Effects of the antidiabetic DPP4/CD26 inhibitors evogliptin and sitagliptin on immune function of H9 T helper cells

윤현이 Greduate School, Korea University 2021 국내박사

Dipeptidyl peptidase (DPP) 4/CD26 is a multifunctional protein containing an ecto-domain with DPP4 enzymatic activity and known to involve in T cell immune response, including T helper 1 cells. Numerous membrane DPP4 (mDPP4) is on the surface of T cells and soluble DPP4 (sDPP4), originating from mDPP4 exists at a high level in blood. Altered DPP4/CD26 expression in DPP4-riched T cell is associated with diverse autoimmune diseases including type 1 diabetes, rheumatoid arthritis (RA), multiple sclerosis (MS), and inflammatory bowel diseases (IBD). DPP4 inhibitors is the most prescribed antidiabetic drugs that suppress the enzymatic activity by direct binding at active site of DPP4. However, their adverse effects are still unclear on the immune function of DPP4-riched T helper cells under the treatment of patients with type 2 diabetes. The aim of this study was to assess whether antidiabetic drugs and other DPP4 inhibitors affect the dipeptidyl enzymatic activity of mDPP4 and intrinsic function of CD4+CD26+ H9 cells in terms of cytokine expression and cellular profiles. The mDPP4 enzymatic activity, T helper cell-specific cytokine expression, and cellular profiles, including cell counts, cell viability, DNA synthesis, and apoptosis, were estimated in pokeweed mitogen (PWM)-activated CD4+CD26+ H9 cells with or without the antidiabetic drugs evogliptin and sitagliptin or DPP4 inhibitors such as diprotin A and berberine. PWM treatment alone stimulated strongly the expression of T helper cell-specific cytokines such as interleukin (IL)-2, IL-10, IL-13, granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-gamma (IFN-ɣ), and tumor necrosis factor-alpha (TNF-α) in CD4+CD26+ H9 cells, albeit induced marginally the expression of CD26. The treated evogliptin or sitagliptin potently inhibited mDPP4 enzymatic activity but did not influence either T helper cell-specific cytokine expression or cellular profiles in PWM-activated CD4+CD26+ H9 cells. In contrast, diprotin A inhibited transiently mDPP4 enzymatic activity and berberine had a little or no inhibitory ability with cytotoxicity to cells. These results imply that mDPP4 enzymatic activity is not essential for cell signaling pathways for T helper cell-specific cytokine expression upon immune stimulation. Moreover, the results implicates that immune functions such as T helper cell-specific cytokine expression are performed normally even after the treatment with evogliptin or sitagliptin which inhibits strongly mDPP4 enzymatic activity.

Finding Regulatory Factors Binding to Rad50 Hypersensitivity Site 7 (RHS7) during T helper type 2 (Th2) cell differentiation

김영욱 서강대학교 대학원 2009 국내석사

생체 내 면역 반응에서 T helper 세포는 여러 가지 면역반응을 통합하여 조절하는 기능을 가지고 있다. T helper 세포가 T helper type 1 (Th1) 또는 Th2 세포로 분화하는 과정에 수반되는 유전자 발현은 이러한 분화 기작을 결정하는 중요한 요소가 된다. 기존 연구에서 Th2 세포 분화시 cytokine의 발현을 조절하는 cis-element로 Locus Control Region (LCR)이 중요한 역할을 한다는 것이 밝혀졌다. 이 Th2 LCR은 RHS4에서 RHS7에 이르는 4개의 DNase I hypersensitive site (HS)로 구성되어 있으며, 이 HS는 Th2 세포 분화와 Th1, Th2 cytokine 발현에 큰 역할을 한다고 알려졌다. 그 중 RHS7 부위가 Th2 세포 분화에 중요한 역할을 한다는 것이 밝혀져 있으므로, 이 RHS7과 상호작용하는 trans-acting factor가 중요한 역할을 할 것으로 예상된다. 따라서, 본 연구에서는 RHS7 부위에 결합 가능한 단백질 후보군을 데이터베이스 검색을 통해서 결정하였고, 여러 후보군들 중에서 Yin Yang 1 (YY-1)이 RHS7 (2)와 RHS7 (11) 부위에 Th2 세포 특이적으로 결합하는 것을 EMSA (electrophoresis mobility shift assay)와 competition과 supershift assay를 통하여 확인하였다. 또한 앞선 후보군과 DNA affinity chromatography를 통하여 얻은 후보군들 중에서 L(3) mbt-like 2가 Th2 세포 특이적으로 발현양이 증가함을 quantitative RT-PCR과 western blot을 통해 확인하였다. T helper cells coordinately control several immune responses. During T helper cells differentiation to Th1 or Th2 cells, differentiation-specific gene regulation is the key to determine the cell's fate. Previous studies have shown that Th2 locus control region (LCR) plays a major role as a cis-element which controls the expression of Th2 cytokines in Th2 cell differentiation. Th2 LCR is composed of 4 DNase I hypersensitive sites, RHS 4 to 7, among which RHS7 has been shown to play a critical role when a Th2 cell differentiates. I selected several putative transcription factor binding sites on RHS7 and performed electrophoresis mobility shift assay (EMSA). I found that Th2 cell specific binding on RHS7(2) and RHS7(11) sites (the site contains putative binding site for CCAAT/enhancer binding protein -alpha, beta, and Yin Yang 1, C/EBP-α, β and YY-1). Among these proteins, I found that YY-1 binds to RHS7(2) and RHS7(11) by EMSA, competition and supershift assay. To identify the protein(s) that bind to the RHS7(2) site, protein elutes were obtained from DNA affinity chromatography and subjected to mass spectrometry (M/S). L(3) mbt-like 2, one of the candidates from the M/S, showed Th2 specific increase of mRNA and protein level by quantitative RT-PCR and western blot. I am currently investigating these candidate proteins' functional role of these bindings.

