Monoclonal cytotoxic T lymphocyte hybridomas capable of specific killing activity, antigenic responsiveness, and inducible interleukin secretion

We have recently described the production of cytotoxic T lymphocyte (CTL) hybridomas that grow continuously in culture, exhibiting constitutive, allospecific (anti-H-2b) killing activity. We now report on the response of these monoclonal CTL hybridomas to specific antigen (H-2Db) and to mitogenic lectins. Both specific antigen and T cell mitogens enhance hybridoma-mediated specific target cell killing. In addition, stimulated, but not unstimulated hybridoma cells secrete considerable amounts of IL 2 into the culture medium. Repeated cloning of the hybridomas provides strong evidence that both killing activity and IL 2 secretion can be attributed to one cell. Unfractionated Con A supernatants, containing IL 2 and other factors known to influence T cell responsiveness, or IL 2-containing media of stimulated hybridomas affect neither the growth nor the lytic activity of the hybridomas. Anti-LFA-1 monoclonal antibody, a potent inhibitor of CTL and CTL hybridoma-mediated target cell lysis, abolishes antigen- or mitogen-induced IL 2 secretion by the CTL hybridomas. Involvement of a single hybridoma receptor in antigen recognition (afferent and efferent) and in initiating IL 2 secretion is proposed. The CTL hybridomas displaying retarded killing activity before the antigenic or mitogenic stimulation appear to represent an intermediate stage in CTL differentiation, reminiscent of "memory" CTL.     

The role of interleukin 2 in inducing Ig production in a pokeweed mitogen-stimulated mononuclear cell system

Cyclosporin A (CsA) has been found previously to block mitogen-stimulated T cell proliferation and production of discrete T cell-derived lymphokines such as interleukin 2 (IL 2) and interferon (IFN)-gamma. In addition, CsA blocks pokeweed mitogen (PWM)-driven T cell-dependent differentiation of B cells into immunoglobulin (Ig)-secreting cells. Recently, we reported that CsA (1 microgram/ml) inhibited PWM-induced T cell production of IL 2 and IFN-gamma, but supernatants retained B cell differentiation factor (BCDF)-like activity. The present study demonstrates the ability of CsA to suppress T cell functions in PWM-driven Ig production in mononuclear cells (MNC), and the capacity of exogenous T cell lymphokines to reverse CsA-induced suppression. CsA profoundly suppressed PWM-driven PFC formation (greater than 95%). However, Ig production was substantially reconstituted by the addition of IL 2 at concentrations of 10 to 50 U/ml. In contrast, no effects were observed by the addition of IFN-gamma or BCGF. The kinetics of CsA inhibition of Ig production and IL 2 secretion were found to be closely related. In addition, to obtain effective reconstitution in the CsA-treated PWM-MNC system it was necessary to add IL 2 at the initiation of culture. T cells themselves were also required for B cell differentiation in this system. However, surface Ig+ cells obtained by cell sorting after 3 days of culture could differentiate in the absence of T cells but only in response to IL 2, not in response to IFN-gamma or BCDF. Thus, in PWM-driven B cell differentiation T cells are necessary early in culture, whereas IL 2 is essential from the initial stage of B cell activation through the final stage of B cell differentiation.     

Minor lymphocyte stimulatory antigen-bearing stimulator cells require lipopolysaccharide activation to induce programmed cell death in T cell hybridomas.

T cell hybridomas respond to conventional peptide Ag associated with self major histocompatibility restriction elements, as well as to alloantigens, activating lectins, and stimulatory forms of mAb by producing lymphokines and undergoing programmed cell death (PCD). We show here that the level of PCD and IL-2 production correlate well in responses to CD3 or allostimulation. The response to minor lymphocyte-stimulatory (Mls) Ag, members of the family of endogenous superantigens, however, are marked by divergence in the levels of the PCD and lymphokine responses. Specifically, PCD in response to Mls activation is achieved poorly despite vigorous IL-2 production. B lymphoma cell stimulators induced PCD in alloreactive T cell hybridomas but not in Mls-reactive T cell hybridomas. This suggests that the absence of PCD in the Mls response is a function of superantigen recognition rather than the stimulator cell type. LPS-preactivated Mls+ stimulators, either splenic B or B lymphoma cells, are shown to trigger PCD in the T cell hybridomas. These results imply that T cell interaction with Mls presented by untreated stimulator cells is not sufficient for induction of PCD and thus is distinct from interactions with conventional Ag.     

