Decision criteria for resolving isotype switching conflicts by B cells

Isotype switching by B cells is highly regulated by a group of cytokines including IL-4, IFN-gamma and TGF-beta. A B cell can only express one isotype at a time; however, during an immune response it may be exposed to combinations of stimuli that provide it with conflicting switching instructions. To determine how such cytokine-induced isotype switch conflicts would be resolved, the responses of B cells exposed to multiple cytokines were investigated. To eliminate complications arising from simultaneous effects of switching cytokines on proliferation, division number was used as a reference framework to monitor switching rate. The results show a clear hierarchy in which IFN-gamma is dominant over IL-4, and both IL-4 and IFN-gamma are dominant over TGF-beta. These studies reveal how B cells possess a set of logical decision criteria for dealing with pathogens that invoke a range of different stimuli.     

Pathophysiological functions of CD30+ CD4+ T cells in rheumatoid arthritis

High levels of soluble CD30 (sCD30) were detected in the serum and synovial fluid of patients with rheumatoid arthritis (RA), indicating the involvement of CD30+ T cells in the pathogenesis. We investigated the induction of CD30 and its functions in CD4+T cells from patients with established RA (disease duration >_2 years). CD4+ T cells from both the peripheral blood (PB) and synovial tissue (ST) of RA patients expressed surface CD30 when stimulated with anti-CD3 antibody (Ab) and anti-CD28 Ab, but their CD30 induction was slower and weaker compared with PB CD4+ T cells of healthy controls (HC). Immunohistochemical analysis showed that only a small proportion of lymphocytes expressed CD30 in the ST (-1%). RA PB CD4+ T cells, after recovery from 6-day stimulation with anti-CD3 Ab and anti-CD28 Ab, showed in intracellular cytokine staining that CD30+ T cells could produce more interleukin-4 (IL-4) but less interferon-gamma. In the culture of RA PB CD4+ T Cells with anti-CD3 Ab and anti-CD28 Ab, blocking anti-CD30 Ab similarly inhibited the cell proliferation and activation of nuclear factor-kappaB on day 4 in RA and HC, but inhibited the apoptotic cell death on day 6 only in RA. These results indicate that despite high-level expression of sCD30, the anti-inflammatory activity of IL-4-producing CD30+ CD4+ T cells may be limited in the ST due to a poor induction of surface CD30 and a susceptibility to CD30-mediated cell death.     

Dysregulation of circulating CD4+CXCR5+ T cells in type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) is characterized by a chronic low‐grade inflammatory state. Follicular helper T cells (Tfh) play critical roles in inducing B‐cell activation and producing various cytokines, whereas circulating CD4+CXCR5+ T cells (CTfh) may act as a counterpart to measure Tfh cell disorders. In this study, we investigated whether Tfh could be involved in the development of T2DM by assessing CTfh in peripheral blood. CTfh and it subtypes were determined by measuring CD3, CD4, CXCR5, CXCR3, and CCR6 in 68 T2DM patients and 60 healthy controls using flow cytometry. Results showed that proportion of CTfh in the peripheral CD4+ T cells was significantly increased in T2DM patients (8.5 ± 0.5%) than in controls (4.5 ± 0.3%) (p < 0.001). Further study revealed that the balance of CTfh subtypes was greatly dysregulated, in which percentage of Th17 subtype was significantly increased in patients. Investigating the correlation between CTfh and risk factors of T2DM demonstrated that proportion of CTfh were significantly elevated in patients with body mass index (BMI) over 24.0 (p = 0.005). Interestingly, patients with abdominal obesity had further increase in CTfh than those without abdominal obesity. This study suggests the involvement of CTfh in T2DM, especially in T2DM‐related obesity.     

Increased interleukin-10 production by ASC-deficient CD4+ T cells impairs bystander T-cell proliferation

