A common precursor for CD4+ T cells producing IL-2 or IL-4.

To investigate whether CD4+ T cells are predetermined to produce a given pattern of lymphokines, we have used a culture system that allows the controlled induction of either IL-2- or IL-4-producing CD4+ T cells. Single, freshly isolated murine CD4+ T cells were activated with Con A, rIL-2, and APC; the developing clones were split and then cultured for an additional 14 days with either rIL-2 alone or with rIL-2 and anti-CD3 stimulation. Subclones expanded in the presence of rIL-2 alone produced predominantly IL-2, although subclones derived from the same precursor and expanded in the presence of rIL-2 and a mitogenic antibody to CD3 released predominantly IL-4. Subclones expanded for 2 wk in the presence of rIL-2 plus a mitogenic mAb to CD3 released up to 60 times more IL-4 but only 1/90 the amount of IL-2 released by subclones derived from the same precursor cell and expanded with rIL-2. Both phenotypes can be derived from IL-2-producing precursor cells. These results demonstrate that IL-2-producing clones can be derived from the same cells as IL-4-producing clones and are most consistent with the view that the IL-2-producing Th1 or the IL-4-producing Th2 phenotype of a T cell clone is acquired during T cell differentiation and is not secondary to the expansion of distinct subpopulations that are predetermined to produce a specific cytokine pattern.     

CD8+ T cells can be primed in vitro to produce IL-4.

IL-4 production by T lymphocytes from naive mice in response to stimulation by plate-bound anti-CD3 is concentrated among CD4+ T cells. In vitro stimulation of lymph node T cells with anti-CD3 plus IL-2 and IL-4 strikingly increases the frequency of cells that produce IL-4 in response to subsequent stimulation with anti-CD3 plus IL-2. Separation of these primed cell populations into CD4+ and CD8+ T cell by cell sorting reveals that the frequency of IL-4-producing cells in both population is similar. Verification that CD8+ T cells produce IL-4 is provided by the capacity of anti-IL-4 mAb to inhibit the response of the indicator cell line to the growth factor produced by the primed cells and by detection of IL-4 by an IL-4-specific ELISA. The in vitro "priming" of CD8+ T cells to produce IL-4 is not dependent on the presence of CD4+ T cells because highly purified CD8+ T cells can be stimulated to develop into cells capable of producing IL-4 by culture with plate-bound anti-CD3 plus IL-2 and IL-4.     

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.     

IL-4 production by T cells from naive donors. IL-2 is required for IL-4 production

Utilizing a sensitive and selective assay for IL-4, it was shown that lymph node T cells from naive mice could produce small amounts of this lymphokine in response to anti-CD3 antibodies adsorbed to culture dishes. The capacity of these cells to produce IL-4 in response to plate-bound anti-CD3 was substantially enhanced by the addition of IL-2 to the culture and was strikingly inhibited by monoclonal anti-IL-2 antibody. Thus, IL-2 appears to be essential for IL-4 production by anti-CD3 antibody-stimulated T cells from naive mice. The effect of IL-2 was not mediated either by preferential proliferation or survival of precursors of IL-4 producing cells, indicating that IL-2 regulates T cell production of IL-4. IL-4 producing capacity of T cells from naive mice was found mainly among CD4+ T cells. Large T cells produced much more IL-4, on a per cell basis, than did small T cells. In contrast, small T cells appeared to be equal or superior to large T cells in producing IL-2. The superiority of large T cells in IL-4-producing capacity was not accounted for by a lack of an accessory cell population from the small T cells as addition of large spleen cells depleted of both B and T cells did not enhance IL-4 production by small lymph node T cells. These results suggest that the bulk of IL-4 production by T cell populations, from normal mice, in response to anti-CD3 depends upon cells that are already activated and that IL-2 is required for such production.     

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.     

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.     

Lectin-mediated induction of IL-4-producing CD4+ T cells.

