京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21020号 / 医博第4366号 / 新制||医||1028(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 椛島 健治, 教授 三森 経世, 教授 小柳 義夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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ABSTRACT Regulatory T cells (Tregs) are CD4+ T lymphocytes that express the gene FOXP3 and suppress other cellular immune responses. Their role in HIV pathogenesis is uncertain. Regulatory T cell (Treg) levels were analysed in peripheral blood of HIV infected patients and controls in South Africa. Immunophenotypic analysis revealed significantly elevated levels of FOXP3 positive Tregs in HIV infected patients (median 6.8%) compared with controls (median 3.7%). Treg levels were inversely correlated with CD4+ T cell count. FOXP3 mRNA expression was dependent on choice of reference gene (GAPDH or 18sRNA) and did not correlate with FOXP3 protein expression analysed flow cytometrically. These findings illustrate that Tregs are elevated in the peripheral blood of patients with late stage HIV disease and suggest a role for Tregs in the clinical immune suppression seen in these patients. Tregs may prove to be useful therapeutic targets for intervention or as a prognostic monitoring tool.

Thymic stromal lymphopoietin (TSLP) is a pleotropic cytokine highly implicated in the pathophysiology of asthma and allergic diseases. Although there are robust data regarding the associations of TSLP polymorphisms with the development of allergy and asthma, there is very little information on how these TSLP variants functionally affect downstream effector pathways and disease phenotype. The overall objective of this thesis was to investigate how TSLP polymorphisms are linked to alterations in TSLP secretion and subsequent downstream cellular events. Initially, we investigated the influence of innate and adaptive stimuli on epithelial-derived TSLP expression and secretion, including effects on dendritic cells (DC). We show that polyinosinic:polycytidylic acid (polyI:C) and allergen-specific T cells induced enhanced TSLP secretion from asthmatic bronchial epithelial cells (BEC) compared to non-asthmatic BEC. Furthermore, activated-BEC culture supernatants induced TSLP-dependent CCL17 production from monocyte-derived DC in relation to clinical asthmatic status (Chapter 2). Next, we examined effects of TSLP on hemopoietic progenitor eosinophil-basophil (Eo/B) differentiation, demonstrating enhanced TSLP-mediated hemopoiesis ex vivo in relation to clinical atopic status. We further demonstrated p38MAPK-dependent autocrine signaling by TNFα in TSLP-mediated human Eo/B differentiation ex vivo (Chapter 3). Lastly, to explore the potential functional consequences of a key variant of the TSLP gene, we investigated associations between the rs1837253 TSLP variant and ex vivo production of TSLP in nasal epithelial cells (NEC). We showed that NEC derived from individuals with the “protective” minor allele have diminished TSLP secretion, which suggests that this rs1837253 polymorphism may be directly involved in the regulation of TSLP secretion (Chapter 4). The novel work presented herein provides further evidence for TSLP regulation of distinct immunological pathways in allergic immune inflammatory airway responses initiated at the epithelial surface, and thus (by implication) of allergic disease. These observations support the concept that TSLP is potentially an important biomarker and therapeutic target for allergic diseases characterized by increased Th2 and/or eosinophilic-basophilic inflammation. Continued investigations into the functional mechanisms linking TSLP variants to allergic disease phenotype are of critical importance. / Dissertation / Doctor of Science (PhD)