The intrinsic role of CD8α- dendritic cell subset in the initiation of effective humoral immunity

Changsik Shin Graduate School of UNIST 2016 국내박사

Dendritic cells (DCs), the most potent antigen presenting cells, have been identified in 1973 by Ralph M. Steinman. DCs. DCs role as an immune initiator as well as regulator, have been intensively studied for their crucial function in regulating cellular and humoral immune responses. Efficiency of human vaccines is mostly dependent on the generation of proper humoral immunity, and recently T follicular helper (Tfh) cells have been identified as a true B cell helper, which significantly advanced our understanding of T cell-dependent B cell humoral immune responses. However, there are still little studies regarding the initial commitment of Tfh cells primed by DCs, particularly by the two major myeloid CD8α+ and CD8α- DC subsets. In this study, we present the undescribed intrinsic features and roles of the two DC subsets in the induction of Tfh cells and subsequent Tfh cell dependent humoral immune responses. We here demonstrate that the CD8α- DC subset critically roles in inducing antigen-specific Tfh cells by up-regulated expressions of Icosl and Ox40l via the non-canonical NF-κB signaling pathway. CD8α- DCs are able to induce functional Tfh cells regardless of an adjuvant type. Tfh cells initially primed by CD8α- DCs function as a true B cell helper that results in dramatically enhanced humoral immune responses against various human pathogenic antigens such as Yersinia pestis LcrV, HIV Gag, and Hepatitis B surface antigen. In addition, we showed that the localization of CD8α- DCs in the marginal zone (MZ) bridging channels is closely related to the induction of CXCR5+CCR7low Tfh cells. We also demonstrated that the major source of IL-6 for inducing Tfh cells is provided from the antigen specifically activated CD4+ T cells primed by CD8α- DCs, and those secreted by the DC subset seem have a minor role. Moreover, CD8α- DCs were superior in inducing functional Tfh cells over other antigen presenting cells majorly B cells. In contrast, CD8α+ DCs localized in the T cell enriched region are superior in inducing CXCR5lowCCR7high CD4+ T cells responsible for the generation of IFN-γ secreting Th1 cells. Taken together, this study reveals the undescribed intrinsic features and mechanistic role of the CD8α- DC subset in priming antigen-specific Tfh cell differentiation and thereby provides the potential of investigating CD8α- DCs to effectively evoke antigen-specific humoral immune responses through the improved therapeutic vaccines.

Regulation of T helper cell responses by costunolide via down-regulation of dendritic cells' maturation

장숙정 Graduate School, Korea University 2014 국내석사

Costunolide, a germacranolide sesquiterpene lactone which is one of the active ingredients in several medicinal plants, is well known with their potent anti-cancer, anti-microbial, anti-fungal and anti-inflammatory properties. Costunolide has the ability to inhibit the production of nitric oxide (NO) and pro-inflammatory cytokines, such as tumor necrosis factor-a (TNF)-a, interleukin (IL)-6 and IL-1b in addition to their suppression on the activation of nuclear factor (NF)-kB and mitogen-activated protein kinase (MAPK) on macrophages. However, the effects of costunolide on dendritic cells (DCs) yet remain to be elucidated. In this study, I determined the effects of costunolide on the maturation and activation of lipopolysaccharide (LPS)-stimulated DCs by determining the expression of cell surface molecules, their cytokine expression and production, as well as T helper (Th) cell responses. Costunolide significantly down-regulated the expression level of cell surface molecules such as CD40, co-stimulatory molecules (CD80 and CD86), and MHC class II. Moreover, costunolide also inhibited the expression and the production of several cytokines on DCs. At the same time, costunolide-treated DCs also inhibited CD4+ T cells proliferation in the presence of antigen by decreasing IL-2-expressing cells. Furthermore, costunolide-treated DCs inhibited LPS-induced IFN-gproduction while increased the production of IL-4 and IL-17 in CD4+ T cells. The production of IFN-g was restored after IL-12 treatment. These findings demonstrate that costunolide could effectively inhibits LPS-induced maturation of DCs, and in the meantime, regulates Th responses by attenuates Th1 via the reduction of IL-12 production and enhances Th2 and Th17 on CD4+ T cells. Thus, costunolide may be able to exhibit its therapeutic use, especially in Th1-mediated immune diseases.

