A study of CD45RO, CD45RA and CD29 antigen expression on human decidual T cells in an early stage of pregnancy

The decidua is the place where the fertilized egg is implanted and where the immunocompetent cells of the mother come into direct contact with genetically disparate cells of the conceptus. Although the T cells in the decidua are exposed to fetal antigens, the fetus is not rejected by maternal immunocompetent cells. In the present study, we examined surface markers to determine whether the T cells in the human decidua are naive T cells without or memory T cells with a history of antigen stimulation. Although few T cells were present in the decidua, as compared to the peripheral blood, CD45RO⁺, CD29⁺ and CD45RA⁻ CD4⁺ T cells as well as CD45RO⁻, CD29⁺ and CD45RA⁻ CD8⁺ T cells, which are considered to be memory T cells, were in the majority, with only small numbers of CD45RO⁻, CD29⁻ and CD45RA⁺ CD4⁺ and CD8⁺ cells, which are naive T cells, present. Also, the decidual mononuclear cells secreted IL-2 and IL-4. Since IL-4 is secreted only by memory T cells, it is suggested that in the decidua memory T cells increase in number and secrete cytokines, thereby in some way influencing the phenomenon of fertility.     

Interleukin-13 is produced by activated human CD45RA+ and CD45R0+ T cells: modulation by interleukin-4 and interleukin-12

Interleukin (IL)-13 is a cytokine originally identified as a product of activated T cells. Little is known, however, about IL-13 production by human T cells and its modulation by other cytokines. Here, we show that IL-13 is produced by activated human CD4⁺ and CD8⁺ CD45R0⁺ memory T cells and CD4⁺ and CD8⁺ CD45RA⁺ naive T cells. In contrast, IL-4, which shares many biological activities with IL-13, is only produced by CD45R0⁺ T cells following activation. Analysis of intracellular cytokine production by single CD45RA⁺ and CD45R0⁺ T cells indicated that IL-13 continued to be produced for more than 24 h after stimulation, whereas IL-4 could not be detected after 24 h. These data were confirmed by measurement of specific mRNA and suggest that IL-13, unlike IL-4, but like interferon-γ (IFN-γ), is a cytokine with long-lasting kinetics. The majority of human CD45R0⁺ T cells produced IL-4 and IL-13 simultaneously. In contrast, IFN-γ protein was generally not co-expressed with IL-4 or IL-13. IL-4 added to primary cultures of highly purified peripheral blood T cells activated by the combination of anti-CD3+anti-CD28 mAb enhanced IL-13 production by CD45RA⁺ and to a lesser extent by CD45R0⁺ T cells. Under these conditions, however, IL-12 inhibited IL-13 production by CD45RA⁺ T cells and to a lesser extent by CD45R0⁺ T cells in a dose-dependent fashion. These inhibiting effects were not related to enhanced IFN-γ production induced by IL-12, since IFN-γ by itself did not affect IL-13 production. Collectively, our data indicate that IL-13 is produced by peripheral blood T cells which also produce IL-4, but not IFN-γ, and by naive CD45RA⁺ T cells which, in contrast, fail to produce IL-4. These observations, together with the long-lasting production of IL-13, suggest that IL-13 may have IL-4-like functions in situations where T cell-derived IL-4 is still absent or where its production has already been down-regulated.     

Distinct involvement of CD45 in antigen receptor signalling in CD4+ and CD8+ primary T cells

We demonstrate that pretreatment of primary CD4⁺, but not CD8⁺ T cells with anti-CD45 inhibits activation signals induced through the T cell receptor for antigen (TCRαβ). Specifically, anti-TCRαβ-mediated tyrosine phosphorylation of phospholipase C-γ1 is inhibited, and this in turn correlates with the inhibition of subsequent Ca²⁺ mobilization and DNA synthesis. In marked contrast, none of these activation parameters are affected by anti-CD45 in CD8⁺ T cells. Perturbation of TCRαβ signalling in CD4⁺ cells is observed in conditions which do not detectably affect the level of CD45 expression, or its membrane distribution. Further, changes in the intrinsic phosphatase activity of CD45 are not detectable. While anti-CD45 ablates TCRαβ signalling, anti-CD3?-mediated activation is unaffected. This suggests that elements of the antigen receptor complex can be functionally uncoupled, and indicates that the requirements for CD45 in signalling through these two elements are different. The results demonstrate that the involvement of CD45 in coupling TCRαβ to second messenger-generating pathways is under distinct physical and/or functional constraints in primary CD4⁺ and CD8⁺ T cells.     

