Agonist-induced T cell receptor down-regulation: molecular requirements and dissociation from T cell activation

T cell receptor (TCR) down-regulation is a consequence of specific receptor engagement and plays an important role in modulating the T cell response. We have investigated the role of protein kinase C (PKC) and protein tyrosine kinases (PTK) in the induction of TCR down-regulation. We report that the mutation of S126 in the CD3-γ chain that is known to inhibit phorbol-12-myristate 13-acetate-induced TCR down-regulation does not affect down-regulation induced by a specific agonist. In addition, agonist-induced TCR down-regulation is not affected by blockade or depletion of PKC, neither by blockade or lack of PTK, while the same treatments efficiently interfere with T cell activation. These results demonstrate that TCR down-regulation is induced by early events which follow specific engagement by an agonist and can be dissociated from those required for full T cell activation.     

T cell immunity in neonates

Typically, neonates exhibit decreased or aberrant cellular immune responses when compared to adults, resulting in increased susceptibility to infection. However, it is clear that newborns are able to generate adult-like protective T cell responses under certain conditions. The focus of our research is to understand the deficiencies within the neonatal immune system that lead to improper cellular responses and how priming conditions can be altered to elicit the appropriate T cell response necessary to protect against development of pathogen-induced disease. With these goals in mind, we are exploring the attributes of neonatal T cells and their development, as well as the conditions during priming that influence the resulting response to immune challenge during the neonatal period.     

Effects of green tea polyphenols on murine transplant-reactive T cell immunity

Green tea polyphenols (GrTP), the active ingredient of green tea, may have immunosuppressive properties, but whether and how GrTP affect transplant-reactive T cells is unknown. To address this, we tested the effects of GrTP on in vitro and in vivo transplant-reactive T cell immunity. GrTP inhibited IFNgamma secretion by cultured monoclonal T cells and by alloreactive T cells in mixed lymphocyte reactions. Oral GrTP significantly prolonged minor antigen-disparate skin graft survival and decreased the frequency of donor-reactive interferon gamma-producing T cells in recipient secondary lymphoid organs compared to controls. In contrast to other hypothesized actions, oral GrTP did not alter dendritic cell trafficking to lymph nodes or affect metalloproteinase activity in the graft. This is the first report of an immunosuppressive effect of GrTP on transplant-reactive T cell immunity. The results suggest that oral intake of green tea could act as an adjunctive therapy for prevention of transplant rejection in humans.     

肿瘤源性热休克蛋白gp96诱导淋巴细胞反应及其抗肿瘤效应

目的：研究肿瘤细胞来源的热休克蛋白gp96-多肽复合物在体外诱导脾淋巴细胞的特异性细胞毒性T淋巴细胞（CTL）反应.方法：利用蛋白纯化技术、SDS-PAGE凝胶电泳及Western blot法分离纯化、鉴定gp96-多肽;通过流式细胞术、免疫荧光技术、CCK-8法等检测经gp96多肽诱导的CD8^＋T细胞及其抗肿瘤效应.结果：经鉴定获得纯化的热休克蛋白;流式细胞仪检测表明,经gp96-肽复合物诱导后的CD8^＋T细胞比例达到近70%,远远高于对照组的35%、26%;该活化的CTL细胞在效靶比为50：1时的肿瘤杀伤率达72%,与对照组相比具有统计学意义;激光共聚焦显微镜观察证实实验诱导组的培养上清能诱导H22肿瘤细胞凋亡的形态学改变.结论：肿瘤来源的热休克蛋白gp96-肽复合物能诱导小鼠脾淋巴细胞的CTL反应,该活化的CTL具有特异性抗H22肿瘤细胞的免疫作用,并能分泌免疫活性物质诱导H22肿瘤细胞凋亡.     

