Single-cell RNA sequencing identifies distinct transcriptomic signatures between PMA/ionomycin- and αCD3/αCD28-activated primary human T cells

Jung Ho Lee,Brian H Lee,Soyoung Jeong,Christine Suh-Yun Joh,Hyo Jeong Nam,Hyun Seung Choi,Henry Sserwadda,Ji Won Oh,Chung-Gyu Park,Seon-Pil Jin,Hyun Je Kim Korea Genome Organization 2023 Genomics & informatics Vol.21 No.2

Immunologists have activated T cells in vitro using various stimulation methods, including phorbol myristate acetate (PMA)/ionomycin and αCD3/αCD28 agonistic antibodies. PMA stimulates protein kinase C, activating nuclear factor-κB, and ionomycin increases intracellular calcium levels, resulting in activation of nuclear factor of activated T cell. In contrast, αCD3/αCD28 agonistic antibodies activate T cells through ZAP-70, which phosphorylates linker for activation of T cell and SH2-domain-containing leukocyte protein of 76 kD. However, despite the use of these two different in vitro T cell activation methods for decades, the differential effects of chemical-based and antibody-based activation of primary human T cells have not yet been comprehensively described. Using single-cell RNA sequencing (scRNA-seq) technologies to analyze gene expression unbiasedly at the single-cell level, we compared the transcriptomic profiles of the non-physiological and physiological activation methods on human peripheral blood mononuclear cell-derived T cells from four independent donors. Remarkable transcriptomic differences in the expression of cytokines and their respective receptors were identified. We also identified activated CD4 T cell subsets (CD55⁺) enriched specifically by PMA/ionomycin activation. We believe this activated human T cell transcriptome atlas derived from two different activation methods will enhance our understanding, highlight the optimal use of these two in vitro T cell activation assays, and be applied as a reference standard when analyzing activated specific disease-originated T cells through scRNA-seq.

Vitamin C is taken up by human T cells via sodium-dependent vitamin C transporter 2 (SVCT2) and exerts inhibitory effects on the activation of these cells in vitro

Jun-Man Hong,Jin-Hee Kim,Jae Seung Kang,Wang Jae Lee,Young-il Hwang 대한해부학회 2016 Anatomy & Cell Biology Vol.49 No.2

Vitamin C is an essential micronutrient that affects immune responses. T cells are one of the main players in acquired immunity and have been reported to be influenced by in vivo vitamin C supplementation. Yet, the way by which T cells uptake vitamin C and what direct effects vitamin C exerts on the cells are not known. To elucidate, we isolated human peripheral blood T cells and analyzed the expression of sodium-dependent vitamin C transporters (SVCT). T cells were activated in vitro in the absence or presence of vitamin C, before or after activation. As results, human T cells expressed SVCT2, but not SVCT1, and the expression level increased following activation. Vitamin C added in the culture media generally did not affect T-cell behaviors following activation, such as proliferation, apoptosis, expression of CD25 and CD69, and interleukin 2 secretion, regardless whether it was added before or after activation. However, exceptionally, high concentration vitamin C, when it was added before activation, but not after activation, did exert toxic effects on cell activation with respect to the above-mentioned parameters. In conclusion, we showed the expression of SVCT2 in human T cells for the first time. Vitamin C exerted toxic effects, at least in vitro, when the concentration was high and when it was given before activation. These toxic effects are not thought to be via anti-oxidant effects of vitamin C.

Vitamin C is taken up by human T cells via sodium-dependent vitamin C transporter 2 (SVCT2) and exerts inhibitory effects on the activation of these cells in vitro

