Akihiko Kitoh

Functional Delineation and Differentiation Dynamics of Human CD4+ T Cells Expressing the FoxP3 Transcription Factor

FoxP3 is a key transcription factor for the development and function of natural CD4(+) regulatory T cells (Treg cells). Here we show that human FoxP3(+)CD4(+) T cells were composed of three phenotypically and functionally distinct subpopulations: CD45RA(+)FoxP3(lo) resting Treg cells (rTreg cells) and CD45RA(-)FoxP3(hi) activated Treg cells (aTreg cells), both of which were suppressive in vitro, and cytokine-secreting CD45RA(-)FoxP3(lo) nonsuppressive T cells. The proportion of the three subpopulations differed between cord blood, aged individuals, and patients with immunological diseases. Terminally differentiated aTreg cells rapidly died whereas rTreg cells proliferated and converted into aTreg cells in vitro and in vivo. This was shown by the transfer of rTreg cells into NOD-scid-common gamma-chain-deficient mice and by TCR sequence-based T cell clonotype tracing in peripheral blood in a normal individual. Taken together, the dissection of FoxP3(+) cells into subsets enables one to analyze Treg cell differentiation dynamics and interactions in normal and disease states, and to control immune responses through manipulating particular FoxP3(+) subpopulations.

Indispensable Role of the Runx1-Cbfβ Transcription Complex for In Vivo-Suppressive Function of FoxP3+ Regulatory T Cells

Naturally arising regulatory T (Treg) cells express the transcription factor FoxP3, which critically controls the development and function of Treg cells. FoxP3 interacts with another transcription factor Runx1 (also known as AML1). Here, we showed that Treg cell-specific deficiency of Cbfbeta, a cofactor for all Runx proteins, or that of Runx1, but not Runx3, induced lymphoproliferation, autoimmune disease, and hyperproduction of IgE. Cbfb-deleted Treg cells exhibited impaired suppressive function in vitro and in vivo, with altered gene expression profiles including attenuated expression of FoxP3 and high expression of interleukin-4. The Runx complex bound to more than 3000 gene loci in Treg cells, including the Foxp3 regulatory regions and the Il4 silencer. In addition, knockdown of RUNX1 showed that RUNX1 is required for the optimal regulation of FoxP3 expression in human T cells. Taken together, our results indicate that the Runx1-Cbfbeta heterodimer is indispensable for in vivo Treg cell function, in particular, suppressive activity and optimal expression of FoxP3.

Perivascular leukocyte clusters are essential for efficient activation of effector T cells in the skin

It remains largely unclear how antigen-presenting cells (APCs) encounter effector or memory T cells efficiently in the periphery. Here we used a mouse contact hypersensitivity (CHS) model to show that upon epicutaneous antigen challenge, dendritic cells (DCs) formed clusters with effector T cells in dermal perivascular areas to promote in situ proliferation and activation of skin T cells in a manner dependent on antigen and the integrin LFA-1. We found that DCs accumulated in perivascular areas and that DC clustering was abrogated by depletion of macrophages. Treatment with interleukin 1α (IL-1α) induced production of the chemokine CXCL2 by dermal macrophages, and DC clustering was suppressed by blockade of either the receptor for IL-1 (IL-1R) or the receptor for CXCL2 (CXCR2). Our findings suggest that the dermal leukocyte cluster is an essential structure for elicitating acquired cutaneous immunity.

IL-17A as an Inducer for Th2 Immune Responses in Murine Atopic Dermatitis Models

Atopic dermatitis (AD) is generally regarded as a type 2 helper T (Th2)-mediated inflammatory skin disease. Although the number of IL-17A-producing cells is increased in the peripheral blood and in acute skin lesion of AD patients, the role of IL-17A in the pathogenesis of AD remains unclear. To clarify this issue, we used murine AD models in an IL-17A-deficient condition. In a repeated hapten application-induced AD model, skin inflammation, IL-4 production in the draining lymph nodes (LNs), and hapten-specific IgG1 and IgE induction were suppressed in IL-17A-deficient mice. Vγ4(+) γδ T cells in the skin-draining LNs and Vγ5(-) dermal γδ T cells in the skin were the major sources of IL-17A. Consistently, in flaky-tail (Flg(ft/ft) ma/ma) mice, spontaneous development of AD-like dermatitis and IgE induction were attenuated by IL-17A deficiency. Moreover, Th2 differentiation from naive T cells was promoted in vitro by the addition of IL-17A. Taken together, our results suggest that IL-17A mediates Th2-type immune responses and that IL-17A signal may be a therapeutic target of AD.

Basophils are required for the induction of Th2 immunity to haptens and peptide antigens

The relative contributions of basophils and dendritic cells in Th2 skewing to foreign antigen exposure remain unclear. Here we report the ability of basophils to induce Th2 polarization upon epicutaneous sensitization with different antigens using basophil conditionally depleted Bas TRECK transgenic mice. Basophils are responsible for Th2 skewing to haptens and peptide antigens, but not protein antigens in vivo. Consistent with this, basophils cannot take up or process ovalbumin protein in significant quantities, but present ovalbumin peptide to T cells for Th2 differentiation via major histocompatibility complex class II. Intriguingly, basophils promote Th2 skewing upon ovalbumin protein exposure in the presence of dendritic cells. Taken together, our results suggest that basophils alone are able to induce Th2 skewing with haptens and peptide antigens but require dendritic cells for the induction of Th2 for protein antigens upon epicutaneous immunization.

