CD45 isoform expression in autoimmune myasthenia gravis

In myasthenia gravis (MG), humoral and cellular immune mechanisms are involved in the autoimmune pathogenesis. In this study, we investigated the role of the CD45 molecule in MG, having recently reported an association in multiple sclerosis. CD45, a protein-tyrosine phophatase receptor type C (PTPRC), is essential for both thymic selection and peripheral activation of T and B cells. Our aims were to determine (a) the prevalence of a functional mutation in the CD45 gene (exon 4 77C --> G; prevalence analysis), and (b) the distribution of memory (CD45RO+) and naive (CD45RA+) T cells in the peripheral blood (subset analysis). T cells from 78 patients with generalised MG were stained with monoclonal antibodies against CD45RO, CD45RA, CD4 and CD8 and quantified by four-colour flow cytometry. The control panel for the prevalence analysis (a) consisted of 303 healthy individuals. (b) From those, 67 age- and sex-matched probands were randomly selected as controls for the subset analysis. Patients were stratified according to their MG onset age, thymic pathology and immunosuppressive treatment. Statistical analysis was performed by Fisher's exact test, asymptotic chi2 test, the two-sided Mann-Whitney test and Spearman's correlation coefficient. As a result, the 77C --> G mutation in exon 4 of the CD45 gene was found in 1 of 78 patients versus none of the 303 controls. Thus, no association was detected with this single nucleotide polymorphism in MG patients overall. Surprisingly, however, ratios of CD45RO+ to CD45RA+ T cells were lower among CD8+ T cells from patients with late-onset MG (P = 0.023). Thymoma patients also showed a similar trend among CD4+ and CD8+ T-cells, as expected. These differences were not related to immunosuppressive drug treatment or thymectomy (in the 67 informative patients). Since there is no other evidence for increased thymopoiesis in late-onset MG, we propose an altered subset balance in the circulation.     

Lymphocyte diversity in a 9-year-old boy with idiopathic CD4+ T cell lymphocytopenia

Since CD4+ lymphocytopenia can be caused by disturbed thymic T-cell maturation, we investigated the T-cell subsets of a 9-year-old boy fulfilling the diagnostic criteria for CD4+ lymphocytopenia in a follow-up period of 4 years. We found (I) reduced CD45RA expression, (II) enhanced CD45RO expression and (III) a significant increase in gamma delta TCR-bearing T cells. An accelerated apoptosis (11%) was observed in the CD45RO+, but not CD45RA+ subset. These findings provide evidence that a disturbed thymic T-cell maturation process might play a role in the pathogenesis of CD4+ lymphocytopenia.     

Abnormalities in the T and NK lymphocyte phenotype in patients with Nijmegen breakage syndrome

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by spontaneous chromosomal instability with predisposition to immunodeficiency and cancer. In order to assess the cellular basis of the compromised immune response of NBS patients, the distribution of functionally distinct lymphocyte subsets in peripheral blood was evaluated by means of double-colour flow cytometry. The study involved the 36 lymphopenic patients with a total lymphocyte count < or =1500 microl (group A) and seven patients (group B) having the absolute lymphocyte count comparable with the age-matched controls (> or =3000 microl). Regardless of the total lymphocyte count the NBS patients showed: (1) profound deficiency of CD4+ and CD3/CD8+ T cell subsets and up to fourfold increase in natural killer (NK) cells, almost lack of naive CD4+ T cells expressing CD45RA isoform, unchanged percentage of naive CD8+ cell subset (CD8/CD45RA+) but bearing the CD8 receptor of low density (CD8low); (2) normal expression of CD45RA isoform in the CD56+ lymphocyte subset, profound decrease in alpha beta but up to threefold increase in gamma delta-T cell-receptor (TCR)-positive T cells; (3) shift towards the memory phenotype in both CD4+ and CD8+ lymphocyte subpopulations expressing CD45RO isoform (over-expression of CD45RO in terms of both the fluorescence intensity for CD45RO isoform and the number of positive cells); and (4) an increase in fluorescence intensity for the CD45RA isoform in NK cells population. These results indicate either a failure in T cell regeneration in the thymic pathway (deficiency of naive CD4+ cells) and/or more dominant contribution of non-thymic pathways in lymphocyte renewal reflected by an increase in the population of CD4+ and CD8+ memory cells, gamma delta-TCR positive T as well as NK cell subsets.     

