Anti-CD3 + IL-2-stimulated murine killer cells. In vitro generation and in vivo antitumor activity

The growth, phenotype, in vitro cytolytic characteristics, and in vivo antitumor activity of murine splenocytes stimulated with anti-murine CD3 mAb in combination with IL-2 as compared with IL-2 alone was investigated. When cultured for 12 days with anti-CD3 mAb + IL-2, murine splenocytes increased 100- to 4000-fold in number compared with only 6- to 20-fold for cultures stimulated with IL-2 alone. Anti-CD3 mAb + IL-2 activated cultures developed high lymphokine-activated killer activity against NK-resistant targets including the P815 mastocytoma cell line and fresh MCA 106 sarcoma. Peak cytotoxicity on a per cell basis developed by day 8 after anti-CD3 mAb + IL-2 activation. A large proportion of the total cytolytic activity of long term anti-CD3 mAb + IL-2-stimulated cultures was related to the presence of anti-CD3 in the assay, indicating enhancement of cytotoxicity by activated CD3+ T cells. Phenotypic analysis indicated that anti-CD3 mAb + IL-2-stimulated cultures contained heterogeneous populations of T cells with increased percentages of both CD4+ and CD8+ phenotypes compared with cultures stimulated with IL-2 alone. Anti-CD3 mAb + IL-2-stimulated cells were tested for their in vivo antitumor activity by using C57BL/6 mice bearing MCA 106 sarcoma pulmonary metastases. IL-2-activated murine killer cells were given in combination with in vivo IL-2 and indomethacin, the latter of which was shown to potentiate the antitumor effect of IL-2. When given on day 5 after tumor inoculation, cell doses as low as 5 x 10(6) anti-CD3 mAb + IL-2-stimulated cells per mouse significantly reduced the number of pulmonary metastases (p less than 0.005). Thus, activation with the combination of anti-CD3 mAb + IL-2 produces rapidly expanding cultures of cytolytic cells with demonstrated in vivo antitumor efficacy.     

The anti-T cell monoclonal antibody 9.3 (anti-CD28) provides a helper signal and bypasses the need for accessory cells in T cell activation with immobilized anti-CD3 and mitogens

CD28 is an antigen of 44 kDa which is expressed on the membrane of the majority of human T cells. The present study examines the functional effects of an anti-CD28 monoclonal antibody (mAb 9.3) on T cell activation induced with immobilized anti-CD3 mAb OKT3 or with mitogens, in the absence of accessory cells. To this end, we used blood resting T cells that were completely depleted of accessory cells (monocytes, B cells, and natural killer cells), and consequently did not respond to recombinant interleukin-2 (rIL-2), to immobilized OKT3, to PHA, or to Con A. Addition of mAb 9.3 to the cultures enhanced IL-2 receptor expression (Tac antigen) on PHA- or immobilized OKT3-stimulated T cells and induced IL-2 receptors on Con A-stimulated T cells. Moreover, addition of mAb 9.3 to cultures of T cells stimulated with PHA, Con A, or immobilized OKT3 resulted in IL-2 production. Soluble mAb 9.3 was a sufficient helper signal for T cell proliferation in response to PHA or immobilized OKT3. Crosslinking of mAb 9.3 by culture on anti-mouse IgG-coated plates enhanced the helper effect and was an essential requirement for the induction of T cell proliferation in response to Con A. No other anti-T cell mAb (anti-CD2, -CD4, -CD5, -CD7, -CD8) was found to provide a complete accessory signal for PHA or Con A stimulation of purified T cells. T cell proliferation induced by the combination of PHA and mAb 9.3 was strongly inhibited by the anti-IL-2 receptor mAb anti-Tac. In conclusion, mAb 9.3 can provide a signal bypassing monocyte requirement in T cell activation with immobilized OKT3, PHA, and Con A, resulting in an autocrine IL-2-dependent pathway of proliferation.     

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.     

