Human CD4+ T cells expressing CD45RA acquire the lymphokine gene expression of CD45RO+ T-helper cells after activation in vitro.

CD4+ T cells were separated into subpopulations according to their expression of different isoforms of the CD45R molecule, i.e. CD45RA and CD45RO. The separated cells were activated with staphylococcal enterotoxin A (SEA) in the presence of formalin fixed Raji cells. Each set of cells was activated twice with a 6-day interval, and the lymphokine gene expression during the first 3 days after initiation of each stimulation was followed by use of polymerase chain reaction (PCR) technology. The lymphokine messenger RNA (mRNA) profiles were found to differ between the subsets, since after the first stimulation the CD45RA+ cells produced mRNA encoding interleukin-2 (IL-2) and IL-1 alpha, whereas the CD45RO+ cells transcribed genes for IL-1 alpha, IL-2, IL-4, IL-5 and interferon-gamma (IFN-gamma). After 6 days of SEA stimulation both populations were mainly CD45RO reactive, and when restimulated displayed the lymphokine mRNA profile restricted to this subset. These results indicate that the CD45RA subset is a precursor of the CD45RO and further strengthen the hypothesis that the former cell population represents naive whereas the latter subset represents memory T cells within the CD4 subset.     

Differential expression of alpha2 integrin separates long-term and short-term reconstituting Lin-/loThy1.1(lo)c-kit+ Sca-1+ hematopoietic stem cells

Self-renewing, multipotent hematopoietic stem cells are highly enriched within the Lin- Thy1.1(lo)c-kit+ Sca-1+ subset of mouse bone marrow. However, heterogeneous expression within this population of certain cell surface markers raises the possibility that it may be further fractionated phenotypically and perhaps functionally. We previously identified alpha2-integrin (CD49b) as a surface marker with heterogeneous expression on Lin(-/lo)Thy1.1(lo)c-kit+ Sca-1+ stem cells. To determine whether differences in alpha2 expression were indicative of differences in stem cell function, we purified alpha2- and alpha2hi stem cells by fluorescence-activated cell sorting and analyzed their function in long- and short-term hematopoietic reconstitution assays. Both alpha2- and alpha2hi cells could give rise to mature lymphoid and myeloid cells after transplantation into lethally irradiated congenic recipients. However, alpha2hi cells supported hematopoiesis for only a short time (<4 weeks), whereas alpha2- cells reproducibly yielded robust, long-term (>20 weeks) reconstitution, suggesting that alpha2- cells represent a more primitive population than do alpha2hi cells. Consistent with this idea, alpha2- Lin(-/lo)Thy1.1(lo)c-kit+ Sca-1+ cells exhibited an approximately sixfold decreased frequency of spleen colony-forming units (day 12) versus alpha2hi cells. Furthermore, bone marrow cells isolated from animals transplanted >20 weeks previously with 20 alpha2- Lin(-/lo)Thy1.1(lo)c-kit+ Sca-1+ cells included both alpha2- and alpha2hi stem cells of donor origin, indicating that alpha2hi cells are likely lineal descendents of alpha2- cells. Interestingly, alpha2 integrin expression is significantly reduced on lineage-restricted oligopotent progenitors in the marrow, suggesting that high level expression of alpha2 selectively marks a subset of primitive hematopoietic cells which retains multilineage reconstitution potential but exhibits reduced self-renewal capacity.     

CD4+ CD45RA+ and CD4+ CD45RO+ T cells differ in their TCR-associated signaling responses.

Peripheral CD4+ T cells can be divided into two different functional populations based on the expression of distinct isoforms of the surface molecule CD45. We have investigated the differences in the proximal signaling induced by anti-CD3 monoclonal antibody in purified populations of "naive" CD45RA+ and "memory" CD45RO+ human CD4+ T cells. Expression of cell surface CD3, CD4 and CD28 was comparable between RA+ and RO+ cells. However, TCR-directed stimulation in the form of anti-CD3 produced markedly different patterns of intracellular signaling. Greater inositol triphosphate generation occurred in naive cells and the rise in intracellular free calcium was also substantially greater in naive than in memory cells. Cells with the naive phenotype were considerably more active in TCR-dependent tyrosine phosphorylation, both at an overall level and specifically in terms of TCR-zeta and ZAP-70 phosphorylation. Despite these differences in phosphorylation, the amounts of TCR-zeta, ZAP-70 and Ick were equivalent between the two subsets. These findings suggest that the TCR-dependent signaling is differentially regulated in naive and memory CD4+ T cells. This may be due to differences in the way that the two isoforms of the CD45 phosphatase regulate the activity of proximal kinases in the TCR signaling pathway, and could be an important means by which the unique functions of differentiated T cell populations are maintained.     