CD 69 expression during selection and maturation of CD4+8+ thymocytes

We have analyzed the inducibility of protein kinase C (PKC)-dependent expression of CD 69 molecules in T cell receptor (TCR) transgenic thymocytes developing in the presence or absence of selecting, class I major histocompatibility complex (MHC) molecules. Small CD4⁺8⁺ thymocytes developing in the absence of selecting MHC molecules could not be induced to express CD 69 by TCR cross-linking even after spontaneous in vitro up-regulation of their TCR level which resulted in enhanced Ca⁺⁺ flux. In contrast, a small proportion of CD4⁺8⁺TCR^low and most TCR^high (CD4⁺8⁺ and CD4⁺8⁺) thymocytes developing in the presence of selecting MHC ligands could be induced to express CD 69 upon TCR cross-linking. Unlike the anti-TCR antibody, phorbol 12-myristate 13-acetate - a direct activator of PKC - induced the expression of CD 69 on all thymocytes. These results suggest that positive selection of CD4⁺8⁺ thymocytes results in coupling of TCR-mediated signals to the CD 69 expression pathway. In vitro analysis of thymocytes before and after positive selection suggests that (1) positive selection does not immediately result in resistance to deletion and (2) that sustained TCR ligation is needed to promote maturation of positively selected CD4⁺8⁺ thymocytes resulting in gradual loss of the sensitivity to deletion and acquisition of the ability to proliferate in response to TCR-mediated signals.     

CD3-induced apoptosis of CD4+CD8+ thymocytes in the absence of clonotypic T cell antigen receptor

Clonal selection of T cells mediated through the T cell antigen receptor (TCR) mostly occurs at the CD4⁺CD8⁺ double positive thymocyte stage. Immature CD4⁺CD8⁺ thymocytes expressing self-reactive TCR are induced to die upon clonotypic engagement of TCR by self antigens. CD3 engagement by antibody of the surface TCR-CD3 complex is known to induce apoptosis of CD4⁺CD8⁺ thymocytes, a process that is generally thought to represent antigen-induced negative selection in the thymus. The present study shows that the CD3-induced apoptosis of CD4⁺CD8⁺ thymocytes can occur even in TCRα^? mutant mice which do not express the TCRαβ/CD3 antigen receptor. Anti-CD3 antibody induces death of CD4⁺CD8⁺ thymocytes in TCRα^? mice either in cell cultures or upon administration in vivo. Interestingly, most surface CD3 chains expressed on CD4⁺CD8⁺ thymocytes from TCRα^? mice are not associated with clonotypic TCR chains, including TCRβ. Thus, apoptosis of CD4⁺CD8⁺ thymocytes appear to be induced through the CD3 complex even in the absence of clonotypic antigen receptor chains. These results shed light on previously unknown functions of the clonotype-independent CD3 complex expressed on CD4⁺CD8⁺ thymocytes, and suggest its function as an apoptotic receptor inducing elimination of developing thymocytes.     

Comparison of lymphokine secretion and mRNA expression in the CD45RA+ and CD45RO+ subsets of human peripheral blood CD4+ and CD8+ lymphocytes

Flow cytometric analysis of human peripheral blood T lymphocytes demonstrated that the majority of the CD4⁺ cells were CD29⁺ or CD45RO⁺ “mature” cells while the CD8⁺ cells were primarily CD45RA⁺ “naive” cells. After an initial separation into CD4⁺ and CD8⁺ cells and a secondary separation into CD45 subsets, lymphokine secretion was assessed after phorbol 12-myristate 13-acetate and ionomycin or fixed anti-CD3 stimulation. Within the respective CD45 subsets, CD4⁺ cells produced more interleukin (IL)-2, IL-4, and IL-6; but the CD8⁺ cells secreted more interferon-γ and granulocyte/macrophage-colony-stimulating factor. Tumor necrosis factor-α secretion was similar in the matched CD45 subsets. Northern analysis revealed a parallel pattern of lymphokine mRNA expression in the four lymphocyte subsets. These results suggest that human CD8⁺ peripheral blood lymphocytes have a significant capacity to secrete lymphokines, and that the low lymphokine production observed in unseparated CD8⁺ cells reflects the higher percentage of less functional CD45RA⁺ cells.     