홍준만,김진희,강재승,이왕재,황영일 대한해부학회 2016 Anatomy & Cell Biology Vol.49 No.2

Vitamin C is an essential micronutrient that affects immune responses. T cells are one of the main players in acquired immunity and have been reported to be influenced by in vivo vitamin C supplementation. Yet, the way by which T cells uptake vitamin C and what direct effects vitamin C exerts on the cells are not known. To elucidate, we isolated human peripheral blood T cells and analyzed the expression of sodium-dependent vitamin C transporters (SVCT). T cells were activated in vitro in the absence or presence of vitamin C, before or after activation. As results, human T cells expressed SVCT2, but not SVCT1, and the expression level increased following activation. Vitamin C added in the culture media generally did not affect T-cell behaviors following activation, such as proliferation, apoptosis, expression of CD25 and CD69, and interleukin 2 secretion, regardless whether it was added before or after activation. However, exceptionally, high concentration vitamin C, when it was added before activation, but not after activation, did exert toxic effects on cell activation with respect to the above-mentioned parameters. In conclusion, we showed the expression of SVCT2 in human T cells for the first time. Vitamin C exerted toxic effects, at least in vitro, when the concentration was high and when it was given before activation. These toxic effects are not thought to be via anti-oxidant effects of vitamin C.

Innate-like Cytotoxic Function of Bystander-Activated CD8⁺ T Cells Is Associated with Liver Injury in Acute Hepatitis A

Kim, Jihye,Chang, Dong-Yeop,Lee, Hyun Woong,Lee, Hoyoung,Kim, Jong Hoon,Sung, Pil Soo,Kim, Kyung Hwan,Hong, Seon-Hui,Kang, Wonseok,Lee, Jino,Shin, So Youn,Yu, Hee Tae,You, Sooseong,Choi, Yoon Seok,Oh, Elsevier 2018 Immunity Vol.48 No.1

<P><B>Summary</B></P> <P>Acute hepatitis A (AHA) involves severe CD8<SUP>+</SUP> T cell-mediated liver injury. Here we showed during AHA, CD8<SUP>+</SUP> T cells specific to unrelated viruses became activated. Hepatitis A virus (HAV)-infected cells produced IL-15 that induced T cell receptor (TCR)-independent activation of memory CD8<SUP>+</SUP> T cells. TCR-independent activation of non-HAV-specific CD8<SUP>+</SUP> T cells were detected in patients, as indicated by NKG2D upregulation, a marker of TCR-independent T cell activation by IL-15. CD8<SUP>+</SUP> T cells derived from AHA patients exerted innate-like cytotoxicity triggered by activating receptors NKG2D and NKp30 without TCR engagement. We demonstrated that the severity of liver injury in AHA patients correlated with the activation of HAV-unrelated virus-specific CD8<SUP>+</SUP> T cells and the innate-like cytolytic activity of CD8<SUP>+</SUP> T cells, but not the activation of HAV-specific T cells. Thus, host injury in AHA is associated with innate-like cytotoxicity of bystander-activated CD8<SUP>+</SUP> T cells, a result with implications for acute viral diseases.</P> <P><B>Highlights</B></P> <P> <UL> <LI> During acute hepatitis A (AHA), non-HAV-specific memory CD8<SUP>+</SUP> T cells are activated </LI> <LI> Non-HAV-specific CD8<SUP>+</SUP> T cells are activated by IL-15 produced by HAV-infected cells </LI> <LI> CD8<SUP>+</SUP> T cells of AHA patients exert TCR-independent, innate-like cytotoxicity </LI> <LI> Innate-like cytotoxicity of CD8<SUP>+</SUP> T cells is associated with liver injury in AHA </LI> </UL> </P> <P><B>Graphical Abstract</B></P> <P>[DISPLAY OMISSION]</P>

Paclitaxel inhibits the hyper-activation of spleen cells by lipopolysaccharide and induces cell death