Cultured murine CD4+ T cells have been shown to differentiate into IL-2 or IL-4-producing subsets. The factors responsible for the development of CD4+ T cells which produce IL-2 but not IL-4 and cells capable of producing IL-4 but not IL-2 are unknown. Here we describe a system that allows the controlled induction of IL-2- or IL-4-producing T cells after one single round of activation. Freshly isolated CD8-depleted T cells were activated with various polyclonal T cell activators for 48 h, washed, and then expanded under different conditions. IL-2 and IL-4 production were induced by restimulation of T cells and were measured with CTLL cells that respond to both cytokines and mAb to IL-2 and IL-4. T cells produced mainly IL-2 and small amounts of IL-4 when restimulated after expansion culture for 12 days with rIL-2 alone. However, after expansion for 12 days in the presence of rIL-2 plus Con A, we observed a 30- to 100-fold up-regulation of IL-4 activity and a 100-fold down-regulation of IL-2 when assessed by responses of CTLL cells incubated with the supernatant of restimulated T cells and by responses of CTLL cells cocultured with restimulated cells. An increase of IL-4 and decrease of IL-2 was also observed when the results were based on the cell numbers at the beginning of the expansion culture. The induction of IL-4 and the down-regulation of IL-2 1) were not reproduced with alpha-methyl-mannoside-treated supernatant of Con A-stimulated spleen cells, 2) were not dependent on the presence of large numbers of APC, 3) did not result from differential consumption of lymphokines after restimulation, 4) were not due to a difference in the time course of IL-2 or IL-4 release in either T cell population, and 5) were obtained regardless of the agents used to activate or to restimulate the T cells. Because Con A remained detectable on the T cell surface and because expansion of activated T cells with IL-2 plus Con A for several days was necessary, our results indicate that mainly IL-4-producing CD4+ T cells can be induced by prolonged engagement of T cell surface molecules.     

Role of CD11a/CD18-CD54 interactions in suppression of human B cell responsiveness by CD4+ suppressor T cells.

The role of leukocyte function-associated Ag-1 (LFA-1, CD11a/CD18) and intercellular adhesion molecule 1 (ICAM-1, CD54) interactions in the suppression of human B cell function by immobilized anti-CD3-activated CD4+ T cells was examined by studying the effects of mAb to these determinants. The suppressive activity was assessed by the effects of CD4+ T cells without mitomycin C treatment activated by immobilized anti-CD3 for 72 hr on the differentiation into Ig-secreting cells of B cells activated for 72 hr with immobilized anti-CD3-stimulated CD4+ T cells that had been treated with mitomycin C (T4 mito). Suppression was not observed when activated CD4+ T cells and B cells were separated by filter membranes, indicating that the suppression requires the direct interactions between anti-CD3-activated CD4+ T cells and activated B cells. In this model system, mAb to either the alpha (CD11a) or beta (CD18) chain of LFA-1 or ICAM-1 (CD54) reversed the suppression of B cell function by suppressor CD4+ T cells significantly. Reversal of suppression of B cell function was most marked when activated B cells were treated with mAb to ICAM-1 and suppressor CD4+ T cells were treated with mAb to LFA-1, but not vice versa. Studies using fluorescence-activated cell sorter revealed marked increase of expression of ICAM-1 on B cells after 72 hr of activation with immobilized anti-CD3-stimulated T4 mito. These results indicate that the interactions between LFA-1 and ICAM-1 play an important role in mediating the suppressive activity of anti-CD3-activated CD4+ T cells to B cells. Moreover, the data are consistent with a model of T-cell-mediated B cell suppression in which interactions between LFA-1 on suppressor T cells and ICAM-1 on activated B cells play a central role in the suppression of B cell function.     

