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.     

Distribution of CD45RA and CD45RO T-lymphocyte subsets in rheumatoid arthritis synovial tissue

We have characterized the lymphocytes in the synovium of patients with rheumatoid arthritis (RA) by immunohistochemistry using monoclonal antibodies directed against B lymphocytes, T lymphocytes, and antibodies directed against CD45RA and CD45RO, which define T-cell subsets. Both CD45RA+ and CD45RO+ T lymphocytes were detected in the perivascular regions. CD45RA+ lymphocytes were present primarily in perivascular areas of moderate to large lymphocytic infiltration. Some synovial perivascular lymphocytic aggregates were organized into focal areas of CD45RA+ B lymphocytes surrounded by CD45RO+ T lymphocytes. In areas of diffuse lymphocytic infiltration, the T lymphocytes were CD45RO+. These data suggest that both CD45RO+ and CD45RA+ T lymphocytes enter the RA synovial tissue via the synovial vasculature and that, once in the tissue, the CD45RA+ T lymphocytes may undergo activation/maturation and acquire the CD45RO phenotype.     

Studies of lymphocyte transendothelial migration: analysis of migrated cell phenotypes with regard to CD31 (PECAM-1), CD45RA and CD45RO.

CD31 is a 130,000 MW cell-surface glycoprotein expressed on endothelial cells, polymorphonuclear leucocytes, monocytes and about 50% of peripheral blood lymphocytes, and it has been proposed that it plays a role in transendothelial migration. If it is involved in endothelial transmigration of lymphocytes then the proportion of CD31+ cells should be increased in the lymphocyte population which has crossed an endothelial monolayer. This was tested using two endothelial types, namely human umbilical vein endothelial cells (HUVEC) and rat high endothelial venule (RHEV) cells. As a control, lymphocyte CD45RA and CD45RO expression was also determined since there is a correlation between lymphocytes bearing these isoforms and different migratory patterns. Double labelling techniques showed a close correlation between CD31 and CD45RA expression. With HUVEC monolayers, the transmigrated lymphocyte population was depleted of CD31+ cells. This depletion was even more marked if the HUVEC monolayers had been stimulated with interleukin-1 beta (IL-1 beta). The migrated lymphocytes were enriched for CD31-CD45RO+ cells but depleted of CD31+CD45RA+ cells. In addition, lymphocyte populations depleted of CD31+ cells by immunopanning were also able to migrate across HUVEC monolayers. Taken together these data suggest that lymphocyte CD31 expression is not necessary for transmigration across HUVEC monolayers and, if anything, is negatively correlated with transmigration. With the second endothelial cell type, RHEV cells, there was no consistent change in the proportion of CD31+ lymphocyte in the transmigrated population, suggesting neither a positive nor a negative correlation between CD31+ expression and lymphocyte transmigration across RHEV cells. However, with both endothelial cell types, the migrated lymphocyte populations were enriched for the marker CD45RO. In conclusion, lymphocyte surface expression of CD31 is not necessary for transmigration across the endothelial cell types used in this study, but with both cell types an enrichment of CD45RO+ lymphocytes is seen in the migrated population.     