Human CD4+CD45R0+ and CD4+CD45RA+ T cells synergize in response to alloantigens.

Alloantigens, unlike recall antigens, activate both CD45RA+ (naive) and CD45R0+ (memory) CD4+ cells to the same extent. These T cell subsets may therefore interact with each other in response to alloantigens on transplanted grafts. We have investigated if the ability of activated CD4+CD45RA+ and CD4+CD45R0+ T cells to produce and respond to interleukin 2 (IL2) and IL4 may be involved in this interaction. After activation, both subsets up-regulate their IL2 receptor (IL2R) and IL4R expression, yet IL4 substantially enhanced the proliferation of the CD4+CD45RA+ but not of the CD4+CD45R0+ T cell subset, while IL2 increased the proliferation of CD4+CD45R0+ but not of the CD4+CD45RA+ T cells. Significantly, the CD4+CD45RA+ T cells synthesized two- to threefold more mRNA for IL2 than the CD4+CD45R0+ subset, while the CD4+CD45R0+ T cells synthesized mRNA for IL4 and interferon-gamma exclusively. The addition of IL2 to alloactivated CD4+CD45R0+ T cells further up-regulated their production of all three lymphokine mRNA; in contrast, IL4 induced an increase in mRNA for IL2 in only the alloactivated CD4+CD45RA+ subset. The reciprocity in the ability of both these CD4+ T cells to synthesize and respond to IL2 and IL4 may provide a rationale for the regulation of lymphokine interactions in vivo. Furthermore, the synergy between these subsets in response to alloantigens, which was directly quantitated by co-culturing CD4+CD45RA+ and CD4+CD45R0+ cells together prior to activation, may potentiate the alloreactivity against transplanted grafts in vivo.     

CD4+CD45RA+T cells from adults respond to recall antigens after CD28 ligation

The leukocyte common antigen isoforms CD45RA and CD45RO havelong been used to discriminate human naive and memory T cellsrespectively. This model was largely based on the observationthat CD45RO⁺ T cells respond preferentially to and show a higherfrequency of precursors specific for recall antigens. However,CD45RA⁺ T cells have more stringent requirements for stimulationand standard in vitro assays may favour CD45RO⁺ cells in thisrespect. We tested the hypothesis that CD45RAf T cells respondpoorly to in vitro stimulation with recall antigens becauseof inadequate stimulation rather than a lack of precursors.Limiting dilution analyses (LDA) for tetanus toxoid (lT)-specificT cells were performed in the presence or absence of exogenousantLCD28 antibody. Addition of antLCD28 yielded no proliferationin the absence of specific antigen. The precursor frequencyfor lT in the CD4⁺ CD45RO⁺ population was –1:4000, whilethe frequency of CD4⁺ CD45RA⁺ T cells specific for lT was 4-to >>20-fold lower. Addition of anti-CD28 antibody didnot significantly alter the apparent precursor frequency forCD45RA⁺ cells but yielded an enhancement of the value for CD45RA⁺cells by 3- to >>5-fold. No enhancement of antigen-specificproliferation by antLCD28 was observed with CD45RA⁺ T cellsderived from cord blood, although phytohemagglutinin responsesof these cells were amplified by CD28 antibody. These resultsindicate that conventional LDA underestimate the true precursorfrequency of antigen-specific cells within the adult CD45RA⁺population and support the possibility that a small number ofcells revert from a primed (CD45RO⁺) to an unprimed (CD45RA⁺)state. The majority of memory T cells, however, appear to residein the CD45RO⁺ population     

Immune memory in CD4+ CD45RA+ T cells.

