T cell receptor repertoire of CD4+ and CD8+ T cell subsets in the allogeneic bone marrow transplant recipient

Allogeneic bone marrow transplantation (BMT) has become a therapy of choice for the treatment of certain malignancies and hematopoietic disorders. However, immunodeficiencies following BMT continue to cause significant morbidity and mortality. We have compared the T cell receptor (TCR) repertoire of BMT patients and healthy control individuals by staining peripheral blood mononuclear cells with fluorochrome-labeled TCR-specific antibodies. Several patients exhibited a biased pattern of TCR expression atypical of the healthy controls, yet no particular TCR bias characterized all patients. For example, we found that 2%–8% of T cell from healthy individuals expressed the V19 TCR. One BMT patient exhibited V19 expression on more than 60% of peripheral T cells, while additional patients expressed V19 on less than 1% of T cells. The patients with the most extreme skewing of TCR types suffered from graft-versus-host disease. The causes of skewed TCR V expression patterns in BMT patients are not fully understood, yet some researchers have suggested that an oligoclonal expansion of CD8⁺ T cell populations may be largely responsible. To test this hypothesis, we examined the TCR V repertoire of CD4⁺ and CD8⁺ T cell populations. We found that biased V expression characterized both CD4⁺ and CD8⁺ T cell populations, sometimes within a single individual. Thus, therapies directed toward CD8⁺ T cells alone may not fully correct repertoire abnormalities following BMT.     

Differential requirements for proliferation of CD4+ and gammadelta+ T cells to spirochetal antigens

Alphabeta+ and gammadelta+ T cells have different mechanisms of epitope recognition and are stimulated by antigens of different chemical nature. An immunization model with antigens from the spirochete Brachyspira hyodysenteriae was used to examine the requirements for proliferation of circulating porcine CD4+ and gammadelta+ T cells in mixed lymphocyte cultures. CD4+ T cells only responded to stimulation with B. hyodysenteriae antigens, whereas gammadelta+ T cells proliferated when cultures were stimulated with either spirochetal antigens or interleukin-2 (IL-2). T cells that had proliferated expressed high levels of IL-2-receptor-alpha (IL-2Ralpha). Furthermore, neutralization of IL-2 at the beginning of the culture period was more efficient in blocking gammadelta+ than CD4+ T cell proliferation. Immunization induced interferon-gamma (IFN-gamma) production by CD4+ T cells, whereas only a small fraction of the antigen-stimulated gammadelta+ T cells produced this cytokine. Our results indicate that, under the same environmental conditions, CD4+ T cell functions are more tightly regulated when compared to gammadelta+ T cells. We conclude that these differences are due, in part, to the enhanced gammadelta+ T cell responsiveness to IL-2.     

GITR ligand-costimulation activates effector and regulatory functions of CD4+ T cells

Engagement of glucocorticoid-induced TNFR-related protein (GITR) enables the costimulation of both CD25⁻CD4⁺ effector (Teff) and CD25⁺CD4⁺ regulatory (Treg) cells; however, the effects of GITR-costimulation on Treg function remain controversial. In this study, we examined the effects of GITR ligand (GITRL) binding on the respective functions of CD4⁺ T cells. GITRL-P815 transfectants efficiently augmented anti-CD3-induced proliferation and cytokine production by Teff cells. Proliferation and IL-10 production in Treg were also enhanced by GITRL transfectants when exogenous IL-2 and stronger CD3 stimulation was provided. Concomitant GITRL-costimulation of Teff and Treg converted the anergic state of Treg into a proliferating state, maintaining and augmenting their function. Thus, GITRL-costimulation augments both effector and regulatory functions of CD4⁺ T cells. Our results suggest that highly activated and increased ratios of Treg reverse the immune-enhancing effects of GITRL-costimulation in Teff, which may be problematic for therapeutic applications using strong GITR agonists.     