Age-related modifications of the human alphabeta T cell repertoire due to different clonal expansions in the CD4+ and CD8+ subsets

We have studied the effects of a life-long antigen stimulation on the clonal heterogeneity of human peripheral T cell subsets, as defined by their CD45 isoform expression. CD4+ or CD8+ T cells were obtained from healthy donors ranging in age from 20 to 100 years, and sorted into CD45RA+ and CD45RO+ populations. A modified PCR-heteroduplex analysis was then used to directly compare the TCR Vbeta clonal make up of either compartment pair. We find that the CD4+ T cell repertoire remains largely polyclonal throughout life, since CD4+ expanded clones are rare and accumulate predominantly in the CD45RO+ compartment of exceptionally old donors (100 years old). In contrast, the CD8+ T cell subset contains expanded clones which are already detectable in young adults and become very frequent in 70- to 75-year-old donors in both CD45RA+ and CD45RO+ compartments analyzed. Interestingly, some expanded clones are detectable in the CD45RA+ or in both CD45RA+ and CD45RO+ compartments of either CD4+ or CD8+ T cells. These results indicate that the age-dependent accumulation of expanded clones starts earlier and is more pronounced in the CD8+ than in the CD4+ T cell subset, reinforcing the concept that clonal expansion in the two subsets is controlled by substantially different mechanisms. Furthermore, whereas the finding of expanded CD45RO+ T cell clones is explained by antigen- driven proliferation, the detection of expanded clones in the CD45RA+ or in both CD45RA+ and CD45RO+ compartments would support the hypothesis of reversion from the CD45RO+ to the CD45RA+ phenotype after antigen encounter.      

Distribution of Alloreactivity Amongst the CD45 Isoforms of Circulating CD4 and CD8 T Lymphocytes

The expression of different isoforms of the CD45 surface molecule allows lymphocytes to be divided into two nonoverlapping categories, CD45RA and CD45RO. Previous studies of CD4 T cells have shown that responses to soluble antigens are present predominantly in the RO subset and to mitogens in the RA, alloreactivity being present in both subsets. Responses of CD8 T cells have not been investigated in such detail, nor have responses been compared to CD4 cells. Here we report the alloreactive responses of both CD45RA and RO subsets amongst both CD4 and CD8 T lymphocytes. Following isolation of CD4 and CD8 cells with immunomagnetic beads, CD45 subsets were separated by negative depletion using specific monoclonal antibodies. CD45RA populations were greater than 97% pure and CD45RO cells greater than 91%. One-way primary mixed lymphocyte reactions were established using the purified responder cells with irradiated allogeneic peripheral blood mononuclear cells as stimulators; experiments were all repeated at least three times. In assays of CD4⁺ RA and RO subsets, reactivity was present in both isoforms, being consistently, but not significantly, greater amongst the RO subset. With CD8⁺ T cells, reactivity was also present in both isoforms, but was significantly greater in the CD45RA subset, with mean proliferation 2.5–3-fold that of the CD45RO cells ( P  < 0.05).     

Selective migration of highly differentiated primed T cells, defined by low expression of CD45RB, across human umbilical vein endothelial cells: effects of viral infection on transmigration.

Low expression of CD45RB on CD45RO+ T lymphocytes defines a subset of highly differentiated T lymphocytes that accumulate in vivo within the affected joints of patients with rheumatoid arthritis (RA). Although it is known that CD45RO+ T lymphocytes migrate to sites of inflammation in vivo, it is not clear whether within this subset the CD45RBlo cells are selectively recruited or develop in situ within the joint. Using a transwell system we show that a small proportion of resting T lymphocytes migrated across unactivated human umbilical vein endothelial cells (HUVEC). These migrating cells were CD45RO+ and enriched for low CD45RB expression. In addition, both the CD45RO+CD45RBlo subset and migrating cells expressed increased levels of beta 1 and beta 2 integrins and CD44. The percentage of CD45RO+CD45RBlo T lymphocytes was increased in the circulation of patients with acute Epstein-Barr virus (EBV) infection. These in vivo activated cells also expressed increased levels beta 1 and beta 2 integrins and CD44, and showed an enhanced rate of transmigration compared with resting T lymphocytes. Transmigration of T lymphocytes was increased using the chemokines RANTES and lymphotactin and the cytokine interleukin-15 (IL-15). In addition, infection of the HUVEC with cytomegalovirus (CMV) led to an enhanced movement of T lymphocytes. In all of these cases the selective migration of the CD45RBlo subset was maintained. Thus although the rate of T-lymphocyte transmigration could be influenced by a number factors, the CD45RO+CD45RBlo subset has a migratory advantage suggesting that more differentiated CD45RO+CD45RBlo T lymphocytes are selectively recruited to sites of inflammation.     