Effect of prostaglandin E2 on the gamma-interferon induction of antigen-presenting ability in P388D1 cells and on IL-2 production by T-cell hybridomas

The effect of prostaglandin E2 on the gamma-interferon (IFN-gamma)-mediated induction of Ia expression and antigen-presenting activity in macrophage cell lines was studied. Using a lymphokine preparation obtained from the T-cell hybridoma FS7-20.6.18 (known to produce interferon) to induce the expression of Ia in P388D1 cells, the influence of PGE2 on this phenomenon was studied. Screening of the cell cultures by indirect immunofluorescence using an anti-I-Ad monoclonal antibody confirmed the inhibitory effect of PGE2 in the induction of I-Ad. However, the inhibition of the antigen-presenting ability of these cells, as measured by their capacity to stimulate interleukin 2 (IL-2) production by antigen-specific, I-region-restricted (Ag/I) T-cell hybridomas, was more difficult to demonstrate and was only evident when using low concentrations of Ia-inducing lymphokines or when using "low avidity" T-cell hybridomas. The latter were distinguished by the limited response (in the form of IL-2 production) that was observed when they were tested with P388D1 cells that had been induced with IFN-gamma. By contrast, PGE2 had profound inhibitory effects on the ability of T-cell hybridomas to secrete IL-2 in response to Ag/I or concanavalin A. These results suggest that although PGE2 inhibits the full induction of Ia on macrophages, it has little effect on the induction of Ag/I presentation by the same cells, probably because most T cells require relatively low levels of Ia on the surface of presenting cells. T-cell responses to Ag/I are inhibited, however, because of the effects of PGE2 on the T cells themselves.     

Retrovirus-mediated immunosuppression. II. FeLV-UV alters in vitro murine T lymphocyte behavior by reversibly impairing lymphokine secretion

Murine splenocytes and cloned murine T cells were used to study the in vitro immunosuppressive effects of UV-inactivated feline leukemia virus (FeLV-UV) on lymphokine secretion. FeLV-UV can significantly depress the accumulation of IL 2 in cultures of Con A-stimulated C57BL/6 splenocytes and in cultures containing the alloreactive helper T cell clone B6D/2-2m plus Con A. Inhibition of lymphokine accumulation in these cultures could not be attributed to absorption or inactivation of IL 2 by the FeLV-UV or to the FeLV-UV-induced production of substances which interfere with the IL 2 bioassay. Thus, FeLV-UV appears to block production and/or secretion of IL 2 by a direct inhibitory effect on IL 2-secreting murine T lymphocytes. Additional studies indicate that FeLV-UV impairs IL 2 production only if added very soon after lymphocyte contact with lymphokine-inducing agents and that IL 2 secretion resumes when FeLV-UV is removed from the culture. FeLV-UV also impairs accumulation of MAF (interferon-gamma?) in cultures of Con A-stimulated C57BL/6 splenocytes and in cultures containing the alloreactive cytotoxic T lymphocyte clone B6D/2-7c plus Con A. The latter observation again suggests that FeLV-UV impairs lymphokine secretion by a direct effect on lymphokine-producing T lymphocytes. Furthermore, it suggests that FeLV-UV does not selectively impair production of IL 2 nor does it have selective inhibitory effects on helper T cells. Rather, FeLV-UV appears to have a general inhibitory effect on lymphokine production by T lymphocytes. Finally, concentrations of FeLV-UV which suppress MAF production by the CTL clone have little influence on the cytolysis mediated by the same cloned T cell population. Thus, the immunosuppressive influence of FeLV-UV is selective for phenomena associated with induction of new T lymphocyte functions, such as lymphokine secretion, and spares other immune functions already expressed by the same cells.     