Zinc inhibits interleukin-1-dependent T cell stimulation

Zinc is a trace element which is essential for immune functions. It directly induces monokine secretion by monocytes; however, effects of zinc on T cells appear contradictory. Apart from enhanced lymphocyte proliferation in peripheral blood mononuclear cells (PBMC), inhibitory properties of high zinc dosages have also been described. In this study, PBMC failed to produce lymphokines like interferon (IFN)-γ after stimulation with zinc in a serum- and LPS-free cell culture system, whereas monokine secretion [interleukin (IL)-1β] occurred. Zinc-uptake studies with the zinc-specific fluorescent probe zinquin revealed that zinc is taken up by PBMC within a few minutes, reaching nearly equal levels in PBMC, isolated monocytes, and T cells. However, if zinc was depleted 1 h after monocyte induction, zinc-free pre-cultured T cells were stimulated to secrete IFN-γ by zinc-induced monokines. Furthermore, the necessity for a cell-cell interaction between monocytes and T cells for IFN-γ induction was elucidated. Zinc ions inhibited the proliferation of the IL-1-dependent T cell line D 10N in a dose-dependent manner, suggesting a direct inhibitory effect of zinc. By immunoprecipitation we revealed a specific inhibition of IL-1 receptor-associated protein kinase (IRAK) by zinc ions. Therefore, in contrast to an indirect stimulation of T cells due to zinc-induced monokines, higher concentrations of zinc directly inhibit T cell functions by means of specific inhibition of IRAK and subsequent signaling events such as NFxB activation. The divergent effects of zinc on different cell populations, depending on the zinc concentration, could explain contradictory results of zinc stimulation. Furthermore, our data suggest new strategies of specific zinc-mediated immune modulation.     

Signal extinction and T cell repolarization in T helper cell-antigen-presenting cell conjugates

We have previously demonstrated that in T cell-antigen-presenting cell (APC) conjugates many T cell receptors (TCR) are serially triggered by a few peptide-MHC complexes, resulting in sustained signaling. Here, we investigate the mechanisms that determine the duration and extent of signaling. We show that in the course of the T helper cell-APC interaction, down-regulation of triggered TCR leads to extinction of signaling. However, T cells that have been activated by a previous encounter with peptide-pulsed APC and have extinguished signaling can swiftly repolarize towards APC displaying higher antigen concentrations and dedicate their help to these cells. These results demonstrate that TCR down-regulation allows T cells to calibrate their response and dedicate their help to APC offering the highest stimulus.     

Protein microarrays for the detection of biomarkers in head and neck squamous cell carcinomas

Protein microarrays are of increasing importance for high-throughput screening of fresh tissues. In our study, protein microarrays were generated by printing antibodies onto membranes to characterize protein profiles expressed by head and neck squamous cell carcinomas (HNSCCs). Cellular proteomes of 30 matched normal squamous epithelial cells and carcinoma specimens were analyzed after tissue microdissection using microarrays composed of 83 different antibodies. As controls, Western blot analysis and tissue microarrays (TMAs) containing 98 HNSCC specimens were used. Of the 83 proteins examined, 14 showed differential expression between HNSCCs and normal epithelium. The protein microarray approach revealed an upregulation of 8 proteins and a downregulation of 6 proteins. Bag-1, Cox-2, Hsp-70, Stat3, pescadillo, MMP-7 (matrilysin), IGF-2, and cyclin D1 were identified to be significantly upregulated, whereas suppressor of cytokine signaling 1, thrombospondin, TGF-beta1, Jun, Fos, and Fra-2 were downregulated. The differential expression of these proteins was confirmed using Western blot and TMA. Upon correlation of differentially regulated proteins with the clinicopathologic data of our patients, MMP-7 (matrilysin) was found to be associated with survival in univariate, but not multivariate, analysis. These data indicate that our protein arrays provide protein information in a systematic, reproducible, and also high-throughput fashion.     