Hyun-Ji Kim,주홍구 대한수의학회 2016 Journal of Veterinary Science Vol.17 No.4

Paclitaxel was isolated from the bark of the Pacific yew, Taxus brevifolia, and used as an anticancer agent. Paclitaxel prevents cancer celldivision by inhibiting spindle fiber function, inducing cell death. A recent study demonstrated that paclitaxel binds to myeloid differentiationprotein-2 of Toll-like receptor 4 and prevents the signal transduction of lipopolysaccharide (LPS). Paclitaxel converts immune cellshypo-responsive to LPS. In this study, we investigated whether paclitaxel can inhibit the phenotype and function of immune cells. Toaccomplish this, we used spleen cells, a major type of immune cell, LPS, a representative inflammatory agent and a mitogen for B lymphocytes. LPS profoundly increased the activation and cytokine production of spleen cells. However, paclitaxel significantly inhibited LPS-inducedhyper-activation of spleen cells. Furthermore, we found that paclitaxel induced cell death of LPS-treated spleen cells. These results suggestthat paclitaxel can inhibit the hyper-immune response of LPS in spleen cells via a variety of mechanisms. These findings suggest that paclitaxelcan be used as a modulating agent for diseases induced by hyper-activation of B lymphocytes. Taken together, these results demonstrate thatpaclitaxel inhibits the function of spleen cells activated by LPS, and further induces cell death.

BMB Reports : Biphasic activation of extracellular signal-regulated kinase (ERK) 1/2 in epidermal growth factor (EGF)-stimulated SW480 colorectal cancer cells

( Donghyun Joo ),( Jong Soo Woo ),( Kwang Hyun Cho ),( Seung Hyun Han ),( Tae Sun Min ),( Deok Chun Yang ),( Cheol Heui Yun ) 생화학분자생물학회 2016 BMB Reports Vol.49 No.4

Cancer cells have different characteristics due to the genetic differences where these unique features may strongly influence the effectiveness of therapeutic interventions. Here, we show that the spontaneous reactivation of extracellular signalregulated kinase (ERK), distinct from conventional ERK activation, represents a potent mechanism for cancer cell survival. We studied ERK1/2 activation in vitro in SW480 colorectal cancer cells. Although ERK signaling tends to be transiently activated, we observed the delayed reactivation of ERK1/2 in epidermal growth factor (EGF)-stimulated SW480 cells. This effect was observed even after EGF withdrawal. While phosphorylated ERK1/2 translocated into the nucleus following its primary activation, it remained in the cytoplasm during late-phase activation. The inhibition of primary ERK1/2 activation or protein trafficking, blocked reactivation and concurrently increased caspase 3 activity. Our results suggest that the biphasic activation of ERK1/2 plays a role in cancer cell survival; thus, regulation of ERK1/2 activation may improve the efficacy of cancer therapies that target ERK signaling. [BMB Reports 2016; 49(4): 220-225]

Resveratrol의 CD4+ T 세포 활성과 분화 억제 효과

서동원,이영주,이상명 한국자원식물학회 2014 한국자원식물학회지 Vol.27 No.5

Resveratrol is a naturally occurring stilbene which is safe and well-described compound with a potent anti-inflammatory activity. Recent studies suggested that resveratrol suppressed various inflammation mediated diseases such as asthma, chronic colitis, rheumatoid arthritis, and type 1 diabetes. These studies indicated that resveratrol might directly modulate CD4 + helper T cells (Th cells)-mediated immune responses. However, it is not fully elucidated whether resveratrol directly regulates CD4 + Th cell activation and differentiation. In the present study, CD4 + Th cells were purified from C57BL/6 and treated with various concentrations of resveratrol. We found that resveratrol directly suppressed CD4 + Th cells activation, leading to a defect in T cell proliferation. When CD4 + Th cells were treated with resveratrol, cytokine production was also significantly reduced in a dose dependent manner. In accordance with these results, resveratrol even inhibited CD4 + Th cells differentiation into Th1, Th2 or Th17, which produces IFN-γ, IL-4 or IL-17 respectively. We also found that resveratrol could induce apoptosis of CD4 + T cells at a high concentration. Our data demonstrated that resveratrol inhibited directly CD4 + Th cells activation and differentiation. It suggests that resveratrol could be an efficient therapeutic strategy for autoimmune diseases in which CD4 + Th cells play a critical role. Resveratrol은 천연 stilbene으로 안전성 있는 항염증 활성을 가진 화합물로 알려져 있다. 최근의 연구들에서 resveratrol이 천식, 만성 대장염, 류마티스성 관절염과 같이 염증에 의해발생하는 다양한 질병을 억제한다고 보고되었다. 이러한 연구들은 resveratrol이 CD4+ helper T cells (Th cells)에 의한 면역반응을 조절할 것이라고 제시하였다. 그러나 resveratrol이 직접적으로 Th cells의 활성화와 분화를 조절하는지 완전히 밝혀지지 않았다. C57BL/6에서 Th cells을 분리하여 다양한 농도의resveratrol을 세포에 처리하였다. 본 연구에서는 resveratrol이 직접적으로 Th cells의 활성화와 증식을 억제하는 것을 확인하였다. Th cells에 resveratrol을 처리하였을 때 IFN-γ, IL-4,IL-17 사이토카인 생성이 농도에 따라 유의하게 감소하였고 또한 Th cells이 이러한 사이토카인들을 분비하는 Th1과 Th2과Th17으로 분화되는 것이 억제되었다. 그리고 고농도의 resveratrol이 Th cells의 세포사멸을 유도하는 것으로 확인되었다. 본 연구에서는 resveratrol이 Th cells의 활성화와 분화를 직접적으로억제하는 것을 확인하였으며, 이는 resveratrol이 CD4+ Thcells에 의해 발생되는 자가면역질환의 효과적인 치료법이 될수 있을 것이라고 제시한다.