1,25-Dihydroxyvitamin D3 inhibits antigen-induced T cell activation

The proliferative response of murine spleen and thymus cells to antigen but not to lectin was inhibited by the active metabolite of vitamin D3, 1,25-(OH)2D3. To directly examine the effect of 1,25-(OH)2D3 on T cell activation in the absence of other complicating interactions, we utilized a panel of cloned Ia-restricted T cell hybridomas that secrete IL 2 on activation by cloned Ia-bearing stimulator cells (TA3) or when stimulated by mitogen. Physiologic concentrations of 1,25-(OH)2D3 (0.01 to 0.1 nm) inhibited the antigen-induced secretion of IL 2 by several of these T cell hybridomas. This inhibition was dependent on the concentration of the free hormone and could be overcome by increasing the number of Ia-bearing stimulator cells used. Pretreatment of the T hybridoma but not the TA3 stimulator cell with 1,25-(OH)2D3 resulted in inhibition of activation. These results are consistent with the finding that specific 1,25-(OH)2D3 receptors are present on the T cell hybridomas but are lacking in TA3 cells. 1,25-(OH)2D3 failed, however, to inhibit the activation of the T cell hybridomas by lectin or by an anti-Thy-1 antibody. These findings suggest that 1,25-(OH)2D3 may be interfering with early events of antigen-induced T cell activation, perhaps by hindering T cell recognition of the relevant antigen on stimulator cell surfaces. This system should prove useful in studying the molecular mechanisms by which 1,25-(OH)2D3 acts to inhibit T cell activation and subsequent IL 2 production.     

Prostaglandin E2 inhibits the activation of cloned T cell hybridomas

To minimize complicating interactions inherent in heterogeneous cell populations, we used a panel of cloned murine autoreactive (E8.A1) and antigen-specific (HEL.C10, HEL.B14) T cell hybridomas to examine the effect of prostaglandin E2 (PGE2) on T cell activation. These T cells secrete interleukin 2 (IL 2) when co-cultured with a cloned population of I region-matched stimulator cells (TA3), or with mitogenic signals in the absence of TA3 stimulator cells. Physiologic concentrations of PGE2 inhibited the induction of IL 2 secretion by the T cell hybridomas tested, when they were activated either by TA3 cells or by mitogenic signals. IL 2 production was inhibited in a dose-dependent manner by concentrations of PGE2 between 10(-7) and 10(-11) M, with 50% inhibition occurring at 10(-10) M. Pretreatment of the T hybridoma cells with 10(-7) M PGE2 for 1 hr before culture also resulted in marked inhibition of IL 2 secretion. Similar pretreatment of the TA3 cells did not affect their ability to activate the T cell hybridomas. PGE2 at 10(-8) M induced a 30-fold increase in cAMP levels within 25 min of addition to culture of the E8.A1 T cell hybridoma, but caused no significant elevation of cAMP levels in TA3 cells. The direct addition of dibutyryl cAMP (dcAMP) to cultures of E8.A1 cells resulted in marked inhibition of IL 2 secretion when stimulated by TA3 or by mitogenic signals, with an average of 80% inhibition occurring at 10(-4) M dcAMP. PGE2 and dcAMP also inhibited the growth of E8.A1 cells. Initially, cell growth was virtually halted, but began to recover between 24 and 48 hr after the addition of either PGE2 or dcAMP. Neither PGE2 nor dcAMP inhibited the division of TA3 cells. High affinity binding sites for PGE2 were detected in the E8.A1 T cell hybridomas with an apparent Kd of 7.6 X 10(-10) M, which is consistent with the functional data. No specific binding was detected in the TA3 stimulator cells. These findings suggest that the immunosuppressive effects of PGE2 are localized to the T cell, are receptor regulated, and may be mediated by the associated increase of cAMP levels in the T cell hybridomas.     