Functional modulation of autoimmunity via blocking the non-classic th1 by ntEomes-TMD and enhancing treg activity by TregL1

신보영 Graduate School, Yonsei University 2022 국내박사

만성 염증성 환경에서 Th17 세포는 쉽게 non-classic Th1 세포 로의 전환이 일어난다. non-classic Th1세포는 기존의 Th17세포보다 훨씬 더 병원성을 나타내며, 전사인자인 Eomes를 높게 발현하는 특성 을 가진다. Eomes는 Th17 관련 유전자의 전사를 억제하고 IFN-γ의 발현을 증가시킴으로써 non-classic Th1 세포의 특성을 유지할 수 있 도록 한다. 또한, Eomes에 의해 유도된 IFN-γ+GM-CSF+는 nonclassic Th1 세포의 병원성을 담당한다. 따라서 본 연구에서는, Eomes 의 기능을 조절하여 고병원성 non-classic Th1 세포의 생성을 억제하 고 궁극적으로 만성 염증성 질환의 발병을 제어하기 위해, 재조합 단백 질인 ntEomes-TMD를 제작하였다. ntEomes-TMD는 Eomes의 전사 조절 부위와 Hph-1-PTD를 융합하여 만든 형태로, 이는 세포의 핵 내로 효과적으로 전달되어 Eomes의 기능을 제어할 수 있는 단백질이 다. ntEomes-TMD는 세포 독성 없이 세포의 핵 내로 전달되어, IFNγ의 전사 및 IL-17A의 전사를 효과적으로 억제할 수 있었다. 또한, Eomes에 특이적으로 작용하여 Eomes의 발현을 저해할 수 있었으며, 이러한 반응을 통해 결과적으로는 non-classic Th1 세포의 생성을 억 제할 수 있었다. 특히, 대표적 만성 자가면역질환 동물 모델인 EAE에 서 ntEomes-TMD는 non-classic Th1의 생성을 억제하여 EAE의 증 상을 확연하게 완화할 수 있음을 확인하였다. 이를 통해, ntEomesTMD는 병원성 세포의 생성을 억제함으로써 만성 염증 반응을 효과적 으로 조절할 수 있는 치료용 단백질임을 입증하였다. Treg 세포는 염증성 T 세포들을 억제하는 기능을 가진 세포로, 자가면 역질환 환경에서는 이러한 Treg의 숫자 및 기능이 현저하게 떨어져 있 어 염증성 T 세포들이 과도하게 활성화되어 있다. 따라서 Treg을 정상 화하는 것이 다양한 자가면역질환을 손쉽게 극복할 수 있는 방법이 될 수 있다. 하지만 Treg 세포 특이적 표면 마커의 부재로 인해 그동안 Treg을 이용한 치료제 개발에 난항이 있었고, 이를 극복하고자 본 실 험실에서는 선행 연구를 통해 TregL1이라는 새로운 Treg 특이적 표 면 마커를 발굴하였다. 본 연구에서는 이러한 TregL1의 기능적 역할을 확인하고 독립된 치료 후보물질로 사용하기 위 새로운 재조합 단백질인 L1-IgG를 새롭게 제작하였다. L1-IgG는 Treg 세포와 염증성 T 세포 모두에서 발현하는 세포 표면 단백질에 붙어 작용할 수 있으며, 특징적 인 것은 L1-IgG에 의해 Treg 세포는 기능이 강화되는 반면 염증성 T 세포는 그 활성이 줄어듦을 확인하였다. 따라서 L1-IgG는 염증성 T 세포는 저해하고 Treg 세포는 강화할 수 있는 역할을 수행하므로 자가 면역질환과 같은 환경에서 좋은 치료 효과를 나타낼 수 있는 치료 후보 물질이 될 수 있고, 이러한 L1-IgG의 치료 효과는 류머티스 관절염 동물 모델을 통해 확인하였다. 뿐만 아니라, TregL1을 타겟하는 agonistic anti-TregL1 Ab (6F01)을 처리하여 Treg의 기능을 상승시 킬 수 있었다. 치료용 항체인 6F01이 TregL1을 인식하면 Treg의 기 능이 향상되었고, 이는 EAE 동물 모델을 통해 그 효과를 확인할 수 있 었다. 그러므로 TregL1을 활용하여 Treg 기능을 향상시키는 것은 효 과적인 치료 전략이 될 수 있을 것이다. Th17-derived non-classic Th1 cells play a critical role in developing late-onset chronic inflammatory diseases, which are more pathogenic than unshifted Th17 cells. Non-classic Th1 cells highly express the transcription factor Eomes compared to Th17 cells. In the non-classic Th1 cells, Eomes is essential to maintain the stability of non-classic Th1 cells by suppressing the Th17-related genes and enhancing the expression of IFN-γ. Also, Eomes induces the generation of IFN-γ+GM-CSF+ cell populations that plays a pathogenic role in non-classic Th1 cells. In this study, to suppress the generation of non-classic Th1 cells by controlling Eomes, a novel therapeutic recombinant protein, ntEomes-TMD, was generated. ntEomes-TMD can be delivered into the nucleus of cells and then control the Eomes-target genes. Eventually, ntEomes-TMD can suppress the non-classic Th1 cells by modulating the function of Eoems. In Experimental Autoimmune Encephalomyelitis (EAE) mouse model, ntEomes-TMD can effectively suppress the development of CD4+Eomes+ and IFNg+GM-CSF+ T cells, leading to ameliorating the diseases during the late phase of EAE. Therefore, ntEomes-TMD can be a novel therapeutic strategy for treating chronic inflammatory diseases associated with non-classic Th1 cells. Regulatory T cells (Treg cells) play an essential role in maintaining immune homeostasis by inhibiting immune responses. Since Treg cells have suppressive activity, Treg cells emerged as attractive therapeutic targets for autoimmune diseases. However, there remain several limitations on the use of Treg cells due to the lack of specific surface markers of Treg cells. Therefore, in our previous study, a novel surface Treg-specific marker, TregL1, was discovered to overcome these limitations. TregL1 is exclusively expressed on the Treg surface, and it plays a critical role in regulating the immuno-suppressive function of Treg cells. In this study, L1-IgG was newly generated to examine the roles of TregL1 on CD4+ T cell responses. L1-IgG is designed by fusing TregL1 ectodomain and mouse IgG2a Fc region. L1-IgG can inhibit the proliferation and activation of effector T cells. However, in Treg cells, L1-IgG plays a positive role, such as maintaining a stable Treg phenotype (CD4+Foxp3+) and enhancing the suppressive function of Treg cells. In addition, consistent with the in vitro results, L1-IgG exhibited therapeutic effects on autoimmune disease. In the Collagen Induced Arthritis mouse model (CIA), L1-IgG alleviated the symptoms and increased CD4+Foxp3+ Treg cells in the spleen. Furthermore, anti-TregL1 Abs were also generated for therapeutic applications. Among the several antibodies, especially agonistic anti-TregL1 Ab could enhance the suppressive function of Treg cells. In EAE, agonistic anti-TregL1 Ab alleviated the EAE symptoms by increasing Treg cell activity and decreasing effector T cells. Therefore, L1-IgG and anti-TregL1 Ab can be an effective therapeutic candidate for autoimmune diseases by controlling the immune response in various ways.

