Association of CD2 and T200 (CD45) in mouse T lymphocytes

A monoclonal antibody (mAb 12-15) reactive with the mouse CD2 was found to co-precipitate a high-molecular-weight glycoprotein from mouse thymocyte, splenic lymphocyte, Con A blast, and T cell tumor detergent lysates which was identified as the leukocyte common T 200 glycoprotein (CD45). The reactivity was specific for CD2 since antibodies to CD3 did not co-precipitate the T200 glycoprotein. mAb 12-15 did not react with immunoaffinity-purified T200 glycoprotein, ruling out the possibility that the antibody detected a cross-reactive epitope. Biochemical data indicated that the association of CD2 with T200 was not generated during lysis of the cell and that the molecular complex was non-covalently linked since it could be destroyed by high salt washing or boiling in SDS. Distribution analysis in Triton X114-H2O revealed that, in contrast to free T200 molecules, the complexed T200 was enriched in the detergents phase. To investigate the CD2-T200 association in more detail at the cell surface, modulation of CD2 and T200 was studied. Modulation could be induced on Con A blasts by monoclonal antibodies followed by cross-linking with a FITC-conjugated second antibody. Within 24 h the expression of CD2 or T200 was reduced to approximately 10-20% of the initial value on the majority of cells. However, two-color fluorescence showed that modulation of CD2 did not lead to co-modulation of CD3 or T200. A possible physiological role of CD2-T200 complexes is discussed.     

Cross-linking of the CAMPATH-1 antigen (CD52) triggers activation of normal human T lymphocytes

The CAMPATH-1 (CD52) antigen is a 21–28 kDa glycopeptidewhich is highly expressed on lymphocytes and macrophages andis coupled to the membrane by a glycosylphosphatidylinositol(GPI) anchoring structure. The function of this molecule isunknown. However, it is an extremely good target for complement-mediatedattack and antibody-mediated cellular cytotoxicity. The humanizedCAMPATH-1H antibody, which is directed against CD52, is veryefficient at mediating lymphocyte depletion in vivo, and iscurrently being used in clinical trials for lymphoid malignancyand rheumatoid arthritis. It is therefore important to examinethe functional effects of this antibody on different lymphocytesub-populations. Because several other GPI-linked moleculesexpressed on the surface of T lymphocytes are capable of signaltransductlon resulting in cell proliferation, we have investigatedwhether the CAMPATH-1 antigen can also mediate these effects.In the presence of phorbol esters and cross-linking anti-lgantibodies, mAbs specific for CD52 induced proliferation andlymphokine production in highly purified resting CD4⁺ and CD8⁺T lymphocytes. The ret lgG2c YTH 361.10 anti-CD52 antibody,however, was able to activate resting CD4⁺ and CD8⁺ T cellsdirectly without cross-linking or phorbol myristate acetatein the absence of Fc-bearlng cells. Anti-CD52 antibodies alsoaugmented the anti-CD3 mediated proliferatlve response of CD4⁺and CD8⁺ T cells when the two antibodies were co-immobilizedonto the same surface or cross-linked in solution by the samesecond antibody. Both CD4⁺CD45RA and CD4⁺CD45RO T cells werestimulated to proliferate by anti-CD52 antibodies in the presenceof appropriate co-stimulatory factors. Antl-CD52 mAbs did not,however, synerglze with anti-CD2 or CD28 mAb to induce CD4⁺T cell proliferation. The activation of CD4⁺ T cells by antl-CD52antibodies was inhibited by cyclosporin A, suggesting a rolefor the calcineurin-dependent signal transduction pathways.Although CD52 could transduce a signal In T cells, anti-CD52antibodies did not inhibit antigen-specific or polyclonal Tcell responses, suggesting this molecule does not play an essentialco-stimulatory role in normal T cell activation.     

Expression of CD45R0 (UCHL1) by CD4+ and CD8+ T cells as a sign of in vivo activation in infectious mononucleosis.

CD45R0 (UCHL1), a member of leucocyte common antigen family, is expressed largely on previously activated or memory T cells. We examined CD45R0 expression of T cell subpopulations in patients with Epstein-Barr virus (EBV) induced infectious mononucleosis (IMN) as a sign of in vivo activation. Consistent with the notion that activated CD8+ T cells expand in acute IMN; the majority of CD8+ T cells in patients with acute IMN expressed CD45R0 to the similar extent to HLA-DR expression. Most CD4+ T cells in these patients also demonstrated marked expression of CD45R0 as well as HLA-DR antigens, compared with age-matched controls. Expression of CD45R0 by CD4+ T cells in patients with acute IMN was more notable than their HLA-DR expression. While predominant CD8+ T cells resulted in decreased percentages of CD4+ T cells, CD4+ T cells expressing CD45R0 were shown to be significantly elevated in absolute number. The results suggest that both CD4+ and CD8+ T cells may be activated by stimulation with EBV infection. The appearance of two T cell subpopulations expressing CD45R0 in acute IMN implies their immunoregulatory roles in the control of EBV-infected cells.     