Differential response of human thymus cells to CD2 antibodies: fragmentation of DNA of CD45RO+ and proliferation of CD45RO- subsets.

Human thymocytes bearing the CD45RO 'memory' cell phenotype do not proliferate in concanavalin A (Con A)-stimulated cultures and may be destined for intrathymic death. To determine whether this subset would exhibit characteristics of programmed cell death (apoptosis), we examined the integrity of the nuclear DNA by gel electrophoresis. DNA fragmentation was restricted to the CD45RO+ subset of human thymocytes following exposure to stimulating concentrations of anti-CD2 antibodies. Both CD45RO- and CD45RO+ subsets mobilized cytoplasmic Ca2+ following cell-surface CD2 ligation, but entry into the cell cycle and vigorous thymidine uptake were restricted to the CD45RO- subset. Our results provide a mechanism which may account for the failure of thymic CD45RO+ cells to respond to stimuli which elicit proliferation by the reciprocal CD45RA+ subset.     

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.     

Locomotor responses of human CD45 lymphocyte subsets: preferential locomotion of CD45RO+ lymphocytes in response to attractants and mitogens.

The CD45RO+ population of lymphocytes from human blood contains a higher proportion of locomotor cells than the CD45RA+ population. Direct from blood there were few locomotor lymphocytes (< 15%), but, among these, a higher proportion of CD45RO+ than of CD45RA+ cells responded to the chemotactic stimuli, foetal calf serum (FCS) and interleukin-2 (IL-2) in polarization assays. Likewise, after overnight culture, a higher proportion of CD45RO+ cells responded to IL-8. Culture for 24-72 hr in activators such as anti-CD3, purified protein derivative (PPD), phytohaemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) or in an allogeneic mixed leucocyte reaction (AMLR) increased the proportion of locomotor lymphocytes to 20-60%, and the CD45RO+ subset showed proportionately more polarized cells than the CD45RA+ subset after culture with all the above activators. Preferential migration of CD45RO+ cells into collagen gels was also seen after culture in antigenic stimuli (PPD or AMLR) but not with polyclonal activators (alpha CD3 or Con A). Double labelling showed that, within the CD4+ and CD8+ subsets, antigen-stimulated CD45RO+ T cells invaded collagen gels in higher proportions than CD45RA+ T cells. Clustering of lymphocytes with accessory cells is an essential prerequisite for locomotion and, after culture in alpha CD3, CD45RO+ lymphocytes were found preferentially in clusters with monocytes. In all of the above populations, CD45RO+ lymphocytes were larger in size. These findings suggest that, not only selective adhesion to vascular endothelium as reported earlier, but also selective locomotion recruits CD45RO+ lymphocytes into sites of inflammation.     

CD45 isoform phenotypes of human T cells: CD4(+)CD45RA(-)RO(+) memory T cells re-acquire CD45RA without losing CD45RO

We have studied the alterations in CD45R phenotypes of CD4(+)CD45RA(-)RO(+) T cells in recipients of T cell-depleted bone marrow grafts. These patients are convenient models because early after transplantation, their T cell compartment is repopulated through expansion of mature T cells and contains only cells with a memory phenotype. In addition, re-expression of CD45RA by former CD4(+)CD45RA(-) T cells can be accurately monitored in the pool of recipient T cells that, in the absence of recipient stem cells, can not be replenished with CD45RA(+) T cells through the thymic pathway. We found that CD4(+)CD45RA(-)RO(+) recipient T cells could re-express CD45RA but never reverted to a genuine CD4(+)CD45RA(+)RO(-) naive phenotype. Even 5 years after transplantation, they still co-expressed CD45RO. In addition, the level of CD45RA and CD45RC expression was lower ( approximately 35 %) than that of naive cells. In contrast, the level of CD45RB expression was comparable to that of naive cells. We conclude that CD4(+)CD45RA(-)RO(+) T cells may re-express CD45(high) isoforms but remain distinguishable from naive cells by their lower expression of CD45RA / RC and co-expression of CD45RO. Therefore, it is likely that the long-lived memory T cell will be found in the population expressing both low and high molecular CD45 isoforms.     