Discovery and functional analysis of surface protein tregS in regulatory T cells

윤지상 Graduate School, Yonsei University 2019 국내석사

Regulatory T cells (Treg cells) are subsets of T cells which express FOXP3 and suppress immune responses in both tumor and autoimmune diseases, thereby a potential target for therapeutics to treat these diseases. Proteins such as Foxp3, GITR, Cd25 are known to be expressed in Treg cells, being in use as Treg cell markers. However, none of these markers are exclusively expressed on the surface of Treg cells, making therapeutic application difficult. This research utilized RNA sequencing data to identify potential Treg cell marker proteins which are exclusively expressed on Treg cells. Using public internet databases, we were able to discard genes which are either previously reported to be markers for Treg cells, or less likely to be expressed on the surface of T cells. Based on the mRNA expression level difference between Treg cells and other T cell subsets and its subcellular localization, 11 candidates were initially selected. Out of these candidates, one protein, TregS was identified to be expressed higher on Treg cells than in most effector T cells. TregS was shown to be expressed when TGF-β, crucial cytokine in Treg differentiation, is applied to naive CD4 T cells under activation condition. Most importantly, CD4+ T cells expressing TregS was shown to successfully suppress T cell activation and proliferation. This suppressive activity did not decrease when treated with antibodies which are expected to block the physical contact of TregS to its ligands. Zinc levels of culture media of regulatory T cells did not show any significance. However, CD4+ TregS+ T cells secreted immune suppressive cytokine IL-10 more than two-folds than CD4+ TregS- T cells. These results suggest a novel therapeutics target to treat cancer and autoimmune diseases using adoptive transfer of CD4+ TregS+ T cells and monoclonal antibodies targeting TregS. 조절자 T 세포는 전사인자 FOXP3를 발현하는 T 세포의 일종으로 암과 자가면역 질환 모두에서 면역 반응을 억제하여 면역 항상성을 유지하는 기능을 수행한다. Foxp3, Ccr4, Ctla4, CD25등의 단백질은 조절자 T 세포에 발현되는 것으로 알려져 있으며 이에 따라 현재 조절자 T 세포의 마커로서 알려져있다. 그러나 상기 단백질은 모두 조절자 T 세포의 표면에 특이적으로 발현되지 않으므로 이러한 단백질을 이용한 치료제 개발은 태생적 한계를 가지고 있다. 본 연구는 RNA 시퀀싱 데이터를 이용하여 잠재적으로 조절자 T 세포의 마커로 쓰일 수 있는 표면단백질을 발굴하였다. 본 연구는 수치화된 발현량을 토대로 조절자 T 세포에서 발현량이 높고 다른 T 세포에서는 발현량이 낮은 후보 단백질을 선정하였다. 이후 선정된 단백질을 인터넷 공용 데이터베이스를 이용하여 세포 내 위치를 예측하여 세포의 표면에서 발견될 가능성이 낮은 단백질을 배제하였으며, 이를 토대로 11개의 단백질을 조절자 T 세포 마커 후보 단백질로 선정하였다. 11개의 마커 후보 단백질 중 TregS 만이 조절자 T 세포에서 특이적으로 발현이 된다는 것이 검증되었다. 미 분화된 CD4+ 세포에 조절자 T 세포 신호 체계 및 분화에 중요한 역할을 하는 사이토카인인 TGF-β를 처리한 결과 TregS의 발현이 TGF-β의 농도에 따라 증가함을 확인했다. 또한 CD4+ T 세포 중 TregS를 발현하는 세포는 면역 억제 사이토카인인 IL-10을 분비함으로 다른 T 세포의 활성과 분화를 저해하는 능력이 있음을 알 수 있었다. 이러한 결과는 RNA 시퀀싱을 통해 찾은 조절자 T 세포의 표면 마커 TregS가 항체 신약 등 암 및 자가면역 질환에 대한 치료제 개발에 쓰일 가능성을 제시한다.

Functional regulation of pathogenic T cell subset by ntTbet-TMD protein or new Treg cell-specific surface marker

Moon, Jae-seung Graduate School, Yonsei University 2018 국내박사