Interleukin 2 up-regulates its own production

It has been previously reported that a combination pair of anti-CD2 monoclonal antibodies (mAb) T11(2)+T11(3) induces a strong proliferation of T cells, which does not require the involvement of accessory cells and exogenous interleukin 2 (IL-2). More recently, we have shown that the requirement for optimal T cell proliferation depends on the combination pairs of anti-CD2 mAb used. Among them, anti-GT2+T11(1) mAb do not allow optimal proliferation of TA4 helper cloned T cells due, at least in part, to a low level of IL-2 production. This observation offered us the opportunity to study the effect of IL-2 on its own production. We show here that stimulation of cloned TA4 cells with anti-GT2+T11(1) mAb induces only a marginal level of IL-2 production. By contrast, significantly higher levels of IL-2 activity are detected in the culture supernatant of TA4 cells preincubated with recombinant IL-2 (rIL-2) before stimulation with anti-GT2+T11(1) mAb. This effect is dose-dependent over a wide range (5 to 50 IU/ml) of rIL-2 concentrations added during preincubation time. In addition, it is not due to carryover of rIL-2 bound during the preincubation time, or to lesser IL-2 consumption by these cells, or to increasing numbers of IL-2-producing cells induced by exogenous IL-2. Moreover, the observation was confirmed with IL-2 mRNA. Although neither rIL-2 nor anti-GT2+T11(1) mAb alone could induce a significant production of IL-2, rIL-2 appears to up-regulate its own production when the TA4 cells are activated by the anti-CD2 mAb-mediated second signal.     

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.     

Lysis of fresh solid tumor targets in the presence of Con A is mediated primarily by Leu 7+ peripheral blood T lymphocytes: blocking by the anti-CD3 monoclonal antibody and comparison with recombinant interleukin 2-induced lysis by natural killer cells

We investigated the lysis of fresh human solid tumor cells by peripheral blood T lymphocytes in the presence of lectins and anti-CD3 monoclonal antibodies (mAb). Addition of certain lectins (Con A, PHA, or WGA) directly into the 4-hr 51Cr-release assay caused significant lysis of (P less than 0.001) noncultured solid tumor targets by enriched populations of granular lymphocytes (GL). Significant levels (P at least less than 0.001) of Con A- or PHA-dependent solid tumor lysis by GL-enriched lymphocytes were observed in 32 of 39 donors (82%) and 14 of 20 donors (70%), respectively. In contrast, the addition of other lectins (PNA, PWM, or LPS) or anti-CD3 mAb did not cause cytotoxicity. The levels of Con A-dependent lysis were comparable to those of interleukin 2 (IL-2)-induced lysis by Leu 11b+ natural killer (NK) cells. The presence of lectins at the effector phase, but not of recombinant IL-2 (rIL-2), was required for the lysis of solid tumor targets. Both Con A-dependent and rIL-2-induced lysis were totally inhibited by treatment of the effector cells with the lysosomotropic agent L-leucine methyl ester (LeuOMe). Effector cells responsible for Con A-dependent lysis of solid tumors expressed T3 (CD3), T8 (CD8), and Leu 7 antigens, but lacked T4 (CD4) and Leu 11 (CD16) antigens as determined by both negative and positive cell selection studies. Con A-dependent lysis was inhibited at the effector phase by anti-CD3 (OKT3 or anti-Leu 4) or anti-CD2 (OKT11) mAb. On the basis of their phenotype (Leu 7+ CD3+ CD8+ CD16-), we hypothesize that these effector cells may contain a population of cytotoxic T cells (CTL) generated in vivo against autologous modified cells that can lyse fresh solid tumor target cells under conditions where the recognition requirements for the CTL are bypassed by lectin approximation.     

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.     

CD28 is an inducible T cell surface antigen that transduces a proliferative signal in CD3+ mature thymocytes