Definition of the HLA-A2 restricted peptides recognized by human CD8+ effector T cells by flow-assisted sorting of the CD8+ CD45RA+ CD28- T cell subpopulation

In response to antigenic stimulation, naive MHC-class I restricted and antigen-specific CD8+ CD45RA+ CD28+ T cells undergo clonal expansion, differentiate into CD8+ CD45RO+ memory T cells and convert to CD8+ CD45RA+ CD28- T cells displaying potent immune effector functions upon re-encounter with the nominal antigen. We show that the effector CD8+ CD45RA+ CD28- T cell subset is expanded in peripheral blood lymphocytes (PBL) from patients with human papilloma virus (HPV)+ cervical lesions as well as in PBL from patients with pulmonary tuberculosis. Flow-cytometric cell sorted CD8+ CD45RA+ CD28- and CD8+ CD45RA+ CD28- T cells were tested for recognition of HLA-A2 restricted peptides derived either from the human papillomavirus (HPV)16-E7 gene product, or from M. tuberculosis antigens. Mostly CD8+ CD45+ CD28- T cells define antigen/peptide-specific and MHC-restricted responses. These data were confirmed in PBL from patients with tuberculosis using HLA-A2 tetramer-complexes loaded with a peptide from the M. tuberculosis Ag85b antigen by flow cytometry. The sorting of this T cell subset enables to determine the fine specificity of CD8+ effector T cells without the need for in vitro manipulation.     

Alterations in levels of CD28-/CD8+ suppressor cell precursor and CD45RO+/CD4+ memory T lymphocytes in the peripheral blood of multiple sclerosis patients.

A comprehensive peripheral blood immunophenotype analysis of 16 multiple sclerosis (MS) patients was performed by three-color flow cytometric analysis, and the results were compared with those for age-matched healthy controls. The cell subsets quantified included T cells (CD3+), B cells (CD19+), NK cells (CD56+), CD4+ and CD8+ T cells, cytotoxic (CD28+) and suppressor precursor (CD28-) CD8+ T cells, CD45RA+ and CD45RO+ T cells (CD4+ and CD8+), and CD5+ T and B cells. Analysis of MS patients' peripheral blood revealed essentially normal levels of total T, B, and NK cells. In agreement with results obtained by other investigators, it was found that MS patients had an increased CD4/CD8 ratio, primarily due to a decrease in CD8+ T cells. MS patients were found to have a significantly decreased level of suppressor precursor (CD28-) CD8+ T cells compared with that of controls but to have normal levels of cytotoxic (CD28+) CD8+ T cells. These data indicate that MS patients do not have a general decrease in CD8+ T cells but that they have a specific decrease in the suppressor precursor subset only and normal levels of cytotoxic CD8+ T cells. MS patients also had a significant increase in memory (CD45RO+) CD4+ T cells and displayed a trend towards a decrease in naive (CD45RA+) T cells in the peripheral blood.     

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.     

CD8+ T-cell subsets defined by expression of CD45 isoforms differ in their capacity to produce IL-2, IFN-gamma and TNF-beta.

