B细胞对T细胞及其亚群CD25、CD30表达的影响

目的 探讨Ｂ淋对Ｔ淋巴细胞的抗原提呈作用方法 分离外周血Ｔ、Ｂ细胞及ＣＤ４￣ＣＤ８Ｔ淋巴细胞,分别用ＬＰＳ、ＰＰＤ、ＰＷＭ刺激Ｂ细胞,选去刺激原后与Ｔ细胞及亚群共同培养。最后用流式细胞仪检测Ｔ细胞及其亚群ＣＤ２５、ＣＤ３０表达水平。结果 ＰＰＤ刺激组Ｔ细胞ＣＤ２５、ＣＤ３０表达水平明显升高,而ＰＷＭ、ＬＰＳ刺激组与对照组间无差异。ＰＰＤ刺激组ＣＤ４＾Ｔ细胞ＣＤ２５、ＣＤ３０表达与对照组间有显著差异     

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。     

Expression and function of Toll-like receptors in T lymphocytes

Toll-like receptors (TLRs) are widely expressed in the innate immune system. They recognize conserved microbial ligands such as bacterial lipopolysaccharide, lipopeptides or viral and bacterial RNA and DNA. TLRs play an essential role in innate immune responses and in the initiation of adaptive immune responses. However, certain TLRs are also expressed in T lymphocytes, and the respective ligands can directly modulate T cell function. TLR2, TLR3, TLR5 and TLR9 act as co-stimulatory receptors to enhance proliferation and/or cytokine production of T-cell receptor-stimulated T lymphocytes. In addition, TLR2, TLR5 and TLR8 modulate the suppressive activity of naturally occurring CD25(+)CD4(+) regulatory T cells. The direct responsiveness of T lymphocytes to TLR ligands offers new perspectives for the immunotherapeutic manipulation of T cell responses.     

锚定蛋白CD59与接头分子Cbp在T细胞上的定位及功能研究

目的 研究CD59、Src羧基末端激酶结合蛋白(Cbp)在细胞膜的定位,及其在细胞活化及增殖中的相互作用.方法 应用免疫荧光细胞化学技术,通过荧光显微镜分析系统对CD59、Cbp的定位进行了分析;Jurkat细胞转染pSUPER-siCD59重组质粒,并行RT-PCR和Western blot法检测转染细胞中CD59的表达及蛋白酪氨酸激酶癌基因家族(fyn)磷酸化水平.利用MTT比色法检测转染各组细胞的增殖效应.结果 CD59、Cbp主要分布在细胞膜上.转染pSUPER-siCD59重组质粒的Jurkat细胞中CD59表达量减少,磷酸化的fyn含量也随之减少,细胞增殖能力也低于其他各组.结论 CD59是一种膜蛋白,在细胞活化及增殖中与Cbp分子共同发挥作用.     

Differential Expression of CD30 on CD3 T Lymphocytes in Patients with Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune systemic disease caused as a result of an imbalance of Th1‐/Th2‐type cytokines. The soluble form of CD30 (CD30s) released from peripheral blood cells has been described as a marker of active disease in Th2‐type immune response as in SLE. However, the expression of CD30 on CD3 T lymphocytes from patients with SLE has not been studied yet. Therefore, we have addressed our study to attempt this issue, studying CD30 expression by flow cytometry on CD3 T lymphocytes and CD4/CD8 subsets in samples from SLE patients mainly with lupus nephritis. In parallel, we have determined the production of the cytokines IL‐4 (Th2), IFNγ (Th1), IL‐10 and TGFβ by intracellular staining. Differences between positive CD30 T cells in healthy controls and patients with SLE were found, with a higher percentage of CD30‐expressing T cells in patients with SLE (P = 0.001). In contrast to healthy controls, CD30 was mainly expressed on CD8 T cells from patients with SLE. The intracellular cytokine staining showed that TGFβ is the main cytokine expressed in CD3 T cells from patients with SLE. In addition to this, we have found a positive correlation between CD30‐expressing T cells and IL‐4, IFNγ, and immunosuppressive cytokines (IL‐10 and TGFβ) (P < 0.05). These results suggest that CD30 could play a role in the pathogenesis of SLE and its expression on CD3 T lymphocytes is not restricted only to Th2‐type response.     