Evidence for differential expression of CD45 isoforms by precursors for memory-dependent and independent cytotoxic responses: human CD8 memory CTLp selectively express CD45RO (UCHL1)

On the basis of differential CD45 expression, human T cells can be separated into approximately reciprocal populations. The mAb UCHL1 detects a 180 kd molecular mass isoform, CD45R0. The CD45RA cluster of mAbs reacts with CD45 isoforms of 205 and 220 kd molecular mass. These reagents subdivide both CD4 and CD8 T cell populations. CD4 T cells that proliferate in response to memory-dependent (recall) antigens have been shown to selectively express CD45R0. We extend these observations to a model for cytotoxic responses that allows the functional analysis of CD8 T cells with differential CD45 expression. Precursors for allo-specific CTL responses are readily detectable in CD45R0 as well as CD45RA populations. In contrast, we find memory CTLp greatly enriched among CD45R0 cells. In combination with earlier work, these results suggest that differential expression of CD45 isoforms is associated with memory formation for different classes of immune responses in both major T cell lineages.     

The monoclonal antibody, UCHL1, recognizes a 180,000 MW component of the human leucocyte-common antigen, CD45.

The leucocyte-common antigen (L-CA or CD45) is a family of high molecular weight glycoproteins, ranging from 180,000 to 220,000 MW that are expressed only on cells of lymphoid and myeloid origin. CD45 monoclonal antibodies (mAbs) recognize epitopes present on all polypeptides of the family, while other mAbs, termed CD45R, recognize determinants found only on the 220,000 MW and 200,000 MW polypeptides. In contrast the mAb UCHL1 recognizes a 180,000 MW antigen. UCHL1-coupled Sepharose beads were used to absorb antigen from lysates of cell lines. CD45 mAbs bound to this immobilized antigen. Antigen immobilized with CD45 mAb-coupled Sepharose beads bound UCHL1. Antigen purified by absorption and elution from the MOLT-4 cell line with CD45 mAb-coupled beads yielded molecules of 180,000 and 190,000 MW. Reprecipitation of the eluted antigen with UCHL1 resulted in a 180,000 MW band only. In a reciprocal experiment, CD45 mAb reprecipitated a 180,000 MW molecule from purified UCHL1 antigen. UCHL1 and the CD45R mAb 2H4 showed a mutually exclusive pattern of reactivity with human T- and B-cell lines, but co-expression of the antigens was seen on two myeloid and one erythroleukaemic cell line. In contrast, epitopes recognized by other putative CD45R mAbs were co-expressed with UCHL1 both on myeloid, erythroid and many T- and B-cell lines. We conclude that UCHL1 recognizes an epitope present only on the 180,000 MW polypeptide of CD45. Expression of this antigen is essentially reciprocal to the epitope detected by the CD45R mAb 2H4.     

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.     

Ontogeny of expression of UCHL1 antigen on TcR-1+ (CD4/8) and TcR delta+ T cells

The frequency of blood mononuclear cells expressing the 180-kDa splice variant of the common leukocyte antigen identified by the UCHL1 monoclonal antibody (a characteristic of post-thymic cells which have been through a cycle of stimulation) rose in a logarithmic relationship to age between birth and 16 years. At birth only a low frequency of CD4+ (negative for CD 25) and no CD8+ T cells expressed UCHL1. The subsequent increase in UCHL1 expression occurred in parallel on T cells expressing alpha/beta receptors and gamma/delta receptors, suggesting that these subsets participated in post-thymic proliferation in proportion to their numbers in blood. The frequency of UCHL1 expression on CD8 cells was 5-15% less than on CD4 cells.     

Expression of a 32-kDa ligand for the CD40 antigen on activated human T lymphocytes

To identify the ligand(s) of the human CD40 antigen, a cDNA encoding the extracellular domain of the CD40 antigen was fused to a cDNA encoding the constant region (Fc) of human IgGl. The CD40-Fc fusion protein was able to specifically bind to CD4⁺ and various CD8⁺ T cell clones activated with immobilized anti-CD3. The ¹²⁵I-labeled CD40-Fc fusion protein bound anti-CD3 activated CD4⁺ T cell clone (MT9) with an equilibrium dissociation constant (Ka) of 10-20 nM. The human CD40-binding protein expressed on the cell surface of activated T lymphocytes is a monomeric protein of ≈ 32 kDa. Minor components of 29 kDa and 17 kDa were also detected. A small proportion of CD4+ and CD8⁺ blood mononuclear T cells activated by anti-CD3 expressed the CD40 ligand but its detection was best observed following depletion of B cells. Addition of B cells to purified T cells abolished the binding of CD40-Fc obtained after anti-CD3 activation.     

