Physical associations between CD45 and CD4 or CD8 occur as late activation events in antigen receptor-stimulated human T cells.

Activation of human PBL T cells with solid phase anti-CD3 mAb or during the course of an MLR response gives rise to the association of CD4 or CD8 molecules with the protein tyrosine phosphatase, CD45, on the cell surface. This paired association of cell-surface molecules occurs late in the activation cycle and appears to be dependent upon Ti-CD3-mediated signaling because mitogen-driven activation does not induce formation of the complex. Maximal association occurred 72 to 96 h after exposure to anti-CD3 mAb on both CD4+ and CD8+ T cells. In contrast, association between CD8 and CD45 during an MLR response did not occur until day 6 of a MLR whereas CD4-CD45 association was detected by 72 h of culture. The kinetics of association between CD4 or CD8 and CD45 was measured by fluorescence resonance energy transfer and confirmed by immunoprecipitation of dithiobis succinimidylpropionate or disuccinimidyl suberate cross-linked 125I-labeled resting or activated T cells. The molecules that co-precipitated with either CD4 or CD8 and had an apparent kDa of 180 to 205 could be immunodepleted with anti-CD45 mAb. Furthermore, CD4 or CD8 immunoprecipitates from 96-h activated T cells contained significant levels of protein tyrosine phosphatase activity whereas corresponding immunoprecipitates from resting or recently activated T cells showed little protein tyrosine phosphatase activity. This association may allow CD45 to engage and dephosphorylate lck or another CD4- or CD8-associated substrate in order to reset the receptor complex to receive a new set of stimuli. Our observations suggest that synergistic signaling provided as a consequence of CD4 or CD8 association with the TCR after antigenic stimulation may develop on a different temporal scale than that observed after soluble anti-CD4+ anti-CD3 heteroconjugate antibody cross-linking.     

The protein-tyrosine phosphatase CD45 reaches the cell surface via golgi-dependent and -independent pathways

CD45 is a receptor protein-tyrosine phosphatase essential for T cell development and lymphocyte activation. It is highly glycosylated, with multiple isoforms and glycoforms expressed on the cell surface depending on the cell type and stage of differentiation. Interestingly, we found two pools of newly synthesized CD45 expressed on plasma membrane, one of which arrived by 5 min after synthesis. The remaining pool of CD45 was fully glycosylated and began to arrive at the cell surface at approximately 15 min. The rapidly expressed population of CD45 possessed exclusively endoglycosidase H-sensitive N-linked carbohydrate. Additionally, this rapidly expressed pool of CD45 appeared on the cell surface in a brefeldin A (BFA)-insensitive manner, suggesting that it reached the cell surface independent of the Golgi complex. The remaining CD45 trafficked through the Golgi complex, and transport proceeded via a BFA-sensitive mechanism. These data suggest that CD45 is able to reach the cell surface via two distinct routes. The first is a conventional Golgi-dependent pathway that allows fully processed CD45 to be expressed. The second utilizes an ill defined mechanism that is independent of the Golgi, is BFA-resistant, and allows for the expression of CD45 with immature carbohydrate on the cell surface.     

CD45-protein tyrosine phosphatase cross-linking inhibits T cell receptor CD3-mediated activation in human T cells

Activation of peripheral blood T cells, and the leukemic T cell line Jurkat, as measured by mobilization of intracellular calcium, by an anti-TCR antibody is blocked by mAb (T191) to the leukocyte common Ag (CD45). T191 also blocked down-regulation of the CD3-TCR complex induced by an anti-CD3 mAb. Vanadate, a phosphotyrosine phosphatase inhibitor, partially blocks the effect of T191 and restored mobilization of intracellular calcium. Assays of the immunoprecipitates of T191 and CD45 from both Jurkat-BM1 and peripheral T cells showed that the immune complexes had intrinsic phosphatase activity. A parallel immunoprecipitate using a mAb (4-10) against HLA class I showed no such activity. Further analysis of the T191 immunocomplex revealed activity against phosphotyrosine, p-nitrophenylphosphate, and [32P-poly-glu-tyr, but not against phosphoserine. Phosphatase activity was inhibited by Vanadate, but not by Zn2+ or F-. These results show that CD45 is a phosphotyrosine phosphatase, and strongly suggest that tyrosine phosphorylation/dephosphorylation is critically involved in activation of T cells through the TCR-CD3 complex.     