Expression of CD21 is developmentally regulated during thymic maturation of human T lymphocytes.

CD21, the C3d/CD23/Epstein-Barr virus (EBV), receptor is expressed at low density on cells of the T lineage. Immature thymocytes express CD21 with high density. In the present study, we have analyzed the expression of CD21 during intrathymic maturation of T cells. An intense staining for CD21 was observed at the double-negative stage and at the stage of early acquisition of CD4. CD21 expression was decreased at the double-positive and single-positive stages, to then reach levels similar to those of peripheral blood T cells. Staining of thymus sections showed a bright fluorescent signal on thymocytes entering the thymus in the cortical region. Taking advantage of the immature phenotype of cells expressing high amounts of CD21 (CD21(++)), we depleted thymocyte suspensions in CD3(+) and CD8(+) cells to study the properties of CD21 on this cell subset. Triggering of CD21 with its ligands iC3b, CD23 and anti-CD21 mAb did not alter the proliferative response of thymocytes to IL-7, and did not induce the differentiation of early cells into CD4(+)CD8(+) thymocytes. Immunoprecipitation did not reveal any molecule associated with CD21 that could play a signaling role in thymocytes. Finally, EBV induced a down-regulation of CD21 and an up-regulation of CD1 in CD21(++) thymocytes. Taken together, our observations demonstrate a regulated expression of CD21 on human thymocytes and suggest that the CD21(++) subset may be a target for EBV. We further suggest that CD21 on early thymocytes acts as a ligand for CD23-expressing cells in the thymus.     

CD8+ memory T lymphocytes from bone marrow--immune function and therapeutic potential

Immunity to previously encountered diseases is provided in large part by memory T lymphocytes, which may be subdivided based on phenotypic and functional differences, as well as the specific cellular compartments in which these cells reside. The bone marrow (BM) is a unique microenvironment that supports robust proliferation and recall responses of both "central" and "effector" memory T cells, particularly within the CD8+ T cell subset. The recent identification within human BM of a population of CD8+ effector memory T cells with hybrid phenotype and enhanced cytotoxic function has important implications for the development of future immunotherapies. Using activated BM CD8+ memory T cells for adoptive transfer or targeting such cells with tailored vaccines may improve the ability of these classic modalities to produce potent and long-lasting antigen-specific responses, ultimately leading to clinically significant control of viral and malignant diseases.     

Surface markers on human activated T lymphocytes. II. CD4 and CD8 differentiation antigens

In order to estimate the dependence of CD4 and CD8 antigen expression upon the cell activation, their presence on resting and activated human T cells was investigated. Peripheral blood lymphocytes of healthy donors derived from late (TEl) and early (TEe) E rosettes were used as resting and presumably in vivo activated T cell subsets, respectively. Expression of the above markers on T cells stimulated in vitro with PHA was also examined. It was found that both lymphocyte subsets contained similar percentage of CD4+ cells, nevertheless, TEe subset was partially enriched in CD8+ cells. PHA stimulation induced in TEe and TEl subsets the considerable increase of proportion of phenotypical opposing cells, CD8+ or CD4+, respectively. The above changes coincided with the time of the maximal DNA synthesis (the 72nd h of cell stimulation).     

Expression of CD2 on porcine B lymphocytes

Remarkable interspecies differences in CD2 expression on B lymphocytes have been reported in mammals. Human and rat B cells lack CD2, whilst B lymphocytes in mice are CD2⁺. In pigs, B cells have been supposed not to express CD2. We show here, however, that CD2 is present at a low level on a prominent subset of porcine B cells. Moreover, we describe changes in the proportions of CD2⁺ and CD2^? B-cell subsets during ontogeny. Before contact with microflora, the majority of peripheral surface immunoglobulin M⁺ (sIgM⁺) B cells express CD2 and sIgM⁺CD2^? B cells are rare. Shortly after colonization of conventional (CV) piglets with complex intestinal microflora, numerous CD2^?B cells appear in the periphery and their relative number increases with age in both CV and specific pathogen-free (SPF) pigs. However, monoassociation of germ-free (GF) piglets with a single Escherichia coli strain does not result in a significant increase of sIgM⁺CD2^?B cells in the periphery. We suggest that CD2 is down-regulated in porcine B lymphocytes upon activation with microflora in mucosa-associated lymphatic tissues. In bone marrow (BM), we identified putative porcine B-cell precursors. These cells express CD2 at low density and do not bear either the common myelomonocytic antigen or T and B-lymphocyte receptors. Similar to mouse and human pre-B cells, this lymphocyte-sized subset expresses CD25 and class II antigens. CD2 positivity of these cells indicates that CD2 is expressed earlier than sIgM during B lymphopoiesis in pigs.     