IFNγ produced by T helper 1 cells (TH1 cells) mediates the onset of systemic lupus erythematosus (SLE) and T-bet is a master inducer of IFNγ transcription in TH1 cells. In lupus patients, excessive expression of T-bet and IFNγ is the evidence of SLE. Therefore, the inhibition of T-bet binding to IFNγ promoter would ameliorate lupus nephritis in (NZB/NZW) F1 mice by reducing IFNγ production in TH1 cells. A nucleus-transducible protein (ntTbet-TMD) consisting of protein transduction domain (PTD) and T-bet transcription modulation domain (Tbox domain) was developed to prevent TH1 cell differentiation. ntTbet-TMD was efficiently delivered into the nucleus of mouse CD4+ T cells and specifically inhibited T-bet-mediated transcription without influencing cellular toxicity and T cell activation. When (NZB/NZW) F1 mice were treated with ntTbet-TMD initially at 23 weeks of age, ntTbet-TMD significantly reduced the severity of lupus nephritis. The symptoms of lupus nephritis such as glomerular damage and deposition of immune complex were considerably alleviated by ntTbet-TMD treatment similarly as methylprednisolone treatment. The proportion of TH1, 2 and 17 cells and serum pro-inflammatory cytokine levels in lupus-prone mice were down-regulated by ntTbet-TMD treatment compared to untreatment. On the contrary to methylprednisolone treatment, the increased population of Treg cells and the secretion of IL-10 cytokine were identified in spleen of (NZB/NZW) F1 mice treated with ntTbet-TMD. Therefore, ntTbet-TMD can alleviate lupus nephritis in (NZB/NZW) F1 mice by regulating overall autoimmune micro-environment and reversing pro-inflammatory T cell subsets toward regulatory T cells. ntTbet-TMD can be a novel immuno-therapeutic agent for TH1-mediated autoimmune diseases. Regulatory T cells (Treg cells) have a role for immunological tolerance and immune homeostasis with suppression of immune reactions, and its therapeutic potential is critical in autoimmune diseases and cancers. However, the lack of specific surface markers of Treg cells has limited their clinical applications. TregL1 was identified as a novel and first-in-class Treg cell-specific surface marker by data-driven and experiments-driven researches. In contrast to the previously identified markers specific to Treg cells, which are also present in other T cell subsets or in activated T cells, TregL1 is exclusively expressed on the surface of Treg cells. Expression of TregL1 is dependent on TGF-β concentration and Foxp3 expression level. CD4+TregL1+ T cells effectively suppressed the proliferation of effector T cells similar to CD4+Foxp3+ T cells. Also, CD4+TregL1+Foxp3- or CD4+TregL1+CD25- T cells had the suppressive function even when Treg markers such as Foxp3 and CD25 were not expressed in T cells. Furthermore, CD4+Foxp3+ or CD4+CD25+ T cells that did not express TregL1 could not suppress the proliferation of effector cells. CD4+TregL1+ T cells or CD4+TregL1hi cells among induced Treg cells significantly showed the therapeutic effect on inflammatory bowel disease (IBD) model and reduced pro-inflammatory cytokines in mouse serum. Therefore, TregL1 is a specific surface marker of Treg cells and adoptive transfer of CD4+TregL1+ T cells or monoclonal antibody specific to TregL1 will be important therapeutic reagents to deliver the functional sub-population of Treg cells and regulate the cell number or functions of Treg cell in autoimmune or tumor microenvironment.