Expression of different isoforms of CD45, the leucocyte common antigen (LCA), on T-cell subsets has permitted distinctions between the functional activities of subpopulations within the major CD4+ T-cell subset. With respect to cytokine production, the expression on CD4+ cells of CD45RA, a high molecular weight isoform, defines a population which produces only interleukin-2 (IL-2) and tumour necrosis factor-beta (TNF-beta) in quantity, with peak production of IL-2 occurring after 24-48 hr stimulation, while the CD4+ population bearing high levels of CD45RO, a low molecular weight isoform, can produce a wide range of cytokines within 24 hr of activation. The literature is conflicting on the capacities for cytokine production of CD8+ subsets divided on the basis of either CD45RA or CD45RO expression. The aim of this study was to attempt to clarify this area by determining the amount and kinetics of production of IL-2, interferon-gamma (IFN-gamma) and TNF-beta in CD8+ cells separated on the basis of both CD45RA and CD45RO isoform expression. The results showed that CD8+ CD45RA- and CD8+ CD45RO+ T lymphocytes produce significantly more of all three cytokines than do CD8+ CD45RA+ or CD8+ CD45RO- T cells. The kinetics for IFN-gamma and TNF-beta production were similar for both subsets, while IL-2 production was delayed by approximately 3 hr in the CD8+ CD45RO- population as compared to the CD8+ CD45RO+ subset. It is suggested that some of the confusion over cytokine production by these CD8+ subsets may be attributable to different conditions for isolation causing pre-activation of positively selected populations. It is also suggested that while CD8+ CD45RA+ cells are shown to acquire CD45RO upon activation, as do CD4+ CD45RA+ cells, the results of the present study argue for a different relationship between CD8+ subsets separated on the basis of CD45 isoform expression than between the corresponding CD4+ subsets.     

Cationic amino acid transport in human T lymphocytes is markedly increased in the CD45RA, CD8+ population after activation.

Membrane transport of cationic amino acids is essential for cells which are actively metabolizing L-arginine or L-lysine. In human cells most of this transport occurs through y+, a transport system which is only now being characterized at the molecular level. We have previously shown that phytohaemagglutinin (PHA) stimulation of peripheral blood E rosette positive (T) lymphocytes specifically activated lysine transport through system y+, whereas Staphlyococcus aureus Cowan A (SAC) stimulation of the E rosette negative fraction did not. We have now analysed this effect in PHA-activated CD4, CD8, CD45RO and CD45RA T-cell subsets. Both PHA-activated CD4+ and CD8+ T cells have increased lysine transport through y+, and in seven out of eight experiments, more activity was seen in the CD8+ fraction. In contrast, marked differences in y+ activity were seen between the PHA-activated CD45RO and CD45RA subsets. Thus in six experiments y+ activity was markedly increased in the CD45RA (naive T cell) population but not in the CD45RO (memory) cells. In one further experiment the activated CD45RO, CD4- population (enriched for CD45RA+, CD8+) was studied and y+ activity was shown to be maximal in this cell subset. Transport of arginine is essential for nitric oxide synthesis. Our findings therefore suggest that activated CD45RA, CD8+ T cells are capable of nitric oxide production.     

慢性乙型肝炎患者外周血CD45RA+、CD45RO+T淋巴细胞亚群的特点及临床意义

目的：探讨慢性乙型肝炎患者外周血CD4^＋CD45RA^＋、CD4^＋CD45RO^＋、CD8^＋CD45RA^＋和CD8^＋CD45RO^＋T淋巴细胞亚群的特点及其与肝病病情的关系。方法：采集46例轻中度慢性乙型肝炎患者、58例重度慢性乙型肝炎患者和30例健康人的外周抗凝血，应用流式细胞技术三色荧光分析法对其外周血中CD4^＋CD45RA^＋、CD4^＋CD45RO^＋、CD8^＋CD45RA^＋和CD8^＋CD45RO＋T淋巴细胞亚群进行检测。结果：轻中、重度慢性乙型肝炎患者与正常人相比，其外周血中CD4^＋、CD8^＋T细胞均无明显改变；CD8^＋CD45RA^＋T细胞均明显降低，CD8^＋CD45RO^＋T细胞均明显增高，而CD4^＋CD45RA^＋、CD4^＋CD45RO^＋T细胞均无明显改变；重度慢性乙型肝炎患者与轻中度慢性乙型肝炎患者相比，CD8^＋CD45RA^＋T细胞明显降低（P〈0.05），CD8^＋CD45RO^＋T细胞明显升高（P〈0.05）。结论：乙型肝炎慢性化过程中，CD8^＋CD45RO^＋T细胞起重要作用且与慢性乙型肝炎患者病情的进展呈正相关；检测CD4^＋CD45RA^＋、CD4^＋CD45RO^＋、CD8^＋CD45RA^＋和CD8^＋CD45RO^＋T淋巴细胞亚群比检测CD4^＋和CD8^＋T细胞亚群更能正确、充分、全面地了解慢性乙型肝炎的发病机制和预后，从而有效地指导临床治疗。     