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an important component of the inflammasome, functioning as an adaptor protein that facilitates the recruitment and activation of procaspases that in turn promote the maturation of interleukin-1β (IL-1β) and IL-18. Despite initial focus on the inflammatory properties of ASC there is emerging evidence that highlights the importance of ASC in facilitating adaptive immune responses. However, the cellular and molecular basis for the involvement of ASC in adaptive immunity remains largely unexplored. We have previously demonstrated that activated ASC-deficient T cells have dampened proliferative responses. We have therefore explored the underlying cellular mechanism(s) by which ASC regulates T-cell proliferation. We show that under activating conditions (anti-CD3/CD28 stimulation) in bulk T-cell cultures the presence of ASC(-/-) CD4(+) T cells is sufficient to suppress the proliferative responses of neighbouring T cells. Furthermore, ASC(-/-) CD4(+) T cells upon activation exhibit a suppressive cytokine profile, with elevated production of IL-10 and reduced secretion of T helper type 1 cytokines, interferon-γ and IL-2. This increase in IL-10 secretion within the activated ASC(-/-) CD4(+) T-cell compartment was not associated with a proportional increase in conventional Foxp3(+) regulatory T (Treg) cells. Interestingly, when equal numbers of fluorescence-activated cell sorted ASC(+/+) and ASC(-/-) Treg cells (CD4(+) CD44(intermediate/high) CD25(+)) were activated in vitro, the ASC(-/-) fraction produced significantly more IL-10 than their wild-type counterparts, suggesting that ASC(-/-) Treg cells have greater suppressive capacity. Collectively, these results imply that the ASC may influence the development and functioning of Treg cells.     

支气管哮喘病人CD4+T细胞CD25、CD30表达状况

目的：通过观察哮喘病人外周血CD4⁺T细胞CD25、CD30表达水平，了解哮喘病人T细胞活化状态。方法：将分离出的CD4⁺T细胞分别用PPD、PHA刺激，最后用流式细胞仪检测抗原刺激前后细胞表面CD25、CD30表达水平。结果：①哮喘病人CD4⁺T细胞CD25、CD30自然表达比率均低于健康对照（P<0.05、P<0.05）。②用PHA刺激哮喘病人CD4⁺T细胞后，CD25表达水平明显高于健康对照（P<0.01），但CD30表达无差异。③PPD刺激组CD25、CD30表达与健康对照间无差异。结论：哮喘病人CD4⁺T细胞活化状态明显异常。哮喘病人的CD4⁺T细胞无刺激因素时，活化水平低下，但接受刺激后表现出高水平的活化状态。     

Selection of CMV-specific CD8+ and CD4+ T cells by mini-EBV-transformed B cell lines

Efficient protocols to generate cytomegalovirus (CMV)-specific T cells are required for adoptive immunotherapy. Recombinant Epstein-Barr virus (EBV) vectors called mini-EBV can be used to establish permanent B cell lines in a single step, which present the CMV antigen pp65 in a constitutive manner. These B cell lines, coined pp65 mini-LCL, were successfully used to reactivate and expand CMV-specific cytotoxic T cells. Here we evaluate this pp65 mini-EBV system in closer detail, focusing on (1) the quantification of T cells with specific effector function and (2) the identification of CMV-specific CD4(+) helper T cells. The co-expansion of various functional CMV epitope specificities was demonstrated by IFN-gamma enzyme-linked immunospot assay (ELISPOT) assays and HLA-peptide tetramer staining. Single-cell cloning resulted in both CD4(+) and CD8(+) T cell clones, the majority of which was CMV specific. Thus, mini-LCL present the pp65 antigen on HLA class I and II, mobilizing both arms of the T cell response. Using a peptide library covering the pp65 sequence for further analysis of T cell clones, we identified new pp65 CD8(+) and CD4(+) T cell epitopes.     

Generation of peptide-specific CD8+ T cells by phytohemagglutinin-stimulated antigen-mRNA-transduced CD4+ T cells

Functional analysis of antigen-specific CD8(+) T cells is important for understanding the immune response in various immunological disorders. To analyze CD8(+) T cell responses to a variety of antigens with no readily defined peptides available, we developed a system using CD4(+) phytohemagglutinin (PHA) blasts transduced with mRNA for antigen molecules. CD4(+) PHA blasts express MHC class I and II, and also CD80 and CD86 and are thus expected to serve as potent antigen presenting cells. EGFP mRNA could be transduced into and the protein expressed by more than 90% of either LCL or CD4(+) PHA blasts. Its expression stably persisted for more than 2 weeks after transduction. In experiments with HLA-A*2402 restricted CD8(+) CTL clones for either EBNA3A or a cancer-testis antigen, SAGE, mRNA-transduced lymphoid cells were appropriate target cells in ELISPOT assays or (51)Cr releasing assays. Finally, using CD4(+) PHA blasts transduced with mRNA of a cancer-testis antigen MAGE-A4, we successfully generated specific CTL clones that recognized a novel HLA-B*4002 restricted epitope, MAGE-A4(223-231). Messenger RNA-transduced CD4(+) PHA blasts are thus useful antigen presenting cells for analysis of CD8(+) T cell responses and induction of specific T cells for potential immunotherapy.     