CD4+T cells do not mediate within-host competition between genetically diverse malaria parasites

Ecological interactions between microparasite populations in the same host are an important source of selection on pathogen traits such as virulence and drug resistance. In the rodent malaria model Plasmodium chabaudi in laboratory mice, parasites that are more virulent can competitively suppress less virulent parasites in mixed infections. There is evidence that some of this suppression is due to immune-mediated apparent competition, where an immune response elicited by one parasite population suppress the population density of another. This raises the question whether enhanced immunity following vaccination would intensify competitive interactions, thus strengthening selection for virulence in Plasmodium populations. Using the P. chabaudi model, we studied mixed infections of virulent and avirulent genotypes in CD4+T cell-depleted mice. Enhanced efficacy of CD4+T cell-dependent responses is the aim of several candidate malaria vaccines. We hypothesized that if immune-mediated interactions were involved in competition, removal of the CD4+T cells would alleviate competitive suppression of the avirulent parasite. Instead, we found no alleviation of competition in the acute phase, and significant enhancement of competitive suppression after parasite densities had peaked. Thus, the host immune response may actually be alleviating other forms of competition, such as that over red blood cells. Our results suggest that the CD4+-dependent immune response, and mechanisms that act to enhance it such as vaccination, may not have the undesirable affect of exacerbating within-host competition and hence the strength of this source of selection for virulence.     

A tolerogenic semimature dendritic cells induce effector T-cell hyporesponsiveness by activation of antigen-specific CD4+CD25+ T regulatory cells that promotes skin allograft survival in mice

Semimature dendritic cells (smDCs) can induce autoimmune tolerance by activation of host antigen-specific CD4⁺CD25⁺ regulatory T (Treg) cells. We hypothesized that donor smDCs injected into recipients would induce effector T-cell hyporesponsiveness by activating CD4⁺CD25⁺Treg cells, and promote skin allograft survival. Myeloid smDCs were derived from C57BL/6J mice (donors) in vitro. BALB/c mice (recipients) were injected with smDCs to generate antigen-specific CD4⁺CD25⁺Treg cells in vivo. Allograft survival was prolonged when BALB/c recipients received either C57BL/6J smDCs prior to grafting or C57BL/6J smDC-derived CD4⁺CD25⁺Treg cells post-grafting, and skin flaps from these grafts showed the highest IL-10 production regardless of rapamycin treatments. Our findings confirm that smDCs constitute an independent subgroup of DCs that play a key role for inducing CD4⁺CD25⁺Treg cells to express high IL-10 levels, which induce hyporesponsiveness of effector T cells. Pre-treating recipients with donor smDCs may have potential for transplant tolerance induction.     

Effects of CD4+ and CD8+ T cells in tumor-bearing mice on antibody production

The function of T cell subsets in tumor-bearing mice was examined using an in vitro culture system of anti-(sheep red blood cell) antibody production, which is known to be dependent on T cells. The helper function of T cells of fibrosarcoma-MethA-bearing mice in antibody production decreased with the tumor stage of the mice. T cells were separated into CD4⁺ and CD8⁺ cells for further analysis of T cell subsets by the panning method using monoclonal antibodies. The helper function of CD4⁺ T cells in antibody production began to decrease significantly in tumor-bearing mice 1 week after the tumor transplantation. On the other hand, the suppressive function of CD8⁺ T cells was retained and had not decreased in the mice even 3 weeks after the transplantation. The same changes in function of CD4⁺ and CD8⁺ T cells were also observed in Methl-bearing mice. These results suggested that this tumor-associated immunosuppression in antibody production is attributable to the decrease in helper activity of CD4⁺ T cells and the maintenance of the suppressive activity of CD8⁺ T cells.     

4-1BB costimulation enhances HSV-1-specific CD8+ T cell responses by the induction of CD11c+CD8+ T cells

Since 4-1BB plays a predominant role in CD8+ T cell responses, we investigated the effects of 4-1BB triggering on the primary and memory CD8+ T responses to HSV-1 infection. 4-1BB was detected on 10-15% of CD4+ and CD8+ T cells following the infection. 4-1BB-positive T cells were in the proliferative mode and showed the enhanced expression of anti-apoptotic proteins. Agonistic anti-4-1BB treatment exerted preferential expansion of CD8+ T cells and gB/H-2Kb-positive CD8+ T cells, and enhanced cytotoxicity against HSV-1 that was mainly mediated by CD11c+CD8+ T cells. CD11c+CD8+ T cells were re-expanded following re-challenge with HSV-1 at post-infection day 50, indicating that CD11c+CD8+ phenotype was maintained in memory CD8+ T cell pool. Our studies demonstrated that 4-1BB stimulation enhanced both primary and memory anti-HSV-1 CD8+ T cell responses, which was mediated by a massive expansion of antigen-specific CD11c+CD8+ T cells.     