Eupatilin inhibits T-cell activation by modulation of intracellular calcium flux and NF-κB and NF-AT activity

Kim, Young-Dae,Choi, Suck-Chei,Oh, Tae-Young,Chun, Jang-Soo,Jun, Chang-Duk Wiley Subscription Services, Inc., A Wiley Company 2009 Journal of cellular biochemistry Vol.108 No.1

<P>Eupatilin, one of the pharmacologically active ingredients of Artemisia princeps, exhibits a potent anti-ulcer activity, but its effects on T-cell immunity have not been investigated. Here, we show that eupatilin has a profound inhibitory effect on IL-2 production in Jurkat T cells as well as in human peripheral blood leukocytes. Eupatilin neither influenced clustering of CD3 and LFA-1 to the immunological synapse nor inhibited conjugate formation between T cells and B cells in the presence or absence of superantigen (SEE). Eupatilin also failed to inhibit T-cell receptor (TCR) internalization, thereby, suggesting that eupatilin does not interfere with TCR-mediated signals on the membrane proximal region. In unstimulated T cells, eupatilin significantly induced apoptotic cell death, as evidenced by an increased population of annexin V<SUP>+</SUP>/PI<SUP>+</SUP> cells and cleavage of caspase-3 and PARP. To our surprise, however, once cells were activated, eupatilin had little effect on apoptosis, and instead slightly protected cells from activation-induced cell death, suggesting that apoptosis also is not a mechanism for eupatilin-induced T-cell suppression. On the contrary, eupatilin dramatically inhibited I-κBα degradation and NF-AT dephosphorylation and, consequently, inhibited NF-κB and NF-AT promoter activities in PMA/A23187-stimulated T cells. Interestingly, intracellular calcium flux was significantly perturbed in cells pre-treated with eupatilin, suggesting that calcium-dependent cascades might be targets for eupatilin action. Collectively, our results provide evidence for dual regulatory functions of eupatilin: (1) a pro-apoptotic effect on resting T cells and (2) an immunosuppressive effect on activated T cells, presumably through modulation of Ca<SUP>2+</SUP> flux. J. Cell. Biochem. 108: 225–236, 2009. © 2009 Wiley-Liss, Inc.</P>