CD4+ CD30+ T cells perpetuate IL-5 production in Dermatophagoides pteronyssinus allergic patients

BACKGROUND: Airway allergic diseases are regulated by interleukin (IL)-5, which causes infiltration of eosinophils into the bronchial epithelium, and by IL-4 which increases serum immunoglobulin E (IgE) production and promotes CD30 expression on Th cells. CD30 generates a costimulatory signal involved in apoptosis or cell proliferation, depending on the microenvironment. Our aims were: (i) to analyze if CD4+ CD30+ T cells from allergic patients proliferate in response to Dermatophagoides pteronyssinus, and (ii) if upon stimulation this cell population produces IL-4 and IL-5. METHODS: Peripheral blood mononuclear cell (PBMC) from 17 allergic rhinitis and mild allergic asthma patients and 12 healthy nonallergic individuals were stimulated with allergen in the presence or absence of anti-IL-4, anti-IL-5 or anti-IL-4Ralpha monoclonal antibodies (mAbs). TdT-mediated dUTP nick end-labeling (TUNEL) assay, 7-aminoactinomycin-D (7-AAD) intercalation, and flow cytometry were used to determine the CD4+ CD30+ blasts percentage, cell proliferation, apoptosis, and intracellular cytokines after 7 culture days. RESULTS: Cell proliferation induced with allergen showed that 90% of the allergen-stimulated blasts were CD4+, 50% of which were CD30+. Allergen-stimulated PBMC showed a progressive increase (mean: from 7% to 23%) of CD4+ CD30+IFN-gamma+ and CD4+ CD30+IL-4+ blasts which diminished (mean: 6%) after 5 culture days. In contrast, CD4+ CD30+IL-5+ blasts showed a continuous progression (from 12% to 24%) that maintained after 7 culture days. The vast majority of CD4+ CD30+ blasts were negative to 7-AAD or TUNEL. Additionally, a significant decrease (34%) was observed in the number of CD4+ CD30+ blasts when IL-4 was neutralized. CONCLUSIONS: These data suggest that specific allergen stimulation of PBMC isolated from allergic patients generates a nonapoptotic CD4+ CD30+ blast subset that produces IL-5.     

Is the activated T helper stimulus able to induce Ig isotype switching in cultured human B cells?

It Is confirmed that large amounts of IgM, IgG, and IgA areproduced when human B cells are cultured with T cells activatedby immobilized CD3 antibody (CD3 system). IL-2 was essential;lowerlevels of Ig production with different isotype ratios were obtainedif IL-4 or IL-6 replaced IL-2. Depletion of sIgG⁺ or sIgA⁺ cellsfrom the B population to be cultured markedly reduced productionof IgG or IgA. Culturesof B cells selected with the pan-B markersCD19, CD72, or CD21 contained similar levels of Ig of all threeisotypes, whereas B cells selected for sIgM or sIgD expressionproduced IgM but very little IgG or IgA indicating that littleisotype switching was occurring. Production of IgG or IgA fromcells expressing these isotypes was more efficient than productionof IgM from IgM⁺ IgD⁺ cells. These results are considered inthe light of the demonstration by others of the production ofmultiple isotypes from single sIgM⁺-selected B cells. Clonedhuman T cells from a single donor induced production of allthree isotypes, but the proportions varied indicating that thepotent T-B cell interactions inducing B cell activation mayoverride and conceal the operation of isotype specific cellinteractions. Some T clones used at an optimal dose were aseffective untreated as X-irradiated, whereas with other clonesmaximumIg production was not achieved without irradiation.     

CD30 in normal and neoplastic cells

In 1982 Stein and coworkers identified a new molecule, CD30 (Ki-1), which is expressed by Reed-Sternberg (RS) cells of Hodgkin's Disease (HD) (1). Although CD30 is not a specific RS cell marker, its characterization has assumed an important role not only in the differential diagnosis of HD, but also in the identification of a morphologically and clinically distinct type of large cell lymphoma, now designated as anaplastic large cell lymphoma (ALCL) (2). The cloning of human and murine CD30 and the utilization of genetically manipulated animal models have rapidly expanded our knowledge on its physiological role in lymphoid development and differentiation. The goal of this review is to present an overview of this rapidly evolving field and discuss the role of CD30 in normal and neoplastic lymphoid cells.     