Simultaneous increased expression of E-rosette receptor (CD2, T11) and T cell growth factor receptor on human T lymphocytes during activation

The E-rosette receptor (CD2, T11) is a differentiation antigen expressed on immature and mature human T lymphocytes. Activation of T cells from human peripheral blood with phytohemagglutinin (PHA) or with monoclonal antibody to the CD3-Ti complex (anti-Leu-4) caused the expression of CD2 to increase 10- to 20-fold. Dual parameter correlated analyses with antibody to the T cell growth factor (TCGF) receptor (anti-Tac) and anti-CD2 antibody demonstrated that the increase in CD2 expression occurred at the same time and on the same cells that expressed the TCGF receptor after stimulation with PHA. The increased expression of CD2 and the initial expression of Tac were totally inhibited by cycloheximide, but were not affected by sufficient actinomycin-D to block the T cell proliferative response. The expression of CD2 was compared with the expression of CD4 and CD8, i.e., T cell differentiation antigens on cytotoxic/suppressor or helper T cells, respectively. Although virtually all of the small percentage of freshly isolated Tac+ peripheral blood cells belonged to the CD4+, CD8- subset, both CD4+ and CD8+ T cells were equivalently activated by PHA to express Tac. By 20-30 hr after activation, the expression of CD4 or CD8 was initially decreased 10-50%. Subsequently, the expression of CD4 and CD8 returned to the levels on resting T cells but did not increase further. Therefore, the increase in CD2 expression does not reflect a universal property of cell surface antigens on activated T lymphocytes.     

Stimulation of human T cells via anti-T cell receptor monoclonal antibody BMA031: distinct cellular events involving interleukin-2 receptor and lymphocyte function antigen 1

We have analyzed activation of resting human T cells by anti-T cell receptor (TCR) monoclonal antibody (mAb) BMA031, a murine mAb of the G2b isotype. Human peripheral blood lymphocytes (PBL) respond to anti-TCR mAb by short-term proliferation in vitro and by acquisition of responsiveness to interleukin 2 (rIL-2) in the absence of detectable IL-2 production. Cell depletion and limiting dilution experiments indicate that anti-TCR mAb +/- rIL-2 stimulation covers a substantial portion of human T cells, including CD4+ and CD8+ cells. Enhancement by rIL-2 of anti-TCR mAb-induced proliferation is blocked by anti-IL-2 receptor (IL-2R, p55) mAb, while anti-TCR mAb-induced proliferation is not. In contrast, anti-TCR mAb-induced proliferation is blocked by anti-lymphocyte function antigen 1 (LFA-1, CD11a) mAb and is not demonstrable in PBL from two patients with severe congenital LFA-1 deficiency, not even in the presence of irradiated LFA-1+ PBL. We conclude that stimulation of resting human T cells by anti-TCR mAb BMA031 enables dissociation of distinct steps in T cell activation that specifically require participation of IL-2R (p55) and LFA-1 cell surface molecules in a mutually exclusive way.     

Treatment with humanized monoclonal antibody against CD154 prevents acute renal allograft rejection in nonhuman primates.

CD154 is the ligand for the receptor CD40. This ligand-receptor pair mediates endothelial and antigen-presenting cell activation, and facilitates the interaction of these cells with T cells and platelets. We demonstrate here that administration of a CD154-specific monoclonal antibody (hu5C8) allows for renal allotransplantation in outbred, MHC-mismatched rhesus monkeys without acute rejection. The effect persisted for more than 10 months after therapy termination, and no additional drug was required to achieve extended graft survival. Indeed, the use of tacrolimus or chronic steroids seemed to antagonize the anti-rejection effect. Monkeys treated with antibody against CD154 remained healthy during and after therapy. The mechanism of action does not require global depletion of T or B cells. Long-term survivors lost their mixed lymphocyte reactivity in a donor-specific manner, but still formed donor-specific antibody and generated T cells that infiltrated the grafted organ without any obvious effect on graft function. Thus, therapy with antibody against CD154 is a promising agent for clinical use in human allotransplantation.     