T cell receptor-induced Fas ligand expression in cytotoxic T lymphocyte clones is blocked by protein tyrosine kinase inhibitors and cyclosporin A

Fas/APO-1 is a member of the tumor necrosis factor receptor family of proteins, that induces apoptosis when cross-linked with monoclonal antibody (mAb) or with its physiological ligand. Recently, both a perforin-based and a Fas-based mechanism have been proposed to account for T cell-mediated cytotoxicity. In the present study we used a murine CD8⁺ cytotoxic T lymphocyte (CTL) clone (KB5.C20) specific for H-2K^b and a T cell receptor (TcR)-negative variant of the same clone (2005^?D4) to test (i) whether the same cell can exert both cytotoxic effector mechanisms and (ii) the role of TcR engagement in the induction of Fas-based cytotoxicity. We demonstrate that both the TcR⁺ and TcR^? clones were able to express the Fas ligand after stimulation with phorbol 12-myristate 13-acetate (PMA)/ionomycin, and that TcR engagement of the KB5.C20 clone by means of antigen-bearing cells or of its anticlonotypic mAb (Désiré-1), which leads to Ca²⁺-dependent, presumably perforin-based, cytotoxicity, was also able to induce Fas-based cytotoxicity. In addition, using inhibitors we investigated the signal transduction pathway(s) involved in the induction of Fas-based cytotoxicity and expression of the Fas ligand mRNA in the CTL clones. The involvement of src-like protein tyrosine kinases (PTK) in Fas ligand induction through TcR engagement, was strongly suggested by inhibition with the src-like PTK inhibitor herbimycin A. Inhibition of Fas ligand induction by genistein, a more general TPK inhibitor, even upon stimulation by PMA plus ionomycin, suggested the possible involvement of PTK activities downstream of protein kinase C (PKC) in Fas ligand induction in CTL. Finally, the implication of the Ca²⁺/calmodulin-dependent protein phosphatase calcineurin in Fas ligand induction was demonstrated by the partial inhibition of Fas ligand induction with cyclosporin A. Thus, in CTL clones, Fas ligand expression is inducible by TcR engagement through a pathway similar to that involved in expression of some lymphokine genes.     

十二指肠球部溃疡患者红细胞免疫功能与T细胞亚群的变化

探讨幽门弯曲菌 (Helicobaclerpylori,Hp)阳性的十二指肠球部溃疡 (DU)患者红细胞免疫和T细胞亚群的变化及其相关关系。应用免疫黏附法和SAP法对 4 0例Hp阳性、6例Hp阴性的DU患者红细胞免疫活性 (RBC C3bRR、RBC ICR和RBC TRR)和外周血T细胞亚群 (CD3+ 、CD4 + 、CD8+ 和CD4 + /CD8+ )进行了检测。结果发现Hp阳性DU患者RBC C3bRR、RBC TRR、CD3+ 、CD4 + 和CD4 + /CD8+ 较Hp阴性患者无明显降低。Hp阳性的DU患者RBC C3bRR、RBC TRR、CD3+ 、CD4 + 和CD4 + /CD8+ 显著低于健康对照组 (P <0.0 1) ,而RBC ICR和CD8+ 显著高于对照组(P <0.0 1)。Hp阳性的DU患者疗程结束后 ,38例Hp被根除 ,根除率为 95 %。Hp根除且溃疡愈合者为 35例 ,愈合率为 86 % ,其红细胞免疫功能和T细胞亚群基本恢复正常。Hp根除但溃疡未愈合者 11例 ,溃疡愈合但Hp未根除者 2例 ,其红细胞免疫功能和T细胞亚群均有所改善。此外 ,红细胞免疫功能 (RBC C3bRR和RBC TRR)与T细胞亚群 (CD4 + /CD8+ )的变化呈明显正相关 (P <0.0 5 )。因此 ,红细胞免疫与T细胞亚群可能均参与了DU的发生、发展和转归。DU和Hp感染可能共同引起了细胞免疫功能的紊乱     

Coronin-1 expression in T lymphocytes: insights into protein function during T cell development and activation