족제비싸리 부위별 추출물의 항암 및 면역활성

김정화,김대호,유진현,권민철,이현정,이학주,이현용 韓國藥用作物學會 2005 한국약용작물학회지 Vol.13 No.1

족제비싸리의 각 부위별 추출물의 정상세포에 대한 독성을 살펴보기 위하여 인간 정장 폐세포인 HEL299를 이용하였으며 각 추출물은 1.0 g/l의 농도에서 최고 22.2%의 세포독성을 나타내었다. 또한 항암활성효과를 살펴보기 위하여 2가지의 암세포를 이용하였는데, A549와 AGS에서 뿌리 추출물의 경우 1.0 g/l의 농도에서 70%정도의 높은 억제활성을 나타내었고, 또한 암세포의 생육활성에 대한 정상 세포의 세포독성의 비로 나타낸 선택적 사멸도는 고농도에서는 모두 1.5이상으로 나타나 모두 암세포에 대한 선택성이 있는 것으로 나타났다. 면역세포의 생육 촉진 실험을 위하적 인간의 B cell과 T cell을 이용하였으며 각 추출물의 농도를 0.5 g/l로 하여 실험을 실시하였다. 생육도는 T cell의 경우 배양 6일째 수피 추출물이 7.5×104 cells/ml의 세포농도를 나타냈으며 B cell의 경우 배양 5일째 4.5×104 cells/ml의 농도를 나타내었다. 면역세포를 이용한 cytokine의 분비량 측정실험에서는 뿌리의 추출물이 배양시간에 따른 cytokine의 분비가 가장 높게 나타나는 것을 확인 할 수 있었다. 각 세포의 TNF-α의 분비량은 6일째 최고의 분비량을 나타내었고 뿌리추출물의 경우 대조구에 비해 7~8배 가량 정도 더 높은 분비량을 나타낸 것으로 확인되었다. IL-6의 경우 5일째까지 감소하다가 6일째부터 증가하는 경향을 나타내었는데 잎과 열매에서 가장 높은 분비량을 나타내었다. 또한 족제비싸리 추출물을 첨가한 배지에 의한 B cell과 T cell의 면역활성 및 cytokine의 분비량에 따른 NK cell의 면역 활성을 측정하였으며 모든 추출물에서 배양 시간에 따라 유의적으로 증가하는 것을 확인하였다. 그 중 족제비싸리의 수피 추출물에서 B cell의 경우 14.2×104 cells/ml, T cell의 경우 14.2×104 cells/ml으로 가장 높은 생육 활성을 나타내었다. This study was performed to experiment about useful biological activities of the parts of the extracts from Amorpha fruticosa LINNE. Experimental studies were progressed through the anticancer activities and immune activities such as cell cytotoxicity, inhibition activities of cell growth, cell growth of human B and T cell, productivity of cytokines and natural killer cell activities. The cell cytotoxicity using human Lung normal cell (HEL299) was showed cytotoxicity of below 22% by extracts of Amorpha fruticosa L. in 1.0 g/l concentration. The anticancer activities were increased in over 70% by roots extracts of Amorpha fruticosa L. in A549 and AGS cells. The immune cell growth using human immune B and T cells was increased against control by barks extracts of Amorpha fruticosa L. The secretion of cytokines (IL-6, TNF-α) from human immune B and T cells was showed secretion of the amount of cytokines by roots extracts of Amorpha fruticosa L. Also NK cell growth was increased against control all of the extracts of Amorpha fruticosa L. From the results, the roots and barks extracts of Amorpha fruticosa L. were showed useful biological activities.

Emerging role of bystander T cell activation in autoimmune diseases

Chae-Hyeon Shim,Sookyung Cho,Young-Mi Shin,Je-Min Choi 생화학분자생물학회 2022 BMB Reports Vol.55 No.2

Autoimmune disease is known to be caused by unregulated selfantigen-specific T cells, causing tissue damage. Although antigenspecificity is an important mechanism of the adaptive immunesystem, antigen non-related T cells have been found in the inflamedtissues in various conditions. Bystander T cell activationrefers to the activation of T cells without antigen recognition. During an immune response to a pathogen, bystander activationof self-reactive T cells via inflammatory mediators such as cytokinescan trigger autoimmune diseases. Other antigen-specificT cells can also be bystander-activated to induce innate immuneresponse resulting in autoimmune disease pathogenesis alongwith self-antigen-specific T cells. In this review, we summarizeprevious studies investigating bystander activation of various Tcell types (NKT, γδ T cells, MAIT cells, conventional CD4+, andCD8+ T cells) and discuss the role of innate-like T cell responsein autoimmune diseases. In addition, we also review previousfindings of bystander T cell function in infection and cancer. Abetter understanding of bystander-activated T cells versus antigenstimulatedT cells provides a novel insight to control autoimmunedisease pathogenesis.