The rearrangement of TCR genes during thymic ontogeny creates a repertoire of T cell specificities that is refined to ensure the deletion of autoreactive clones and the MHC restriction of T cell responses. Signals delivered via the accessory molecules CD2, CD4, and CD8 have a crucial role in this phase of T cell differentiation. Recently, CD28 has been identified as a signal transducing molecule on the surface of most mature T cells. Perturbation of the CD28 molecule stimulates a novel pathway of T cell activation regulating the production of a variety of lymphokines including IL-2. We have studied the expression and function of CD28 during thymic ontogeny, and in resting and activated PBL. A variable percentage of resting thymocytes were CD28+ (3 to 25%, n = 8), but it was found in high density only on mature CD3+(bright) CD4/CD8 cells. Both unseparated thymocytes and isolated CD3-CD28-/dull cells proliferated when stimulated with PMA plus IL-2 or PMA plus ionomycin. PMA treatment also rapidly up-regulated CD28 expression in the CD3- subset as these cells became CD3-CD28+(bright). Despite the ability of PMA to induce high density CD28 expression in CD3- cells, CD3- thymocytes did not proliferate in response to PMA plus anti-CD28 mAb, in contrast to unseparated cells. CD3+ thymocytes stimulated with immobilized anti-CD3 mAb also failed to proliferate in culture. However, the addition of either IL-2 or anti-CD28 mAb supported proliferation, suggesting that only CD3+ cells could respond to CD28 signaling. The comitogenic effect of anti-CD3 and anti-CD28 mAb was IL-2 dependent as it was abrogated by an anti-IL-2R mAb. Interestingly, the expression of CD28 on the cell surface of CD3+ cells was also inducible, as flow cytometric analysis demonstrated a 10-fold increase in cell surface CD28 by 24 to 48 h after anti-CD3 stimulation of both CD3+ thymocytes and peripheral blood T cells. This increase was accounted for by a commensurate increase in CD28 mRNA levels. Together, these results suggest that CD28 is an inducible T cell antigen in both CD3- and CD3+ cells. In addition, stimulation of the CD28 pathway can provide a second signal to support the growth of CD3+ thymocytes stimulated through the TCR/CD3 complex, and may therefore represent a mechanism for positive selection during thymic ontogeny.     

The role of CD11a/CD18-CD54 interactions in human T cell-dependent B cell activation.

The role of leukocyte function-associated Ag-1 (LFA-1, CD11a/CD18) and intercellular adhesion molecule 1 (ICAM-1, CD54) interactions in human T cell and B cell collaboration was examined by studying the effect of mAb to these determinants on B cell proliferation and differentiation stimulated by culturing resting B cells with CD4+ T cells activated with immobilized mAb to the CD3 molecular complex. In this model system, mAb to either the alpha (CD11a) or beta (CD18) chain of LFA-1 or ICAM-1 (CD54) inhibited B cell responses significantly. The mAb did not directly inhibit B cell function, inasmuch as T cell-independent activation induced by formalinized Staphylococcus aureus and IL-2 was not suppressed. Moreover, DNA synthesis and IL-2 production by immobilized anti-CD3-stimulated CD4+ T cells were not suppressed by the mAb to LFA-1 or ICAM-1. Although the mAb to LFA-1 inhibited enhancement of IL-2 production by co-culture of immobilized anti-CD3-stimulated CD4+ T cells with B cells, addition of exogenous IL-2 or supernatants of mitogen-activated T cells could not abrogate the inhibitory effects of the mAb to LFA-1 or ICAM-1 on B cell responses. Inhibition was most marked when the mAb were present during the initial 24 h in culture. Immobilized anti-CD3-stimulated LFA-1-negative CD4+ T cell clones from a child with leukocyte adhesion deficiency could induce B cell responses, which were inhibited by mAb to LFA-1 or ICAM-1. These results indicate that the interactions between LFA-1 and ICAM-1 play an important role in mediating the collaboration between activated CD4+ T cells and B cells necessary for the induction of B cell proliferation and differentiation, and for enhancement of IL-2 production by CD4+ T cells. Moreover, the data are consistent with a model of T cell-B cell collaboration in which interactions between LFA-1 on resting B cells and ICAM-1 on activated CD4+ T cells play a critical role in initial T cell-dependent B cell activation.     

Activation of cord T lymphocytes. I. Evidence for a defective T cell mitogenesis induced through the CD2 molecule