Coronin has been described as an actin-binding protein of Dictyostelium discoideum, and it has been demonstrated to play a role in cell migration, cytokinesis and phagocytosis. Coronin-related proteins are found in many eukaryotic species, including Coronin-1 in mammals whose expression is enriched in the hematopoietic tissues. Here, we characterize Coronin-1 gene and protein expression in mouse embryonic and adult T lymphocytes. Coronin-1 is expressed throughout T cell ontogeny and in peripheral alphabeta T cells. Expression varies along thymic cell development, with maximum levels observed in embryonic early thymocytes and, in the adults, the selected TCRalphabeta(+) single-positive thymocytes. Subcellular localization analysis indicates that Coronin-1 is in equilibrium between the cytosol and the cell cortex, where it accumulates in F-actin-rich membrane protrusions induced by polarized activation of TCR-CD3-stimulated T cells. These data are consistent with a role of Coronin-1 in T cell differentiation/activation events involving membrane dynamisms and the cortical actin cytoskeleton.     

Obesity and CD8 T cell metabolism: Implications for anti-tumor immunity and cancer immunotherapy outcomes

Obesity is an established risk factor for many cancers and has recently been found to alter the efficacy of T cell–based immunotherapies. Currently, however, the effects of obesity on immunometabolism remain unclear. Understanding these associations is critical, given the fact that T cell metabolism is tightly linked to effector function. Thus, any obesity-associated changes in T cell bioenergetics are likely to drive functional changes at the cellular level, alter the metabolome and cytokine/chemokine milieu, and impact cancer immunotherapy outcomes. Here, we provide a brief overview of T cell metabolism in the presence and absence of solid tumor growth and summarize current literature regarding obesity-associated changes in T cell function and bioenergetics. We also discuss recent findings related to the impact of host obesity on cancer immunotherapy outcomes and present potential mechanisms by which T cell metabolism may influence therapeutic efficacy. Finally, we describe promising pharmaceutical therapies that are being investigated for their ability to improve CD8 T cell metabolism and enhance cancer immunotherapy outcomes in patients, regardless of their obesity status.     

Rate of neurite outgrowth in sympathetic neurons is highly resistant to suppression of protein synthesis: role of protein degradation/synthesis coupling

Neurites projecting to their target tissues during embryogenesis are subject to many perturbations that could influence their rate of growth. For example, environmental influences such as supply of neurotrophic factor or electrical activity profoundly influence the rate of neuronal protein synthesis. Because accumulation of protein is necessary for outgrowth to proceed normally, a perturbation in protein synthesis could cause a net change in the rate of accumulation of proteins with the result that neurite outgrowth rate increases or decreases. That neurite outgrowth does not normally seem to be subject to such perturbations suggests involvement of a homeostatic system controlling the rate of outgrowth. Consistent with this hypothesis, we show here that the rate of growth of neurites of sympathetic neurons is highly resistant to decreased rates of protein synthesis. Chronic suppression of protein synthesis by 60% had no significant effect on neurite outgrowth over a 2-day period while complete suppression halted it almost immediately. By the 3rd day of exposure, 60% suppression slowed outgrowth. Sustained suppression of protein synthesis rate by 33% had no effect on rate of outgrowth even after 7 days. We show that the ability of the growing neurites to resist protein synthesis suppression appears to be caused, at least in part, by a parallel decrease in the rate of protein degradation. The result of this coupling between degradation and synthesis is that proteins can continue to accumulate even when protein synthesis rate decreases, allowing normal rates of neurite outgrowth.     

Differential activation of T cell cytokine production by the extracellular signal-regulated kinase (ERK) signaling pathway