An antigen expressed by murine CD8+ T cells and activated B cells

A monoclonal antibody, H1F5, is described that reacts with a subset of Lyt-2 (CD8) mouse T cells and LPS-activated B cells. In both lymph nodes and spleen of BALB/c mice, the H1F5 antigen is coexpressed approximately on 20%-30% of the CD8+ T cells and approximately on 91% of LPS-activated B cells. In the thymus, few cells (less than 1%) are positive for the marker, but no correlation could be demonstrated with markers for mature T cells such as MEL-14 and PNA expression. Elimination of H1F5+ cells by complement lysis led to a 30%-50% reduction of specific lysis as measured in a primary allo CTL, indicating that the cytotoxic effector cells are injured. The relationship of this marker and other antigenic determinants on lymphocytes is discussed.     

CD4+CD25+ T cells as immunoregulatory T cells in vitro

We have further characterized the in vitro phenotype and function of anergic and suppressive CD4(+)25(+) T cells. Following TCR ligation, DO.11.10 CD4(+)25(+) T cells suppress the activation of OT-1 CD8(+)25(-) T cells in an antigen nonspecific manner. Although suppression was seen when using a mixture of APC from both parental strains, it was very much more marked when using F1 APC. APC pretreated with, and then separated from CD4(+)25(+) T cells did not have diminished T cell costimulatory function, suggesting that APC are not the direct targets of CD4(+)25(+) T cell regulation. CTLA-4 blockade failed to abrogate suppression by CD4(+)25(+) T cells in mixing experiments. Although CD4(+)25(+) T cells failed to respond following cross-linking of TCR, they could be induced to proliferate following the addition of exogenous IL-2, allowing the generation of a T cell line from CD4(+)25(+) T cells. After the first in vitro restimulation, CD4(+)25(+) T cells were still anergic and suppressive following TCR engagement. However, after three rounds of restimulation, their anergic and suppressive status was abrogated.     

老年初诊急性髓细胞白血病患者CD4+CD25+调节性T细胞的表达

背景：研究证实,很多恶性肿瘤患者体内CD4⁺CD25⁺调节性T细胞存在高表达,近期也有研究发现,急性髓细胞白血病患者外周血CD4⁺CD25⁺调节性T细胞同样表现出高比例表达。 目的：分析老年初诊急性髓细胞白血病患者CD4⁺CD25⁺调节性T细胞的表达特点。 方法：纳入初诊急性髓细胞白血病患者92例,将年龄在60岁以下者设为中青年组(n=22),年龄在60岁以上者设为老年观察组(n=70)。在老年观察组中,32例经规范化疗后完全缓解,设为完全缓解组;将余下38例设为老年组,依据FAB分型标准,分为M2 6例、M3 19例、M4 7例、M5 6例。另选择同期体检健康人群42名作为正常对照组。抽取受试者外周静脉血,检测CD4⁺CD25⁺调节性T细胞表达情况。 结果与结论：老年组、完全缓解组CD4⁺CD25^highFOXP3⁺调节性T细胞比例高于正常对照组(P < 0.01),并且老年组CD4⁺CD25^high FOXP3⁺调节性T细胞比例高于完全缓解组(P < 0.01)。老年组、完全缓解组CD4⁺FOXP3⁺T细胞比例高于正常对照组(P < 0.01),并且老年组CD4⁺ FOXP3⁺T细胞比例高于完全缓解组(P < 0.01)。老年组CD4⁺CD25^high FOXP3⁺调节性T细胞与CD4⁺ FOXP3⁺T细胞比例高于中青年组(P < 0.01)。老年组不同分型间CD4⁺CD25^high FOXP3⁺调节性T细胞和CD4⁺ FOXP3⁺T细胞比例比较差异均无显著性意义(P > 0.05)。Pearson相关性检验结果显示,老年初诊急性髓细胞白血病患者外周血CD4⁺CD25^high FOXP3⁺调节性T细胞比例和CD4⁺ FOXP3⁺T细胞比例呈正相关(r=0.87,P=0.019)。表明老年初诊急性髓细胞白血病患者CD4⁺CD25⁺调节性T细胞比例高于健康人群和中青年急性髓细胞白血病患者。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