Thymic hormone modulation of CD38 (T10) antigen on human cord blood lymphocytes

We studied the in vitro effect of three different thymic factors on the expression of CD38 (T10) antigen on cord T-lymphoid cell surface. The results showed that cord mononuclear cell populations contain variable percentages of CD38+ cells. The CD38 molecule was expressed on cord T and B lymphocyte and monocyte surfaces. Incubation with thymic agents induced a significant increases in the CD38+ cell percentage only in the samples with low CD38 antigen expression, and this modulation was mainly attributable to the T-cell subset. The effect seems to be specific and not correlated with the known high spontaneous DNA synthesis rate of cord mononuclear cells.     

Expression of leukocyte common antigen (CD45) on various human leukemia/lymphoma cell lines

CD45 antigen (leukocyte common antigen), a unique and ubiquitous membrane glycoprotein with a molecular mass of about 200 kDa, is expressed on almost all hematopoietic cells except for mature erythrocytes. However, the biological function of this glycoprotein still remains to be resolved. In order to clarify the role of CD45 antigen in hematopoietic cell differentiation and function, its expression on human leukemia/lymphoma cell lines was studied by membrane immunofluorescence. Thirty-eight established cell lines were analyzed using T29/33, a monoclonal antibody (MoAb) that recognizes the common epitopes of this glycoprotein molecule. Conventional cell marker studies were also carried out on these cell lines to compare their CD45 expression. It was shown that CD45 expression varies among B-lineage cells depending on cell differentiation, in contrast to its stable expression on leukemic T cell (6/6, positive) and myeloid (5/5, positive) lineage cell lines. On the other hand, only two out of six histiomonocytoid lineage cell lines were positive. Human T cell leukemia/lymphoma virus type I (HTLV-I)-associated T cell lines derived from peripheral blood leukocytes of patients with adult T cell leukemia/lymphoma (ALT/L) in Japan did not express CD45 on their cell surface. Taken together, these observations suggest that CD45 has a functional role in hematopoietic cell activation and differentiation.     

Expression of CD31 epitopes on human lymphocytes: CD31 monoclonal antibodies differentiate between naive (CD45RA+) and memory (CD45RA-) CD4-positive T cells

The CD31 antigen, a member of the immunoglobulin superfamily with a possible cell adhesion function, is expressed on approximately 50% of peripheral blood lymphoid cells at relatively low intensity (10-20% of the level on monocytes). In the accompanying paper we showed that a mAb, 5A2.G5, which identifies a glycosylation-dependent epitope of the CD31 antigen, bound to fewer lymphocytes than two other CD31 mAb, B2B1 and 2BD4, although the 3 antibodies bound equally well to monocytes. We have now analyzed the pattern of expression of epitopes of the CD31 antigen on lymphoid cell subpopulations using two-color immunofluorescence and flow cytometry. Large granular lymphocytes (CD16+), CD8-positive T cells and B cells (SMIg+) were mostly CD31-positive as indicated by the binding of mAb B2B1 and 2BD4. Single populations displaying some overlap with the negative control were obtained in each case. In contrast, CD4-positive T cells fell into two discrete populations with respect to CD31 antigen expression. mAb 5A2.G5 displayed weaker binding to all lymphoid cell types, indicating that the pattern of glycosylation of the CD31 antigen differs between lymphocytes (of all types) and cells of the myeloid lineages. The heterogeneity of CD31 antigen expression by CD4-positive cells was further examined by dual-labelling of purified CD4 cells with mAb B2B1 and CD45RA or CD29 mAb which identify naive and memory T cells respectively. The CD31 antigen was found to be preferentially expressed by the CD45RA-positive, naive cell population.     

Ligation of the CD23,p45 (BLAST-2,EBVCS) antigen triggers the cell-cycle progression of activated B lymphocytes

CD23,p45 (BLAST-2,EBVCS) is a 45-kDa lineage-restricted antigen which appears on the surface of human B cells shortly after activation. A monoclonal antibody (MHM6) to CD23,p45, as well as a polyclonal rabbit antibody raised against the purified antigen were found to promote DNA synthesis in purified tonsillar B cells which had been activated with phorbol ester. Interleukin 1, which was not, by itself, stimulatory for either resting or activated B cells, significantly augmented the growth-promoting properties of MHM6. Kinetic studies indicated that while MHM6 exerted its influence in early G1, interleukin 1 acted later in the cycle just prior to the entry of cells into S phase. The findings demonstrate a role for CD23,p45 in triggering the progression of activated B lymphocytes through the G1 phase of the cell cycle. The possibility that this antigen serves as a receptor for a B cell stimulatory factor is discussed.     