Specific CD45 isoforms differentially regulate T cell receptor signaling.

Multiple isoforms of T cell CD45 tyrosine phosphatase are expressed as a result of alternative RNA splicing among extracellular exons. To discern the presence and identity of distinct functions among CD45 isoforms, we compared thymic T cell activation responses by elevating expression of two CD45 isoforms normally found on quiescent T cells. We report that CD45RABC significantly increased CD4+ thymic T cell proliferation in both a mixed lymphocyte reaction and following anti-T cell receptor (TCR) antibody stimulation. Additionally, CD45RABC enhanced Ca2+ mobilization and phosphotyrosine accumulation, and suppressed the inhibitory effect of anti-CD4 antibodies. By contrast, CD45R0 did not enhance TCR signaling or phosphotyrosine levels in CD4+ thymic T cells and required a TCR co-stimulus to augment cellular proliferation. These studies provide genetic evidence that alternative CD45 isoforms are functionally distinct and disclose a unique mechanism by which T cell immunologic responsiveness can be modified.     

A novel pathway of human T lymphocyte activation. Identification by a monoclonal antibody generated against a rheumatoid synovial T cell line

Substantial evidence indicates that compartmentalized infiltrates of T lymphocytes are central to the pathogenesis of autoimmune diseases such as rheumatoid arthritis, but the mechanisms by which such cells become activated remain unknown. To define surface components of activation pathways important in the function of these cells, we have generated mAb against a rheumatoid synovial T cell line. One such antibody, termed anti-UM4D4, reacts with an Ag, termed UM4D4, which is strongly expressed on most rheumatoid synovial T cell lines and clones, and on a subset of peripheral blood T cells, resting or activated. Anti-UM4D4 is mitogenic in soluble form for PBMC and certain T cell clones, and is comitogenic with the phorbol ester PMA for purified resting T lymphocytes. These functional effects are similar to those previously observed with antibodies to epitopes of CD2 and CD3, surface Ag involved in two well defined pathways of human T cell activation. Binding of anti-UM4D4 to T cells is not, however, blocked by antibodies directed at various epitopes of CD2 and CD3. Moreover, UM4D4 does not comodulate with CD3, and is expressed on a T cell line that lacks CD2, CD3, and CD28. The data, therefore, indicate that anti-UM4D4 identifies a T cell activation pathway, distinct from those previously described, that could play a role in the pathogenesis of T cell-mediated autoimmune diseases.     

Retinoic acid disrupts the Golgi apparatus and increases the cytosolic routing of specific protein toxins

All-trans retinoic acid can specifically increase receptor mediated intoxication of ricin A chain immunotoxins more than 10,000 times, whereas fluid phase endocytosis of ricin A chain alone or ricin A chain immunotoxins was not influenced by retinoic acid. The immunotoxin activation by retinoic acid does not require RNA or protein synthesis and is not a consequence of increased receptor binding of the immunotoxin. Vitamin D3 and thyroid hormone T3, that activate retinoic acid receptor (RAR) cognates, forming heterodimers with retinoid X receptor (RXR), do not affect the potency of immunotoxins. Among other retinoids tested, 13-cis retinoic acid, which binds neither RAR nor RXR, also increases the potency of the ricin A chain immunotoxin. Therefore, retinoic acid receptor activation does not appear to be necessary for immunotoxin activity. Retinoic acid potentiation of immunotoxins is prevented by brefeldin A (BFA) indicating that in the presence of retinoic acid, the immunotoxin is efficiently routed through the Golgi apparatus en route to the cytoplasm. Directly examining cells with a monoclonal antibody (Mab) against mannosidase II, a Golgi apparatus marker enzyme, demonstrates that the Golgi apparatus changes upon treatment with retinoic acid from a perinuclear network to a diffuse aggregate. Within 60 min after removal of retinoic acid the cell reassembles the perinuclear Golgi network indistinguishable with that of normal control cells. C6-NBD-ceramide, a vital stain for the Golgi apparatus, shows that retinoic acid prevents the fluorescent staining of the Golgi apparatus and eliminates fluorescence of C6-NBD-ceramide prestained Golgi apparatus. Electron microscopy of retinoic acid-treated cells demonstrates the specific absence of any normal looking Golgi apparatus and a perinuclear vacuolar structure very similar to that seen in monensin-treated cells. This vacuolization disappears after removal of the retinoic acid and a perinuclear Golgi stacking reappears. These results indicate that retinoic acid alters intracellular routing, probably through the Golgi apparatus, potentiating immunotoxin activity indepedently of new gene expression. Retinoic acid appears to be a new reagent to manipulate the Golgi apparatus and intracellular traffic. As retinoic acid and immunotoxins are both in clinical trials for cancer therapy, their combined activity in vivo would be interesting to examine.     