(The) effects of crude antigen of Caenorhabditis elegans on the interaction between dendritic cells and CD4+ T cells of mice

이지민 서울대학교 대학원 2015 국내석사

Helminth infections have been known to modulate the immune response of the host and several helminth-derived molecules were suggested to have protective effects against allergic diseases and autoimmune diseases. In the previous study, crude extracts of Caenorhabditis elegans (CEC) have been shown to suppress airway inflammation in a murine asthma model. The present study was undertaken to investigate the effects of CEC on the interaction between bone marrow-derived dendritic cells (BMDCs) and CD4+ T helper cells of mice. CEC treatment on BMDCs markedly attenuated the expression of MHC class II molecules and co-stimulatory molecules including CD80, CD86, and CD40 compared to BMDCs stimulated by LPS alone (P<0.01). Production of pro-inflammatory cytokines including IL-12p70, IL-6 and TNF-α, was significantly decreased and that of an anti-inflammatory cytokine, IL-10, was elevated by stimulation of BMDCs with CEC and LPS than those stimulated by LPS alone (P<0.05). Moreover, LPS+CEC-pulsed BMDCs suppressed proliferation of CD4+ T cells. CD4+ T cells cultured with LPS+CEC-pulsed BMDCs produced significantly higher amounts of Th1 cytokine, interferon-γ (IFN-γ), and lower Th2 cytokines including IL-4, IL-5 and IL-13 compared with those with LPS-pulsed BMDCs (P<0.05). Taken together, the present results suggest that CEC activates BMDCs and induces naïve CD4+ T cells to differentiate into Th1 cells and also suppresses the proliferation of CD4+ T cells. Therefore, CEC may modulate the immune response of hosts by interacting with BMDCs, resulting in the differentiation of naïve T cells to Th1 cells.

Dissection of Immunomodulatory Mechanisms in Chronic Lung Disease Under Various Conditions