Role of IL-2 in the generation of CD4+ suppressors of human B cell responsiveness

The capacity of human T4 cells stimulated by immobilized monoclonal antibodies to the CD3 molecular complex (64.1 and OKT3) to induce and regulate B cell responsiveness was examined. T4 cells stimulated by low concentrations of immobilized 64.1 (3.0 ng/well) and all concentrations of immobilized OKT3 supported B cell proliferation and differentiation. High concentrations of immobilized 64.1 (200 ng/well) failed to stimulate help but rather induced suppression by T4 cells. Suppression was prevented by treating the T4 cells with mitomycin C. Suppression could not be accounted for by deprivation of IL-2. In contrast, induction of suppressor T4 cell activity was closely related to the amount of IL-2 produced by anti-CD3 stimulated T4 cells. Moreover, IL 2 appeared to facilitate the generation of suppressor T4 cell activity. Suppressor cell activity could be generated from unseparated T4 cells as well as from highly purified T4 cell subsets, including Leu 8-, CD45R+, and CD45R- T4 cells, after stimulation with immobilized 64.1. A primary action of suppressor T4 cells appeared to be the direct inhibition of B cell function, as evidenced by the finding that immobilized anti-CD3 activated T4 cells directly suppressed B cell responses stimulated by Staphylococcus aureus and IL-2. Anti-CD3 activated T4 cells did not inhibit initial B cell activation, but suppressed the capacity of the activated B cells to differentiate into ISC. The suppressive influence of anti-CD3 activated T4 cells was reversible as evidenced by the finding that removal of the activated T4 cells from the culture permitted B cell differentiation to proceed. Moreover, anti-CD3-activated T4 cells were able to stimulate initial B cell activation that became apparent when the T cells and B cells were separated. Inhibition of B cell responsiveness by 64.1-activated T4 cells was the result of a block at the G1-S interphase of the cell cycle. The data indicate that anti-CD3-stimulated T4 cells directly and reversibly suppress human B cell function. Moreover, IL 2 appears to play an important role in the differentiation of functionally effective suppressor cells from activated T4 cells.     

Cytochalasins enhance the proliferation of CD4 cells through the CD3-Ti antigen receptor complex or the CD2 molecule through an effect on early events of activation.

Cytochalasins are known to inhibit or enhance the proliferation of T cells induced by mitogens in a concentration-dependent fashion. To clarify the mechanism by which cytochalasins enhance T cell proliferation, we examined which activation pathways and events in signal transduction were affected by cytochalasins. We also examined subsets of CD4 cells for a preferential response to cytochalasins. Cytochalasins enhanced the proliferation of CD4 cells induced by optimal doses of anti-CD3 antibody or suboptimal doses of anti-CD2 antibodies. Cytochalasins, at low concentrations, enhanced the rise in intracellular Ca2+ and production of IP3 in CD4 cells activated by anti-CD2 or CD3 antibodies. Cytochalasins also enhanced the modulation of CD3 induced by anti-CD3 antibody. These results suggest that cytochalasins enhance the proliferation of CD4 cells by affecting early events in signal transduction after activation through the CD3-Ti Ag-receptor complex or CD2 molecule. At the doses used, cytochalasins appear to interact with cytochalasin-binding sites in the cell membrane. Cytochalasins predominantly enhanced CD3-mediated proliferation in the CD29-subset of CD4 cells.     

Suppression of antibody synthesis by CD4+ T cell clones and normal T cells stimulated with monoclonal anti-CD3 antibody

CD4+ve Th1 clones, as well as normal splenic T cells, were found to suppress LPS-driven antibody secretion in a non-Ag-specific and non-MHC-restricted manner when the T cells were activated with the anti-CD3 mAb, 145-2C11. Suppression was observed with both primed and naive B cells, as well as with purified hapten-specific B cells, a result that suggests a direct effect of anti-CD3-activated T cells on B cell differentiation. Th1 clones activated by cognate Ag also suppressed LPS-driven antibody secretion. Furthermore, suppression of LPS-driven antibody secretion could be achieved across a cell-impermeable porous membrane when T cells were activated with anti-CD3. Suppression by Th1 clones and by normal T cells could not be attributed to a concomitant decrease in B cell proliferation or to a shift in the kinetics or isotype of the antibody response. These data demonstrate that CD4+ve Th1 clones, as well as normal T cells, can effect suppression of polyclonal antibody formation.     