CD44-initiated cell spreading induces Pyk2 phosphorylation, is mediated by Src family kinases, and is negatively regulated by CD45

CD44 is a cell adhesion molecule implicated in leukocyte adhesion and migration, co-stimulation of T cells, and tumor metastasis. CD45 is a leukocyte-specific protein tyrosine phosphatase that dephosphorylates the Src family kinases, Lck and Fyn, in T cells. Positive regulation of Lck by CD45 is required for its effective participation in T cell receptor signaling events. Here, immobilized CD44 antibody induced a distinctive cell spreading in CD45(-), but not CD45(+), T cells, and this correlated with the induction of tyrosine-phosphorylated proteins. Two focal adhesion family kinases, Pyk2 and, to a lesser extent, FAK were inducibly phosphorylated, as was a potential substrate, Cas. CD44-mediated cell spreading and induced tyrosine phosphorylation were prevented by the Src family kinase inhibitor, PP2. Furthermore, 2-fold more Lck associated with CD44 in the low density sucrose fraction from CD45(-) T cells compared with CD45(+) T cells, suggesting that CD45 may regulate the association of Lck with CD44 in this fraction. Therefore, in CD45(-) T cells, CD44 signaling is mediated by Src family kinases, and this leads to Pyk2 phosphorylation, cytoskeletal changes, and cell spreading. This implicates CD45 in the negative regulation of Src family kinase-mediated CD44 signaling leading to T cell spreading.     

The catalytic activity of the CD45 membrane-proximal phosphatase domain is required for TCR signaling and regulation.

Cell surface expression of CD45, a receptor-like protein tyrosine phosphatase (PTPase), is required for T cell antigen receptor (TCR)-mediated signal transduction. Like the majority of transmembrane PTPases, CD45 contains two cytoplasmic phosphatase domains, whose relative in vivo function is not known. Site-directed mutagenesis of the individual catalytic residues of the two CD45 phosphatase domains indicates that the catalytic activity of the membrane-proximal domain is both necessary and sufficient for restoration of TCR signal transduction in a CD45-deficient cell. The putative catalytic activity of the distal phosphatase domain is not required for proximal TCR-mediated signaling events. Moreover, in the context of a chimeric PTPase receptor, the putative catalytic activity of the distal phosphatase domain is not required for ligand-induced negative regulation of PTPase function. We also demonstrate that the phosphorylation of the C-terminal tyrosine of Lck, a site of negative regulation, is reduced only when CD45 mutants with demonstrable in vitro phosphatase activity are introduced into the CD45-deficient cells. These results demonstrate that the phosphatase activity of CD45 is critical for TCR signaling, and for regulating the levels of C-terminal phosphorylated Lck molecules.     

Lipopolysaccharide rapidly traffics to and from the Golgi apparatus with the toll-like receptor 4-MD-2-CD14 complex in a process that is distinct from the initiation of signal transduction

Mammalian responses to LPS require the expression of Toll-like receptor 4 (TLR4), CD14, and MD-2. We expressed fluorescent TLR4 in cell lines and found that TLR4 densely localized to the surface and the Golgi. Similar distributions were observed in human monocytes. Confocal imaging revealed rapid recycling of TLR4-CD14-MD-2 complexes between the Golgi and the plasma membrane. Fluorescent LPS followed these trafficking pathways in CD14-positive cells. The TLR4- adapter protein, MyD88, translocated to the cell surface upon LPS exposure, and cross-linking of surface TLR4 with antibody induced signaling. Golgi-associated TLR4 expression was disrupted by brefeldin A, yet LPS signaling was preserved. We conclude that LPS signaling may be initiated by surface aggregation of TLR4 and is not dependent upon LPS trafficking to the Golgi.     

