5-Fluorouracil enhances apoptosis sensitivity of T lymphocytes mediated by CD3 epsilon

Previous studies by our laboratory have reported that the T cell receptor (TCR) TCR/CD3 complex could mediate activation as well as apoptosis of T lymphocytes. Two tyrosine residues in the ITAM (immuno-receptor tyrosine-based activation motifs) of CD3 epsilon were required for apoptosis signalling of Jurkat T lymphocytes. Stable cell lines TJK and T3JK produced from CD8(-) Jurkat T lymphocytes by transfection with wild-type and mutant CD8 epsilon (fusion of the extracellular and transmembrane domains of human CD8 alpha to the intracellular domain of mouse CD3 epsilon), were used with CD8(-) Jurkat T lymphocytes for studying the role of single intact CD3 epsilon. 5-Fluorouracil (5-FU), a chemotherapeutic drug can induce cell death of many tumour cell lines. In the present experiments, we examined the expression of caspase-3, p53 and Bid in the three cell lines induced by 5-FU and/or anti-CD8 antibody. We found high expression of p53 during activation-induced cell death of TJK cells mediated by anti-CD8 antibody and apoptosis of TJK and T3JK induced by 5-FU, implicating p53 involvement in apoptosis of leukemia cells induced by anti-CD8 antibody and 5-FU. We also detected the active form of caspase-3 and Bid in apoptotic leukemia cells after treatment with 5-FU and/or anti-CD8 antibody, indicating that the drug and antibody induced cell death through caspase-3 and the signal pathway may involve the Bcl-2 protein family. Our results showed that combined treatment with 5-FU and anti-CD8 antibody could enhance the rate of apoptosis induced by 5-FU or anti-CD8 antibody through increased expression of p53 and by promoting activation of caspase-3 and Bid. This suggests that the combination of 5-FU and anti-CD8 antibody may play an important role in inducing apoptosis of leukemia cells.     

T cell receptor aggregation, but not dimerization, induces increased cytosolic calcium concentrations and reveals a lack of stable association between CD4 and the T cell receptor.

Exposure of T94, a CD4+ V beta 8-expressing murine Th cell clone, or immediately ex vivo CD4+ T cells to deaggregated, bivalent antibodies specific for either the TCR or CD3 failed to induce an increase in [Ca2+]i, or activation of phosphatidylinositol hydrolysis unless cross-linked with a secondary anti-Ig antibody. In contrast, we show that a combination of two mAb directed against different components of the TCR/CD3 complex (145.2C11, anti-CD3 epsilon and F23.1, anti-V beta 8) successfully induce second messenger formation, that is, without any requirement for a secondary antibody. This requirement for either a secondary antibody or two independent bivalent antibodies to activate second messenger production in T cells suggested that the signal transduction apparatus may be activated by multiple TCR/CD3 complexes being brought together on the T cell surface. This was supported by the observation that conditions inducing increased T cell [Ca2+]i through the TCR/CD3 complex also resulted in aggregation of the TCR/CD3 complex on the T cell surface. Conversely, binding of anti-TCR/CD3 antibodies to the T cell under conditions that did not induce increased [Ca2+]i also failed to induce surface TCR/CD3 redistribution. Cross-linking of the CD4 accessory molecule on T94 also resulted in increased [Ca2+]i, with kinetics similar to those observed after TCR/CD3 oligomerization. CD4 is involved in the recognition of invariant regions of MHC class II during Ag presentation and has been proposed to be associated with TCR/CD3 in the absence of Ag. Aggregation of TCR/CD3 and subsequent second messenger formation was achieved by combinations of mAb to distinct determinants within the complex due to the stable association of these determinants within the T cell membrane. We therefore assessed the functional association of CD4 with the TCR/CD3 complex by examining whether a combination of mAb directed against CD4 and CD3 or TCR induced second messenger formation. We found that anti-CD4 in combination with F23.1 or with 145.2C11 failed to induce increases in [Ca2+]i. Furthermore, mAb to CD4 failed to inhibit the increase in [Ca2+]i observed with the combination of 145.2C11 and F23.1. We therefore conclude that CD4 is not stably associated with TCR or CD3 in the absence of Ag/MHC class II composites.     