Interleukin-4 promotes human CD8 T cell expression of CCR7

Despite strong evidence supporting a pathway of human T cell differentiation characterized by changes in the expression of CCR7, CD28, CD27 and CD62L, few studies have addressed the mechanisms of pathway regulation. Cutaneous lymphocyte-associated antigen (CLA)-positive skin-homing CD8(+) T cells expressed significantly elevated levels of activation markers compared with CLA(-) CD8(+) T cells in individuals (n = 27) with cutaneous atopic disease. Despite such an activated phenotype, CLA(+) T cells expressed significantly higher levels of CCR7 than a CLA(-) T cell subset. Interleukin (IL)-4 was found to dramatically promote CCR7 expression by antigen-specific CD8(+) cells. Furthermore, skin-homing CD8(+) T cells from individuals with severe disease produced significantly less IL-10 than those derived from mildly affected atopic subjects. Thus in a T-helper 2 dominated disease, tissue-specific CD8(+) T cells show altered CCR7 expression and cytokine production, which may contribute to continued lymph node homing, antigen presentation and disease. IL-4 promotes expression of CCR7, a marker linked to existing models of CD8(+) T cell differentiation.     

CD45RA antibodies split the CD3bright T cell subset.

Thymocyte subsets have been well characterized on the basis of CD4 and CD8 antigen expression. Recently, the use of anti-CD3 antibodies has allowed more precise phenotyping of these subsets. The most immature T cell precursors are largely CD3-CD4-CD8-, while the most mature are CD3brightCD4+CD8- or CD3brightCD4-CD8+. Moreover, the expression of CD45RA on thymocytes appears to define a progenitor population and may define a continuous lineage of cells. Using a panel of CD45RA antibodies, we have further characterized the CD45RA+ thymocyte population in the murine system. The size of this subset is greatly enhanced in cortisone-treated mice and in sublethally irradiated mice. Moreover, the CD45RA+ population is present early in foetal life and is maintained thereafter. Using three-colour immunofluorescence, we show that (i) while most CD45RA+ cells are present amongst the CD4-CD8- thymocyte subset in the normal thymus, after cortisone treatment or irradiation, all four thymocyte subsets co-express significant amounts of CD45RA. This suggests that not only progenitor cells but also the mature population which can survive such manipulation are CD45RA+; and (ii) a large proportion of CD45RA+ cells are CD3bright and this subset is represented in the thymus at all stages of maturation tested. These data suggest that a proportion of TCR-gamma delta + CD3+ cells in the fetus as well as of TCR-alpha beta+ CD3+ cells in the adult co-express CD45RA.     

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     

The acquisition of CD4 and CD8 during the differentiation of early thymocytes in short-term culture

Subpopulations of thymocytes known to represent early stages of T cell development were isolated from the adult mouse thymus, and their ability to differentiate during short periods of culture was assessed by their acquisition of surface CD4 and CD8. Virtually all cells of the most mature of the CD4-CD8- thymocyte subpopulations (other surface markers CD3- HSA++ IL-2R-Pgp-1-) and of the immature CD4-CD8+ thymocyte subpopulation (other surface markers also CD3- HSA++ IL-2R- Pgp-1-) became CD4+CD8+ in less than 1 day of culture without added stimuli or growth factors. This suggested they had already received signals initiating CD4 and CD8 acquisition. However, stimulation of these precursor cells with phorbyl ester and ionomycin prevented this acquisition of CD4 and CD8. No distinct CD4-CD8+ intermediate was detected as the CD4-CD8- cells became CD4+CD8+ in the non-stimulated cultures, thus questioning the assumption that these three groups of cells are sequential steps in one lineage. In contrast to this pre-programmed acquisition of CD4 and CD8, the less mature CD4-CD8- IL-2R+ subpopulation did not progress to the CD4+CD8+ stage in culture, although it is able to develop further on intrathymic transfer. It is likely that this subpopulation represents a control point requiring specific differentiation signals for further development.     

The CD45RA molecule is expressed in naive murine CTL precursors but absent in memory and effector CTL.