Macrophage generation of reactive nitrogen intermediates (RNI) represents a major effector mechanism in anti‐microbial immunity and non‐septic inflammatory reactions. The induction of macrophage RNI production has been demonstrated to require at least two signals which in microbial infections can be provided by interferon (IFN)‐γ and lipopolysaccharide (LPS). The current study demonstrates that, in the absence of LPS, T lymphocytes can provide cognate signal(s) which synergize with IFN‐γ in stimulating macrophage RNI production, as evidenced by the ability of plasma membranes from T cell clones to activate IFN‐γ‐primed macrophages. Although viable resting T cells can activate IFN‐γ‐primed macrophages by an interaction that is antigen specific, plasma membranes from resting T cells do not activate macrophages. Plasma membranes from T cells activated by immobilized anti‐CD3 were able to effectively induce RNI production in IFN‐γ‐primed macrophages. However, in contrast to the antigen‐specific interaction of macrophages with viable resting T cells, the activation of IFN‐γ‐primed macrophages by membranes from activated T cells does not display antigen specificity. Plasma membranes from activated T helper TH2 and from activated TH1 cells were equally effective in activating IFN‐γ‐primed macrophages, suggesting that the dominance of TH1 over TH2 cells in cell‐mediated responses involving macrophage effectors is not a reflection of differences in their ability to interact with macrophages but rather is a reflection of their different pattern of cytokine production. These results suggest that the T cell‐macrophage interaction involves reciprocal activation of both cells ‐ an antigen‐specific activation of the T cells which results in the acquisition of T cell membrane components involved in antigen‐nonspecific stimulation of the macrophages.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Matrix metalloproteinases (MMPs) are known for their role in extracellular matrix remodeling, but their role in regulating intracellular immune cell function is unknown. We reported that MMP inhibition down regulated T cell proliferation in response to alloantigens and autoantigens; but the direct role of MMP involvement in T cell activation has not been reported. Methods: MMP deficient or MMP sufficient wild-type CD4+ or CD8+ T cells from C57BL/6 mice were treated with SB-3CT, a specific inhibitor of MMP2 and MMP9, stimulated with anti-CD3 Ab, alone, or with IL-2 or CD28. Cellular activation and cytokine profiles were examined. A mouse model of antigen specific T cell mediated lung injury was used to examine MMP inhibition in antigen-specific T cell mediated lung injury. Results: SB-3CT (1-25μM) induced dose-dependent reductions in anti-CD3 Ab-induced proliferation (p<0.0001). Compared to wild-type, MMP9-/- CD4+ and CD8+ T cells proliferated 80-85% less (p<0.001) in response to anti-CD3 Ab. Compared to untreated or wild-type cells, anti-CD3 Ab-induced calcium flux was enhanced in SB-3CT-treated or MMP9-/- CD4+ and CD8+ T cells. Cytokine transcripts for IL-2, TNF-α and IFN-γ were reduced in both CD4+ and CD8+ MMP9-/- T cells, as well as in SB3CT treated CD4+ T cells. MMP inhibition dampened antigen-specific T cell mediated lung injury. Conclusions: Although known to be functional extracellularly, the current data suggest that MMPs function inside the cell to regulate intracellular signaling events involved in T cell activation. T cell targeted MMP inhibition may provide a novel approach of immune regulation in the treatment of T cell-mediated diseases. - David S. Wilkes, M.D., Chair.

Indiana University-Purdue University Indianapolis (IUPUI) / T helper cells play a crucial role in providing protection against a wide variety of pathogens. The differentiation and effector function of T helper cell subsets is dependent on cytokine activation of Signal Transducer and Activator of Transcription (STAT) family members. The development of Th17 cells, which are important for immunity to fungi and extracellular bacteria, relies on STAT3. We show that IL-23 in combination with IL-1β promotes maintenance of the Th17 phenotype following multiple rounds of stimulation. However, IL-23 does not promote commitment of Th17 cells, and when Th17 cells are cultured with IL-12 or IL-4 they switch to a Th1 and Th2 phenotype, respectively. The maintenance of the Th17 phenotype by IL-23 also requires STAT4. STAT4-deficient memory cells cultured with IL-23 have reduced IL-17 production following stimulation with either anti-CD3 or IL-18+IL-23 stimulation compared to wild type memory cells. Furthermore, STAT4-deficient mice have impaired in vivo Th17 development following immunization with ovalbumin. This challenges a one-STAT/one-subset paradigm and suggests that multiple STAT proteins can contribute to a single phenotype. To test this further we examined whether STAT3 is required for the development of Th2 cells, a subset known to depend upon the IL-4-induced activation of STAT6 for immunity to parasites and promoting allergic inflammation. We demonstrate that in the absence of STAT3, the expression of Th2-associated cytokines and transcription factors is dramatically reduced. STAT3 is also required for in vivo development of Th2 cells. Moreover, allergic inflammation is diminished in mice that have T cells lacking expression of STAT3. STAT3 does not affect STAT6 activation, but does impact how STAT6 functions in binding target genes. Thus, multiple STAT proteins can cooperate in promoting the development of specific T helper subsets.

Indiana University-Purdue University Indianapolis (IUPUI) / CD4 T cells are critical regulators of inflammatory diseases and play an important role in allergic airway diseases (AAD) by producing type 2 cytokines including IL-4, IL- 13, IL-5 and IL-9. In chronic AAD models, IL-9 producing CD4 T-helper (TH9) cells lead to accumulation of eosinophils and mast cells in the airway, increase levels of type-2 cytokines, stimulate ILC2 cell proliferation, and induce mucus production from airway epithelium. However, the transcriptional network that governs the development of TH9 cells and their function during allergic responses is not clearly understood. Naïve CD4 T cells differentiate into TH9 cells in the presence of IL-2, IL-4 and TGFβ, activating a complex network of transcription factors that restricts their development to TH9 lineage. In this study a variety of approaches were utilized, including characterizing Il9 reporter mice, to identify an additional Ets-transcription factor termed ERG (Ets-related gene) that is expressed preferentially in the TH9 subset. Knock-down of Erg during TH9 polarization led to a decrease in IL-9 production in TH9 cells in vitro. Deletion of Erg at the later stage of TH9 induced pathogenesis resulted in reduced IL-9 production in the airways in chronic AAD. Chromatin immunoprecipitation assays revealed that ERG interaction at the Il9 promoter region is restricted to the TH9 lineage and is sustained during TH9 polarization. In the absence of PU.1 and ETV5, ERG regulated IL-9 production independent of other Ets-transcription factors and the deletion of Erg further lead to a decrease in IL-9 production by lung-derived CD4-T cells in chronic AAD model. Lastly, I also identified IL-9 secreting CD4 tissue resident memory cell population that play an instrumental role in allergic recall responses. In summary, in this study novel transcription factors were identified that can regulate TH9 function and the role of IL-9 in allergic airway recall responses. / 2022-12-28