This study addresses the question of whether human peripheral CD4+ CD45RA+ T cells possess antigen-specific immune memory. CD4+ CD45RA+ T cells were isolated by a combination of positive and negative selection. Putative CD4+ CD45RA+ cells expressed CD45RA (98.9%) and contained < 0.1% CD4+ CD45RO+ and < 0.5% CD4+ CD45RA+ CD45RO+ cells. Putative CD45RO+ cells expressed CD45RO (90%) and contained 9% CD45RA+ CD45RO+ and < 0.1% CD4+ CD45RA+ cells. The responder frequency of Dermatophagoides pteronyssinus-stimulated CD4+ CD45RA+ and CD4+ CD45RO+ T cells was determined in two atopic donors and found to be 1:11,314 and 1:8031 for CD4+ CD45RA+ and 1:1463 and 1:1408 for CD4+ CD45RO+ T cells. The responder frequencies of CD4+ CD45RA+ and CD4+ CD45RO+ T cells from two non-atopic, but exposed, donors were 1:78031 and 1:176,903 for CD4+ CD45RA+ and 1:9136 and 1:13,136 for CD4+ CD45RO+ T cells. T cells specific for D. pteronyssinus were cloned at limiting dilution following 10 days of bulk culture with D. pteronyssinus antigen. Sixty-eight clones were obtained from CD4+ CD45RO+ and 24 from CD4+ CD45RA+ T cells. All clones were CD3+ CD4+ CD45RO+ and proliferated in response to D. pteronyssinus antigens. Of 40 clones tested, none responded to Tubercule bacillus purified protein derivative (PPD). No difference was seen in the pattern of interleukin-4 (IL-4) or interferon-gamma (IFN-gamma) producing clones derived from CD4+ CD45RA+ and CD4+ CD45RO+ precursors, although freshly isolated and polyclonally activated CD4+ CD45RA+ T cells produced 20-30-fold lower levels of IL-4 and IFN-gamma than their CD4+ CD45RO+ counterparts. Sixty per cent of the clones used the same pool of V beta genes. These data support the hypothesis that immune memory resides in CD4+ CD45RA+ as well as CD4+ CD45RO+ T cells during the chronic immune response to inhaled antigen.     

Sca-1 + bone marrow mesenchymal stem cells transplantation for the alleviation of delayed type hypersensitivity in mouse

目的 探讨骨髓Sca-1+间充质干细胞(BMSCs)在迟发型超敏反应(DTH)小鼠中的免疫调控作用.方法 BALB/c小鼠随机分为对照组及MSCs注射组.注射组于0、2及6d腹腔注射Sca-1+ BMSCs,对照组注射0.9％氯化钠注射液.所有小鼠在7和14 d时分别于背部皮下与足跖注射C57BL/6小鼠脾细胞.24h后用千分尺测量小鼠足跖的肿胀,ELISA法测外周血细胞因子及FACS法测脾脏免疫细胞比例.结果 注射组小鼠足跖的肿胀程度[ (0.368 *0.126) mm]显著轻于对照组[(0.731±0.111) mm,P＜0.01].注射组外周血中IL-10和TGF-β含量显著高于对照组(P＜0.05);而IL-12和TNF-α水平显著低于对照组(P＜0.05).注射组小鼠脾脏调节型T细胞和树突细胞的比例均明显高于对照组(P＜0.05).结论 BMSC通过上调抑炎因子和调节型免疫细胞的数量而减轻DTH小鼠的免疫反应.     