Stimulation of T cells via the T cell receptor (TCR) activates a number of signaling pathways that are potentially involved in the elicitation of physiological responses, such as the production of cytokines. The extracellular signal-regulated kinases (ERK) are a group of molecules activated in response to TCR ligation, whose role in T cell cytokine production is controversial. In this study, we have asked whether ERK activation is coupled to the production of a number of T cell-derived cytokines, and whether particular cytokines are differentially affected by ERK activation. To address these questions, we have utilized a constitutively active version of the immediate upstream activator of both ERK1 and ERK2, mitogen-activated/extracellular signal-regulated kinase 1 (MEK1), to activate ERK signaling selectively in the absence of other TCR-activated signaling pathways. The effect of constitutive MEK/ERK activation on T cell cytokine production was measured by transiently co-transfecting newly activated mouse T cells with DNA encoding constitutively active MEK1 (CA-MEK1) and the human interleukin-2 (IL-2) receptor α chain (hCD25), purifying hCD25⁺ transfectants by flow-cytometric cell sorting, and measuring the production of IL-3, IL-4, interferon (IFN)-γ and granulocyte/macrophage-colony-stimulating factor (GM-CSF) either in the presence or absence of ionomycin stimulation. Newly activated T cells were used in these experiments as they more closely resemble T cells activated in vivo than do transformed T cells or long-term established T cell clones. CA-MEK1 expression led to constitutive ERK activation, which acted synergystically with ionomycin treatment to stimulate cytokine production. Furthermore, these experiments revealed a hierarchy of cytokine responsiveness to MEK/ERK activation, such that the production of IL-3 was most affected, followed by GM-CSF, IFN-γ, and IL-4.     

抗T细胞免疫毒素的T细胞清除效率及其对造血细胞的影响

抗T细胞免疫毒素主要用于同种异体骨髓移植中清除T细胞,减轻移植物抗宿主反应(GVHD),提高移植成功率,也可用于白血病自体骨髓移植中清除残留白血病细胞,减少移植后白血病复发的可能性。本实验结果表明抗T细胞免疫毒素在10~(-8)M浓度对靶细胞的杀伤达3.0log以上,即可清除99.99%的靶细胞,并可有效地抑制周血T细胞转化功能和骨髓T细胞分泌IL—2的功能,同时对骨髓粒单系和红系造血细胞的增殖无明显的抑制作用。     

T cell immunity to SARS-CoV-2

Exceptional efforts have been undertaken to shed light into the biology of adaptive immune responses to SARS-CoV-2. T cells occupy a central role in adaptive immunity to mediate helper functions to different arms of the immune system and are fundamental to mediate protection, control, and clearance of most viral infections. Even though many questions remain unsolved, there is a growing literature linking specific T cell characteristics to differential COVID-19 severity and vaccine outcome. In this review, we summarize our current understanding of CD4⁺ and CD8⁺ T cell responses in acute and convalescent COVID-19. Further, we discuss the T cell literature coupled to pre-existing immunity and vaccines and highlight the need to look beyond blood to fully understand how T cells function in the tissue space.     

Nature and nurture in Foxp3(+) regulatory T cell development, stability, and function

Foxp3(+) regulatory T lymphocytes (Treg) are critical homeostatic regulators of immune and inflammatory responses. Their absence leads to fulminant multiorgan autoimmunity. This review explores recent studies that have altered our emerging view of the development, stability, and plasticity of these cells. Treg appear not to be a single entity, but a family of immunomodulatory cell types with shared capabilities. On a first level, Treg may alternatively form in response to developmental cues in the thymus as a distinct lineage of CD4(+) T cells or adaptively, in response to environmental cues received by mature conventional CD4(+) T lymphocytes. These 2 populations bear distinct specificity, stability, and genetic profiles and are differentially used in immune responses. Secondarily, in a manner analogous to the generation of T helper (Th)-1, Th2, and other T cell subsets, Treg may further specialize, adapting to the needs of their immunologic surroundings. Treg therefore comprise developmentally distinct, functionally overlapping cell populations that are uniquely designed to preserve immunologic homeostasis. They combine an impressive degree of both stability and adaptability.     