The perivascular adventitia (PA) senses and responds to injuries in blood vessels and the tissues they feed. Cells in the PA form the outermost vascular layer, joining the circulatory system to other organs. Housing hematopoietic, mesenchymal and neuronal cells allows flexible adventitial responses to diverse perturbations. However, the PA response can also be pathogenic. Thickening of the adventitia may drive ischemia and hypertension. It can also be a niche for local lymphocyte priming in diseases such as idiopathic pulmonary arterial hypertension. Despite their importance, PA contributions to skin diseases were understudied. The hypothesis that contrasting two cutaneous diseases, scleroderma and discoid lupus erythematosus (DLE), would illuminate discrete PA alterations was explored. Vascular changes are prominent, but distinct, in both diseases. Studying perivascular adventitial changes in these diseases may yield insights into both dermal and vascular pathologies. PA fibroblasts in healthy human skin were phenotypically distinct from the surrounding dermal fibroblasts. In both scleroderma and DLE, PA fibroblasts expanded and expressed surface markers not observed in healthy skin including vascular cell adhesion molecule 1 (VCAM1), podoplanin (PDPN) and the p75 low affinity nerve growth factor receptor (NGFR). Elaborated networks of PA fibroblasts in DLE expressed VCAM1 and enmeshed dense, T cell-rich infiltrates. Transcriptional analyses indicated positive correlations between VCAM1, T cell chemoattractants and interleukin (IL)-15, which promotes their survival. Activated PA fibroblasts in DLE likely create a supportive niche for T cells infiltrating the skin. In contrast, enlarged PA fibroblast networks in scleroderma expressed NGFR in the absence of leukocyte infiltrates. This PA fibroblast phenotype was shared among reparative and pathologic scarring, and four dermal tumors. NGFR is a mesenchymal stem cell (MSC) marker, and expanded NGFR+ mesenchymal cells were immediately adjacent to cluster of differentiation (CD)34+ and CD73+ PA MSC. Expression of NGFR by PA fibroblasts is likely associated with reparative responses. Different stimuli induced VCAM1 and NGFR on cultured human dermal fibroblasts, supporting these as discrete activation states. In conclusion, these studies demonstrated the responsive and plastic nature of human dermal PA mesenchymal cells, and pointed to connections with vascular alterations in skin diseases.

Tumors evade immune system tumor-controlling functions. T cells critical to antitumor immunity are tolerogenic in tumor-burdened animals, and fail to lyse neoplastic cells. Our goal was to investigate the kinetics of immune dysfunction related to tumor-burdened host (TBH) memory T cell responses (or the lack thereof). We demonstrate tumor growth impairs T cell activation by modulating CD4+ T cell infiltration and systemic CD44 and CD62L activation marker expression, and by downregulating TBH TH1 cytokine production by splenic CD4+ T cells. Since chemotherapeutic treatments have potent cytostatic effects, we posited they enhance T cell dysfunctionality; which leads to limited therapeutic efficacy. Paclitaxel is a potent chemotherapeutic agent currently being administered in Stage III clinical trials; however, it reduces T cell proliferative capacity and interferon-γ (IFN-γ) production. In contrast, our data suggest that administration of low dose paclitaxel prolongs adaptive immunity in a limited capacity. We show paclitaxel enhances CD4highCD62Llow cell populations that drive TH1 cytokine production and prolongs the production of interleukin-2 (IL-2) in TBHs. We hypothesize that the initiation and maintenance of activated TH1 cell populations in patients during therapy serves as a reliable prognostic indicator of a favorable therapeutic response. Paclitaxel's limited therapeutic effects are due, in part, to its suppression of T cell activities; but the administration of low dose chemotherapy in combination with immunotherapeutic agents temporally takes advantage of paclitaxel's immunostimulatory capabilities. Our work will enhance current understanding of immune dysregulation during cancer development, and promote advances in the monitoring and development of combinatorial cancer treatments. / Master of Science