Expression of CD8alpha identifies a distinct subset of effector memory CD4+ T lymphocytes

Circulating CD4+ CD8+ T lymphocytes have been described in the peripheral blood of humans and several animal species. However, the origin and functional properties of these cells remain poorly understood. In the present study, we evaluated the frequency, phenotype and function of peripheral CD4+ CD8+ T cells in rhesus macaques. Two distinct populations of CD4+ CD8+ T cells were identified: the dominant one was CD4hi CD8lo and expressed the CD8alphaalpha homodimer, while the minor population was CD4lo CD8hi and expressed the CD8alphabeta heterodimer. The majority of CD4hi CD8alphalo T cells exhibited an activated effector/memory phenotype (CCR5lo CD7- CD28- HLA-DR+) and expressed relatively high levels of granzyme B. Intracellular cytokine staining assays demonstrated that the frequency of cytomegalovirus-specific T cells was enriched five-fold in CD4hi CD8alphalo T cells compared to single-positive CD4+ T cells, whereas no consistent enrichment was observed for simian immunodeficiency virus (SIV)-specific T cells. Cross-sectional studies of SIV-infected animals demonstrated that the frequency of CD4hi CD8alphalo T cells was lower in wild-type SIV-infected animals compared to uninfected controls, although prospective studies of SIV-infected animals demonstrated depletion of CD4hi CD8alphalo lymphocytes only in a subset of animals. Taken together, these data suggest that CD4+ T cells expressing CD8alpha represent an effector/memory subset of CD4+ T cells and that this cell population can be depleted during the course of SIV infection.     

Intracellular expression of CD1 molecules on PHA-activated normal T lymphocytes.

In this study we have analyzed, using immunoperoxidase (IPx) and indirect immunofluorescence (IIF), the intracellular and cell surface expression of CD1 antigens on PHA activated human T cells. By IIF, CD1 isotypes were not detected on the surface of 3 days PHA activated cells. Conversely, CD1a, CD1b and CD1c molecules were found by IPx in the cytoplasm of normal activated cells from 13 different donors. Kinetic studies showed that, while CD25 was already observed 24 h after activation, all 3 isotypes of CD1 molecules started to be detected 48 h after PHA activation, with a peak expression at 72 h.     

Masking of veto function in vivo by activated CD4+ T lymphocytes

Donor CD8+ T lymphocytes injected into recipient mice incompatible at major histocompatibility complex (MHC) class I genes induce donor-specific CTL nonresponsiveness, attributed to the veto function of donor cells. Here we show that conditions leading to strong activation of CD4+ T cells, namely the presence in the recipient of foreign MHC class II determinants, lead to the apparent loss of veto function of donor cells. This "masking" of veto function is dependent on the dose of foreign MHC class II present. Veto function can be partially restored by treatment of recipients in vivo with CD4-specific antibody, a measure which has been shown to eliminate the function of CD4+ T cells in vivo. We conclude that CD4+ T cells activated by contact with antigen can interfere with the veto function of CD8+ T cells. Consequences of this finding are: (a) veto function of a sample cell population can be overlooked when activation of CD4+ T cells occurs simultaneously. (b) The balance between veto function of recipient cells and its abrogation might be responsible for the kind of graft-vs.-host reaction generated (CD8+ T cell-mediated and frequently lethal or CD4+ T cell-mediated and not lethal) when parental T cells are injected into recipients incompatible at MHC class I and class II genes. (c) A possible contribution of veto cells should be considered in several protocols in which donor hemopoetic cells were used in conjunction with CD4-specific antibodies to induce transplantation tolerance. (d) Veto function in vivo does not require a contribution of CD4+ T cells.     

Expression of CD45 isoforms by fresh and activated human gamma delta T lymphocytes and natural killer cells.