Activation of resting T lymphocytes by anti-CD3 (T3) antibodies in the absence of monocytes

The antigen receptor molecules on human T lymphocytes are noncovalently associated on the cell surface with the CD3 (T3) molecular complex. Perturbation of this complex with anti-CD3 monoclonal antibodies induces T cell activation. Previous studies have demonstrated that this process requires the participation of monocytes. In the present report, we demonstrate that purified, resting (G0 phase) T cells incubated with monoclonal anti-CD3 antibodies proliferate in response to purified interleukin 2 (IL 2), in a lymphokine dose-dependent fashion. Anti-CD3 antibody or IL 2 alone did not trigger cell division. The effect was specific for anti-CD3 antibodies because monoclonal antibodies reactive with other surface molecules (OKT4, OKT8, L368) were inactive. Furthermore, the same phenomenon was observed when anti-CD3 antibody Leu-4 (IgG1) was incubated with cells of individuals whose monocytes cannot process antibodies of the IgG1 subclass (Leu-4 nonresponders). In addition, both F(ab')2 and Fab fragments of anti-CD3 antibody OKT3 were also capable of rendering T cells receptive to the IL 2 growth signal. These data indicate that neither monocytes nor CD3 receptor cross-linking are required absolutely for resting T cell activation, provided that IL 2 is supplied exogenously. T lymphocytes treated with anti-CD3 antibodies proliferated in response to both purified mitogen-induced and recombinant IL 2. Antibodies to the IL 2 receptor (anti-Tac) inhibited the proliferation. Thus, the most likely mechanism for anti-CD3 antibody-mediated triggering is induction of IL 2 receptors.     

Development of a monoclonal antibody, anti-6C2, which is involved in the interaction of CD4 T helper cells and activated B cells.

We have developed a mAb anti-6C2, by immunizing mice with T cell line derived from the Callithrix jacchus (common marmoset). Anti-6C2 is reactive with approximately 50% of unfractionated T cells, 50% of CD4+ cells, and 40% of CD8+ cells. Regarding CD4+ cells, anti-6C2-reactive cells substantially overlap with the CD29+CD45RO+ Th cell population. Moreover, anti-6C2 can divide these T cells into 6C2+ and 6C2- subpopulations. The CD4+CD45RO+6C2+ cells maximally respond to soluble Ag such as tetanus toxoid and provide strong helper function for PWM-driven B cell IgG synthesis. Most interestingly, anti-6C2 was also reactive against activated B cells but not resting B cells; furthermore, this epitope was inducible through activation of resting B cells or B cell line. Biochemical characterization showed that anti-6C2 precipitated two glycoproteins with the relative molecular weights of 180,000 and 95,000 from 125I-surface labeled cell lysate. Sequential immunoprecipitation studies demonstrated that these two glycoproteins were the lymphocyte function-associated antigen (LFA-1) Ag complex (CD11a/18). Significantly, although this antibody did not inhibit cytotoxic killer T cell responses and Ag-induced T cell proliferation as did conventional anti-LFA-1, it did inhibit PWM-driven B cell IgG synthesis. Because 6C2 expression was induced after B cell activation, the above results strongly suggest that the 6C2 molecule may play a role in the interaction of CD4 helper cells and activated B lymphocytes.     