Evaluation of Sca-1 and c-Kit as selective markers for muscle remodelling by nonhemopoietic bone marrow cells

Bone marrow (BM)-derived cells (BMCs) have demonstrated a myogenic tissue remodeling capacity. However, because the myoremodeling is limited to approximately 1%-3% of recipient muscle fibers in vivo, there is disagreement regarding the clinical relevance of BM for therapeutic application in myodegenerative conditions. This study sought to determine whether rare selectable cell surface markers (in particular, c-Kit) could be used to identify a BMC population with enhanced myoremodeling capacity. Dystrophic mdx muscle remodeling has been achieved using BMCs sorted by expression of stem cell antigen-1 (Sca-1). The inference that Sca-1 is also a selectable marker associated with myoremodeling capacity by muscle-derived cells prompted this study of relative myoremodeling contributions from BMCs (compared with muscle cells) on the basis of expression or absence of Sca-1. We show that myoremodeling activity does not differ in cells sorted solely on the basis of Sca-1 from either muscle or BM. In addition, further fractionation of BM to a more mesenchymal-like cell population with lineage markers and CD45 subsequently revealed a stronger selectability of myoremodeling capacity with c-Kit/Sca-1 (p < .005) than with Sca-1 alone. These results suggest that c-Kit may provide a useful selectable marker that facilitates selection of cells with an augmented myoremodeling capacity derived from BM and possibly from other nonmuscle tissues. In turn, this may provide a new methodology for rapid isolation of myoremodeling capacities from muscle and nonmuscle tissues. Disclosure of potential conflicts of interest is found at the end of this article.     

骨髓Sca-1+间充质干细胞移植减轻小鼠迟发型超敏反应

 目的 探讨骨髓Sca-1+间充质干细胞（BM-MSCs）在迟发型超敏反应（DTH）小鼠中的免疫调控作用。方法 小鼠随机分为对照组及MSCs注射组。注射组于0、2及6 d腹腔注射Balb/c小鼠Sca-1+ BM-MSCs,对照组注射生理盐水。所有鼠在7和14 d时分别于背部皮下与足跖注射C57BL/6小鼠脾细胞。24 h后用千分尺测量小鼠足跖的肿胀,ELISA法测外周血细胞因子及FACS法测脾脏免疫细胞比例。结果 注射组小鼠足跖的肿胀程度（0.368 ? 0.126 mm）显著轻于对照组（0.731 ? 0.111 mm,p < 0.01）。注射组外周血中IL-10和TGF-β含量显著高于对照组（p < 0.05）;而IL-12和TNF-α水平显著低于对照组（p < 0.05）。注射组小鼠脾脏调节型T细胞、树突细胞的比例明显高于对照组。结论 BM-MSC通过上调抑炎因子和调节型免疫细胞的数量而减轻DTH小鼠的免疫反应。     

Identical expression of CD45R isoforms by CD45RC+ 'revertant' memory and CD45RC+ naive CD4 T cells.

Naive and memory CD4 T cells are frequently defined by exon-specific monoclonal antibodies (mAb) which stain (or not) high- or low-molecular-weight (MW) isoforms of the leucocyte common antigen CD45. The link between isoform and the naive/memory designation is complicated by the fact that CD4 T cells with a 'memory' phenotype (CD45RA-, RB-, RC-, or CD45RO+) may revert ('revertants') and re-express the high mw isoform (CD45RA+, RB+, RC+). Isoform expression also changes during normal T-cell development. Furthermore, the picture may be incomplete since an exon-specific mAb will not detect all possible isoforms on a cell. We have used molecular techniques to determine whether revertant CD4 memory T cells were different from naive T cells with respect to CD45R isoform expression. Using the anti-CD45RC mAb OX22 to purify rat lymphocyte subsets, CD45R isoform expression was examined at the mRNA level in CD4 T cells at different stages of development and compared with that of B cells and unseparated lymphocytes. B cells contained abundant message for the highest MW 3-exon isoform ABC, the 2-exon isoforms AB and BC, and the null isoform O. Both immature CD45RC- (i.e. CD4+8- 'single positive' thymocytes, and peripheral Thy-1+ recent thymic emigrants) and mature CD45RC- 'antigen-experienced' CD4 T cells had message for single-exons B, possibly C and for the O exon. In contrast, CD45RC+ CD4 T cells contained mRNA coding for ABC (low level), AB, BC, B, C (low level) and O (low level). Importantly, there was no difference between CD45RC+ T cells that had not seen antigen ('truly native') and CD45RC+ antigen-experienced revertant memory T cells. This observation has implications for understanding long-term immunological memory.     