Loss of N-terminal charged residues of mouse CD3 epsilon chains generates isoforms modulating antigen T cell receptor-mediated signals and T cell receptor-CD3 interactions

The antigen T cell receptor (TCR)-CD3 complexes present on the cell surface of CD4(+) T lymphocytes and T cell lines express CD3 epsilon chain isoforms with different isoelectric points (pI), with important structural and functional consequences. The pI values of the isoforms fit the predicted pI values of CD3 epsilon chains lacking one, two, and three negatively charged amino acid residues present in the N-terminal region. Different T cells have different ratios of CD3 epsilon chain isoforms. At a high pI, degraded CD3 epsilon isoforms can be better recognized by certain anti-CD3 monoclonal antibodies such as YCD3-1, the ability of which to bind to the TCR-CD3 complex is directly correlated with the pI of CD3 epsilon. The abundance of CD3 epsilon isoforms can be modified by treatment of T cells with the proteinase inhibitor phenanthroline. In addition, these CD3 epsilon isoforms have functional importance. This is shown, first, by the different structure of TCR-CD3 complexes in cells possessing different amounts of isoforms (as observed in surface biotinylation experiments), by their different antigen responses, and by the stronger interaction between low pI CD3 epsilon isoforms and the TCR. Second, incubation of cells with phenanthroline diminished the proportion of degraded high pI CD3 epsilon isoforms, but also the ability of the cells to deliver early TCR activation signals. Third, cells expressing mutant CD3 epsilon chains lacking N-terminal acid residues showed facilitated recognition by antibody YCD3-1 and enhanced TCR-mediated activation. Furthermore, the binding avidity of antibody YCD3-1 was different in distinct thymus populations. These results suggest that changes in CD3 epsilon N-terminal chains might help to fine-tune the response of the TCR to its ligands in distinct activation situations or in thymus selection.     

Inhibition of CD4+ T cell activation and adhesion by peptides derived from the gp160.

It has been previously demonstrated that the HIV envelope glycoprotein gp160 can inhibit the activation of T cells triggered by phytohemagglutinin, anti-CD3 antibody and Ag, caused in part by the modulation of the expression of CD4. In this study, we show that gp160 is also able to inhibit the Ag-independent adhesion of CD4+ T cells to B cells as anti-CD4 antibodies do. In addition, synthetic peptides (14 to 21 mer) derived from the gp160 sequence and analogous to the putative binding site of gp160 to CD4 (residues 418-460), and also covering residues 460 to 474 inhibit the capacity of both CD4+ T cell proliferation induced by tuberculin and anti-CD3 antibody and adhesion. This was not associated with inhibition of Ca2+ flux in T cell activation. These inhibitory activities are specific because a) CD4+ T cells but not CD8+ T cells are susceptible to their effects, and b) soluble CD4 neutralizes the inhibitory activities. Peptides are, however, about 100- to 1000-fold less potent inhibitors than the native gp160. In addition, they do not induce CD4 modulation. It is thought therefore that at least part of the gp160 inhibitory activity is not secondary to CD4 modulation but may rely either upon steric hindrance of CD4-MHC class II interaction, of CD4/CD3 TCR complex interaction, or upon negative signaling through binding to CD4. The latter hypothesis is suggested by the inhibition by gp160, gp160-derived peptides, and anti-CD4 antibodies of the Ag-independent adhesion of CD4+ T cells. This adhesion process has been previously shown to be mediated by the LFA-1 and CD2 molecules and not by the TCR/CD3 complex and by CD4. Together, these results support the role of part of the 418-460 region of gp160 as a binding site to CD4, and suggest that binding of part of this region to CD4 can alter T cell proliferation and adhesion. It is proposed that these effects are mainly mediated by negative signaling through CD4.     

Activation requirements for CD4+ T cells differing in CD45R expression.

Murine CD4+ T cells can be subdivided into naive and memory T cells based on surface phenotype, on recall response to Ag, and on differences in activation requirements. Furthermore, several studies have shown that two signals are required for CD4+ T cell activation; one signal is provided by occupancy of the TCR and the other signal is provided by the APC. In this report, analysis of naive and memory CD4 T cells, separated on the basis of CD45 isoform expression, has shown that their requirements for two signals differ. Activation of memory CD4 T cells to proliferate and secrete IL-2/IL-4 only required occupancy of the TCR complex, whereas activation of naive CD4 T cells required an APC-derived signal as well. Moreover, the signal induced by anti-CD3 antibodies differs from the signal provided by anti-V beta cross-linking of the TCR because both antibodies activate memory CD4 T cells but only anti-CD3 activates naive CD4 T cells. Together these data suggest that the consequence of stimulation through the TCR/CD3 signal complex differs between memory and naive CD4 T cells.     