CD4 ligation promotes the IL-4-independent development of IL-4-producing clones from naive CD4(+) T cells.

The signals that trigger IL-4-independent IL-4 synthesis by conventional CD4(+) T cells are not yet defined. In this study, we show that coactivation with anti-CD4 mAb can stimulate single naive CD4(+) T cells to form IL-4-producing clones in the absence of APC and exogenous IL-4, independently of effects on proliferation. When single CD4(+) lymph node cells from C57BL/6 mice were cultured with immobilized anti-CD3epsilon mAb and IL-2, 65-85% formed clones over 12-14 days. Coimmobilization of mAb to CD4, CD11a, and/or CD28 increased the size of these clones but each exerted different effects on their cytokine profiles. Most clones produced IFN-gamma and/or IL-3 regardless of the coactivating mAb. However, whereas 0-6% of clones obtained with mAb to CD11a or CD28 produced IL-4, 10-40% of those coactivated with anti-CD4 mAb were IL-4 producers. A similar response was observed among CD4(+) cells from BALB/c mice. Most IL-4-producing clones were derived from CD4(+) cells of naive (CD44(low) or CD62L(high)) phenotype and the great majority coproduced IFN-gamma and IL-3. The effect of anti-CD4 mAb on IL-4 synthesis could be dissociated from effects on clone size since anti-CD4 and anti-CD11a mAb stimulated formation of clones of similar size which differed markedly in IL-4 production. Engagement of CD3 and CD4 in the presence of IL-2 is therefore sufficient to induce a substantial proportion of naive CD4(+) T cells to form IL-4-producing clones in the absence of other exogenous signals, including IL-4 itself.     

Different effects of IL-2 addition or antibody crosslinking on T-cell subset stimulation by CD3 antibodies

The effect of exogenous recombinant interleukin-2 (IL-2) or of antibody crosslinking on the activation of human T-cell subsets by IgG2a (OKT3/BMA030), IgG1 (Leu4 and UCHT1), or IgG2b (BMA031) anti-T3 antibodies (CD3) was investigated. In so-called nonresponder cultures as well as in monocyte-depleted cell cultures addition of IL-2 increased the CD3-induced activation and proliferation of T4 and T8 cell subsets. Relatively more T8 than T4 cells were stimulated by antibody binding and IL-2. Crosslinking the cell-bound CD3 antibodies by plastic bound goat anti-mouse antibodies activated both T-cell subsets optimally and increased the IL-2 production of the IgG1-CD3 stimulated cultures. The data show that T cells (T8 greater than T4) can be stimulated by CD3 antibody binding and IL-2, but that crosslinking the cell-bound CD3 antibodies is crucial for optimal T4 cell stimulation and IL-2 production.     

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.     

Resting human peripheral blood lymphocytes can be activated to cytolytic function by antibodies to CD3 in the absence of exogenous interleukin-2

We investigated the ability of anti-CD3 antibodies to activate resting human peripheral blood lymphocytes (PBL) to a cytolytic function. We found that two anti-CD3 antibodies, but not an anti-CD4, anti-CD8, or anti-CD2 antibody, could activate resting unseparated PBL to become killer cells in the absence of exogenous interleukin-2 (IL-2), although exogenous recombinant IL-2 (rIL-2) synergized with anti-CD3. We also found that these anti-CD3 antibodies were active in the absence of rIL-2 only when linked to a solid surface such as a Sepharose bead or a plastic tissue culture plate. Cytolytic activity was measured in several ways: (i) by the ability of activated PBL to lyse the NK-sensitive line K562, and (ii) by the ability of these cells to lyse a CD10+ (CALLA+), NK-resistant target in the presence of either concanavalin A (lectin-dependent lysis) or an anti-CD10-anti-CD3 heterodimer. At least two different types of cytolytic cells were activated by anti-CD3 antibodies, an NK-like cell, which was CD2+CD3-CD4-CD8-CD16+-NKH1a+, and a CTL-like cell, which was CD2+CD3+CD4-CD8+CD16-NKH1a-. The former cell lysed the K562 line and the latter cell lysed Namalwa in the presence of the anti-CD10-anti-CD3 heterodimer or concanavalin A. The NK-like cell was probably activated by endogenous IL-2 produced by the anti-CD3-activated CD3+ cells and both the NK and CTL-like cells required the presence of adherent cells for maximal activity. The dose response and the kinetics of anti-CD3 activation of PBL to cytolytic activity were also studied. The use of the anti-CD3-activated cytolytic cells as effectors in anti-CD3 heterodimer-mediated lysis of tumor cells may be a novel approach to the therapy of cancer, and a comparison with the well-studied rIL-2/lymphokine-activated killer (LAK) system is discussed.     

