが報告されている.本研究において研究代表者は、CCL26が細胞傷害性リンパ球に高発現するCX3CL1受容体/CX3CR1の新しい機能的リガンドであることを見出した.CCL26は好酸球だけでなく細胞傷害性リンパ球も同時に炎症巣に誘導することにより、アレルギー性疾患の病態形成においてこれまで理解されていた以上の役割を果たすことが示唆された.

CCR4のプロモーター解析によりATLでの転写活性化サイトとしてGATA-3サイトとAP-1サイトを同定した。GATA-3はTh2細胞で選択的に発現し、CCR4のTh2選択的発現に関係すると考えられた。一方、AP-1はFosファミリーとJunファミリーのホモダイマーあるいはヘテロダイマーとして機能する。そこでAP-1ファミリーのATLでの発現を検討した。その結果、ATLの臨床検体ではFra-2、JunD、JunBの構成的発現が見られた。さらにCCR4プロモーターの活性化はFra-2/JunDおよびFra-2/JunBのヘテロダイマーがきわめて特異的に誘導することを見出した。さらにFra-2の発現はHTLV-1の転写因子Taxでは誘導されないことも確認した。発がん遺伝子Fra-2の正常T細胞での発現やATLでの構成的活性化はいまだ報告されていない。そこでATLでのFra-2、JunD、JunBの役割をsiRNAノツクダウンで検討した。その結果、Fra-2とJunDのsiRNAがCCR4発現および細胞増殖を抑制した。またFra-2あるいはJunDをJurkatで強制発現すると細胞増殖を促進した。そこでATLでのFra-2の下流標的遺伝子をFra-2 siRNAによるノックダウンとマイクロアレイ解析で検討した。その結果、Fra-2のsiRNAによりATL細胞株で少なくとも49の遺伝子が3倍以上の低下を示した。この中には発がん遺伝子c-Myb、Bc1-6、MDM2も含まれていた。そしてこれらの発がん遺伝子の発現はJunDのsiRNAでも抑制され、Fra-2/JunDヘテロダイマーの下流遺伝子であることが示された。さらにATLの臨床検体でもこれらの発がん遺伝子の構成的発現が確認された。発がん遺伝子Fra-2、c-Myb、Bc1-6、MDM2のATLでの構成的発現は初めての知見である。

CCR10は皮膚特異的に発現するケモカインCCL27の受容体として同定され、皮膚指向性T細胞での発現が報告されたが、さらに各種の粘膜組織で発現しているCCL28の受容体であることも明らかにされた。そして我々はCCR10がB細胞では形質細胞の段階で発現してくることを世界に先駆けて報告した。またCCR10の発現はIgA産生細胞の腸管粘膜への帰巣に重要であることも報告した。形質細胞の分化にはXBP-1やBlimp-1などの転写因子が重要な役割をはたす。しかしながらCCR10のプロモーター領域にはこれらの転写因子の認識配列は存Eしない。そこで本研究では形質細胞におけるCCR10発現に関わる転写因子を同定し、それによって形質細胞分化における新たな転写制御機構を明らかにすることを目的とした。 活性化ヒトB細胞をIL-21の存在下で培養すると形質細胞に分化することが報告された。そこでこの分化系を用いてCCR10の発現を検討した。その結果、IL-21のみではCCR10の発現は誘導されないが、活性型ビタミンD3(VD3)を加えるとCCR10の発現が誘導された。そこでCCR10の開始コドンから上流-1500bpまでをクローニングし、pGL3に挿入してCCR10を発現しているヒト骨髄腫細胞株に導入してレポーターアッセイを行った。その結果、-71〜-62bpの領域に存在するEts-1認識配列がCCR10の発現誘導に基本的に重要であることが示された。そしてEts-1の結合を確認した。さらに-124〜-110bpにVD3応答サイトと見なしえる配列を見出し、そこに点変異を導入することによりVD3に対する反応性の消失を確認した。そしてVD3レセプターのVD3依存的結合を確認した。VD3は粘膜免疫を誘導することが報告されており、形質細胞でのCCR10発現誘導もそのメカニズムのひとつと考えられる。

