CD45 isoform expression during T cell development in the thymus.

Various isoforms of leukocyte common antigen, or CD45, are expressed differentially on T cells at different stages of development and activation. We report studies on CD45 isoform expression on various subsets of human T cells using two- and three-color flow cytometry and cell depletion. Bone marrow cells that were depleted of CD3+ and HLA-DR+ cells were CD45RA-RO-. The earliest CD3-CD4-CD8-CD19- thymocytes were CD45RO- with 20%-30% CD45RA+ cells. The most prominent population of CD4+CD8+ double-positive thymocytes were CD45RA-RO+. Even the CD4+CD8+ blasts were greater than 90% CD45RO+. About 80% of single-positive thymocytes (CD4+CD8- or CD4-CD8+) were also CD45RO+. Only 4.3% of CD4+ and 18% of CD8+ single-positive thymocytes were CD45RA+. In contrast, cord blood T cells which represent the stage that immediately follows single-positive thymocytes, contained 90% CD45RA+ cells. Thus, in terms of CD45 isoform expression, single-positive thymocytes are more like double-positive cells than cord blood T cells. These results suggest the following sequence of CD45 isoform switching during T cell development: CD45RA-RO- or RA+RO- (double-negative thymocytes)----RA-RO+ (double-positive and most single-positive thymocytes)----RA+RO- (cord blood T cells), the last switch from CD45RO to CD45RA occurring as a final step of maturation in the thymus.     

CD45 isoform switching precedes the activation-driven death of human thymocytes by apoptosis.

A recently described chimaeric organ culture system is employed to investigate the effects of antibodies to the CD3 and CD2 cell surface structures on the fate of developing human thymocyte populations. Engagement of CD3 as well as CD2 with reagents that stimulate peripheral human T cell results in the activation-induced death of human thymocytes in organ culture. Death is preceded by a characteristic phenotypic change; thymocytes that bear CD45RA (determinants associated with high-molecular-mass isoforms of the leukocyte common antigen family, CD45) are first induced to co-express CD45RO (the low-molecular-mass isoform of CD45), and subsequently lose CD45RA expression. This antigenic change is followed by cellular DNA fragmentation, characteristic of apoptosis. We conclude that engagement of CD3 as well as CD2 can recruit human CD45RA+ thymocytes into the CD45RO+ population, where cell death occurs. Our results suggest that this phenotype conversion is a marker for thymocytes destined for programmed cell death.     

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.     

Activation, survival and apoptosis of CD45RO+and CD45RO−T cells of human immunodeficiency virus-infected individuals: effects of interleukin-15 and comparison with interleukin-2

HIV infection is associated with increased representation of T cells bearing an activated, memory (CD45RO+) phenotype. Although administration of antiretroviral agents and interleukin-2 (IL-2) augment depleted CD4+ T-cell numbers, such therapies have been preferentially beneficial for CD45RO+ T cells. Interleukin-15 (IL-15) exhibits many biological activities in common with IL-2, including promoting T-cell survival and proliferation. The present study found that these two cytokines differed in their ability to induce proliferation, enhance survival, and control apoptosis of CD45RO+ and CD45RO- T-cell populations of human immunodeficiency- (HIV) infected individuals. When used at equivalent concentrations in vitro, IL-15 was more potent than IL-2 in activating and stimulating proliferation of CD4+CD45RO+, CD8+CD45RO+ and CD8+CD45RO- cells, but failed to be more effective than IL-2 in reducing apoptosis. Poor activation of CD4+CD45RO- cells by IL-15 and to IL-2 appeared to be attributable to low expression of the beta receptor chain utilized by both cytokines. However, IL-15 was more effective than IL-2 in enhancing survival of the CD4+CD45RO- population, suggesting a greater protective effect of IL-15 for naive CD4+ T cells, which are preferentially lost in HIV-infected individuals.     

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.     

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.     

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.     

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.     

A reduction in the number of peripheral CD28+CD8+T cells in the acute phase of influenza

Influenza patients show a high incidence of T lymphocytopenia in the acute phase of the illness. Since CD8+ T cells play an important role in influenza virus infection, we investigated which subset of CD8+ T cells was involved in this lymphocytopenia. CD8+ T cells from eight patients with influenza A were studied for lymphocyte count, surface marker, and intracellular IFN-gamma production in the acute (days 1-3) and recovery phases (days 9-12). Total and T lymphocyte counts in the acute phase were approximately three times less than in the recovery phase; however, the CD4/8 ratio was the same in both phases. The cell count reduction in the acute phase was attributed predominantly to the CD28+ CD8+ subset, compared with the CD28- CD8+ subset. The memory/activation marker CD45RO on the CD8+ T cells was assessed. The CD28+ CD45RO- subset, a naive phenotype, was reduced significantly in number in the acute phase compared with the recovery phase. The CD28+ CD45RO+ subset, a memory phenotype, was also reduced in the acute phase, but the reduction was not statistically significant. Intracellular IFN-gamma in the CD8+ subset after mitogenic stimulation was measured by flow cytometry; the percentage of CD28+ IFN-gamma-/CD8+ subset in the acute phase was significantly less than in the recovery phase. These results indicated that the predominant reduction of peripheral CD8+ T cells in the acute phase of influenza was from naive-type lymphocytes, suggesting that these quantitative and qualitative changes of CD8+ T cells in influenza are important for understanding the immunological pathogenesis.     