The T cell activation marker CD150 can be used to identify alloantigen-activated CD4(+)25+ regulatory T cells

We have been investigating whether alloantigen-specific CD4(+)25+ regulatory T cells can be identified for use in treating graft-versus-host disease. CD150, which is upregulated on the surface of all activated T lymphocytes, was identified as a candidate marker for alloantigen-activated CD4(+)25+ regulatory T cells by gene chip analysis. Freshly isolated CD4(+)25+ cells had only low cell-surface expression of CD150, comparable to that of CD4(+)25- T cells. Increased CD150 expression was observed on all T cells after coculture with allogeneic stimulator cells. When purified CD4(+)25+ cells were precultured with allogeneic stimulator cells, then sorted into CD150+ and CD150- subsets, allosuppressive activity was contained primarily in the CD150+ fraction. These cells also suppressed the proliferation of alloantigen-activated autologous T cells, and they could be expanded in vitro without loss of their suppressive capacity. These results suggest that CD150 can be used as a marker for the identification of purified alloantigen-activated CD4(+)25+ regulatory T cells.     

狼疮性肾炎患者血清NETs、TWEAK、外周血CD4+T/CD8+T比例与疾病活动度及肾脏预后的关系

摘要 目的：探讨狼疮性肾炎（LN）患者血清中性粒细胞胞外诱捕网（NETs）、肿瘤坏死因子样凋亡微弱诱导剂（TWEAK）、外周血分化簇（CD）4⁺T/CD8⁺T比例与疾病活动度及肾脏预后的关系。方法：选取2021年8月～2022年8月川北医学院附属医院肾内科收治的LN患者137例（LN组）,根据系统性红斑狼疮疾病活动指数（SLEDAI）-2000评分分为轻度活动组（52例）、中度活动组（45例）、重度活动组（40例）。随访1年,根据肾脏相关终点事件发生情况分为预后不良组（43例）和预后良好组（94例）,另选取同期76名体检健康志愿者（对照组）。采用酶联免疫吸附法检测血清NETs、TWEAK水平,流式细胞术检测外周血CD4⁺T/CD8⁺T比例。Spearman相关性分析LN患者血清NETs、TWEAK和外周血CD4⁺T/CD8⁺T与SLEDAI-2000评分的相关性,多因素Logistic回归分析LN患者预后不良的因素,受试者工作特征曲线分析血清NETs、TWEAK和外周血CD4⁺T/CD8⁺T对LN患者预后不良的预测价值。结果：与对照组比较,LN组血清NETs、TWEAK水平升高,外周血CD4⁺T/CD8⁺T降低（P＜0.05）。轻度活动组、中度活动组、重度活动组血清NETs、TWEAK依次升高,外周血CD4⁺T/CD8⁺T依次降低（P＜0.05）。LN患者SLEDAI-2000评分与血清NETs、TWEAK呈正相关,与外周血CD4⁺T/CD8⁺T呈负相关（P＜0.05）。慢性肾脏病分期4期、SLEDAI-2000评分升高、NETs升高、TWEAK升高为LN患者预后不良的独立危险因素,估算肾小球滤过率升高、CD4⁺T/CD8⁺T升高为独立保护因素（P＜0.05）。血清NETs、TWEAK和外周血CD4⁺T/CD8⁺T联合预测LN患者预后不良的曲线下面积为0.943,大于血清NETs、TWEAK和外周血CD4⁺T/CD8⁺T单独预测的0.790、0.788、0.799（P＜0.05）。结论：LN患者血清NETs、TWEAK水平升高,外周血CD4⁺T/CD8⁺T降低,与疾病活动度及肾脏预后不良密切相关,血清NETs、TWEAK联合外周血CD4⁺T/CD8⁺T预测LN患者肾脏预后的价值较高。     

Ex vivo activated OVA specific and non-specific CD4+CD25+ regulatory T cells exhibit comparable suppression to OVA mediated T cell responses