蕈样肉芽肿患者外周血单一核细胞CD45RA及CD45RO表达的研究

目的探讨蕈样肉芽肿（Mycosis fungoides,MF）患者外周血单个核细胞CD45RA及CD45RO的表达及其与MF发病的关系。方法应用双荧光抗体标记、流式细胞仪检测15例MF患者外周血单个核细胞CD45RA及CD45RO的表达。结果（1）MF患者外周血CD3^＋、CD3^＋CD8^＋细胞与正常对照比较差异不显著（P〉0.05）。（2）MF患者外周血CD4^＋细胞低于正常对照,差异非常显著（P〈0.001）。（3）MF患者外周血T细胞CD3^＋CD4^＋/CD3^＋CD8^＋比值低于正常对照,差异显著（P〈0.001）。（4）MF患者外周血CD45RA^＋细胞低于正常对照,差异非常显著（P〈0.001）,CD45RO^＋细胞高于正常对照,差异非常显著（P〈0.001）。（5）MF患者外周血CD45RO^＋/CD45RA^＋比值高于正常对照,差异非常显著（P〈0.001）。（6）MF患者外周血CD4^＋CD45RA^＋细胞低于正常对照,差异非常显著（P〈0.001）。（7）MF患者外周血CD4^＋CD45RO^＋细胞及CD8^＋CD45RO^＋细胞均高于正常对照,差异非常显著（均P〈0.001）。（8）MF患者外周血CD4^＋CD45RO^＋/CD4^＋CD45RA^＋比值及CD8^＋CD45RO^＋/CD8^＋CD45RA^＋比值均高于正常对照,差异非常显著（P〈0.01及P〈0.001）。结论MF患者外周血中,不仅存在CD4^＋亚群失调和CD4^＋/CD8^＋比值降低,而且在CD4^＋和CD8^＋亚群中也存在CD45RA^＋、CD45RO^＋亚群失调和CD45RO^＋/CD45RA^＋比值升高,从而导致的机体免疫功能紊乱,可能与MF的发病或病情加剧有关。     

结直肠癌患者与健康受试者外周血CD45RA+/CD45RO+淋巴细胞表型分析

目的 分析结直肠癌患者与健康受试者外周血CD45RA+/CD45RO+系列T淋巴细胞表达的差异.方法 运用流式细胞术(FCM)检测2010年1月至2013年12月解放军总医院收治的109例结直肠癌患者(试验组)与64例健康受试者(对照组)外周血CD45RA+、CD45RO+、CD4+ CD45RA+、CD4+ CD45RO+T淋巴细胞亚群表达情况,统计分析试验组和对照组性别和年龄的分布是否存在差异,然后进一步分析CD45 RA等T淋巴细胞亚群与结直肠癌临床分期的关系.结果Ⅰ+Ⅱ期、Ⅲ期和Ⅳ期结直肠癌患者外周血CD45 RA+细胞百分率[三者分别为(56.23±7.75)％、(58.86±7.66)％和(59.02±9.71)％]明显高于对照组[(48.94±12.66)％],差异具有统计学意义(F=11.128,P＜0.001);Ⅲ期和Ⅳ期患者的CD45RO+细胞百分率[分别为(47.19±8.30)％和(45.41±10.45)％]则明显低于对照组[(53.43±11.75)％],差异具有统计学意义(F=5.817,P=0.00083);Ⅲ期和Ⅳ期患者CD45RA+/CD45RO+的比值(分别为1.32 ±0.46和1.43±0.63)明显高于对照组(1.00±0.47),差异具有统计学意义(F=6.986,P=0.000185);Ⅰ+Ⅱ期患者CD4+ CD45 RO+细胞百分率[(31.37±6.39)％]明显高于对照组[(27.49±7.19)％],差异具有统计学意义(F=2.368,P=0.009);Ⅳ期患者CD4+ CD45RA+/CD4+ CD45RO+的比值(0.66±0.39)明显高于Ⅰ+Ⅱ期的患者(0.49±0.23),差异具有统计学意义(F=1.812,P=0.029);各组之间CD4+ CD45RA+细胞百分率无明显统计学差异(F=0.637,P=0.592).结论 随着临床分期的增加,结直肠癌患者外周血CD45RA+细胞逐渐增加而CD45RO+细胞逐渐减少,反映出结直肠癌患者随着肿瘤的进展其免疫功能逐渐抑制、逐渐降低的动态过程;CD4+ CD45RA+细胞和CD4+ CD45RO+细胞在反映结直肠癌患者机体免疫功能方面不如CD45RA+细胞和CD45RO+细胞敏感.     

Loss of CD45RA and gain of CD45RO after in vitro activation of lymphocytes from HIV-infected patients.