Naive and primed alpha beta T cells can be distinguished on the basis of their differential expression of CD45RA and CD45RO, respectively. The present study indicates that these CD45-isoforms also identify naive and primed maturational stages of gamma delta T cells and natural killer (NK) cells. In peripheral blood, all V gamma 9-V delta 2 gamma delta T cells reportedly express CD45RO whereas all V delta gamma delta T cells lack CD45RO. Here, we show that these CD45RO- V delta gamma delta T cells all express CD45RA and the CD45RO+ V.9-V delta 2 gamma delta cells lack expression of CD45RA. The V delta T cells acquired CD45RO expression and lost part of their surface CD45RA, following in vitro activation with phytohaemagglutinin or IL-2. Also the CD3-CD16+ NK cells in peripheral blood that are uniformly CD45RA+ CD45RO- completely converted to the CD45RA-CD45RO+ phenotype upon in vitro activation. Moreover, all cloned V.9-V delta 2 and V delta 1 T cells and NK cells express CD45RO and lack expression of CD45RA. Our results strongly suggest that CD45RA and CD45RO are genuine markers for naive and primed lymphocytes that represent distinct differentiation lineages.     

Expression of a 32-kDa ligand for the CD40 antigen on activated human T lymphocytes

To identify the ligand(s) of the human CD40 antigen, a cDNA encoding the extracellular domain of the CD40 antigen was fused to a cDNA encoding the constant region (Fc) of human IgGl. The CD40-Fc fusion protein was able to specifically bind to CD4⁺ and various CD8⁺ T cell clones activated with immobilized anti-CD3. The ¹²⁵I-labeled CD40-Fc fusion protein bound anti-CD3 activated CD4⁺ T cell clone (MT9) with an equilibrium dissociation constant (Ka) of 10-20 nM. The human CD40-binding protein expressed on the cell surface of activated T lymphocytes is a monomeric protein of ≈ 32 kDa. Minor components of 29 kDa and 17 kDa were also detected. A small proportion of CD4+ and CD8⁺ blood mononuclear T cells activated by anti-CD3 expressed the CD40 ligand but its detection was best observed following depletion of B cells. Addition of B cells to purified T cells abolished the binding of CD40-Fc obtained after anti-CD3 activation.     

Expression of CD45RB functionally distinguishes intestinal T lymphocytes in inflammatory bowel disease

The importance of CD45RB expression on T cells was already shown in mice where CD45RB(high) expression determines pathogenic potential. In this study, we analyzed the expression of CD45RA, CD45RB, and CD45RO on CD4(+) T lymphocytes in the intestinal mucosa and in the circulation of patients with inflammatory bowel disease (IBD). In addition, we studied the cytokine profile of these cells. In the circulation, virtually all CD4(+)CD45RB(high) T cells expressed the naive marker CD45RA, and circulating CD4(+)CD45RB(low) cells expressed the memory marker CD45RO in IBD patients and a control patient population. In contrast, the intestinal CD4(+) CD45RB(high) T cells are in normal controls for 90% CD45RO(+). However, in IBD, 27.7% [Crohn's disease (CD)] and 49% [ulcerative colitis (UC)] of the intestinal CD4(+) CD45RB(high) T cells are CD45RA(+). This special CD4CD45RA(+) T cell in IBD can be found in the lamina propria as well as in lymphoid follicles (confocal laser-scanning microscopy). The CD4(+)CD45RB(high) T lymphocytes produce significantly less interleukin (IL)-10 and IL-4 and produce more tumor necrosis factor alpha than CD45RB(low) T lymphocytes in control patients. CD4(+)CD45RB(low) T cells from IBD patients produced less IL-10 than CD4(+)CD45RB(low) T lymphocytes of controls, and interferon-gamma production by both T lymphocyte subsets was decreased in IBD. These data indicate that CD and UC are characterized by an influx of CD4(+)CD45RB(high) T lymphocytes. These CD4(+)CD45RB(high) T lymphocytes seem to be important in the pathogenesis of IBD, as they produce more proinflammatory cytokines and less anti-inflammatory cytokines compared with CD4(+)CD45RB(low) T lymphocytes.     