Pathomechanisms of paraneoplastic myasthenia gravis

Thymic T cell development is characterized by sequential selection processes to ensure generation of a self-tolerant, immuncompetent mature T cell repertoire. Malfunction of any of these selection processes may potentially result in either immunodeficiency or autoimmunity. Myasthenia gravis (MG) is a typical autoimmune manifestation of thymic epithelial tumors (thymomas) and is related to the capacity of these tumors to generate and export mature T cells. Analysis of the factors that lead to autoimmunization in thymomas will help to understand the mechanisms that prevent MG under physiological conditions in humans. In a comparison of MG(+) and MG(-) thymomas, we could show that only thymomas capable of generating mature CD45RA+CD4+ T cells are associated with MG (p < 0.0001), while terminal thymopoiesis was abrogated in MG(-) thymomas. In particular, acquisition of the CD27+CD45RA+ phenotype appears to be a critical checkpoint of late T cell development in the human thymus and may play an important role in the prevention of autoimmunity. Moreover, MG(-) thymomas were virtually depleted of regulatory (CD4+CD25+) T cells (regT), while regT were readily detectable in MG(+) thymomas, albeit at significantly reduced numbers compared to control thymuses. Thus, in MG(+) thymoma patients, thymectomy apparently also results in removal of a regulatory T cell pool and may explain the frequent temporary postoperative deterioration of MG in these patients.     

Ex vivo characterization of human thymic dendritic cell subsets

Interactions between thymic dendritic cells (DC) and thymocytes are critical for proper development of T-cells. We identified human thymic DC populations on the basis of CD123, CD11c and CD14 expression. High levels of CD123 (IL-3R) and CD45RA defined the plasmacytoid DC (pDC) subset. Human thymic CD11c(+) DC expressed CD45RO and myeloid-related markers (CD13, CD33 and CD11b). CD11c(+) DC could be separated into two main subsets based on differential expression of CD14: CD11c(+) CD14(-) and CD11c(+) CD14(+) cells. Spontaneous production of IL-10 and IFNgamma without exogenous stimulation, was observed in the three DC subsets. Important phenotype modifications were observed in pDC cultures supplemented with IL-3. A down-regulation of CD123 and appearance of myeloid markers such as CD11b and CD11c on CD45RA(+) cells was noticed within the first 48h; at a later time there was a shift from CD45RA to CD45RO expression, as well as appearance of CD14 expression. CD11c(+) cells emerging in pDC culture did not express high levels of HLA-DR, CD83 and co-stimulatory molecules. This suggests an in vitro evolution of human thymic pDC toward a myeloid phenotype found in the CD11c(+) subset of thymic DC.     

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.     

桥本氏甲状腺炎患者外周血中CD4+ CD45RO+ 记忆性T 细胞的表达及意义

目的:检测初诊桥本氏甲状腺炎(Hashimoto’s thyroiditis,HT)患者外周血CD4+ CD45RO+ 记忆性T 细胞比例,探讨其在HT 发病中的意义。方法:收集初诊的HT 患者作为病例组(HT,n =53),另外选取年龄、性别相匹配的正常人作为对照组(HC,n =43),并根据甲状腺功能状态将HT 组分为甲状腺功能正常组(HT-A,n =15)、亚临床甲减组(HT-B,n =14)和临床甲减组(HT-C,n =24)。流式细胞仪检测外周静脉血中CD4+ CD45RO+记忆性T 细胞比例,酶联免疫吸附法检测血清中IFN 、IL-17 水平,化学发光法检测甲状腺功能及甲状腺特异性抗体滴度。结果:HT 组外周血中CD4+ CD45RO+ 记忆性T 细胞比例、IFN 、IL-17、TPOAb 与TgAb 水平均显著高于HC 组,P<0.01。双变量相关分析显示HT 患者外周血CD4+ CD45RO+记忆性T 细胞的比例与IFN 、TPOAb、TgAb 均呈正相关(P<0.01,P =0.015,P<0.01)。结论:CD4+ CD45RO+ 记忆性T 细胞在HT 患者外周血中呈高表达,CD4+ CD45RO+记忆性T 细胞可能参与了HT 的发病过程。