IL 1 induction by murine T cell clones: detection of an IL 1-inducing lymphokine

T cell activation is widely believed to depend on interleukin 1 (IL 1) provided by antigen (Ag)-presenting cells (APC). Because IL 1 is not a constitutive product of APC, we examined the features of its production during the interaction of murine T cell clones and APC. We observed that IL 1 was detectable in supernatants of most myoglobin-specific T cell clones grown with APC and Ag. Two of these T cell clones induced exceptionally high levels of IL 1 in their supernatants, and these same clones demonstrated the unusual restriction to I-Ek, which is a low responder type for sperm whale myoglobin. One of these clones was characterized additionally as to the mechanism of IL 1 induction. This clone rapidly stimulated IL 1 production in the APC population (detectable at 4 hr of co-culture) or in macrophages (M phi) or a M phi-like cell line. IL 1 induction was Ag dependent and H-2 restricted. Induction was radioresistant, both on the part of the T cell and of the IL 1 producer. The IL 1-induction process was attributable to a lymphokine produced by the T cell clone. This lymphokine was distinct from IFN-gamma, TNF and CSF-1 and may account for a principal mechanism of T----APC signalling. The induced IL 1 was the same in size, co-mitogenicity, and pyrogenicity as lipopolysaccharide-induced IL 1.     

Binding of antigen-specific, H-2-restricted T cell hybridomas to antigen-pulsed adherent cell monolayers

Two methods were investigated for studying the binding of radiolabeled hybridoma T cells to antigen (Ag) and H-2 products for which they bore receptors. In both cases hybridoma T cells were labeled with tritiated thymidine. In one method labeled cells were added to adherent splenic cells prepulsed with antigen, and the mixture was incubated overnight at 37 degrees C before nonadherent cells were gently washed away. The percent of adherent hybridoma T cells was then estimated by harvesting the adherent monolayers and measuring tritium counts bound. In a second method radiolabeled hybridoma T cells were added to adherent antigen-pulsed B cell lymphomas or hybridomas for between 15 min and 1 hr at 37 degrees C before removal of nonadherent cells and harvesting of the adherent monolayers. In both cases binding was both antigen- and I-region specific. In the second case binding was also rapid; significant binding could be measured after 15 min incubation. These techniques were used to study subclones of one of our T cell hybridomas that were thought by a functional assay (interleukin 2 release) to have lost receptors for Ag/H-2. It was found that subclones of the hybridoma that no longer secreted interleukin 2 in response to Ag/H-2, even though they continued to secrete interleukin 2 in response to concanavalin A, also no longer bound specifically to Ag-pulsed monolayers of the appropriate H-2 type. This confirmed the idea that these subclones had lost the ability to synthesize receptors for Ag/H-2. It is hoped that assays of this type will be useful in the future for the study of Ag/H-2 receptors on T cells.     

Specific lymphocyte-target cell conjugate formation between tumor-specific helper T-cell hybridomas and IA-bearing RCS tumors and IE-bearing allogeneic cells. I. Role of Ia and both L3T4 and LFA-1 antigens in recognition/binding

The studies reported here describe the feasibility of using single cell techniques with nonadherent target cells for the formation of T helper lymphocyte-target cell conjugates in an Ia recognition system. We have taken advantage of four tumor-specific T cell hybridomas lines, two of which respond only to IA-bearing RCS tumor cells of SJL/J (H-2s) origin, and the other two that respond to both RCS and IA- or IE-bearing allogeneic cells of H-2k,d haplotypes. The conjugate frequency between the T cell hybridomas and target cells was scored microscopically and was facilitated by labeling the lymphocyte with fluorescein. The frequency of conjugate formation ranged from 20 to 40% above background. Conjugate formation was antigen specific and correlated well with the hybridoma specificity determined by IL 2 responses after antigenic stimulation. The cross-reactive hybridomas formed conjugates with RCS and LPS blasts derived from CBA or DBA/2 origin, but not with cells of syngeneic or other allogeneic strains. Conjugate formation with RCS was inhibited greater than 50% with mAb directed against IAs determinants on the RCS tumor cells, and conjugate formation with allogeneic cells was blocked only with mAb directed to either IA/IEk or IA/IEd specificities directed against the alpha or beta polypeptide chain. Blocking of conjugate formation was also achieved by various mAb directed against surface membrane molecules associated with the T cell hybridomas. LFA-1 mAb inhibited significantly the formation of conjugates. However, L3T4 mAb blocked only partially the conjugates. Other antibodies directed against Lyt-1 or Thy-1.2 antigens were without blocking effect. The poor blocking observed with L3T4 mAb did not correlate with the almost complete blocking observed in the IL 2 response by the same hybridomas. These studies of the syngeneic anti-RCS tumor response directed against IA-bearing RCS showed that the conjugate assay permits mapping of tumor-associated Ia epitopes. In addition, the results of these studies demonstrate the feasibility of conjugate formation in determining the antigenic specificity of the T helper system. This assay system can be used to establish the minimal frequency of antigen-reactive cells and can divide the T helper response into multiple steps (i.e., recognition/binding, activation, proliferation, and lymphokine release) and determine the surface membrane molecules involved in recognition.     