A study was carried out on cord blood T cell activation via the CD2-mediated pathway. Despite similar percentages of circulating CD3+ and CD2+ cells in adult and cord blood, the proliferation of cord PBMC to the anti-CD3 mAb and cord T cells to anti-CD2 mAb were defective. The T cell CD3-surface structure was normally able to control CD2-mediated activation, as its modulation by a non-mitogenic anti-CD3 mAb blocked cord PBMC proliferation induced by anti-CD2 mAb. CD2-stimulated cord T cells did not proliferate and did not produce a significant amount of IL-2 in culture, although they expressed the IL-2R. This observation was confirmed by the optimal proliferation of CD2-induced cord T cells when rIL-2 was added. Despite the alternative T cell activation pathway is monocyte-independent in adults, the defective cord T cell activation via the CD2 molecule could also be bypassed by the addition of PMA, small amounts of either autologous or allogeneic adult and cord AC or simply rIL-1 alone. Our findings provide evidence for an intrinsic functional defect in cord CD2-mediated T cell activation, which is linked to an impaired increase of free cytoplasmic calcium, as confirmed by the effectiveness of calcium ionophore A23187 in restoring a good CD2-induced cord T cell proliferation and by measurement of cellular calcium uptake after activation via the CD2 molecule. The characteristics of cord T cells revealed by this study recall the thymocyte functional pattern and may represent functional expression of the previously described phenotypic immaturity of cord T 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.     

IL-6 is an accessory signal in the alternative CD2-mediated pathway of T cell activation

Recent studies have demonstrated that IL-1 and IL-6 are synergistic accessory signals for activation of T cells. In this study, highly purified human T cells were cultured with either a stimulating pair of anti-CD2 mAb or with immobilized anti-CD3 mAb. Monocytes, a cellfree monocyte culture supernatant or IL-1 were required for anti-CD2-stimulated T cell proliferation, and they each strongly enhanced anti-CD3-induced T cell growth. IL-6 was synergistic with IL-1 as a helper factor for T cell growth after activation via CD2, but we could not demonstrate any effect of IL-6 in the CD3 pathway. The mechanism of the synergistic helper activity of IL-1 and IL-6 on T cell activation in the CD2 pathway was further examined. IL-1 (but not IL-6) was required for induction of IL-2 production. Both IL-1 and IL-6 enhanced IL-2R (p55) expression and the proliferative response to IL-2. T cell proliferation after stimulation with anti-CD2 and IL-1 or IL-1/IL-6 proceeded through an autocrine IL-2-dependent pathway. Moreover we found that, in the absence of IL-1, IL-6 still supported a transient and limited proliferation of anti-CD2- (but not of anti-CD3-) stimulated T cells, which apparently was independent of the autocrine growth factors IL-2 or IL-4. Our data suggest that IL-6 is important as an accessory signal for T cell growth in the CD2 pathway of T cell activation.     

Central role for TCR/CD3 ligation in the differentiation of CD4+ T cells toward A Th1 or Th2 functional phenotype.

Activated CD4+ T cells can be classified into distinct subsets; the most divergent among them may be considered to be the IL-2 and IFN-gamma-producing Th1 clones and the IL-4 and IL-5-producing Th2 clones. Because Th1 and Th2 clones can usually be detected only after several months of culture, we used conditions that modulate the IL-2 and IL-4 production in short term culture. Here we show that freshly isolated and subsequently in vitro-activated CD4+ T cells that were cultured for 11 days with rIL-2 and restimulated showed a IFN-gamma+ IL-2+ IL-3+ IL-4- IL-5- pattern. Because these cells were not capable of providing B cell help for IgG1, IgG2a, or IgE in an APC- and TCR-dependent T-B cell assay, they expressed a phenotype typical for most Th1 clones. In contrast, activated T cells that were cultured for 11 days with IL-2 plus a mAb to CD3 and then restimulated produced a IFN-gamma- IL-2- IL-3+ IL-4+ IL-5+ pattern. These cells were capable of providing B cell help for IgG1, IgG2a, and IgE synthesis and thus presented a phenotype typical for Th2 clones. Similar results were observed when mitogenic mAb to Thy-1.2 or to framework determinants of the alpha beta TCR were used. The induction of Th1- and Th2-like cells did not depend on the relative expression of CD44 or CD45 by the T cells before activation in vitro. Because the incubation of activated T cells with anti-CD3/TCR mAb induced high unrestricted lymphokine production, the latter might be responsible for the Th2-like lymphokine pattern observed after restimulation. To address this point, TCR V beta 8+ and V beta 8- T cell blasts were co-cultured in the presence of mAb to V beta 8. After restimulation, V beta 8+ cells had a IL-4high IL-2low phenotype and V beta 8- cells had a IL-4low IL-2high phenotype. This demonstrates that TCR ligation but not lymphokines alone are capable of inducing Th2-like cells, and this points out a central role for the TCR in the generation of T cell subsets.     