We have studied the expression of the CD45RA molecule in murine cytotoxic T lymphocytes (CTL) specific for the allogeneic H-2Kb molecule at different stages of differentiation. The CD45RA phenotype of naive H-2Kb-specific CTL precursors has been determined using primary in vitro CTL responses. For the analysis of memory CTL we have immunized mice in vivo followed by restimulation in vitro. We have also determined the CD45RA expression at the CTL effector stage. Our results show that among naive CD8+ T cells both the CD45RA+ and the CD45RA- subpopulations can mount Kb-specific CTL responses. In contrast, memory CTL responses are mediated only by the CD8+ CD45RA+ T cell subpopulation. Similarly, effector CTL are CD8+ CD45RA- while the CD8+ CD45RA+ subpopulation does not exhibit specific cytolytic activity. The data indicate that CD45RA expression changes during CTL differentiation and that memory as well as effector CTL lack this marker.     

The tetraspanin CD9 is preferentially expressed on the human CD4(+)CD45RA+ naive T cell population and is involved in T cell activation

Human CD4+ T cells can be divided into reciprocal memory and naive T cell subsets based on their expression of CD45 isoforms and CD29/integrin beta1 subunit. To identify unique cell surface molecules on human T cells, we developed a new monoclonal antibody termed anti5H9. Binding of anti5H9 triggers a co-stimulatory response in human peripheral blood T cells. Retrovirus-mediated expression cloning has revealed that the antigen recognized by anti5H9 is identical to the tetraspanin CD9. We now show that human CD9 is preferentially expressed on the CD4(+)CD45RA+ naive T cell subset, and that CD9(+)CD45RA+ T cells respond preferentially to the recombinant beta2-glycoprotein I, compared to CD9-CD45RA+ T cells. Furthermore, anti5H9 inhibits both the recombinant beta2-glycoprotein I- and the recall antigen tetanus toxoid-specific T cell proliferation. These results suggest that the tetraspanin CD9 plays an important role in T cell activation.     

Antigen-independent adhesion of CD4 CD45RA T cells from cord blood

Antigen-independent adhesion of resting adult CD4⁺ CD45RO⁺ T cells to B lymphocytes has been shown to be transient and can be down-regulated by CD4 major histocompatibility complex (MHC) class II molecule interactions. Conversely, adhesion of adult CD4+ CD45RA+ subpopulation to B cells is not regulated by ligands of CD4. We have investigated the regulation of adhesion of cord blood CD45RA⁺ CD4⁺ T lymphocytes. In contrast to adult CD45RA⁺ CD4⁺ T cells, cord blood CD45RA⁺ CD4⁺ T cells were strongly sensitive to the down-regulation of adhesion mediated by the CD4-HLA class II interaction, since adhesion to MHC class II(⁺) B cells was transient and inhibited by an anti-CD4 antibody. In addition, human immunodeficiency virus gpl60, synthetic gpl06-derived peptides encompassing a CD4 binding site inhibited conjugate formation between cord blood CD45RA⁺ CD4+ T cells and B cells. Following activation of the cord blood CD4 T cells by an anti-CD3 antibody, a conversion from a transient to a stable adhesion pattern of cord blood CD4 T cells to B cells occurred in 2 days. The reversal to a transient adhesion occurred at day 8 following anti-CD3 activation in correlation with a complete shift to a CD45RO phenotype of the cord blood CD4 T cells. These data suggest that CD4 T cell adhesion can be developmentally regulated.     

CD45 expression by murine B cells and T cells: Alteration of CD45 isoforms in subpopulations of activated B cells

The CD45 family of high molecular weight cell surface glycoproteins is abundantly expressed by virtually all hematopoietic cells. CD45 molecules exist as multiple isoforms whose extracellular portions vary in protein structure and carbohydrate content but whose intracellular portions are highly conserved and possess tyrosine phosphatase activity. In this review we summarize current studies describing CD45 isoform expression on peripheral and thymic lymphocytes. Further, we analyze changes in CD45 isoform expression by selective populations of activated B cells.     