急性淋巴细胞白血病的免疫分型及CD4+CD25+T调节细胞检测

背景：在急性淋巴细胞白血病发病过程中,CD4⁺CD25⁺T调节细胞对机体免疫反应可能起着一定的调节作用。 目的：观察急性淋巴细胞白血病患者的免疫分型及外周血CD4+CD25+T调节细胞的变化情况。 方法：采用流式细胞仪对35例急性淋巴细胞白血病患者进行免疫分型,并检测外周血CD4⁺CD25⁺T调节细胞的数目,与18名健康对照作比较。 结果与结论：急性B细胞淋巴细胞白血病22例,急性T细胞淋巴细胞白血病13例;22例急性B细胞淋巴细胞白血病中CD19的阳性表达率最高(100%),而13例急性T细胞淋巴细胞白血病中CD7阳性表达率最高(100%)。急性B细胞淋巴细胞白血病患者外周血CD4⁺CD25⁺T调节细胞和急性T细胞淋巴细胞白血病患者差异无显著性意义(P > 0.05),但均高于健康对照(P < 0.05)。表明急性B细胞淋巴细胞白血病中CD19阳性表达率最高,急性T细胞淋巴细胞白血病中CD7阳性表达率最高,同时急性淋巴细胞白血病患者外周血CD4⁺CD25⁺T调节细胞水平显著增高。     

CD4+CD25+免疫调节性T细胞对CD4+T细胞介导的移植物抗宿主病预防作用的研究

目的研究CD4+CD25+免疫调节性T(Treg)细胞在小鼠骨髓移植后,对移植物抗宿主病的预防作用及其作用机制.方法用C3H(H-2k)小鼠骨髓作为供体,提取C3H(H-2k)小鼠CD4+T及CD4+CD25+T细胞,C3H×B6(H-2k/b)F1小鼠为骨髓移植的受者.在受者接受致死量全身放射后,输注供者去除T细胞的骨髓(ATBM),使其造血功能重建(ATBM组).于不同的实验组给予CD4+(CD4组)T细胞,CD4+CD25+T(CD25组)或二者同时输注(CD4/CD25组).观察各组小鼠移植物抗宿主病(GVHD)的发生率.结果所有10只ATBM组小鼠至骨髓移植后60天仍全部存活,无GVHD发生;所有10只CD4组小鼠在骨髓移植10天内全部死于GVHD(P＜0.01);所有5只CD25组小鼠于骨髓移植后60天仍全部存活,无明显GVHD发生(P＞0.05);同样,所有6只CD4/CD25组小鼠至骨髓移植后仍全部存活,无明显GVHD发生(P＞0.05).结论在同种异基因小鼠的骨髓移植模型中,CD4+CD25+T不诱导GVHD的发生,并有预防CD4+T细胞介导的GVHD发生的作用.     

CD4+CD25+Treg在急性淋巴细胞白血病患者外周血中的表达及其体外实验研究

目的初步探讨CD4 CD25 调节性T细胞(CD4 CD25 regulatory T cells,CD4 CD25 Treg)在急性淋巴细胞白血病(acute lymphocytic leukemia,ALL)患者化疗前及化疗缓解后外周血中的表达水平,并研究患者血清能否诱导外周血CD4 CD25-T细胞转化为CD4 CD25 Treg。方法①采用流式细胞术分别检测ALL初诊组、化疗完全缓解或部分缓解组及正常对照组外周血中CD4 CD25 T细胞所占比例,然后通过荧光定量RT-PCR检测各组外周血中转录因子Foxp3mRNA的表达水平,并逐层分析比较。②采集正常人外周血单个核细胞后,对照组用正常人血清,实验组用患者血清并分别设浓度梯度进行培养,72h后采用流式细胞术、荧光定量RT-PCR分别检测CD4 CD25 T细胞和Foxp3mRNA表达。结果ALL化疗缓解组CD4 CD25 T细胞及Foxp3mRNA表达水平均明显高于ALL初诊组和正常对照组(P<0.05),后两者之间CD4 CD25 T细胞水平无统计学差异(P>0.05),但ALL初诊组Foxp3mRNA含量较正常对照组明显升高(P<0.01),差异具有统计学意义;并且血清培养对照组CD4 CD25 T细胞水平及Foxp3mRNA含量均明显低于实验组(P<0.05),且其表达并不随血清浓度的增加而升高。结论CD4 CD25 Foxp3 Treg在ALL初诊组及化疗缓解组患者外周血中比例明显升高,且初步表明患者血清中的可溶性物质可诱导外周血CD4 CD25 T细胞转化为CD4 CD25 Treg,提示CD4 CD25 Treg可能是ALL免疫抑制的一个重要原因。