Maximal interferon-gamma production and early synthesis of interleukin-2 by CD4+ CDw29- CD45R- p80- human T lymphocytes

Functionally distinct subsets within the T-helper (CD4+) cell population have been described in man, rat and mouse. We have shown previously that the CD4+ 45R- human lymphocytes are producers of IL-2 within 24 hr of polyclonal stimulation and are the major interferon (IFN) producers. The mAbs 4B4 (CDw29) and Leu 8 (p80) are here used together with Leu-18 (CD45R) to characterize the subpopulations of the CD4+ human lymphocytes in more detail. The majority of the CD45R+ cells were CDw29- and the majority of the CDw29+ cells were CD45R-. Most of the CD45R+ cells (78%) were also p80+. A significant number of cells (12%) were CDw29- CD45R-. The predominant subpopulations were defined to be CDw29- CD45R+ p80+ (31%), CDw29+ CD45R- p80- (24%) and CDW29+ CD45R- p80+ (18%). Within the CD4+ CD45R- subpopulation different subsets vary in their capacities to produce IFN and to produce IL-2 within 24 hr after activation. CD4+ CDw29- CD45R- p80- T lymphocytes produced the largest quantities of IFN and IL-2.     

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.     

CD5 (OKT1) augments CD3-mediated intracellular signaling events in human T lymphocytes

CD5 is expressed on thymocytes, all mature T cells, and a subset of mature B cells, and probably contributes to T-cell–B-cell adhesion. We assessed whether CD5-crosslinking by mAb augments T-cell stimulation. Plate-bound anti-CD5 or anti-CD3 mAb alone had no effect on any of the assessed activation parameters of resting T cells. However, concomitant signaling through both CD5 and CD3 by plate-bound antibodies resulted in marked increases in T-cell surface CD69 expression and T-cell metabolism, as assessed by the T cell's ability to reduce 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxylmethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) to formazen. In addition, simultaneous cross-linking of CD5 and CD3 caused a significant (p < 0.001) increase in phosphatidylinositol hydrolysis in resting T cells compared to stimulation with anti-CD3 mAb alone or anti-CD3 mAb plus anti-CD5 isotype control antibody. These results indicate that CD5 augments signaling through CD3 and consequently functions as a costimulatory molecule for resting T cells.     

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.     

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.     

Isoform-specific associations of CD45 with accessory molecules in human T lymphocytes.

Association of CD45 with surface molecules was investigated in human T lymphocytes by co-capping. CD45 appeared to be associated with the CD3/T cell receptor complex and with CD4 or CD8 molecules in memory, but not in naive T cells, as previously reported in the mouse. Associations of CD45 isoforms with accessory molecules were then identified with seven anti-CD45R monoclonal antibodies (mAb). An isoform-specific association pattern was observed: CD2 co-capped with CD45 molecules recognized by UCHL1 mAb (CD45R0). LFA-1 with molecules bound by 2H4 mAb (CD45RA), and both CD4 and CD8 with molecules reacting with MCA.347 mAb (whose isoform specificity was not known). Further information on the CD45 isoform(s) associated to CD4 and CD8 was sought by assessing the isoform specificity of MCA.347. Cross-competition experiments showed that it reacts with an epitope clearly different from those recognized by 2H4 and UCHL1, and only partially overlapping the PD7/26 epitope (CD45RB). Moreover, the competition between MCA.347 and PD7/26 was maximal in naive T cells and minimal both in memory T cells and in a subset expressing CD11b, a marker of granular lymphocytes. Immunoprecipitation experiments showed that MCA.347 binds to CD45 molecules with a molecular mass of 220, 205 and 190 kDa, the 190-kDa molecules not being recognized by 2H4, PD7/26 or UCHL1. These data indicate that MCA.347 recognizes amino acid sequences different from those coded by the exon A or B of the gene, and not expressed by CD45R0, suggesting that it binds to sequences coded by the exon C. In conclusion, this work shows that in human T cells different CD45 isoforms are associated to different surface molecules: LFA-1 is associated to CD45RA, CD2 to CD45R0 and CD4 and CD8 presumably to CD45RC. This peculiar behavior of CD45 suggests that it may play a crucial role in lymphocyte activation, probably by modulating the signals delivered to the cell by different receptor systems.