T-lymphocyte activation is accompanied by a loss of CD45RA determinant and gain of CD45RO marker. Previous work suggests that the CD45RO population could represent the T-cell memory pool since it contains most of the cells that can respond to recall soluble antigens. A selective loss of memory T cells was recently found in early stages of human immunodeficiency virus (HIV) infection, which could contribute to the qualitative defects observed in HIV-infected patients. In order to investigate whether HIV infection could induce an alteration in the process of differentiation from naive cells to cells with a memory phenotype, we studied the expression of CD45RA and CD45RO in CD4+ and CD8+ lymphocytes, by flow cytometric analysis, after mitogenic stimulation of mononuclear cells from patients with HIV infection. These preliminary results suggest that after in vitro activation the CD4+ and CD8+ populations from HIV-infected patients lose the CD45RA marker and concomitantly acquire the CD45RO determinant in the same way as shown for the healthy control population studied simultaneously. Thus, it appears that an alteration in the process of conversion of CD45RA into CD45RO does not contribute to the defects observed in the memory T-cell population in HIV infection.     

CD45RO and CD45RA positive cell populations in idiopathic membranous and IgA glomerulopathy.

AIMS: To immunophenotype and quantitate glomerular and interstitial inflammatory cells in cases of idiopathic membranous and IgA glomerulopathy; to correlate cell numbers with aspects of clinical data and renal function. METHODS: Routine indirect immunoperoxidase staining was performed on frozen section renal biopsy specimens for T and B lymphocyte related antigens, macrophages and MHC class II antigens. Double immunohistochemical staining was performed to identify CD45RO+ and CD45RA+ cells. RESULTS: In IgA glomerulopathy correlations were found relating interstitial cell numbers to creatinine concentration at biopsy (CD45RO+ and CD45RA+ cells) and follow up creatinine concentration (CD3+, CD4+, CD8+, CD45RO+, and CD45RA+ cells). Also in IgA glomerulopathy mean arterial pressure at biopsy correlated with interstitial cell numbers and most recent follow up creatinine concentration. There were no correlations between glomerular inflammatory cells and renal function in either disease. Double staining showed that although most glomerular CD45RO+ and CD45RA+ cells were macrophages, positive cells in the interstitium were lymphocytes. The interstitial CD45RO+:RA+ ratio in normal renal biopsy specimens was approximately 5:1; for IgA glomerulopathy it was 1.5 and was 1.0 in idiopathic membranous glomerulopathy. CONCLUSIONS: This study demonstrates that interstitial, and not glomerular, inflammatory cell numbers correlate with renal function in primary glomerular disease and that double staining is necessary to interpret positive immunostaining for antigens located on more than one type of inflammatory cell. Detailed investigation of the interstitial CD45RO+ and CD45RA+ cells may give an insight into the pathogenesis of glomerular disease.     

Changes in CD45 isoform expression vary according to the duration of T-cell memory after vaccination.

Healthy young (< 40 years) and elderly (< 60 years) adults were immunized with the 1992-1993 preparation of trivalent influenza vaccine, and changes in CD45 isoform expression on peripheral blood lymphocytes were measured in the pre- and postvaccination periods. Fluorescence-activated cell sorter analysis was used to study T-cell subsets in fresh peripheral blood lymphocytes (day 0) and after 6 days of culture with live influenza virus. We have reported previously that the interleukin-2 response to the stimulating strain of virus, A/Texas/16/89, did not decline until 26 weeks postvaccination. In ex vivo CD4+ subsets, this interleukin-2 response was paralleled by a > 10% increase in the proportion of cells expressing the CD45RO+ phenotype following vaccination (p < 0.0001). In vitro stimulation had no effect on CD4+ subsets prior to vaccination but, after vaccination, was associated with a > 10% increase in CD45RA+RO+ cells (P < 0.0001). In addition, we have identified a change in the population of cells that express a CD45 isoform that is neither CD45RA nor CD45RO (CD45RA-RO-). At 26 weeks postvaccination, the proportion of CD45RA-RO- cells in ex vivo CD4+ peripheral blood mononuclear cells increased by approximately 15% from that measured at the earlier postvaccination time points (P < 0.0001). In vitro stimulation with influenza virus resulted in a further 20% increase in the proportion of CD45RA-RO- cells (P < 0.0001). The CD45RA-RO- phenotype may identify a population of cells undergoing apoptosis (programmed cell death) that limits the duration of helper T-cell (CD4+) memory after vaccination.     