Measurement of protein synthesis and degradation in C2C22 myoblasts using extracts of muscle from hormone treated bovine

A detailed methodology is described for determination of treatment effects on muscle cell protein synthesis and muscle cell protein degradation in a cell culture system. C₂C₂₂ mouse myoblasts were treated with growth media containing muscle extracts from bovine treated with different pharmaceutical agents. Radiolabeled amino acids were added to the growth media to determine treatment effects on protein synthesis and protein degradation. Percent protein synthesis was calculated by measuring amino acid uptake as a percentage over internal control. Percent protein degradation was measured using a pulse chase technique. These procedures will allow researchers to determine treatment effects on overall protein synthesis and degradation in vitro in a relatively short amount of time without excessive costs. A second benefit is that animals do not have to be fed radiolabeled feedstuffs. These procedures are not intended to elucidate the mechanisms behind pharmaceutical enhancement of muscle cell protein synthesis or protein degradation.     

再生障碍性贫血患者骨髓及外周血T、NK细胞膜分子和可溶性分子动态变化

目的研究T淋巴细胞及免疫分子在再生障碍性贫血（AA）致病中的作用及其与临床疗效的相关性。方法应用免疫荧光染色技术和流式细胞仪分析AA骨髓及外周血T淋巴细胞表型,ELISA测定骨髓和外周血白介素-2及其可溶性受体（IL-2,sIL-2R）、可溶性Fas配体（sFas-L）和Flt3配体（FL）的水平。结果（1）AA病人骨髓和外周血CD8+细胞百分率增加,CD4/CD8比例下降;骨髓血CD25+和HLA-DR+细胞百分率增多,急性AA增加尤为显著（P<0.01）;骨髓血中CD16+或CD56+细胞百分率也明显增多（P<0.05）。（2）所有AA患者骨髓及外周血IL-2含量均显著升高,绝大部分患者的sFas-L含量增加,FL水平升高尤为显著,高达正常水平的20倍。IL-2、sFas-L和FL的含量与临床疗效密切相关,经治疗有效的患者骨髓和外周血的IL-2、sFas-L和FL水平明显下降,但FL仍不能降至正常水平。结论T细胞的异常活化,多种免疫分子表达的异常升高,以及产生针对自身造血干/祖细胞的细胞毒效应,是AA造血功能衰竭的主要原因。     

妊娠17～37周正常和感染胎儿的T细胞免疫

目的探讨妊娠17-37周正常和宫内感染胎儿的T细胞免疫。方法超声引导下行脐带穿刺术收集129例胎儿外周血,其中97例为正常对照组胎儿血,32例为宫内感染组胎儿血(单纯疱疹病毒感染、弓形虫感染、风疹病毒感染),采用双色免疫荧光标记流式细胞仪技术测定胎儿外周血T细胞亚群百分率,3H-TdR掺入法检测T细胞增殖能力,分析生理状态下胎儿的T细胞亚群和T细胞增殖情况、宫内感染时胎儿T细胞亚群和增殖反应的变化。结果生理状态下胎儿CD3 、CD4 、CD8 T细胞百分率随孕龄增长而增加(P<0.01),相关系数为:r(CD3 )=0.63、r(CD4 )=0.45、r(CD8 )=0.27。CD4 /CD8 比值,CD8 HLA-DR 、CD3 CD45RO 、CD3 CD45RA T细胞百分率,T细胞增殖能力与孕龄无相关。CD3 CD45RA T细胞百分率为(96±2)%。宫内感染时,胎儿CD3 、CD4 、CD8 HLA-DR T细胞百分率减少,CD8 T细胞百分率增多,CD4 /CD8 比值降低,T细胞增殖能力下降,与正常对照组相比差异显著。CD3 CD45RO 、CD3 CD45RA T细胞百分率无明显变化。结论妊娠17-37周胎儿各T细胞亚群百分率随孕龄增长情况不同,但多数仍处于“初始状态”;宫内感染时,胎儿容易产生T细胞免疫抑制。     

Polarization and asymmetry in T cell metabolism

T cell activation results in a rapidly proliferating T cell endowed with a metabolic phenotype necessary for growth and division. However, before the cell can proceed towards this burst of cell division a phase of quiescence occurs, during which the basic mechanisms governing regulation of metabolic reprograming are established. This review focuses on key cellular processes controlling early metabolic regulation and how these circuits of metabolic control dictate distinct cellular fates upon the first asymmetric division.