The CD3-gamma and CD3-delta subunits of the T cell antigen receptor can be expressed within distinct functional TCR/CD3 complexes.

The T cell receptor for antigen (TCR) consists of two glycoproteins containing variable regions (TCR-alpha/beta or TCR-gamma/delta) which are expressed on the cell surface in association with at least four invariant proteins (CD3-gamma, -delta, -epsilon and -zeta). CD3-gamma and CD3-delta chains are highly homologous, especially in the cytoplasmic domain. The similarity observed in their genomic organization and their proximity in the chromosome indicate that both genes arose from duplication of a single gene. Here, we provide several lines of evidence which indicate that in human and murine T cells which expressed both the CD3-gamma and CD3-delta chains on their surface, the TCR/CD3 complex consisted of a mixture of alpha beta gamma epsilon zeta and alpha beta delta epsilon zeta complexes rather than a single alpha beta gamma delta epsilon zeta complex. First, a CD3-gamma specific antibody failed to co-immunoprecipitate CD3-delta and conversely, several CD3-delta specific antibodies did not coprecipitate CD3-gamma. Secondly, analysis of a panel of human and murine T cell lines demonstrated that CD3-gamma and CD3-delta were expressed at highly variable ratios on their surface. This suggested that these chains were not expressed as a single complex. Thirdly, CD3-gamma and CD3-delta competed for binding to CD3-epsilon in transfected COS cells, suggesting that CD3-gamma and CD3-delta formed mutually exclusive complexes. The existence of these two forms of TCR/CD3 complexes could have important implications in the understanding of T cell receptor function and its role in T cell development.     

Differential Src family kinase activity requirements for CD3 zeta phosphorylation/ZAP70 recruitment and CD3 epsilon phosphorylation

The current model of T cell activation is that TCR engagement stimulates Src family tyrosine kinases (SFK) to phosphorylate CD3zeta. CD3zeta phosphorylation allows for the recruitment of the tyrosine kinase ZAP70, which is phosphorylated and activated by SFK, leading to the phosphorylation of downstream targets. We stimulated mouse CTLs with plate-bound anti-CD3 and, after cell lysis, recovered proteins that associated with the CD3 complex. The protein complexes were not preformed, and a number of tyrosine-phosphorylated proteins were inducibly and specifically associated with the TCR/CD3 complex. These results suggest that complex formation only occurs at the site of TCR engagement. The recruitment and tyrosine phosphorylation of most proteins were abolished when T cells were stimulated in the presence of the SFK inhibitor PP2. Surprisingly, CD3zeta, but not CD3epsilon, was inducibly tyrosine phosphorylated in the presence of PP2. Furthermore, ZAP70 was recruited, but not phosphorylated, after TCR stimulation in the presence of PP2, thus confirming the phosphorylation status of CD3zeta. These data suggest that there is a differential requirement for SFK activity in phosphorylation of CD3zeta vs CD3epsilon. Consistent with this possibility, ZAP70 recruitment was also detected with anti-CD3-stimulated, Lck-deficient human Jurkat T cells. We conclude that TCR/CD3-induced CD3zeta phosphorylation and ZAP70 recruitment do not absolutely require Lck or other PP2-inhibitable SFK activity, but that SFK activity is absolutely required for CD3epsilon and ZAP70 phosphorylation. These data reveal the potential for regulation of signaling through the TCR complex by the differential recruitment or activation of SFK.     