Identification of the anti-CD3-unresponsive subpopulation of CD4+, CD45RA+ peripheral T lymphocytes.

The majority of peripheral CD4+ T lymphocytes proliferate in vitro in response to anti-CD3 in presence of autologous APC. The present study describes a subpopulation of CD4+ T cells that cannot be activated and progress into cell cycle by stimulation with anti-CD3 plus APC or with mitogenic combinations of anti-CD2. The in vitro responses of these anti-CD3-unresponsive CD4+ T cells were investigated with a panel of mAb to CD2, CD3, and CD28, and found to be similar to those previously observed for mature thymocytes: only the combination of anti-CD2 plus anti-CD28 produced cell proliferation. Anti-CD3-unresponsive T cells were CD45RA+, but represented only 14 to 22% of the CD4+, CD45RA+ T cell population. Activation with anti-CD2 plus anti-CD28 mAb resulted in major changes in the cell surface phenotype and functional properties: a loss of CD45RA+ occurred and an increased expression of CD45RO, CD29, and CD58 (LFA3), as well as a gain in responsiveness to anti-CD3 and anti-CD2. This change in CD45 phenotype from CD45RA to CD45RO occurs in both the anti-CD3-responsive and in the anti-CD3-unresponsive subsets of the CD45RA+, CD4+ cells after cell proliferation. The anti-CD3-unresponsive subset may represent a pool of not yet fully differentiated peripheral T cells. The acquisition of anti-CD3 responsiveness could occur as a consequence of Ag priming or by an Ag-independent mechanism. Involvement of the CD28 Ag in this process is suggested from the present study.     

Frequency analysis of lymphokine-secreting CD4+ and CD8+ T cells activated in a graft-versus-host reaction

Lymphokine secretion by in vivo-activated T cells was analyzed at the population and single-cell levels in lymphocytes from mice undergoing an acute allogeneic graft-vs-host reaction (GVHR). Three observations were made. First, constitutive lymphokine production by these cells was very low but could be dramatically up-regulated by TCR ligation. Thus, even when harvested at the peak of the GVHR, fewer than 0.1% of lymphocytes secreted detectable granulocyte-macrophage (GM)-CSF, IFN-gamma, or IL-3 in the first 24 h in vitro, and average production of these lymphokines in bulk cultures was less than 10(-5) U/cell. However, when cultured for 24 h with anti-CD3 antibody under conditions which activated less than 0.1% of normal cells, about 30% of GVHR T cells secreted GM-CSF, IFN-gamma, and/or IL-3, and average production levels were increased by 10(3)- to 10(4)-fold. Together with evidence that host alloantigen-induced lymphokine secretion was 10 to 100 times lower than the anti-CD3 response, these data suggest that physiologic lymphokine synthesis by most T cells is low (less than 10(-18) mol of IL-3 per cell) but can be raised above the threshold of detection by TCR cross-linking. Second, individual GVHR lymphocytes varied markedly in their total and relative production of different lymphokines in response to anti-CD3 stimulation, with some cells secreting IL-3 alone, some secreting IL-3 accompanied by other lymphokines (GM-CSF and/or IFN-gamma), and some secreting other lymphokines without detectable IL-3. Finally, both CD4+ and CD8+ T cells from GVHR mice responded to anti-CD3 antibody by secreting IL-3 and other lymphokines: purified CD4+ cells contained an average of 16% and CD8+ cells an average of 10% anti-CD3-inducible lymphokine-secreting cells. By contrast, only 2 to 3% of cells of either subset formed clones in cultures with host allogeneic cells and IL-2, suggesting that clonogenic alloreactive cells were a minority of the T cells activated in the GVHR.     