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).     

The bacterial superantigen Staphylococcal enterotoxin B stimulates lymphocyte locomotor capacity during culture in vitro.

The bacterial superantigen Staphylococcal enterotoxin B (SEB) was investigated for its effects on lymphocyte locomotion in vitro. Culture of peripheral blood mononuclear cells (PBMC) for 24-72 hr in SEB (1-100 micrograms/ml) increased the proportion of lymphocytes in locomotor (polarized) morphology and capable of invading collagen gels, to the same extent as the established locomotor activator, anti-CD3 (alpha-CD3), though the conventional antigen, tetanus toxoid was ineffective. The cells responding to SEB were predominantly T cells. SEB had no effect on lymphocyte locomotion in short-term (45 min) assays, thus its effect is to stimulate growth-related locomotor capacity and it does not act as a chemoattractant. During culture of PBMC in SEB, the chemokines interleukin-8 (IL-8) and macrophage chemotactic protein-1 (MCP-1) were released into the culture medium. The presence of anti-IL-8, but not of anti-MCP-1, either during culture or added to SEB culture supernatants and tested in short-term assays, inhibited the development of polarization suggesting that IL-8, which is a lymphocyte chemoattractant, also plays a key role in SEB-induced locomotor activation. Among SEB-activated lymphocytes, CD45RO+CD45RA- lymphocytes showed enhanced locomotor responses, but a relation was not found between locomotor activity and the presence of cell surface CD69.     

Loss of activation-induced CD45RO with maintenance of CD45RA expression during prolonged culture of T cells and NK cells.

The results of the present study show that activation-induced changes in CD45RA and CD45RO expression on T cells and natural killer (NK) cells are not unidirectional for all cells during a 5-week culture period. T cells and NK cells were generated from a resting subpopulation of peripheral blood mononuclear cells (PBMC) defined by sedimentation at Percoll high buoyant densities (p greater than 1.0640 g/ml) and unresponsiveness to IL-2. T cells were activated by a combination of PHA, sheep erythrocytes and IL-2-conditioned medium (IL-2-CM), and NK cells were activated by co-culture with gamma-irradiated malignant melanoma (MM-170) cells and IL-2-CM. Both T-cell and NK-cell cultures were maintained by subculture in IL-2-CM. NK cells and the CD45R(Abright)RO(dim/neg) subpopulation of T cells gained CD45RO following activation and this was accompanied by a two-fold decrease in CD45RA expression. In different cultures, CD45RO expression was not stable on 28-80% of T cells and 10-55% of NK cells. Cells with decreased CD45RO expression showed increased expression of CD45RA. Instability of CD45RO expression on cultured T cells and NK cells occurred at a time following the period of rapid cell growth when the cells were entering a quiescent phase. Both the CD4+ and CD8+ T-cell subpopulation showed similar changes in CD45 isoform expression. In contrast to the results obtained with the CD45R(Abright)RO(dim/neg) resting T cells, the CD45RO(bright)RA(dim/neg) subpopulation of resting T cells when activated and cultured under identical conditions retained CD45RO expression and remained CD45RAdim/neg. Thus a significant proportion of resting CD45R(Abright)RO(dim/neg) T cells is not related in a differentiation sequence to resting CD45RObrightRAdim/neg T cells, and therefore resting CD45RAbrightROdim/neg T cells and resting NK cells may be heterogeneous with respect to their activation history.     

CD45RA and CD45RO isoforms in infected malnourished and infected well-nourished children.