Defect of CD2- and CD3-mediated activation pathways in T cells of atopic patients: role of interleukin 2.

In the present work we analyzed the proliferative response of T lymphocytes from 11 atopic patients stimulated in vitro via either the CD2 or the CD3 pathway of cell activation. In both cases we found a significant decrease of thymidine incorporation in cell DNA in comparison with T cells from normal donors. The mechanism of this impaired proliferative response was analyzed. Atopic patients' T cells were found to secrete low quantities of interleukin 2 (IL2) and to express low amounts of Tac antigen, measured as both a percentage of Tac-positive cells and a mean fluorescence intensity of Tac antigen per cell. Addition of recombinant IL2 to cultures completely restored both cell proliferative response and Tac antigen expression. This effect was specific of IL2 since addition of IL1 or IL4 did not significantly affect T cell proliferative response. We conclude that atopic patients' T lymphocytes have a defect in both CD2 and CD3 pathways of cell activation relying on impairment of IL2 production, without involving IL2 responsiveness or other lymphokine defects.     

Release of lymphokines after Epstein Barr virus infection in vitro. I. Sources of and kinetics of production of interferons and interleukins in normal humans

Infection of human lymphocytes with Epstein Barr virus (EBV) activates the release of lymphokines. Previous experiments have emphasized the ability of interferon-gamma (IFN-gamma) to prevent EBV-induced B cell transformation. However, the factors that regulate IFN-gamma synthesis and release during in vitro EBV infection are controversial. In the present investigation we have systematically evaluated the kinetics of production, cellular origins, and accessory cell requirements for IFN-alpha and IFN-gamma and for IL 1 and IL 2, after EBV infection. Our data indicate that IFN-alpha is released entirely by natural killer (NK) cells and B cells, in the absence of accessory cells, independently of the other lymphokines and within 24 hr of infection. In contradistinction, IFN-gamma secretion is exclusively of T cell origin, is absolutely dependent on the prior elaboration of IL 1 and IL 2, and is maximal 8 days after EBV infection. IL 2 secretion by T cells peaks on day 5 and requires the earlier release of IL 1. Both NK cells and monocytes are a source of IL 1. Secretion of IL 2 and IFN-gamma occurs in the presence of either one of these cell types but not in the absence of both. Antibody against IL 1 blocks EBV-induced IL 2 and IFN-gamma generation, and antibody against IL 2 decreases production of IFN-gamma. Thus, the production of IFN-gamma, the lymphokine that prevents EBV-induced B cell transformation, is the final outcome of a cascade of lymphokine-mediated events that involve interactions between virus-infected B lymphocytes that serve as antigen-presenting cells, NK cells and monocytes as sources of IL 1, and T lymphoblasts. Dysfunctions of any or all of these cell types would be expected to impair the regulation of EBV transformation.     

Characterization of T helper 1 and 2 cell subsets in normal mice. Helper T cells responsible for IL-4 and IL-5 production are present as precursors that require priming before they develop into lymphokine-secreting cells

We have shown that the requirements for the production of IL-4 and IL-5 by normal L3T4+T cells from murine spleen are very different from those for the production of IL-2. Secretion of detectable quantities of IL-4 and IL-5 and induction of the mRNA for each lymphokine occurs in vitro only after cells are primed and re-stimulated. This priming can be achieved by mitogens (Con A), by antibodies to the TCR (anti-T3) or by stimulation with alloantigen. In contrast, requirements for induction of lymphokine production after priming resemble those for initial production of IL-2. Thus the majority of T cells of helper phenotype that have the potential to become IL-4- and IL-5-secreting T cells, exist in the form of precursors requiring stimulation and several days of culture as well as re-stimulation with mitogen or Ag before they become detectable as lymphokine-secreting cells. In contrast, among fresh CD4+T cells, secretion of IL-2, IL-3, granulocyte/macrophage CSF, and IFN-gamma is easily detected within 24 h of stimulation with mitogen or Ag. These observations establish that distinct phenotypes of Th cells are found at different times after stimulation and support the concept that synthesis and secretion of different lymphokines or groups of lymphokines are regulated independently. Furthermore the patterns of lymphokines secreted by fresh vs primed Th cells, which largely correspond to the patterns that have been used to define the Th1 and Th2 subsets among Th cell lines, provides evidence that different subsets of normal T cells exist that may correspond to these designations. Secretion of different lymphokines by two subsets of Th cells at different times in an immune response, and perhaps in different places, suggests a model in which the ratio of the two T cell subsets (Th1 vs Th2) and state of differentiation of each (precursor vs effector), influence or determine the direction of the response, with variations in these parameters leading to differing responses.     