Expression of CD5 regulates responsiveness to IL-1

The role of the CD5 surface molecule in T cell responsiveness to IL-1 was examined. A CD5-mutant Jurkat cell line was generated from a CD5+ parent cell line. This CD5- mutant subclone was infected with a defective retrovirus containing the CD5 cDNA and/or the neo gene encoding G418 resistance. The CD5+ wild type Jurkat produced IL-2 in response to anti-CD3 mAb, OKT3, cross-linked to a solid surface. IL-2 production was enhanced by co-culture with IL-1 or anti-CD5 Mab. Neither the CD5- mutant nor the CD5- G418-resistant infectant responded to anti-CD5 mAb or to IL-1. Responsiveness to IL-1 was restored by cell surface expression of CD5 in the CD5+ infectant. Both the CD5+ wild type Jurkat and the CD5+ infectant responded equivalently to purified IL-1, IL-1 alpha and rIL-1 beta. Optimal concentrations of IL-1 and anti-CD5 mAb had an additive effect on the enhancement of IL-2 production stimulated with cross-linked anti-CD3 mAb suggesting that IL-1 and CD5 act through distinct, complementary pathways to augment T cell activation. The correlation of CD5 expression and specific binding of rIL-1 beta was examined in these cell lines. Both the specific binding (at 4 degrees C) and subsequent internalization (at 37 degrees C) of 125I labeled rIL-1 beta was equivalent in the CD5+ infectant and the CD5+ wild type Jurkat cell, whereas specific binding of 125I-labeled rIL-1 beta was markedly decreased in the CD5-G418-resistant infectant. These observations strongly suggest that cell surface expression of CD5 regulates binding of and responsiveness to IL-1.     

T cell regulation of human B cell proliferation and differentiation. Regulatory influences of CD45R+ and CD45R- T4 cell subsets

The immunoregulatory functions of human T4 cell subpopulations defined by mAb to the CD45R molecule (2H4) were examined. Both CD45R- and CD45R+ T4 cells that had been treated with mitomycin C (CD45R- and CD45R+ T4-mito) provided help for the generation of Ig-secreting cells (ISC) in cultures stimulated by PWM or by immobilized mAb to CD3 (64.1). IL-2 enhanced the generation of ISC in PWM-stimulated cultures and in anti-CD3-stimulated cultures containing CD45R+ T4-mito. The generation of ISC was maximal in cultures containing anti-CD3-activated CD45R- T4-mito and was not increased by IL-2. By contrast, CD45R+ T4 cells that had not been treated with mitomycin C suppressed B cell responses in cultures stimulated with PWM or anti-CD3, whereas CD45R- T4 cells suppressed the generation of ISC only in cultures stimulated with anti-CD3. IL-2 enhanced suppression by anti-CD3, but not PWM, activated CD45R- T4 cells. Suppression by CD45R+ T4 cells was maximal and not increased by IL-2. CD45R+ T4-mito were more effective suppressor-inducers in PWM-stimulated cultures, promoting the differentiation of suppressor-effector cells from CD8+ T cells. However, both CD45R+ and CD45R- T4-mito exerted comparable suppressor-inducer function in anti-CD3-stimulated cultures. Moreover, in anti-CD3-stimulated cultures, T8 cells could function as both suppressor-effector cells and suppressor-inducer cells. One of the functions of suppressor-inducer cells in this system appeared to involve the production of IL-2. Thus, the addition of IL-2 facilitated the induction of suppressor-effector T8 cells by CD45R- T4-mito in PWM-stimulated cultures. Although IL-2 production by the T cell subsets varied widely depending on the nature of the stimulus, these differences could not entirely explain their capacity to function as helper cells, suppressor-effector cells or suppressor-inducer cells. These results indicate that both CD45R+ and CD45R- T4 cells can help or suppress B cell responses, as well as induce suppressor-effector T8 cells. Moreover, suppressor-inducer function of T cells is not limited to the T4 cell population, but rather can also be accomplished by T8 cells. The results indicate that both T4 cell subsets and T8 cells exert multiple regulatory effects on human B cell function, with the nature of the activating stimulus playing a major role in determining the functional capacity of various T cell subsets.     