A fraction of CD3 epsilon subunits exists as disulfide-linked dimers in both human and murine T lymphocytes

In a T cell antigen receptor complex (TCR), the clonotypic disulfide-linked Ti heterodimer is noncovalently associated with the invariant CD3 polypeptides. The latter are composed of three monomeric subunits (gamma, delta, epsilon) and either a disulfide-linked homodimer (zeta zeta) or a disulfide-linked heterodimer (zeta eta). The exact stoichiometry of the Ti-CD3 subunits in a given complex is still largely unknown. Here, we report the presence of a CD3 epsilon dimer in a fraction of the TCR. When TCRs from both human and murine T lymphocytes were immunoprecipitated with monoclonal antibodies against either CD3 epsilon or Ti, a 40-kDa disulfide-linked dimer was coprecipitated with the other TCR subunits from digitonin lysates. Amino acid sequence analysis of peptides obtained by in situ CNBr cleavage of the 20-kDa product blotted to polyvinyl difluoride membranes from reducing/nonreducing two-dimensional gels identified human CD3 epsilon. Assuming this CD3 epsilon to derive from a homodimer, then either some TCRs contain more than one CD3 epsilon chain or several TCRs are covalently associated with one another via their CD3 epsilon subunits. Although it has been suggested that a putative TCR association with CD2 exists under similar conditions to those utilized to detect CD3 epsilon dimers, the CD2 molecule was not coimmunoprecipitated with the TCR by any of a series of anti-CD3 epsilon monoclonal antibodies. In conjunction with the fact that CD2 and the TCR do not colocalize during conjugate formation between T cells and antigen-presenting cells (Koyasu, S., Lawton, T., Novick, D., Recny, M. A., Siliciano, R. F., Wallner, B. P., and Reinherz, E. L. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2603-2607), we conclude that CD2 and the TCR are not physically associated on the T cell surface.     

CD3.TCR1, a human CD3 epitope expressed on viable gamma/delta lymphocytes exclusively.

T lymphocytes express either the alpha/beta or the gamma/delta receptor (TCR) in a mutually exclusive fashion. Both structures are associated on the cell membrane with the CD3 proteins which are thought to transduce signals resulting from antigen recognition. The CD3 complex is present in both alpha/beta and gamma/delta cells and includes at least five proteins (designated gamma, delta, epsilon, zeta and eta). We have developed here a novel mAb, anti-CD3.TCR1, which immunoprecipitates the CD3 molecules from both alpha/beta and gamma/delta cells lysates following solubilization with Triton X-100. While the SDS-PAGE migration profile of the material recognized by either anti-CD3.TCR1 or anti-OKT3 are superimposable in both cell types, this mAb recognizes viable untreated gamma/delta T lymphocytes exclusively. These findings further support the view that molecular interactions within the TCR/CD3 protein complex are distinct in the two T lymphocyte populations.     

A conformational epitope expressed upon association of CD3-epsilon with either CD3-delta or CD3-gamma is the main target for recognition by anti-CD3 monoclonal antibodies.

We have performed immunofluorescence analysis of COS cells transfected with human CD3 genes and detergent permeabilized to define the specificity of several anti-CD3 antibodies. We have found that the mAb OKT3, WT31, UCHT1, and Leu-4 did not stain COS cells singly transfected with the CD3-epsilon chain. However, these antibodies very strongly stained COS cells doubly transfected with a combination of CD3-epsilon plus either CD3-gamma or CD3-delta. By contrast, the antibodies SP34 and APA 1/1, which were raised against isolated SDS-denatured CD3-epsilon protein, gave a strong staining of COS cells singly transfected with CD3-epsilon as well as of the double transfectans. The recognition by this panel of anti-CD3 antibodies of CD3-gamma/epsilon and CD3-delta/epsilon complexes and not of CD3-epsilon alone was assessed by immunoprecipitation. These findings suggest that the most widely used mAb specific for the CD3 complex recognize conformational epitopes on CD3-epsilon, which are expressed when this chain is bound to either CD3-gamma or CD3-delta. It should also be highlighted that antibody WT31 clearly recognizes the CD3 moiety of the TCR/CD3 complex.     

Valency of CD3 binding and internalization of the CD3 cell-surface complex control T cell responses to second signals: distinction between effects on protein kinase C, cytoplasmic free calcium, and proliferation