Lee Seunghyun 연세대학교 일반대학원 2025 국내석사

결핵(TB)과 천식과 같은 만성 폐질환은 구조 세포와 면역세포 간의 복잡한 상호작용에 의해 매개되는 만성 염증을 특징으로 한다. 다양한 면역 경로를 표적으로 하는 면역 조절 접근법은 질환 완화의 가능성을 제공할 수 있으며, 만성 염증을 효과적으로 조절하고 조직 손상을 최소화하는 전략은 이러한 폐질환을 관리하는 데 필수적이다. 본 연구에서는 서로 다른 폐질환 마우스 모델을 활용하여 면역 조절 기전을 탐색하였다. 첫 번째 모델은 NADPH oxidase 계열의 구성원인 NOX4가 결핵 감염과 천식에서 면역 반응을 어떻게 조절하는지를 평가하였고, 두 번째 모델에서는 항염증 효과로 알려진 콜히친(colchicine)이 천식에서 적응 면역 기전에 영향을 줄 수 있는지를 분석하였다. 결핵 마우스 모델에서 NOX4 결핍은 폐 내 세균 부하 감소 및 염증 완화와 연관되었으며, Nox4⁻/⁻ 마우스에서는 수지상세포(DC)의 침윤과 IFN-γ를 분비하는 CD4⁺ T 세포의 비율이 증가하였다. 기전적으로, Nox4⁻/⁻ 유래 DC는 AKT/GSK-3β 경로를 통한 IRF1 활성화에 의해 IL-12 발현이 증가하였고, 이로 인해 Th1 분화가 촉진되어 효과적인 Mtb 면역 조절이 유도되었다. 또한 Bacillus Calmette-Guérin(BCG) 백신은 Nox4⁻/⁻ 마우스에서 더 효과적으로 작용하였으며, 이는 NOX4가 결핵 감염에서 보호 면역을 음성적으로 조절함을 시사하며, 백신 보조제 타깃으로서의 가능성을 나타낸다. 한편 천식 모델에서는 NOX4 결핍이 기관지 염증 및 점액 생성은 완화하였지만, Th2 사이토카인 수치나 호산구 유입에는 유의미한 영향을 주지 않았다. 반면 콜히친은 자극된 CD4⁺ T 세포에서 IL-4 및 IL-13 분비를 유의미하게 억제하였고, 폐조직과 기관지폐포세척액(BALF) 모두에서 호산구 침윤을 현저히 감소시켰다. 그러나 콜히친은 이미 형성된 조직 병리학적 변화에는 뚜렷한 개선 효과를 보이지 않았으며, 이는 사이토카인 조절과 조직 재형성 간의 분리 가능성을 시사한다. 이러한 결과는 효과적인 천식 조절을 위해서는 선천 면역 경로와 적응 면역 경로를 동시에 표적으로 삼는 전략이 필요함을 강조한다. 종합적으로, 본 연구는 NOX4가 결핵과 천식이라는 서로 다른 폐질환 환경에서 선천면역세포 기능과 조직 염증을 조절한다는 점을 보여준다. 반면, 콜히친은 Th2 매개 사이토카인 반응에 선택적으로 작용하였으나 구조적 병리 개선에는 제한적인 효과를 보였다. 이와 같은 결과는 산화 스트레스를 조절하고 콜히친과 같은 비스테로이드성 항염증제를 대안적 접근법으로 탐색하는 것이 만성 폐질환의 치료 가능성을 제시할 수 있음을 시사한다. 특히, 면역 반응을 조절하여 염증을 완화하는 접근은 효과적인 치료 옵션이 될 수 있으며, 이 과정에서 NOX4는 유망한 면역조절 표적으로 작용할 가능성이 있다. Chronic lung diseases such as tuberculosis (TB) and asthma involve chronic inflammation mediated by complex interactions between structural and immune cells. Alternative approaches to immune regulation may offer potential for disease amelioration by targeting multiple immune pathways. Developing immune regulatory approaches that can control chronic inflammation and minimize tissue injury is essential in managing lung diseases such as tuberculosis and asthma. In this study, I examined immunoregulatory mechanism using different disease models. One focused on the role of NOX4, a member of the NADPH oxidase family, in controlling immune responses during Mycobacterium tuberculosis (Mtb) infection and asthma. The other of the study explored whether colchicine, known for its anti-inflammatory effects, could modulate adaptive immune mechanisms involved in asthma. In a mouse model of TB, a diminished bacterial burden and reduced pulmonary inflammation were observed in the context of NOX4 deficiency. Nox4⁻/⁻ mice exhibited increased infiltrated dendritic cells (DCs) and increased proportions of CD4⁺ T cells secreting IFN-γ. Mechanistically, DCs from Nox4⁻/⁻ mice showed augmented IL-12 expression, mediated by IRF1 activation through the AKT/GSK-3β pathway. This enhanced IL-12 response facilitated more effective Type 1 helper cell polarization and immune control of Mtb. Moreover, Bacillus Calmette-Guérin vaccination was more efficacious in Nox4⁻/⁻ mice, indicating that NOX4 negatively regulates protective immunity under Mtb infection and potentially indicate a promising adjuvant target for TB vaccines. In a separate model of asthma, NOX4 deficiency alleviated bronchial inflammation and mucus production but did not significantly affect Type 2 helper cell (Th2) cytokine levels or eosinophil recruitment. In contrast, colchicine significantly suppressed the secretion of IL-4 and IL-13 from stimulated CD4⁺ T cells and led to a significant reduction in eosinophil infiltration within both lung tissue and bronchoalveolar lavage fluid in a mouse model of conventional asthma. However, colchicine failed to reverse established histopathological changes, suggesting a dissociation between cytokine modulation and tissue remodeling. These results underscore the necessity of targeting both innate epithelial signaling and adaptive immunity for effective asthma control. Taken together, this dual-chapter investigation illustrates that NOX4 regulates innate immune cell function and tissue inflammation across two distinct pulmonary disease contexts. Meanwhile, colchicine exerts selective effects on Th2-mediated cytokine responses without fully resolving structural pathology. These findings suggest that targeting oxidative stress or employing anti-inflammatory agents such as colchicine may represent a meaningful therapeutic approach for regulating inflammation in chronic lung diseases. In particular, modulating immune responses to alleviate inflammation may serve as an effective treatment option, with NOX4 emerging as promising immunoregulatory target in this context.