Deficiency of the expression of CD45RA isoform of CD45 common leukocyte antigen in CD4+ T lymphocytes in children with infantile cholestasis

The immunological background of the pathological changes that appear in infantile cholestasis (infections, inflammatory process in the liver) is largely unknown. With the use of double color flow cytometry, we assessed the distribution of functionally different lymphocyte subpopulations in the peripheral blood of 29 infants with extra and intra-hepatic cholestasis (12 and 17 patients, respectively), aged from 1 to 8.6 months. Control group consisted of 15 age-matched, healthy infants. We examined: (1) the expression of CD3, CD4, CD8, CD19 lymphocyte surface receptors; and (2) the distribution of lymphocyte subsets with distinctive surface Ag characteristics of 'naive' (CD45RA+) and 'memory' (CD45RO+) cells in both CD4+ and CD8+ cell populations. The surface markers expression was evaluated in terms of percentage of positive cells and receptor density. The following changes in the expression of lymphocyte surface markers are described: (1) a decrease in the percentage of total CD3+, CD4+ cells but normal percentage of CD8+ cells and elevated proportion of CD19+ B cells; (2) a reduction of the proportion of 'naive' CD4+ lymphocytes but normal percentage of 'naive' CD8+ as well as 'memory' CD4+ and CD8+ cell subsets; (3) a decrease in density of CD3, CD4+, CD8 receptors, and D45RA isoform in a subset of 'naive' CD4+ cells. We conclude that deficiency of 'naive' CD4+ T cell subset which possess important effector and immunoregulatory functions, and low expression of certain lymphocyte receptors known to be engaged in T cell activation, possibly reflect a defect of cell mediated immunity that may account for viral and bacterial infections, often observed in infants with cholestasis.     

CD45 enhances positive selection and is expressed at a high level in large, cycling, positively selected CD4+CD8+ thymocytes.

T-cell development is arrested at the CD4+CD8+ (DP; double-positive) stage of thymocyte development in CD45 null mice. However, the mechanism by which CD45 participates in the positive selection of T cells remains to be investigated. In this report we describe a DP thymocyte population that associates positive selection with expression of high levels of CD45, CD4 and CD8. DP thymocytes of this phenotype are large, cycling cells and represent approximately 20% of DP thymocytes in normal mice. In mice expressing a transgenic T-cell receptor (TCR) specific for the male antigen presented by H-2Db (H-Y TCR), the up-regulation of TCR, CD5 and CD69 in this large DP population occurred in a major histocompatibility complex (MHC)-restricted manner. To investigate further the role of CD45 in positive selection, we determined whether thymocytes that expressed a transgenic CD45RO molecule under the control of the proximal lck promoter can influence the positive selection of T cells in H-Y TCR transgenic mice. It was found that in female H-Y TCR transgenic mice, MHC-restricted positive selection of CD4- CD8+ H-Y TCR+ thymocytes was enhanced by increased CD45RO expression. Thus, CD45 increases the efficacy of positive selection of CD4- CD8+ thymocytes that express H-Y TCR.     

Definition of the thymic generative lineage by selective expression of high molecular weight isoforms of CD45 (T200)

Selective expression of CD45 isoforms distinguishes naive and memory T cells in peripheral blood. Paradoxically, although the most recent thymic emigrants are CD45R+ CD45 p180-, the majority of thymocytes are CD45 p180+. Speculating that the small subset of thymocytes selectively expressing only the high molecular weight isoforms of CD45 constitute the thymic generative lineage giving rise to peripheral T cells, we characterized the phenotypic and functional properties of CD45 p180- thymocytes. All cells bearing CD45 p180 were removed by rigorous depletion or all CD45R+ thymocytes were removed in a parallel depletion. CD45R- thymocytes were essentially the same in phenotype and CD4/CD8 subset distribution as unfractionated thymus, and dissimilar to naive peripheral blood lymphocyte (PBL) T cells. In contrast, CD45 p180- thymocytes, mainly CD45R+, were CD1- CD38- pgp 1+, corresponding closely to the phenotype of naive CD45R+ PBL T cells. This subset is enriched in CD4+ or CD8+ single positives, includes a high proportion of CD4-8- thymocytes which are predominantly CD3-, and appears to have a medullary location. Approximately 40%-50% of CD45 p180- thymocytes expressed a high density of CDw29 (4B4), which in the periphery is expressed at high density only on CD45 p180+ memory T cells and at low density on CD45R+ naive T cells. However, the expression of high density CDw29 in the absence of CD45 p180 indicates a close resemblance to fetal lymphocytes and suggests an essential role for CDw29 in both the least and the most mature of T cells. If CD45 p180- thymocytes constitute the generative lineage and CD45 p180+ cells are commited to intrathymic death, then the CD45 p180- subset should have enhanced proliferative potential. By combining depletion methods with a limiting dilution assay for clonogenic potential, we found that 100% of the clonogenic precursors present in unfractionated thymus were CD45R+ CD45 p180- cells. This indicates that the CD45 p180+ majority of thymocytes has a very limited capability for proliferation consistent with a commitment to intrathymic death. The clonogenic potential of CD45 p180- thymocytes indicates a greater functional resemblance to PBL T cells than to CD45 p180+ thymocytes. In so far as clonogenic potential in vitro reflects generative potential in vivo, expression of high molecular weight CD45 isoforms appears to define the generative thymic lineage. Our working hypothesis proposes that expression of CD45 p180 implements the mechanism for eliminating thymocytes with self-reactive receptor specificities.     