Dual expression of CD45RA and CD45RO isoforms on myelin basic protein-specific CD4+ T-cell lines in multiple sclerosis

Myelin basic protein (MBP)-specific T-cell lines from patients with multiple sclerosis (MS) and healthy controls were analyzed for the expression of CD45 isoforms and adhesion molecules. In the multiple sclerosis group, 22 of 24 MBP-specific T-cell lines were CD4+. Two distinct patterns were observed with regard to CD45 isoform expression. Pattern I showed dual expression of CD45 isoforms (CD4+CD45RA+CD45RO+CD29+) and Pattern II included cells with a single CD45 isoform (CD4+CD45RA–CD45RO+CD29+). All 10 cell lines from healthy controls were CD4+ and displayed Pattern II (CD4+CD45RA–CD45RO+CD29+). The dual expression of CD45 isoform in T-cell lines from MS was stable, did not represent a transition stage from CD45RA to CD45RO, and was cell-cycle independent. All cell lines from MS and controls expressed increased levels of LFA-1 (CD11a), LFA-2 (CD2), LFA-3 (CD58), ICAM-1 (CD54), and VLA-4 (CDw49d). These data show the presence of unique MBP-specific T cells (CD4+CD45RA+CD45RO+CD29+) that might play a role in the pathogenesis of MS.     

Co-expression of the CD45RA and CD45RO antigens on T lymphocytes in chronic arthritis

The site of T lymphocyte activation in chronic arthritis is unknown. Peripheral blood (PB) lymphocytes from chronic arthritis patients are in a ‘naïve’ or non-activated state, as defined by expression of the CD45RA antigen and lack of HLA class II expression. In contrast, most synovial fluid (SF) T lymphocytes express a ‘memory’ or activated phenotype, as defined by the CD45RO antigen and high HLA class II expression. Following stimulation, naive cells lose CD45RA and gain CD45RO expression to become memory cells with a transitional stage of dual CD45RA, CD45RO antigen expression. To localize where this change in phenotype occurs we used dual colour immunofluorescence labelling to compare the percentage of dual CD45RA, CD45ROpositive T lymphocytes in PB and SF from chronic arthritic patients and from normal PB, assuming this population would be increased at the primary site of T lymphocyte activation. Expression of the intermediate and late activation marker. HLA-DR, was also analysed using dual colour immunofluorescence labelling. The percentage of dual positive T lymphocytes was similar between arthritic PB, SF. and normal PB, as was the density of both CD45RA and CD45RO antigens. Thus, CD45 isoform expression did not indicate where T lymphocytes were activated. However, we identified a previously unreported population of CD45RA⁺ CD45RO⁺ HLA-DR^- T lymphocytes in arthritic and normal PB. In SF, this population was absent, but a substantial number of dual CD45RA, CD45RO-positive HLA-DR⁺ T lymphocytes were identified. This population would not be predicted by the current model of T lymphocyte activation. Division of T lymphocytes into functional groups on the basis of CD45 isoform expression is likely to be more complicated than previously thought. Based on our findings we propose an alternative model of T lymphocyte differentiation.     

Delineation of chicken thymocytes by CD3-TCR complex, CD4 and CD8 antigen expression reveals phylogenically conserved and novel thymocyte subsets.

To further define the relationship between thymocyte subsets and their developmental sequence, multi-parameter flow cytometry was used to determine the distribution of the CD3-TCR complex and the accessory molecules CD4 and CD8 on chicken thymocytes. As in mammals, adult thymocytes could be subdivided into CD3-, CD3lo, and CD3hi staining populations. CD4 and CD8 distribution on such populations revealed the presence of CD3-CD4+CD8- and CD3-CD4-CD8+ thymocytes, putative precursors to CD4+CD8+ cells, detectable in the adult and at high frequency during ontogeny. Of particular interest was the existence of CD3lo expression on CD4+CD8- and CD4-CD8+, and in some instances, on CD4-CD8- thymocytes. Such phenotypes are not easily detectable in the mammalian thymus but were readily observed in both adult and embryonic chicken thymus from 16 days of embryogenesis. Further analysis of the TCR lineage of these CD3lo cells revealed that they were essentially all of the alpha beta TCR type. Mature CD3hi thymocytes were found within the CD4+CD8+ and CD4+CD8- and CD4-CD8+ subsets. Both alpha beta and gamma delta TCR lineage thymocytes were detected within all CD4- and CD8-defined subsets, thus identifying novel thymocyte subsets in the chicken thymus, namely alpha beta TCR+CD4-CD8- and gamma delta TCR+ CD4+CD8- cells. Hence, this analysis of chicken thymocytes, while confirming the phylogenically conserved nature of the thymus, has revealed novel T cell subsets, providing further insight into the complexity of mainstream thymocyte maturation pathways.