Cooperation between an anti-T cell (anti-CD28) monoclonal antibody and monocyte-produced IL-6 in the induction of T cell responsiveness to IL-2

CD28 is an Ag of 44-kDa Mr that is expressed on the membrane of the majority of human T cells and that is recognized by mAb 9.3. The functional effects of mAb 9.3 on peripheral blood T cells were studied. mAb 9.3 was not mitogenic, unless it was combined with PMA. When CD28 was cross-linked after binding of mAb 9.3 to the T cell by immobilized or soluble anti-mouse IgG, T cells proliferated in response to rIL-2, provided that monocytes were also present. The additional signal required for IL-2 responsiveness after cross-linking of CD28 could also be delivered in cultures of purified T cells by a cellfree monocyte culture supernatant. Expression of IL-2R on about 10% of the T cells was demonstrated by staining with an anti-IL-2R mAb, and was found to be largely restricted to CD4+ cells. The active compound responsible for the helper signal in the monocyte culture supernatant was identified as IL-6 because purified IL-6 (but not IL-1 beta) had similar activity and because an antiserum to IL-6 (but not an antiserum to IL-1 beta) neutralized the activity of the monocyte supernatant and blocked T cell proliferation. An anti-IL-2R antibody also completely inhibited T cell proliferation induced by the combination of mAb 9.3, IL-2, and IL-6. Our results provide evidence that cross-linking of CD28 induces functional IL-2R and that this activity is dependent on a helper signal provided by monocytes, more specifically IL-6. Moreover, our results indicate that IL-6 (previously called B cell stimulatory factor-2) is active on T cells. If a natural ligand for CD28 can be identified, the mechanism of induction of IL-2 responsiveness described here might explain how T cells become nonspecifically involved in an ongoing cellular immune reaction.     

Both naive and memory T cells can provide help for human IgE production, but with different cytokine requirements.

Most in vitro systems for the induction of IgE production by human B cells require both IL-4 and the presence of T cells. Little is known about the mechanism of T cell help or the ability of different T cell subsets to provide this helper activity. In the present study we demonstrate that, in the presence of exogenous IL-4, anti-CD3 stimulated naive T cells (CD4+CD45RA+) are potent helper cells for human IgE production. In their presence, as little as 750 autologous B cells can produce up to 100 ng/ml IgE. This response was found over a broad range of anti-CD3 concentrations. IgE helper activity by naive T cells was inhibited by IL-2. Under all conditions tested, naive T cells were unable to provide help for IgM production. This is in contrast to activated memory T cells (CD4+CD45RO+), which are very efficient helper cells for IgM or IgE production, provided that IL-2 or IL-2 plus IL-4 are present respectively.     

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.     

p38 mitogen-activated protein kinase regulates human T cell IL-5 synthesis.

Involvement of p38 mitogen-activated protein (MAP) kinase in human T cell cytokine synthesis was investigated. p38 MAP kinase was clearly induced in human Th cells activated through the TCR. SB203580, a highly selective inhibitor of p38 MAP kinase, inhibited the induction of p38 MAP kinase in human Th cells. Major T cell cytokines, IL-2, IL-4, IL-5, and IFN-gamma, were produced by Der f 2-specific Th clones upon stimulation through the TCR. IL-5 synthesis alone was significantly inhibited by SB203580 in a dose-dependent manner, whereas the production of IL-2, IL-4, and IFN-gamma was not affected. The proliferation of activated T cells was not affected. IL-5 synthesis of human Th clones induced upon stimulation with rIL-2, phorbol ester plus anti-CD28 mAb, and immobilized anti-CD3 mAb plus soluble anti-CD28 mAb was also suppressed by SB203580 in the same concentration response relationship. The results clearly indicated that IL-5 synthesis by human Th cells is dependent on p38 MAP kinase activity, and is regulated distinctly from IL-2, IL-4, and IFN-gamma synthesis. Selective control of IL-5 synthesis will provide a novel treatment devoid of generalized immune suppression for bronchial asthma and atopic dermatitis that are characterized by eosinophilic inflammation.