CD4+CD25+ regulatory T cells (Tr) are important in maintaining immune tolerance to self-antigen (Ag) and preventing autoimmunity. Reduced number and inadequate function of Tr are observed in chronic autoimmune diseases. Adoptively transferred Tr effectively suppress ongoing autoimmune disease in multiple animal models. Therefore, strategies to modulate Tr have become an attractive approach to control autoimmunity. Activation of Tr is necessary for their optimal immune regulatory function. However, due to the low ratio of Tr to any given antigen (Ag) and the unknown nature of Ag in many autoimmune diseases, specific activation is not practical for potential therapeutic intervention. It has been shown in animal models that once activated, Tr can exhibit immune suppression in a bystander Ag-non-specific fashion, suggesting the effector phase of Tr is Ag independent. To investigate whether the immune suppression by activated bystander Tr is as potent as that of the Ag specific Tr, Tr cells were isolated from BALB/c or ovalbumin (OVA) specific T cell receptor (TCR) transgenic mice (DO11.10) and their immune suppression of an OVA specific T cell response was compared. We found that once activated ex vivo, Tr from BALB/c and DO11.10 mice exhibited comparable inhibition on OVA specific T cell responses as determined by T cell proliferation and cytokine production. Furthermore, their immune suppression function was compared in a delayed type hypersensitivity (DTH) model induced by OVA specific T cells. Again, OVA specific and non-specific Tr exhibited similar inhibition of the DTH response. Taken together, the results indicate that ex vivo activated Ag-non-specific Tr are as efficient as Ag specific Tr in immune suppression, therefore our study provides additional evidence suggesting the possibility of applying ex vivo activated Tr therapy for the control of autoimmunity.     

组蛋白去乙酰化酶3对外周CD4+ T细胞分化的影响

目的 探讨组蛋白去乙酰化酶3（HDAC3）对外周CD4⁺ T细胞分化及功能的调控作用。方法 采用CD4cre酶介导Hdac3杂合基因缺失小鼠（Hdac3^fl/flCD4^cre+/-）及其野生型正常对照小鼠（Hdac3^fl/fl，WT），流式细胞术检测HDAC3缺失对外周CD4⁺和CD8⁺ T细胞比例和数量的影响；在体外佛波酯（PMA）和离子霉素（Ionomycin）刺激条件下，流式细胞术检测HDAC3缺失对CD4⁺ T细胞中IFN-γ、IL-4和IL-17A的表达以及Tfh细胞产生的影响；采用ELISA检测HDAC3缺失对小鼠血清IFN-γ、IL-4和IL-17表达的影响；分选Hdac3^fl/flCD4^cre+/-和WT小鼠外周初始CD4⁺ T细胞，分别在Th1和Th2分化条件下培养，细胞内染色检测HDAC3缺失对Th1、Th2以及Th17相关细胞因子及其特异转录因子表达的影响；采用Microarray检测HDAC3缺失对CD4⁺ T细胞分化亚群相关基因表达的影响；采用链脲佐菌素（STZ）处理小鼠构建I型糖尿病（TIDM）疾病模型，检测HDAC3缺失对T1DM发病的影响。结果 与WT小鼠相比，Hdac3^fl/flCD4^cre+/-小鼠外周CD4⁺和CD8⁺ T细胞的比例和数量显著降低。Hdac3^fl/flCD4^cre+/-小鼠CD4⁺ T细胞及血清中IFN-γ的表达显著降低，而IL-4和IL-17A的表达显著增加，Tfh细胞比例也显著增加；HDAC3缺失抑制体外培养CD4⁺ T细胞向Th1分化但促进其向Th2分化；Microarray检测发现HDAC3缺失导致Th1型细胞谱系基因表达降低，而Th2、Th17以及Tfh细胞谱系基因表达增加；在STZ诱导条件下，HDAC3缺失抑制小鼠T1DM的发生和CD4⁺ T细胞向Th1分化。结论 HDAC3促进外周CD4⁺ T细胞向Th1细胞分化并加重T1DM的发生。     