CD30 ligation differentially affects CXCR4-dependent HIV-1 replication and soluble CD30 secretion in non-Hodgkin cell lines and in gamma delta T lymphocytes

We studied whether signaling through CD30, a member of the TNF receptor family, affected acute infection with HIV-1, encompassing its entire replicative cycle. Several non-Hodgkin cell lines, targets of CXCR4-dependent (X4) HIV-1 infection, were positive for CD30 expression. CD30 ligation induced up-regulation of viral replication only in certain CD30+ cell lines. Enhancement of X4 virus replication by CD30 engagement inversely correlated with both CD30 surface density and constitutive NF-kappaB activation. Conversely, expression of CD30, but not of other members of the TNF receptor family, was proportional to constitutive NF-kappaB binding. Concomitantly, secretion of soluble (s) CD30 increased in all cell lines by CD30 ligation. sCD30 release was enhanced by engagement of CD30 alone and, to a greater extent, by co-engagement of CD3 also in primary gamma delta T lymphocytes, along with complementary modulations of their surface CD30 expression. sCD30-containing supernatant specifically inhibited HIV-1 expression induced by CD30 engagement in chronically infected ACH-2 T cells; thus sCD30 may act as a negative feed-back molecule. In conclusion, we have delineated novel features of CD30 biology and underline the peculiar link of CD30 expression to constitutive NF-kappaB activation which is pivotal to both HIV replication and cell survival.     

亚急性衰老小鼠脾脏T细胞CD137表达的研究

目的：探讨CD137分子在衰老小鼠脾脏T细胞表面表达的规律。方法：通过注射D－半乳糖建立亚急性衰老小鼠模型。分离衰老模型组小鼠及正常对照组小鼠的脾脏T细胞，经Con A刺激后进行培养，采用流式细胞术检查Con A刺激后不同培养时间的T细胞上CD137的表达，并进行比较。结果：小鼠脾脏T细胞经过Con A刺激后，正常对照组小鼠T细胞上CD137分子的表达高峰出现于刺激后第6天，平均表达率为48．5％，之后迅速下降。1个月和2个月衰老模型组小鼠T细胞上CD137分子在表达高峰的表达率平均分别为39．1％和32．8％，均显著低于正常对照组小鼠（P＜0．01），其中1个月模型组小鼠T细胞上CD137分子表达高峰的出现时间及下降规律与正常对照组小鼠相同；而2个月衰老模型组小鼠T细胞上CD137分子的表达高峰出现于刺激后第4天，第9天缓慢下降至与正常对照组相同时间的水平。结论：亚急性衰老小鼠T细胞上CD137分子的表达低于正常水平。至衰老过程的后期，T细胞上CD137分子的表达高峰提前出现，表达水平达到峰值后下降速度较正常小鼠缓慢，提示CD137分子在机体抗衰老过程中，具有调节T细胞功能的作用。     

小鼠CD4+记忆性T淋巴细胞的产生、纯化、鉴定及皮肤移植的检测

目的 建立CD4+记忆T细胞(memory T lymphocytes,Tm)的免疫磁珠分离方法,并检测其体外部分功能.方法 借助小鼠皮肤移植模型,制备受鼠脾细胞悬液,运用免疫磁珠方法分离CD4+Tm,用台盼蓝染色法和流式细胞仪检测分选所得细胞的活性和纯度,并在体外用ELISPOT技术检测其不同抗原刺激时IFN-γ活性分泌频数.结果 小鼠皮肤移植皮片平均存活时间为(13.6±0.9)d;分离所得CD4+Tm的活细胞百分率为(98.4±0.7)%;流式细胞术检测表型,其中CD4+CD8-细胞比例占(96.06±2.49)%,CD44+CD62L-细胞比例占(94.13±2.36)%,CD4+CCR7-细胞比例占(96.39±1.93)%;在无刺激物作用或者大鼠脾细胞、供体脾细胞、刀豆素A等刺激物作用时,IFN-γ活性分泌频数分别为3±1、6±3、339±14、108±9个/104,差异有统计学意义.结论 通过小鼠皮肤移植模型及免疫磁性分离技术,可制备高纯度无菌的CD4+Tm,其细胞活力不受影响,作用具有良好的供体特异性.这为深入研究Tm在移植物免疫方面的作用提供了技术资料.