Resolvin E1 inhibits dendritic cell migration in the skin and attenuates contact hypersensitivity responses

Sawada et al. report that Resolvin E1 (RvE1) down-regulates DC motility in both steady state and inflammatory conditions in the skin and exerts its antiinflammatory effects in contact hypersensitivity. They propose the LTB4-BLT1 signaling blockade as a possible major mechanism through which RvE1 exerts its regulatory effects.

CD8+tumor-infiltrating lymphocytes at primary sites as a possible prognostic factor of cutaneous angiosarcoma

Tumor‐infiltrating lymphocytes (TILs) have been reported as a prognostic factor in various cancers and are a promising target for immunotherapy. To investigate whether TILs have any impact on the prognosis of angiosarcoma patients, 55 non‐treated patients (40 patients at stage 1 with cutaneous localized tumors, 4 patients at stage 2 with lymph node metastases and 11 patients at stage 3 with distant metastases) with angiosarcoma were evaluated retrospectively by immunohistochemistry stained CD4, CD8, FOXP3 and Ki67. The Kaplan–Meier method was used to estimate overall survival with patients at stage 1. Survival differences were analyzed by the log‐rank test. Patients with higher numbers of CD8+ TILs in their primary tumors survived significantly longer compared with patients with lower values. Moreover, the number of CD8 in TILs was positively correlated with a distant metastasis‐free period. The total number of primary TILs (CD4 plus CD8) and CD8+ primary TILs of stage 3 patients with distant metastases was positively correlated with their overall survival. To evaluate whether CD8+ effector T cells are activated or differentiated, flow cytometric analysis of peripheral blood mononuclear cells (PBMC) was performed. The percentages of CD8+ T cells producing IFN‐γ in PBMC were significantly higher in patients with angiosarcoma (n = 10) compared not only with that of healthy controls (n = 20) but also patients with advanced melanoma (n = 11). These results suggest that anti‐tumor immunity is clinically relevant in angiosarcoma.

Perturbations of both nonregulatory and regulatory FOXP3+ T cells in patients with malignant melanoma

Background  ‘FOXP3+ regulatory T cells’ (Tregs) are reported to be increased in tumour‐bearing hosts including patients with melanoma, leading to tumour immune suppression. However, this idea is challenged by recent evidence that the ‘FOXP3+ Treg’ fraction in fact contains activated ‘nonregulatory’ T cells. Also, FOXP3+ T cells are reported to have functionally and kinetically distinct subsets.

Natural killer T cells are essential for the development of contact hypersensitivity in BALB/c mice.

Contact hypersensitivity (CHS) has been widely used to study cutaneous immune responses, as a prototype of delayed-type hypersensitivity. Although natural killer T (NKT) cells have been assumed to have an important role in CHS, their role is controversial. Here, we report the role of NKT cells in the sensitization phase of CHS, by promoting the survival and maturation of dendritic cells (DCs) in the draining lymph nodes (LNs). The CHS response was attenuated with Cd1d1(-/-) and Traj18(-/-) BALB/c mice in which NKT cells were absent. In the draining LNs, the number of effector T cells and cytokine production were significantly reduced with NKT cell-deficient mice. NKT cells activated and colocalized with DCs in the draining LNs after sensitization. The number of migrated and mature DCs was reduced in NKT cell-deficient mice 72 hours after FITC application. In in vitro experiments, activated NKT cells enhanced bone marrow-derived DC (BMDC) survivability via tumor necrosis factor (TNF) production from BMDCs. In addition, TNF production from BMDCs was partially suppressed by the neutralizing anti-CD54 or CD154 antibodies. Our data demonstrate that DC-NKT interaction has a pivotal role in the sensitization phase of CHS.

Peripheral blood Th9 cells are a possible pharmacodynamic biomarker of nivolumab treatment efficacy in metastatic melanoma patients

ABSTRACT Although nivolumab is associated with a significant improvement in overall survival and progression-free survival, only 20 to 40% of patients experience long-term benefit. It is therefore of great interest to identify a predictive marker of clinical benefit for nivolumab. To address this issue, the frequencies of CD4+ T cell subsets (Treg, Th1, Th2, Th9, Th17 and Th22), CD8+ T cells, and serum cytokine levels (IFNγ, IL-4, IL-9, IL-10, TGF-β) were assessed in 46 patients with melanoma. Eighteen patients responded to nivolumab, and the other 28 patients did not. An early increase in Th9 cell counts during the treatment with nivolumab was associated with an improved clinical response. Before the first nivolumab infusion, the responders displayed elevated serum concentrations of TGF-β compared to non-responders. Th9 induction by IL-4 and TGF-β was enhanced by PD-1/PD-L1 blockade in vitro. The role of IL-9 in disease progression was further assessed using a murine melanoma model. In vivo IL-9 blockade promoted melanoma progression in mice using an autochthonous mouse melanoma model, and the cytotoxic ability of murine melanoma-specific CD8+ T cells was enhanced in the presence of IL-9 in vitro. These findings suggest that Th9 cells, which produce IL-9, play an important role in the successful treatment of melanoma patients with nivolumab. Th9 cells therefore represent a valid biomarker to be further developed in the setting of anti-PD-1 therapy.