The aim of this study was to determine if the distribution in vivo of CD4(+)CD45RA(+)/CD45RO(-) (naive), CD4(+)CD45RA(+)/CD45RO(+) (Ddull) and CD4(+)CD45RO(+) (memory) lymphocytes differs in malnourished infected and well-nourished infected children. The expression of CD45RA (naive) and CD45RO (memory) antigens on CD4(+) lymphocytes was analysed by flow cytometry in a prospectively followed cohort of 15 malnourished infected, 12 well-nourished infected and 10 well-nourished uninfected children. Malnourished infected children showed higher fractions of Ddull cells (11.4 +/- 0.7%) and lower fractions of memory cells (20.3 +/- 1.7%) than the well-nourished infected group (8.8 +/- 0.8 and 28.1 +/- 1.8%, respectively). Well-nourished infected children showed increased percentages of memory cells, an expected response to infection. Impairment of the transition switch to the CD45 isoforms in malnourished children may explain these findings, and may be one of the mechanisms involved in immunodeficiency in these children.     

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.     

Differential infiltration by CD45RO and CD45RA subsets of T cells associated with human heart allograft rejection.

Subsets of T cells express different isoforms of the leukocyte common antigen CD45; those expressing the glycoprotein 220 isoform (CD45RA) have been characterized as naive in their response to antigens, and those expressing the glycoprotein 180 isoform (CD45RO) as memory T cells. The association between the rejection status of human cardiac allograft recipients and the relative infiltration of the CD45 subsets of both CD8+ and CD4+ T cells was examined using two-color immunohistological labeling techniques on 33 heart transplant biopsies, categorized by routine histological and clinical criteria as mild (requiring no treatment) or moderate (requiring antirejection therapy) rejection. Double-labeling was performed using pairs of monoclonal antibodies to define the following populations: CD4+ CD45RA+, CD4+ CD45RO+, CD8+ CD45RA+, and CD8+-CD45RO+. The number of cells per high-power field (HPF) for each of these cell subsets was counted in every biopsy. In cases with mild rejection, infiltration was predominant for CD4+ CD45RA+ cells (median = 5.0 cells/HPF) relative to CD4+ CD45RO+ (3.12 cells/HPF), CD8+ CD45RA+ (2.14 cells/HPF), and especially CD8+ CD45RO+ (1.22 cells/HPF) populations. In cases with moderate rejection, all four subpopulations increased but were essentially equivalent in intensity, such that in comparison to cases with mild rejection, the smallest increase was seen for CD4+ CD45RA+ cells (6.67 cells/HPF, P < 0.09) and the greatest for CD8+ CD45RO+ cells (7.00 cells/HPF, P < 0.002). A majority of CD8 cells expressed CD45RA in 14 of 16 (88%) cases of mild rejection compared to only 2 of 17 cases of moderate rejection. Moreover, the ratio of CD45RO+ to CD45RA+ cells in each biopsy was higher in moderate versus mild rejection for both CD4 (median ratios = 1.13 versus 0.68, respectively; P < 0.008) and CD8 (1.43 versus 0.58, respectively; P < 0.005) subsets. A majority of T cells expressed CD45RO in cases of moderate rejection (11 of 14 or 79%), compared to only 1 of 13 (8%) cases of mild rejection. These findings indicate that during generally self-limited mild acute cardiac allograft rejection there is a predominance of naive CD45RA+ T cells, especially of the CD4 phenotype, whereas during moderate rejection there is a significant shift toward activated CD45RO+ T cells, especially in the CD8 population.     

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.     

Differentiation of T Lymphocytes in the Human Adenoid as Measured by the Expression of CD45 Isoforms