Characterization of a novel murine T cell-activating factor

Purified resting peripheral lymph node T cells can be activated to produce interleukin 2 (IL 2) and to proliferate in the presence of Concanavalin A (Con A) and an apparently novel lymphokine that we call T cell activating factor (TAF). TAF is biochemically distinct from IL 1, IL 2, IL 3, and other colony stimulating factors, IL 4 (BSF-1) and interferons. Furthermore, of the recombinant and natural cytokines tested, only IL 2 and TAF are active in the TAF assay. In the presence of Con A, TAF stimulates an increase in the steady-state level of IL 2 mRNA in T cells, the secretion of active IL 2 into the culture medium, and the proliferation of the T cells. We propose that TAF is a previously undescribed molecule the function of which is to stimulate IL 2 production by T cells that have encountered antigen, and we propose that TAF has an important role in primary T cell immune responses.     

Production of T-T hybrids from T cell clones. Direct comparison between cloned T cells and T hybridoma cells derived from them

It has been assumed, without direct evidence, that T cell hybridomas and non-transformed T cell clones are both good models of normal Ag-specific T cells. To compare directly the difference in activation of cloned normal T cells and T hybridoma cells with the same TCR, cloned T hybridoma cells were obtained by fusing pre-established, myoglobin-specific, Iad-restricted T cell clones (14.5 and 9.27) with BW5147 cells. T cell clones were pre-activated with IL-2 as well as specific Ag before fusion. Cloned T hybridoma A3.4C6 was derived from Lys 140-specific and I-Ed-restricted clone 14.5. The other cloned T hybridoma, C7R14, was a fusion product of Glu 109-specific and I-Ad-restricted clone 9.27. Both T hybridomas showed the same Ag specificity and Ia restriction as the parental cloned T cells. However, C7R14 showed higher apparent affinity and broader cross-reactivity than 9.27. Clone 14.5, but not hybridoma A3.4C6, appeared to stimulate splenic cells to secrete cytokines inhibiting HT-2A cell proliferation. The most striking difference between the clones and hybridomas was that both clones, but neither of the matched hybridomas, were induced to synthesize IL-1 on stimulation with Ag. Finally, both cloned T cells and T hybridomas killed Ag-pulsed Iad-bearing B lymphoma target cells. This evidence suggests that killing function can be inherited from clones to hybridomas. However, the clones were much more efficient at killing than the hybridomas, and the hybridomas were more efficient at IL-2 production than the clones. Thus, matched pairs of clones and hybridomas differ in their capacity to mediate the two functions or may tend to be selected differently during cloning. Thus, although our results generally support the validity of T cell hybridomas as faithful models of the corresponding T cell clones, a number of subtle and not-so-subtle differences indicate that caution must be used in such an extrapolation.     