We have studied the relationship of valency of CD3 stimulation and modulation of the CD3 receptor complex with biochemical and proliferative responses of T cells. Anti-CD3 Fab, as well as F(ab')2 and whole antibody caused rapid modulation of the CD3 antigen, whereas anti-CD3 conjugated to Sepharose did not. In the absence of monocytes, T cells stimulated with anti-CD3 Fab, F(ab')2, or F(ab')2-Sepharose showed differences in their ability to respond to second signals given by PMA, IL 1, IL 2, or antibodies to Tp67 and Tp44. None of the anti-CD3 signals alone caused resting T cells to produce IL 2, and only the Sepharose-bound anti-CD3 F(ab')2 caused T cells to express high levels of functional IL 2 receptors. Anti-CD3 F(ab')2-Sepharose-stimulated T cells produced IL 2 and proliferated in response to each of the second signals. Because anti-CD3-Sepharose did not cause modulation of the CD3 antigen, the ability of the Sepharose-bound antibody to induce T cells to express IL 2 receptors and to respond to individual second signals may be related to lack of modulation rather than valency of binding. Anti-CD3 Fab-stimulated T cells responded to PMA but required combinations of other second signals. T cells stimulated with unmodified anti-CD3 antibody or F(ab')2 fragments responded to PMA but did not respond to any other second signals alone or in combination. Stimulations that resulted in modulation (i.e., anti-CD3 whole antibody, anti-CD3 F(ab')2, or anti-CD3 Fab fragments) caused an increase in cytoplasmic calcium levels in resting T cells but blocked proliferation of T cells in response to mitogenic lectins or CD2 stimulation. Anti-CD3 F(ab')2 on Sepharose, however, did not block T cell proliferation. Whole bivalent anti-CD3 antibody or F(ab')2 fragments, but not monovalent Fab fragments, caused a rapid translation of protein kinase C activity from cytosol to membrane in the Jurkat T cell line. Because all of these modulate the receptor, these data indicate that the functional difference between monovalent and bivalent binding to CD3 is related to antibody valency and not to antigenic modulation. The use of Fab anti-CD3 stimulation that requires combinations of second signals for proliferation allowed an analysis of the functional relationships between IL 1, anti-Tp67, and anti-Tp44.     

Stimulation via the CD3 and CD28 molecules induces responsiveness to IL-4 in CD4+CD29+CD45R- memory T lymphocytes

Although both IL-2 and IL-4 can promote the growth of activated T cells, IL-4 appears to selectively promote the growth of those helper/inducer and cytolytic T cells which have been activated via their CD3/TCR complex. The present study examines the participation of CD28 and certain other T cell-surface molecules in inducing T cell responsiveness to IL-4. Purified small high density T cells were cultured in the absence of accessory cells with various soluble anti-human T cell mAb with or without soluble anti-CD3 mAb and their responsiveness to IL-4 was studied. None of the soluble anti-T cell mAb alone was able to induce T cell proliferation in response to IL-4. A combination of soluble anti-CD3 with anti-CD28 mAb but not with mAb directed at the CD2, CD5, CD7, CD11a/CD18, or class I MHC molecules induced T cell proliferation in response to IL-4. Anti-CD2 and anti-CD5 mAb enhanced and anti-CD18 mAb inhibited this anti-CD3 + anti-CD28 mAb-induced T cell response to IL-4. In addition, anti-CD2 in combination with anti-CD3 and anti-CD28 mAb induced modest levels of T cell proliferation even in the absence of exogenous cytokines. IL-1, IL-6, and TNF were each unable to replace either anti-CD3 or anti-CD28 mAb in the induction of T cell responsiveness to IL-4, but both IL-1 and TNF enhanced this response. The anti-CD3 + anti-CD28 mAb-induced response to IL-4 was exhibited only by cells within the CD4+CD29+CD45R- memory T subpopulation, and not by CD8+ or CD4+CD45R+ naive T cells. When individually cross-linked with goat anti-mouse IgG antibody immobilized on plastic surface, only anti-CD3 and anti-CD28 mAb were able to induce T cell proliferation. These results indicate that the CD3 and CD28 molecules play a crucial role in inducing T cell responsiveness to IL-4 and that the CD2, CD5, and CD11a/CD18 molecules influence this process.     