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.     

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.     

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.     

Apoptosis among CD45RA−/low CD3+ Progeny Accompanies Differentiation of Human Multinegative Thymocytes

Apoptotic cell death is widely believed to be the fate of negatively selected cells in the thymus. In this work we have used multiparameter flow cytometry to analyse reductions in DNA content that occur among differentiating human CD3^?4^?8^? multinegative (MN) thymocytes as they acquire CD3/TCR during in vitro culture. DNA content was measured as the intensity of the DNA-binding dye DAPI. The position of the diploid peak was identified by comparison to chicken red blood cells and to unfractionated uncultured thymocytes which have a sharply defined diploid peak. Apoptosis, was defined as a reduction in DNA content. Apoptotic cell death occurred continuously throughout the 7 day culture period and at the latest stages of culture DNA fragmentation was apparent on gels. Although both CD3^? and CD3⁺ progeny became apoptotic, it was more frequent among the CD3⁺progeny of the MN thymocytes. Apoptotic progeny included 60–70% CD3⁺ cells, while progeny remaining diploid were 8–36% CD3⁺. Fifty five per cent of CD3⁺ TCRδl⁺ progeny had less than 75% of the diploid DNA content, while for CD3⁺ TCRδ1^? progeny only 28% were in this category. CD3⁺ TCRδ1⁺ progeny also comprised 66% of the cycling cells at days 6–7 of culture, suggesting a pattern of rapid cell division followed by apoptotic cell death for this subset. A lack of positive selection in culture may trigger apoptosis among the TCRδ1 progeny. In contrast, TCRαβ progeny arising in culture appear to be less susceptible to apoptosis, perhaps due to their lack of CD4 and CD8. The expression of CD45RA and CD45R0 isoforms were assessed on the progeny of MN thymocytes based on their DNA content. Although 30% of apoptotic progeny expressed CD45RA, it was present at relatively low density compared to that on diploid or cycling cells, 55% of which were CD45RA^hi. The majority of apoptotic cells expressed neither CD45RA or CD45R0, but were CD45⁺, indicating the presence of an isoform not detected by our monoclonal antibodies (MoAbs). This is consistent with speculations that apoptotic cell death among thymocytes is preceded by a transition in CD45 isoform expression. These conditions may model early selective events resulting from high avidity TCR engagement that is independent of CD4 and/or CD8.     

Characterization of constitutive and strain-dependent subsets of CD45RA+ cells in the thymus.

We have previously shown that the occurrence of CD45RA+ adult mouse thymocytes is strain-dependent, e.g. constituting approximately 0.6% in C57BL/Icrf and approximately 2.5% in BALB/c (Huby, R. and Goff, L., 1992. Eur. J. Immunol. 22:1659). Here we show that irrespective of strain, the thymus contains approximately 0.6% CD45RA+ cells which are composed of slg+ B cells (approximately 0.4%), slg- CD4-CD8- cells (< 0.2%), and CD4+ CD8+ cells (< 0.2%). In some strains an additional CD45RA+ population, representing up to approximately 2% of all thymocytes, is present and has a CD4-CD8+ phenotype. It is this CD4-CD8+CD45RA+ subset which is responsible for the observed strain difference. In BALB/c mice, this additional population comprises approximately 90% of the CD45RA+ thymic cells. They are larger than the majority of thymocytes, with a size typical of mature, single positive cells (CD4+CD8- or CD4-CD8+). Further phenotyping for co-expression of other maturation markers showed them to be distinctive; they are CD3int-hi, i.e. as bright as other CD8 single positives, which are dimmer than CD4 single positives. In addition they are CD44hi, MEL-14dim and hi, Thy-1lo, HSAlo/-, and PNAlo, suggesting them to be amongst the most mature cells in the thymus. This was corroborated by their phenotypic similarity to CD45RA+ lymph node T cells. Furthermore, in BALB/c adult thymus sections, CD45RA+ cells are localized mainly in the medulla, consistent with a mature phenotype. Comparable with most mature thymocytes, cell cycle analysis revealed this subset to be composed of resting (G0/G1) cells. The CD4-CD8+CD45RA+ cells are amongst the most mature thymocytes and yet are indistinguishable from peripheral T cell counterparts; the possibilities that they are mature thymocytes due to exit the thymus, or that they may represent recirculating peripheral T cells, are discussed.     

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.