经阿托伐他汀治疗的急性冠脉综合征患者CD4+T、CD4+CD28+T水平变化及与预后的关系

目的:探讨经阿托伐他汀治疗的急性冠脉综合征(ACS)的CD4~+T、CD4~+CD28~+T水平变化及与预后的关系。方法:选择128例ACS患者,随机分为对照组(64例)和观察组两组(64例),其中对照组患者给予常规治疗,观察组患者在上述基础上外加阿托伐他汀治疗。比较两组患者的细胞因子、CD4~+T及CD4~+CD28~+T水平变化,随访6个月,观察两组患者预后终点事件发生情况。并将观察组患者根据预后是否并发终点事件,将其分为预后良组(未并发终点事件)和预后不良组(并发终点事件)两组,分析不同预后ACS患者CD4~+T、CD4~+CD28~+T水平变化及与预后发生终点事件的相关性。结果:治疗后两组的高敏C反应蛋白(hs-CRP)、干扰素-γ(IFN-γ)水平均明显降低,白细胞介素-10(IL-10)、转化生长因子β1(TGF-β1)水平均明显升高,且观察组改善更为明显,差异均有统计学意义(P0.05)。两组患者治疗后CD4~+T明显升高,CD8~+T、CD4~+CD28~+T明显降低,且观察组上述指标改善更为明显,差异均有统计学意义(P0.05)。随访6月中,对照组患者预后有23例终点事件发生,观察组患者预后有26例终点事件发生,差异无统计学意义(P0.05)。与预后良好组相比,预后不良组患者的CD4~+T降低,CD4~+CD28~+T升高,差异均有统计学意义(P0.05)。经阿托伐他汀治疗的ACS患者预后发生终点事件与CD4~+T水平呈现负相关(r=-0.682,P=0.000),与CD4~+CD28~+T水平呈现正相关(r=0.733,P=0.000)。结论:经阿托伐他汀治疗的ACS患者预后发生终点事件与CD4~+T水平呈现负相关,与CD4~+CD28~+T水平呈现正相关,可为临床ACS患者预后的预测提供参考。     

Trichinella spiralis Infection Suppressed Gut Inflammation with CD4+CD25+Foxp3+ T Cell Recruitment

In order to know the effect of pre-existing Trichinella spiralis infection on experimentally induced intestinal inflammation and immune responses, we induced colitis in T. spiralis-infected mice and observed the severity of colitis and the levels of Th1, Th2, and regulatory cytokines and recruitment of CD4⁺CD25⁺Foxp3⁺ T (regulatory T; T_reg) cells. Female C57BL/6 mice were infected with 250 muscle larvae; after 4 weeks, induction of experimental colitis was performed using 3% dextran sulfate sodium (DSS). During the induction period, we observed severity of colitis, including weight loss and status of stool, and evaluated the disease activity index (DAI). A significantly low DAI and degree of weight loss were observed in infected mice, compared with uninfected mice. In addition, colon length in infected mice was not contracted, compared with uninfected mice. We also observed a significant increase in production of pro-inflammatory cytokines, IL-6 and IFN-γ, in spleen lymphocytes treated with DSS; however, such an increase was not observed in infected mice treated with DSS. Of particular interest, production of regulatory cytokines, IL-10 and transforming growth factor (TGF)-β, in spleen lymphocytes showed a significant increase in mice infected with T. spiralis. A similar result was observed in mesenteric lymph nodes (MLN). Subsets of the population of T_reg cells in MLN and spleen showed significant increases in mice infected with T. spiralis. In conclusion, T. spiralis infection can inhibit the DSS-induced colitis in mice by enhancing the regulatory cytokine and T_reg cells recruitment.     

The role of CD4+FoxP3+ regulatory T cells in the immunopathogenesis of COVID-19: implications for treatment

The severe cases of Coronavirus Disease 2019 (COVID-19) frequently exhibit excessive inflammatory responses, acute respiratory distress syndrome (ARDS), coagulopathy, and organ damage. The most striking immunopathology of advanced COVID-19 is cytokine release syndrome or “cytokine storm” that is attributable to the deficiencies in immune regulatory mechanisms. CD4⁺FoxP3⁺ regulatory T cells (Tregs) are central regulators of immune responses and play an indispensable role in the maintenance of immune homeostasis. Tregs are likely involved in the attenuation of antiviral defense at the early stage of infection and ameliorating inflammation-induced organ injury at the late stage of COVID-19. In this article, we review and summarize the current understanding of the change of Tregs in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and discuss the potential role of Tregs in the immunopathology of COVID-19. The emerging concept of Treg-targeted therapies, including both adoptive Treg transfer and low dose of IL-2 treatment, is introduced. Furthermore, the potential Treg-boosting effect of therapeutic agents used in the treatment of COVID-19, including dexamethasone, vitamin D, tocilizumab and sarilumab, chloroquine, hydroxychloroquine, azithromycin, adalimumab and tetrandrine, is discussed. The problems in the current study of Treg cells in COVID-19 and future perspectives are also addressed.     