Sustained linked stimulation via CD3 and CD4 is required for the IL-4-independent development of IL-4 synthesizing CD4+ T cells

Previous work has shown that CD4 engagement can promote the development of interleukin-4-producing cells from naive CD4+ T cells activated with anti-CD3 antibody and interleukin-2 in the absence of other exogenous signals, including interleukin-4 itself. When CD44low CD4+ T cells were activated with immobilized anti-CD3 antibody and interleukin-2, they proliferated and produced interferon-gamma but not interleukin-4. Co-immobilization of antibodies to CD3 and CD4 enhanced cell recoveries and induced interleukin-4 as well as interferon-gamma synthesis. Here we show that these effects of CD4 ligation were not observed when anti-CD4 antibody was replaced with another CD4 ligand, interleukin-16, or when the anti-CD3 and anti-CD4 antibodies were spatially separated by immobilization on different beads. Removal of the anti-CD4 antibodies within the first three days of stimulation also prevented the development of detectable interleukin-4-producing cells. The data suggest that interleukin-4-independent priming of interleukin-4-producing cells in this system requires sustained stimulation via both the T cell receptor and CD4 with close physical association between the ligands for these two receptors.     

The role of CD4 in antigen-independent activation of isolated single T lymphocytes

The membrane molecule CD4 (L3T4) is thought to facilitate activation of Class II H-2-restricted T cells by binding to Ia determinants on antigen-presenting cells. Recent reports suggest that CD4 can also contribute to antigen-independent activation by anti-T cell receptor (TCR) antibodies. An assay which measures the secretion of two lymphokines, granulocyte-macrophage colony-stimulating factor and interleukin 3 (IL-3), by single T cells activated with an anti-TCR antibody, F23.1, was used to analyze the effects of anti-CD4 antibodies on antigen-independent T cell activation. Single cells of a CD4+F23.1+ clone were micromanipulated into wells to which F23.1 had been immobilized, and their lymphokine secretion was measured 24 hr later. The frequency of lymphokine-secreting cells was consistently reduced up to 10-fold in the presence of soluble anti-CD4 antibody (GK1.5) but only up to 2.5-fold by an antibody to the cell adhesion molecule, LFA-1. In both bulk and single-cell cultures, responses to suboptimal concentrations of F23.1 were more susceptible to inhibition by GK1.5 than responses to optimal F23.1. The failure of GK1.5 to inhibit IL-2-stimulated lymphokine synthesis in bulk cultures suggested that CD4 ligation did not deliver a negative signal to the clone. By contrast, when either anti-CD4 or anti-LFA-1 was immobilized on the same surface as F23.1, the frequency of lymphokine-secreting cells could be increased up to 10-fold. It is concluded that anti-CD4 antibodies can act directly on the responding T cell to affect TCR-dependent activation, in the absence of interaction with antigen-presenting cells or any other cell type.     

The CD4 molecule transmits biochemical information important in the regulation of T lymphocyte activity

The role of the CD4 molecule in the transmission and regulation of the biochemical signals involved in T cell activation was investigated using an anti-CD4 monoclonal antibody termed 6B10. 6B10 immunoprecipitated the 55-kDa CD4 molecule and detected an epitope of CD4 that overlapped with that detected by OKT4A, B, and D. 6B10, 6B10 Fab fragments and recombinant HIV envelope glycoprotein (gp120) induced calcium mobilization in PBMC. 6B10 stimulation also resulted in calcium mobilization in murine L cells expressing transfected CD4 gene products, indicating that CD4-mediated calcium mobilization occurred independently of the CD3/T cell receptor (TCR) complex. 6B10 induced a phosphatidylinositol response, but the response resulted in reduced inositol phosphate production compared to levels obtained using OKT3. Though 6B10 caused calcium mobilization and a phosphatidylinositol response, 6B10 did not induce DNA synthesis. The amount of inositol phosphates produced by 6B10 may be below the threshold necessary for cell cycle progression. We hypothesized that 6B10-mediated calcium mobilization is important in the regulation of T cell proliferation. 6B10, but not 6B10 Fab fragments, inhibited OKT3-induced DNA synthesis. Furthermore, 6B10 but not 6B10 Fab fragments inhibited OKT3-induced calcium mobilization, suggesting that crosslinking of CD4 may be an important factor determining whether signals result in both the up- and down-regulation of CD3/TCR complex function. The implication of this work is that signals generated via the CD4 molecule are important in the regulation of T cell function and that the signals generated as a result of HIV gp120 binding to CD4 can contribute to the mechanism by which HIV inhibits T cell function.     

Kinetics of anti-CD4-induced T helper cell depletion and inhibition of function. Activation of T cells by the CD3 pathway inhibits anti-CD4-mediated T cell elimination and down-regulation of cell surface CD4.