上皮細胞でのケモカイン発現に対するEBV感染の影響を明らかにするため、上咽頭癌(NPC)細胞株、EBV陽性と陰性の上皮細胞株を用いてケモカインの網羅的発現解析を行ってきた。これまでに、これらのEBV感染上皮細胞とEBV感染B細胞におけるケモカイン発現プロファイルの比較解析により、EBV感染B細胞ではTh2型ケモカインTARC/CCL17とMDC/CCL22の発現が特異的に誘導され、EBV感染上皮細胞では対照的にTh1型ケモカインMIG/CXCL9とIP-10/CXCL10の発現が特異的に誘導されることを明らかにしている。 今回、EBV関連上皮系腫瘍の病態形成におけるケモカインの役割を臨床検体で明らかにするため、NPC腫瘍組織を用いてケモカインおよびケモカインレセプターの免疫染色を行った。その結果、NPCの腫瘍細胞はMIG/CXCL9とIP-10/CXCL10を強く発現していた。さらに、腫瘍細胞の周囲には、それらのレセプターであるCXCR3陽性の多数のT細胞浸潤が認められた。以上の結果から、MIG/CXCL9とIP-10/CXCL10の産生はCXCR3を介してTh1細胞を誘導することが示唆された。また、腫瘍細胞の周囲には、Foxp3^+IL-10^+CD69^-制御性T細胞の多数の浸潤が認められ、これらの細胞は従来報告されていたCCR4に加えてCXCR3を発現していた。NPC組織では、CCR4のリガンドであるTARC/CCL17とMDC/CCL22の発現は認められなかったため、制御性T細胞はCXCR3を介してNPC組織に誘導され腫瘍細胞に対する免疫反応の抑制に関与すると考えられた。

我々は、ATLではケモカイン受容体CCR4が高頻度・強陽性であること、CCR4発現はATLの高頻度皮膚浸潤を説明するとともに、ATLの起源がTh2あるいは制御性T細胞であることを示唆することを報告した(Blood, 2002)。このようなATLでのCCR4高頻度・高レベル発現は、ATLの発がん機構と転写レベルで密接に関係している可能性がある。そこで本研究では、ATLでのCCR4発現に関わる転写因子を明らかにし、それらのATL発がんへの関与を検討する。まずATL細胞におけるCCR4の転写開始部位を5'-RACEによってマップした。その結果、ATL細胞でのCCR4転写はエクソン1の上流と第1イントロン内の2領域で開始されており、それぞれ第1プロモーターと第2プロモーターと命名した。そこで第1プロモーター1-kbおよび第2プロモーター2-kbをルシフェレースレポータープラスミドにクローニングし、レポーターアッセイを行った。その結果、これらのプロモーターはATL細胞で強い転写活性を示した。そこで、それぞれのプロモーター領域を5'側から削っていってレポーターアッセイを繰り返し、発現に必要な領域を絞り込んだ。そしてこれらの必要領域に存在する候補エレメントに次々と点変異を導入し、必要エレメントの同定を行っている。第1プロモーターおよび第2プロモーターともにTh2分化に関わるGATA-3の認識配列が重要であることをみいだしたが、それ以外にも共同して作用するエレメントが存在することが明らかになってきた。現在、これらのエレメントに結合する転写因子の同定とクローニングを進めている。今後はCCR4の発現に関与することが明らかになった転写因子について、それらのATL発がんにおける役割を強制発現系やsiRNAによるノックダウン実験によって明らかにする。さらに、ATL細胞でのこれらの転写因子の構成的な発現および機能亢進のメカニズムを分子レベルで明らかにする。それによっていまだ不明なところの多い遺伝子レベルでのATLの発がん機構に関して新たな知見をもたらす。

1.マウス肺がんのリンパ節転移に対するCCR7の発現とCCL21/Secondary Lymphoid-tissue Chemokine (SLC)による運動能の亢進 本研究では、同様にC57BL/6マウスにリンパ節転移を引き起こす肺がん細胞(LLC)を用いて、CCL21及びCCR7とリンパ節転移との関連性について検討した。その結果、LLC細胞におけるCCR7の発現がmRNAレベルで確認され、運動能についてもCCL21の濃度に依存して亢進することが明らかとなった。更に、CXCR5、CXCR6の発現も確認され、それぞれの受容体に対するケモカインにより、濃度依存的に運動能の亢進が確認出来た結果から、CCR7以外にもリンパ節転移に関与する新たな受容体を同定するに到った。種々がん細胞のリンパ節転移は、リンパ管新生の惹起により亢進することが明らかとなってきている。(発表論文1.) 2.リンパ節転移性マウス肺がん細胞(LLC)に対するCCL21によるシグナル伝達系の解明 上記(1)でリンパ節転移性マウス肺がん細胞(LLC)にCCR7の発現が確認されたことで、次に、CCL21によるシグナル伝達系の解明を、行った。その結果、細胞増殖や生存に関与するシグナル伝達分子であるAkt及びErkのリン酸化の亢進が確認できた。今回、我々はAkt及びErk分子により、マウス結腸がんの肺転移が制御されている事を報告した。(発表論文2-3.) 3.がん細胞におけるケモカイン受容体、CCR7の発現亢進メカニズムの解明 現在までに、ケモカイン受容体の発現とがん転移の関連性に関する研究は、著しい知見の集積がなされている現在ではあるが、がん細胞におけるケモカイン受容体発現亢進のメカニズムは全く解明されていない。最近我々は、サイトカインががん転移を含むがん細胞の増悪化を引き起こすことを報告した。(発表論文3.)