T-cell stimulation through the T-cell receptor/CD3 complex regulates CD2 lateral mobility by a calcium/calmodulin-dependent mechanism

T lymphocyte activation through the T cell receptor (TCR)/CD3 complex alters the avidity of the cell surface adhesion receptor CD2 for its ligand CD58. Based on the observations that activation-associated increases in intracellular [Ca2+] ([Ca2+]i) strengthen interactions between T cells and antigen-presenting cells, and that the lateral mobility of cell surface adhesion receptors is an important regulator of cellular adhesion strength, we postulated that [Ca2+]i controls CD2 lateral mobility at the T cell surface. Human Jurkat T leukemia cells were stimulated by antibody-mediated cross-linking of the TCR/CD3 complex. CD2 was labeled with a fluorescently conjugated monoclonal antibody. Quantitative fluorescence microscopy techniques were used to measure [Ca2+]i and CD2 lateral mobility. Cross-linking of the TCR/CD3 complex caused an immediate increase in [Ca2+]i and, 10-20 min later, a decrease in the fractional mobility of CD2 from the control value of 68 +/- 1% to 45 +/- 2% (mean +/- SEM). One to two hours after cell stimulation the fractional mobility spontaneously returned to the control level. Under these and other treatment conditions, the fraction of cells with significantly elevated [Ca2+]i was highly correlated with the fraction of cells manifesting significantly reduced CD2 mobility. Pretreatment of cells with a calmodulin inhibitor or a calmodulin-dependent kinase inhibitor prevented Ca2+-mediated CD2 immobilization, and pretreatment of cells with a calcineurin phosphatase inhibitor prevented the spontaneous reversal of CD2 immobilization. These data suggest that T cell activation through the TCR/CD3 complex controls CD2 lateral mobility by a Ca2+/calmodulin-dependent mechanism, and that this mechanism may involve regulated phosphorylation and dephosphorylation of CD2 or a closely associated protein.     

A 44 kilodalton cell surface homodimer regulates interleukin 2 production by activated human T lymphocytes

We previously described a cell surface antigen, termed Tp44, detected by monoclonal antibody 9.3 on approximately 80% of mature human T lymphocytes. Analysis by SDS-polyacrylamide gel electrophoresis and isoelectric focusing demonstrated that this antigen consists of two identical 44 kilodalton glycopeptides that form a disulfide-linked homodimer. Competitive binding experiments showed that antibody 9.3 and an anti-CD3 antibody (64.1) recognize distinct antigenic determinants; furthermore, the binding of antibody 9.3 was unaffected by prior modulation of CD3. Thus, Tp44 has no detectable cell surface association with CD3. By itself, antibody 9.3 had no detectable effect on either IL 2 receptor expression or IL 2 release, and did not cause T cell proliferation even when monocytes were present and exogenous IL 2 was provided, indicating that binding of antibody 9.3 does not provide a primary signal for T cell activation. However, the proliferative responses of T lymphocytes activated by phytohemagglutinin, concanavalin A, or an anti-CD3 monoclonal antibody were strikingly enhanced in the presence of antibody 9.3, an effect associated with increased IL 2 receptor expression and increased IL 2 secretion. Antibody 9.3 enabled anti-CD3-Sepharose-activated T cells and anti-CD3 antibody-activated Jurkat cells to release IL 2 in the absence of monocytes. Fab fragments of antibody 9.3 had no effect on anti-CD3-induced IL 2 release by Jurkat cells, whereas F(ab')2 fragments had activity comparable to that of unmodified antibody, indicating that bivalent binding of Tp44 molecules is required for IL 2 secretion. Together, these results suggest that TP44 may function as a receptor for accessory signals in the activation of T cells.     

Tissue distribution and ultrastructure of B lymphocytes reacting with the monoclonal antibody anti-Y29/55

The reactivity of normal tonsilar cells with the monoclonal antibody anti-Y29/55 is characterized at the tissue and ultrastructural cytological level. Using an indirect immuno-alkaline phosphatase method on frozen sections the antibody labels mantle zone and germinal center lymphocytes. This staining reaction is more generalized in B-lymphocyte areas than that obtained with antibodies to IgM and IgD. By indirect immunoperoxidase staining, as well as by an indirect rosetting procedure in cell suspensions, the reactive cell population were either small resting lymphocytes or activated lymphocytes corresponding to centrocytes, centroblasts, immunoblasts and plasmoblasts; some plasma cells were also labeled. These results characterize the monoclonal antibody anti-Y29/55 as a pan-B-marker antibody, useful for labeling resting and activated peripheral B-lymphocytes in frozen tissue sections and cell suspensions.     