In vivo treatment with anti-CD4 antibody profoundly suppresses a number of T cell-dependent responses and is clinically useful in the treatment of certain mouse models of autoimmune disease. Treatment with anti-CD4 antibody will inactivate and can deplete CD4 T cells, but the mechanisms responsible for these effects are incompletely understood. When mouse spleen cells were exposed in vitro to both SRBC and monoclonal anti-CD4, there was 55% reduction of the anti-SRBC response. If cultures were preincubated with anti-CD4 for 48 h before in vitro challenge, the reduction was greater than 80%. When unfractionated spleen cells were cultured with anti-CD4 for 96 h, there was actual elimination of CD4 cells in these cultures since virtually all CD3+ cells were CD8+. Activation of T cells by exposure to anti-CD3 rendered them resistant to antibody-mediated CD4 depletion. This resistance to CD4 depletion was seen even in cultures that were pretreated with anti-CD4 for as long as 24 h before anti-CD3 exposure. In cultures of purified T cells, anti-CD4 did not eliminate CD4 T cells. However, culture of T cells with macrophage-rich adherent cells and anti-CD4 resulted in elimination of CD4 T cells. Thus, it appears that macrophages play a role in anti-CD4-induced T cell elimination. While anti-CD4 did not eliminate CD4 cells from a population of purified T cells, there was profound down-regulation of cell surface CD4. Activating T cells with immobilized anti-CD3 before addition of anti-CD4 prevented down-regulation of CD4. These experiments demonstrate that T cell activation by anti-CD3 renders the activated cells resistant to antibody-induced CD4 down-regulation and to antibody-induced CD4 T cell depletion. These findings may have relevance to the application of anti-CD4 therapy in human diseases that are mediated by activated Th cells.     

Thymic nurse cells exclusively bind and internalize CD4+CD8+ thymocytes.

Thymic nurse cells (TNC) contain 20-200 thymocytes within specialized vacuoles in their cytoplasm. The purpose of the uptake of thymocytes by TNCs is unknown. TNCs also have the capacity to present self-antigens, which implies that they may serve a function in the process of thymic education. We have recently reported the development of thymic nurse cell lines that have the ability to bind and internalize T cells. Here, we use one of these TNC lines to identify the thymocyte subpopulation(s) involved in this internalization process. TNCs exposed to freshly isolated thymocytes bind and internalize CD4 and CD8 expressing thymocytes (CD4+CD8+ or double positives) exclusively. More specifically, a subset of the double-positive thymocyte population displayed binding capacity. These double-positive cells express cell surface alpha beta type T cell antigen receptor (TCR), as well as CD3 epsilon. Binding was not inhibited in the presence of antibodies against CD3, CD4, CD8, Class I antigens, or Class II antigens. These results describe two significant events in T cell development. First, TNCs exclusively bind and internalize a subset of alpha beta TCR expressing double-positive T cells. Also, binding is facilitated through a mechanism other than TCR recognition of major histocompatibility complex antigens. This suggests that thymocyte internalization may be independent of the process used by TNCs to present self-antigen.     

CD8-independent tumor cell recognition is a property of the T cell receptor and not the T cell

The CD8 coreceptor enhances T cell function by stabilizing the TCR/peptide/MHC complex and/or increasing T cell avidity via interactions with the intracellular kinases Lck and LAT. We previously reported a CD4(+) T cell (TIL 1383I), which recognizes the tumor-associated Ag tyrosinase in the context of HLA-A2. To determine whether CD8 independent tumor cell recognition is a property of the TCR, we used retroviral transduction to express the TIL 1383I TCR in the CD8(-) murine lymphoma, 58 alpha(-)/beta(-). Immunofluorescent staining of TCR-transduced cells with human TCR V beta subfamily-specific and mouse CD3-specific Abs confirmed surface expression of the transferred TCR and coexpression of mouse CD3. Transduced effector cells secreted significant amounts of IL-2 following Ag presentation by tyrosinase peptide-pulsed T2 cells as well as stimulation with HLA-A2(+) melanoma lines compared with T2 cells alone or HLA-A2(-) melanoma cells. Further analysis of TCR-transduced clones demonstrated a correlation between T cell avidity and cell surface expression of the TCR. Therefore, the TIL 1383I TCR has sufficient affinity to mediate recognition of the physiologic levels of Ag expressed by tumor cells in the absence of CD8 expression.