Athymic nude CD4+8- T cells produce IL-2 but fail to proliferate in response to mitogenic stimuli

A comparative study of immune functions of CD4+8- T cells isolated from normal and athymic nude mice by electronic cell sorting was performed. Athymic nude CD4+8- T cells expressed the TCR-associated CD3 molecule but the level of expression was significantly lower than that of normal CD4+8- T cells. Proliferative responses were studied upon stimulation by 1) the T cell mitogen Con A; 2) anti-CD3 mediated cross-linking of the CD3:TCR complex, and 3) the combined action of PMA + ionomycin. All three mitogenic stimuli caused readily detectable cell division in normal (euthymic) CD4+8- T cells. In marked contrast, none of the mitogenic stimuli induced significant proliferation in athymic nude CD4+8- T cells. The failure of athymic nude CD4+8- T cells to proliferate occurred over a wide range of mitogen concentrations and over a 4-day observation period. Neither exogenously supplied rIL-2 or mixed lymphocyte culture supernatant had any effect on the impaired proliferative response by athymic nude CD4+8- T cells. Although IL-2 was produced by athymic nude CD4+8- T cells at a reduced level when compared to normal CD4+8- T cells, it was nevertheless readily detected upon stimulation with either Con A or anti-CD3. Furthermore, stimulation of athymic nude CD4+8- T cells by anti-CD3 induced the expression of the p55 chain of IL-2R on the cell surface. Therefore, despite production of IL-2 and induced expression of IL-2R, athymic nude CD4+8- T cells failed to undergo cell division.     

Generation propagation, and targeting of human CD4+ helper/killer T cells induced by anti-CD3 monoclonal antibody plus recombinant IL-2. An efficient strategy for adoptive tumor immunotherapy.

We developed a culture system for the rapid generation of CD4+ T cells that have both helper and killer functions. CD4+ T cells isolated from human PBL did not proliferate or develop significant cytotoxicity when treated with rIL-2 because of the lack of p75 IL-2R expression. However, culture of isolated CD4+ T cells with immobilized anti-CD3 mAb plus rIL-2 resulted in a marked proliferation (500-fold increase in 14 days) of CD4+ T cells. The proliferating CD4+ T cells produced IL-2 (92 U/ml) and showed strong cytotoxicity against OKT3 hybridoma cells and Daudi, K562, and U937 tumor cells in an anti-CD3 mAb-dependent manner. The CD4+ T cells contained significant amounts of cytolytic granule-related proteins such as serine esterase and perforin. Activated CD4+ helper/killer cells can be generated from both healthy donors and tumor patients and can be propagated in vitro for 14 to 35 days by biweekly restimulation with immobilized anti-CD3 mAb plus rIL-2. This culture yielded about 20,000-fold increase in cell number after a 21-day culture. Bispecific antibody containing anti-CD3 and anti-glioma Fab components enhanced the cytotoxicity of activated CD4+ helper/killer cells against IMR32 glioma cells. Moreover, the activated CD4+ helper/killer cells showed both helper and antitumor activity in vivo and prevented growth of anti-CD3 hybridoma cells in nude mice whether or not IL-2 was administered. These results indicate that anti-CD3 mAb plus IL-2-activated CD4+ helper/killer cells may provide an effective strategy for adoptive tumor immunotherapy of cancer.     

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.