The coexpression of CD45RA and CD45RO isoforms on T cells during the S/G2/M stages of cell cycle.

The tyrosine phosphatase CD45 is alternatively spliced to generate isoforms of different molecular weights (180-220 kDa) which are differentially expressed on hematopoietic cells. Monoclonal antibodies reacting with either the 180-kDa (UCHL-1, CD45RO) or the 200- to 220-kDa (2H4, CD45RA) isoform have been used to subdivide T cell populations based on their expression of one or the other of these two epitopes. CD45RA T cells have "naive" characteristics of unresponsiveness to recall antigens and prominence in cord blood, while CD45RO T cells are considered "memory" T cells because they proliferate to recall antigens and increase following PHA activation of cord blood. However, we have recently demonstrated the expression of the CD45RA isoform on a subpopulation of CD45RO+ T cell clones, suggesting that CD45RA is not a universal marker for naive T cells. Using propidium iodide staining of the DNA to determine cell cycle stage, we now show that CD45RA expression is significantly higher on T cell clones during the S, G2, and M stages of cell cycle when compared to CD45RA expression on cells in Go and G1. Furthermore, CD45RA expression on cells undergoing mitosis is not limited to long-term activated T cell clones, as uncultured peripheral blood T cells in the S/G2/M phase express significantly more CD45RA. The percentage of T cells coexpressing CD45RA and CD45RO also increases following PHA activation, indicating that T cells in the process of division express both isoforms. These results suggest a potential role of the CD45RA isoform during the stages of cell cycle leading to mitosis.     

CD4 molecules are associated with the antigen receptor complex on activated but not resting T cells

To explore the relationship between CD4 and CD3/Ti on the T cell surface, we have studied a panel of Ag-specific Th cell lines and clones, as well as resting and mitogen-activated CD4+ cells. Our results show that exposure of Th cells to their specific antigenic stimuli, but not to irrelevant stimuli, induced the rapid disappearance of approximately 20 to 35% of CD3 and CD4 molecules. The modulation of these molecules was detected in less than 1 h, became maximal at 12 h, and recovered thereafter in parallel. Treatment of Th cells with anti-CD4 antibody prevented Ag-induced modulation of CD3, and treatment with anti-CD3 blocked modulation of CD4. In the absence of Ag, treatment of these cells with an antibody (WT-31) directed at a conformational determinant within CD3/Ti or with the combination of anti-CD3 antibody and goat anti-mouse Ig, also resulted in significant modulation of CD4. Similar treatment of PHA-activated CD4+ T cells with anti-CD3/Ti antibodies also induced CD4 modulation; however, the same antibodies failed to affect CD4 expression on fresh resting T cells. These results indicate that on activated, but not resting T cells, CD4 molecules can be physically associated with CD3/Ti. We postulate that this association is essential for efficient Th cell activation, and further that the ability of anti-CD4 antibodies to inhibit helper functions is due to their prevention of CD4-CD3/Ti interaction on the T cell surface.     

Supernatant from a cloned helper T cell stimulates resting B cells to express transferrin and IL-2 receptors

We describe the properties of the supernatant from a murine cloned helper T cell (clone 52.3) which is able to polyclonally activate most resting B cells in the absence of any additional stimulus. We hypothesize that an activity which we call BCAF (B-cell-activating factor(s)) exists in our supernatant which can activate resting B cells alone or in conjunction with other lymphokines. In the present report, we investigate changes in the surface antigen pattern induced on resting B cells by BCAF-containing supernatant. Analysis of the cells by flow cytometry shows that transferrin receptor and IL-2 receptor expression increase on a large fraction of B cells after 2 days of activation by the T-helper-cell clone supernatant. Monoclonal anti-transferrin receptor antibody inhibits cell division but does not affect blastogenesis, while IL-2 has no effect in our experimental system. Our present results confirm that BCAF-containing supernatants can act on most resting B cells and replace helper T cells in inducing B-cell activation and proliferation.