Encounter of antigen by T lymphocyte on antigen-presenting cells results in changes in the expression of several cell surface molecules, including the abundant cell surface glycoprotein CD45. We have characterized the expression of the CD45 isoforms CD45RA and CD45RO in CD4⁺ and CD8⁺ T lymphocytes in the adenoids and peripheral blood of young children. We found that the relative proportions of CD45RA⁻,CD45RO⁺ antigen-experienced T cells was higher in the adenoids than in peripheral blood, and that the proportion of naive or resting CD45RA⁺,CD45RO⁻ T cells was lower in the adenoids than in peripheral blood. The frequency of bright double-positive CD45RA⁺,CD45RO⁺ T cells, which represent cells in transition from the CD45RA⁺ to CD45RO⁺ phenotype, was higher in the adenoids than in peripheral blood. The frequency of another double-positive cell population, but with unknown ontogeny, expressing both CD45RA and CD45RO at a low level, was higher in peripheral blood than in adenoidal T cells. It was found that the frequency of adenoidal antigen-experienced CD45RA⁻,CD45RO⁺ T lymphocytes increased with increasing age of the child. These results are consistent with the model that the adenoids serve as a site for conversion of CD45RA⁺ to CD45RO⁺ T lymphocytes, and that the maturation of the immune system in young children is associated with phenotypic changes in T lymphocytes residing in secondary lymphoid organs.     

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.     

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.     

Phenotypical and Functional Differentiation of CD4+ CD45RA+ Human T Cells Following Polyclonal Activation

Human CD4+ T cells differ in their expression of the leucocyte common antigen. Antibodies detecting certain forms (CD45RA and CD45RO) of this antigen have been used to identify and isolate subpopulations of the CD4+ T cells. These isolated subsets have been shown to have different abilities concerning lymphokine production and provision of help to B cells for Ig production. When these T-cell subsets were activated in vitro with polyclonal activators, the production. When these T-cell subsets were activated in vitro with polyclonal activators, the CD45RA+ cells lost this marker and gained the expression of CD45RO. This was true for all mitogens used in this report, i.e. accessory cell-dependent stimulation with SEA and accessory cell-independent activation with PMA or PHA. A correlation between proliferation and differentiation was observed, but this was probably not causative as stimulation with PMA in the absence of DNA synthesis resulted in the acquisition of CD45RO and loss of the CD45RA antigen. Moreover, cells proliferating vigorously for long periods of time expressed both markers at significant levels, which suggests that proliferation did not automatically result in complete loss of the CD45RA marker. The phenotypical differentiation was associated with a functional differentiation which induced the stimulated cells' ability to act as helper cells for Ig production and to produce gamma interferon (IFN-gamma). The results obtained in this study support the contention that the CD45RA+ cells are precursors of the CD45RO+ cells and that the two subsets represent different maturational stages of the same lineage.     

Locomotor phenotypes of unstimulated CD45RAhigh and CD45ROhigh CD4+ and CD8+ lymphocytes in three-dimensional collagen lattices.

The spontaneous locomotion of immunomagnetically isolated resting CD4+ and CD8+ lymphocytes in three-dimensional collagen gels was recorded by time-lapse videomicroscopy. Two-dimensional projections of the paths of randomly selected individual cells were digitized, plotted and quantitatively analysed. Among five different donors 46 +/- 10% of CD4+ and CD8+ lymphocytes (n = 180) showed initial spontaneous locomotion (individual speed 5-25 microns/min; mean speed CD4+ 5.2 +/- 3.6 microns, CD8+ 3.23 +/- 2.72 microns). Active CD4+ cells were constantly migrating for more than 4 hr, whereas CD8+ lymphocytes significantly slowed down after 60-90 min in the collagen gel (P < 0.003). Quantitative analysis of the paths indicated at least three migratory phenotypes: (1) spontaneously locomoting cells exhibiting high speed and low frequency of stopping; (2) a major non-motile fraction without significant displacement; and (3) a subpopulation within CD4+ and CD8+ cells with intermediate activity of speed and stopping. Further subtype analysis of immunomagnetically isolated CD45RAhigh/ROlow or CD45RAlow/ROhigh lymphocytes showed that more than 90% of CD4+ CD45RAhigh/ROlow cells were actively locomoting. In contrast, only 20% of the CD4+ CD45ROhigh/RAlow phenotype showed spontaneous motility to a limited degree. The data indicate that resting CD4+ and CD8+ lymphocytes comprise further locomotory subpopulation related to the expression of different CD45 isoforms.