小鼠干细胞抗原阳性胎肝细胞在受体鼠脑组织中的分化

为了解胎肝干细胞是否具有向神经组织细胞分化的潜能 ,本研究采用免疫磁珠法分离雄性胎鼠肝组织中干细胞抗原 1(stemcellantigen ,Sca 1)阳性细胞 ,尾静脉注射Sca 1+ 细胞 (2 0× 10 3个 小鼠 )到经致死剂量 (10 0Gy)放射线照射的 10～ 12周C5 7BL 6J雌性小鼠。免疫组化和FISH双染色检测结果显示移植 2、4、6月后受体雌鼠脑组织内存在大量Y染色体阳性细胞 ,约占脑组织总细胞为 4 5 %± 0 5 % ,分布包括 :侧脑室脉络膜丛、脑室室管膜、大脑白质灰质、小脑等。这些Y染色体阳性细胞表达神经组织细胞特异标志 ,如神经元特异核蛋白 (Neuron specificnucleiprotein ,Ne uN)、神经纤维细丝蛋白M(neurofilament 16 0Ka,NF M)、微管蛋白Ⅲ (tubulinⅢ ,TuJ 1)、或者胶质纤维酸性蛋白 (Glialfibrillaryacidicprotein,GFAP)等 ,其中Y染色体和NeuN均阳性的细胞为 1 2 %± 0 3%、Y染色体和GFAP均阳性的细胞为 1 0 %± 0 2 %。这说明胎肝Sca 1+ 细胞能迁移进入脑组织 ,并且分化成神经细胞和星形胶质细胞。     

Phenotypic changes associated with activation of CD45RA+ and CD45RO+ T cells.

Resting CD45RO+, mature/memory, T cells are phenotypically distinct from intermediate CD45RO+/CD45RA+ and CD45RA+, immature/virgin, T cells, and are characterized by high levels of expression of a number of adhesion molecules, such as CD2, CD18, CD58 and CD29. The kinetics of up-regulation of molecules, like CD25 and CD54 associated with activation, were similar in both subsets and suggested that their high level expression was associated with later events rather than initial recognition and signal transduction. CD45RA+ T cells, unlike CD45RO+ T cells, were unable to proliferate in response to mitogenic combinations of CD2 monoclonal antibodies (mAb), although in combination with submitogenic doses of PMA both up-regulation of cell-surface molecules and proliferation occurred. In addition, recruitment of CD45RA+ T cells by CD2 mAb-activated CD45RO+ T cells can occur.     

胚胎小鼠肝脏Sca-1+细胞横向分化的研究

目的:研究和探索胚胎小鼠肝脏细胞的横向分化潜能。方法:将2×10³ C57BL/6j雄性胚胎小鼠的肝脏Sca-1⁺细胞从尾静脉注射到受致死性γ射线(10 Gy, ⁶⁰Co)全身照射的同种成年雌性小鼠体内;于移植后2个月, 用荧光原位杂交和免疫组织化学技术, 检测雄性胚胎小鼠肝脏Sca-1⁺细胞在雌性受体小鼠肾脏和脑组织内的分化情况。结果:在雌性受体小鼠的肾脏和脑组织内, 分别检测到Y染色体阳性的供体来源的肾小管上皮组织细胞样和神经组织细胞样的细胞, 其细胞表型分别为RCA⁺/CD^-₄₅F^-_4/80和NueN⁺/CD^-₄₅F^-_4/80。结论:胚胎小鼠肝脏Sca-1⁺细胞具有向肾脏和脑组织细胞横向分化的能力。     

CD3+CD20+ T cells and their roles in human diseases

CD3⁺CD20⁺ T cells are a population of CD3⁺ T cells co-expressing CD20 that make up to ∼3–5% of the CD3⁺ T-cell compartment in the peripheral blood of human beings. In healthy individuals, CD3⁺CD20⁺ T cells are heterogeneous for containing a lower proportion of CD4⁺ cells, but produce higher levels of IL-17A and/or IFN-γ than those of CD3⁺CD20⁻ T cells. Recently, emerging studies have shown a pathogenic behavior of CD3⁺CD20⁺ T cells in autoimmune diseases and CD20⁺ T-cell malignancies, and patients with the diseases may benefit from anti-CD20 immunotherapy to deplete these cells. However, CD3⁺CD20⁺ T cells may also play a protective role in ovarian cancer and HIV infection for their strong propensity to IFN-γ production. In this review, we will describe the current knowledge about CD3⁺CD20⁺ T-cell biology, and discuss their functional roles in autoimmune diseases as well as cancer and infectious diseases.     