Cytokine- and Transcription Factor-Mediated Mechanisms of T Follicular Helper Cell Regulation

Read, Kaitlin Adele The Ohio State University ProQuest Dissertations & 2023 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

CD4+ T "helper" populations comprise a key subset of adaptive immune cells that are critical for orchestrating antigen-specific immune responses for both the clearance of pathogens and elimination of cancers. This population responds to insult-specific environmental signals, including those from cytokines, by differentiating into a number of functionally distinct subsets, which produce cytokines and interact with additional immune cells to effect their diverse functions. Of these, T follicular helper (TFH) cells are established coordinators of humoral immune responses, as they engage in bi-directional signaling with B cells, via both cell surface receptors and cytokine signals. Ultimately, this interaction is critical for the germinal center reaction, during which B cells are activated, proliferate, and are ultimately selected to support the generation of high-affinity B cell clones, and thus, high-affinity antibodies. This process is also required for the formation of long-lived plasma cell populations, which are a key part of both natural and vaccine-induced immunological memory. In contrast to this important role, TFH cells have also been implicated in autoimmune disorders, including rheumatoid arthritis, systemic lupus erythematosus, and others, for which the production of autoantibodies is a key aspect of pathogenesis. To date, the full scope of mechanisms underlying TFH cell differentiation are incompletely understood. Complicating this process, TFH cells are not comprised of a single, monolithic population, and numerous studies support the existence and function of 'polyfunctional' TFH populations which exhibit characteristics of other CD4+ T cell subsets (recently reviewed in (1)). Thus, it will be important for TFH-focused work to identify not only shared, but also TFH-subset-specific, regulatory mechanisms. Here, I present findings regarding both cytokine- and transcription-mediated factor mechanisms by which TFH populations are regulated.First, we identify the contribution of cytokine signals to the differentiation of T helper 1 (TH1)-biased TFH (TFH1) cells, which exhibit characteristics of TH1 cells, including the production of IFN-γ, and have recognized roles in both immune responses to pathogens and the pathogenesis of autoimmune disease. Given their apparent importance to human health, there is interest in understanding the mechanisms that regulate TFH1 cell formation and function. However, their origin and the molecular requirements for their differentiation are unclear. Here, we describe a population of murine TH1-derived, TFH1-like cells that express the chemokine receptor Cxcr3 and produce both the TH1 cytokine interferon-? and the TFH-associated cytokine interleukin-21 (IL-21). These TFH1-like cells promote B cell activation and antibody production at levels indistinguishable from conventional IL-6-derived TFH-like cells. Regarding their regulatory requirements, we find that IL-12 signaling is necessary for the differentiation and function of this TFH1-like cell population. Specifically, IL-12-dependent activation of STAT4, and unexpectedly STAT3, promotes increased expression of IL-21 and the TFH lineage-defining transcription factor Bcl-6 in TFH1-like cells. Taken together, these findings provide insight into the potential origin and differentiation requirements of TFH1 cells.Second, we identify Aiolos as a reciprocal regulator of TFH and cytotoxic programming. Effective immunity against intracellular pathogens such as influenza and SARS-CoV-2 requires the generation of CD4+ T cell subsets that coordinate diverse aspects of the immune response, including TFH and TH1 cells. A third population, CD4+ cytotoxic T lymphocytes (CD4-CTLs), facilitates clearance of infection via mechanisms normally associated with CD8+ T cells. The mechanisms that guide the differentiation of these complementary cell populations are incompletely understood. Here, we identify the transcription factor Aiolos as a reciprocal regulator of TFH and CD4-CTL programming. We demonstrate that Aiolos-deficient cells exhibit increased production of cytotoxic effector molecules during influenza infection, including perforin and granzyme B. We further find that loss of Aiolos results in global disruptions to the TFH transcriptome. This includes reduced expression of key TFH-associated transcription factors, such as Bcl-6 and Zfp831, which we identify as direct Aiolos targets. We further demonstrate that Aiolos deficiency allows for elevated expression of IL-2R subunits, enhanced sensitivity to IL-2, and increased STAT5 association with key CD4-CTL gene targets. These include CTL-associated transcription factors (Eomes, Blimp-1), effector molecules (granzyme B, perforin, IFN-γ), and IL2Rα itself. Thus, our collective findings identify Aiolos as a pivotal regulator of CD4-CTL and TFH programming and highlight its potential as a target for manipulating CD4+ T cell cytotoxic and humoral responses.Together, the work presented in this dissertation provides novel i) insights into TFH cell biology, and ii) potential targets for the generation of immunotherapies to support vaccine efficacy and the treatment of human disease.