Calcium Homeostasis Change in CD4<Superscript>+</Superscript> T Lymphocytes from Human Peripheral Blood during Differentiation <Emphasis Type="Italic">in vivo</Emphasis>

Resting naive CD4⁺CD45R0^?CD45RA⁺ T cells are sensitive to ionomycin. In contrast, resting CD4⁺CD45RA^?CD45R0⁺ memory T cells show resistance to this Ca²⁺ ionophore. In the present study, the ability of activated T lymphocytes to respond to ionomycin during the transition from naive precursors into memory T cells has been analyzed. Activated CD4⁺CD45RA⁺CD45R0⁺ T cells are always present both in human peripheral blood (HPB) and in the ionomycin-resistant (IR) fraction. Therefore, some activated T cells are resistant toward the Ca²⁺ ionophore. CD69 molecules are markers of the very early stage of T cell activation. However, CD4⁺CD69⁺ T cells have never been found in the IR fraction. Thus, the majority of CD4⁺ T lymphocytes at the early stage of activation are ionomycin-sensitive cells. The proportion of CD4⁺CD25⁺ T cells did not differ significantly in HPB and in the IR fraction. The presence of CD4⁺CD25⁺ T lymphocytes in the IR fraction reflects changes in the Ca²⁺-signaling pathway at this differentiation step of activated cells. Depending on the expression level of CD25 molecules, the population of CD4⁺CD25⁺ cells is divided in T-regulatory (CD25^high) and proliferating (CD25^low) subpopulations. The action of ionomycin results in a decrease in the portion of the CD4⁺CD25^low T-cells, but it leads to an increase in the proportion of the CD4⁺CD25^high T lymphocytes. Consequently, greater portion of CD4⁺CD25^high T lymphocytes and smaller portion of CD4⁺CD25^low T cells are IR cells. Expression of HLA-DR molecules can be used as the marker for the late activation step. The IR fraction is significantly rich in CD4⁺HLA-DR⁺ T lymphocytes in comparison to the blood of the same donor. The link between different differentiation steps of CD4⁺ T-lymphocytes and alterations in calcium ion homeostasis is discussed.     

Influence of CD4+CD25+ T cells on Plasmodium berghei NK65 infection in BALB/c mice

CD4(+) T cells co-expressing CD25 (CD4(+)CD25(+) T cells) have been identified as immunoregulatory suppressors modulating autoimmune response. Beside that, autoimmune response was supposed to be associated with malaria infection. Based on these data, we hypothesised that CD4(+)CD25(+) T cells may influence protective immunity to malaria parasites, while suppressing autoimmune response arising throughout the course of malarial infection. To test this possibility, we evaluated the kinetics of CD4(+)CD25(+) T cells during malaria infection and investigated the influence of CD25 depletion by anti-mouse CD25 monoclonal antibody (PC61) on the infection, using a mouse model of premunition to Plasmodium berghei NK65 malaria. The results showed that, during exacerbation of P. berghei NK65 infection, the proportion of CD4(+)CD25(+) T cells among CD4(+) T cells decreased, although that of CD4(+) T cells increased. CD25 depletion clearly delayed the growth of parasitaemia during parasite challenge, particularly in immunised mice. These findings demonstrated that CD4(+)CD25(+) T cells are able to influence protective immunity underlying premunition to P. berghei NK65 parasites.     

Mathematical analysis of the global dynamics of a model for HIV infection of CD4+ T cells

A mathematical model that describes HIV infection of CD4(+) T cells is analyzed. Global dynamics of the model is rigorously established. We prove that, if the basic reproduction number R(0) < or = 1, the HIV infection is cleared from the T-cell population; if R(0) > 1, the HIV infection persists. For an open set of parameter values, the chronic-infection equilibrium P* can be unstable and periodic solutions may exist. We establish parameter regions for which P* is globally stable.