CD45 epitope mapping of human CD1a+ dendritic cells and peripheral blood dendritic cells.

The authors studied the pattern of leukocyte common antigen (CD45) epitope expression on dendritic cells in sections of human epidermis, tonsillar epithelium, dermatopathic lymph nodes, and in isolates from blood. The monoclonal antibodies (MAb) used were specific for all known CD45 epitopes, including the seven different CD45 common epitopes as well as the four known CD45R epitopes (two CD45RA, one CD45RB, and one CD45RO). Dendritic cells in all sites were uniformly reactive for the CD45 common epitopes tested except 2B11, which may recognize a CD45R rather than CD45 epitope. By single-label immunoperoxidase and double-label immunofluorescence epitope mapping of CD1a+ dendritic cells in tissue sections, it was generally difficult or impossible to detect expression of CD45RA, CD45RB, CD45RO, or 2B11. In blood dendritic cells, however, low levels of these CD45R epitopes were detected consistently using single-label immunoperoxidase staining of cytocentrifuge preparations. Monocytes were similar to blood dendritic cells except that the staining with MAb to CD45RO and 2B11 was slightly stronger. The authors conclude that dendritic cells differ from most subpopulations of lymphocytes in that CD45 common epitopes are readily detectable but the existing RA, RB, and RO epitopes are either undetectable or expressed at relatively low levels. These studies raise the possibility that CD1a+ dendritic cells may express a novel dominant CD45 isoform.     

CD4+CD25+调节性T细胞在肿瘤免疫中的作用

CD4+CD25+调节性T细胞(Tr)是体内自然发生的调节性T细胞的重要亚群,具有无反应性和免疫抑制两大特性,主要通过与靶细胞的直接接触而起作用,其在体内不仅参与自身免疫性疾病、移植排斥反应等,还在肿瘤的发生、发展及免疫治疗中发挥重要作用.近几年来,Tr在肿瘤免疫中的作用倍受关注.     

Haploidentical stem cell transplantation with purified CD34 cells after a chemotherapy-alone conditioning regimen.

We investigated whether a novel chemotherapy-alone conditioning regimen would permit durable engraftment of standard doses of CD34+ purified stem cell grafts from full-haplotype mismatched related donors. We also examined the role of infusing limited doses of donor leukocytes for prevention of leukemia relapse. Our conditioning regimen consisted of thiotepa, fludarabine, rabbit antithymocyte globulin, melphalan, cyclosporin, and prednisolone. Since October 1998, 14 patients with high-risk leukemia were treated; 13 donor-patient pairs shared 3 of 6 HLA antigens, and 1 pair shared 5 of 6 HLA antigens. A median of 5.4 x 10(6) CD34+ cells per kilogram, 1.62 x 10(4) CD3+ cells per kilogram, and 9.32 x 10(4) CD19+ cells per kilogram were infused. T-cell depletion was the only graft-versus-host disease (GVHD) prophylaxis. All patients had prompt engraftment, and no late graft rejections were observed. All surviving patients received at least 1 infusion of donor whole blood containing 5, 7, 10, 25, or 50 x 10(3) CD3+ cells per kilogram between days 25 and 95 after transplantation, after which 8 developed acute GVHD (3 grade I, 2 grade II, 2 grade III, and 1 grade IV) and 2 developed a bronchiolitis obliterans-like syndrome. After attaining complete remission, 5 patients relapsed and died with active leukemia. The estimated relapse-related mortality at 4 years is 38.1%. As of June 15, 2003, 6 of 14 patients have survived a median of 43.5 months after transplantation with 100% donor cells. All 6 surviving patients developed acute GVHD and had a natural killer cell mismatch with their donors in the direction of graft versus host. The estimated overall survival and event-free survival for the 14 patients at 4 years is 41.7% +/- 13.5%.