Alloreactive cloned T cell lines. VI. Multiple lymphokine activities secreted by helper and cytolytic cloned T lymphocytes

Culture supernatants generated by alloantigenic or lectin stimulation of a cloned helper T lymphocyte, designated L2, contain interleukin 2 (IL 2), granulocyte/macrophage colony-stimulating factor (CSF), B cell stimulating factor (BCSF), macrophage (Ia+)-recruiting factor (MIRF), (Ia+)-inducing activity, gamma-interferon, Fc receptor-enhancing activity, macrophage migration inhibitory factor (MIF), macrophage activation factor (MAF), interleukin 3 (IL 3), and a factor responsible for prolonging the synthesis and secretion of the fourth and second components of complement by guinea pig peritoneal macrophages. Erythropoietin was not detected. A spontaneously arising variant of L2, designated L2V, produces much lower quantities of macrophage-stimulating activities, IL 2, and interferon. However, when compared to L2, L2V produces much higher levels of BCSF, equivalent amounts of IL 3, and slightly smaller amounts of CSF. Unlike L2V, a cytolytic clone, designated L3, secretes lymphokines that primarily affect macrophage function. The time course of lymphokine production by L2 cells indicates that for the six lymphokine activities studied there are three different times at which maximal or near maximal levels are reached, as follows: 1) IL 2, 12 to 24 hr; 2) IL 3 and CSF, 24 to 48 hr; and 3) (Ia+)-inducing activity, MAF, and interferon, 48 hr or later. Only IL 2 activity disappears during the 8-day culture cycle. The time course data and the differential production of activities by the three types of lymphocyte clones suggest that at least four terminal effector lymphokine molecules account for the ten biologic activities tested.     

Differential effect of cyclosporin A on the expression of T and B lymphocyte activation antigens

We have used a monoclonal antibody (Mab) to the interleukin 2 (IL 2) receptor as well as a Mab to the transferrin receptor to analyze the effects of cyclosporin A (CsA) on the induction and expression of these activation antigens on mitogen-stimulated murine T and B lymphocytes. The same concentration (0.25 microgram/ml) of CsA that produced optimal inhibition of the T cell proliferative response to concanavalin A (Con A) was also very effective at inhibiting IL 2 production and the induction of IL 2 responsiveness, as well as the expression of the IL 2 and transferrin receptors when measured 72 hr after mitogen activation. Surprisingly, CsA only minimally inhibited expression of these receptors 24 hr after the addition of mitogen; however, T cell blasts recovered from these cultures failed to respond to IL 2 even though IL 2 receptor expression was only modestly decreased. These results suggest that inhibition of the maturation of receptor expression is secondary to an early effect of CsA in blocking the induction of IL 2 responsiveness or to an arrest in the sequence of events required for maturation of T cells that bear high densities of these receptors. In contrast to the results observed with T lymphocytes, CsA had no effect on the B cell proliferative response to lipopolysaccharide (LPS) or on the induction of the IL 2 and transferrin receptors on activated B lymphocytes.     

The AKR thymoma BW5147 is able to produce lymphokines when stimulated with calcium ionophore and phorbol ester

We produced the T cell hybridoma D9C1.12.17 by fusing an IL-4-producing T cell clone D9.1Hi with the AKR thymoma BW5147. The resulting hybridoma produced IL-2 as well as IL-4 even though none of the parental cells produced IL-2 after stimulation with Con A. The production of IL-2 was confirmed at the mRNA level by using an S1 nuclease protection assay. Further analysis indicated that Con A-induced IL-2 production was a common phenomenon among T cell hybridomas derived from this fusion. Although BW5147 does not produce detectable lymphokines after Con A stimulation, this line was able to produce IL-2, granulocyte-macrophage colony stimulating factor, and small amounts of IL-3 and IFN-gamma when stimulated with calcium ionophore and phorbol ester. The latter agents are thought to mimic the activating signal(s) delivered through the Ag:MHC TCR. This observation indicates that BW5147 has the ability to produce lymphokines but may lack component(s) which couple the extracellular signal to lymphokine production, and suggests that in T cell hybridomas, part of the spectrum of lymphokines produced may be contributed by BW5147.     

Phenotypic and functional heterogeneity of human peripheral blood T lymphocytes producing colony stimulating factor. A clonal and precursor frequency analysis

In this report we describe the precursor frequency and the subset distribution of peripheral blood human T cells producing lymphokine(s) acting on the proliferation and differentiation of bone marrow precursors of the granulocyte/macrophage series, as assessed in a liquid microculture assay. Because the sensitivity of this system was similar to that of the classic colony formation assay in semi-solid (methylcellulose) medium, it is likely that the lymphokine activity measured in this assay corresponds to the colony-stimulating factor (CSF) activity. Single human T lymphocytes, isolated from peripheral blood by E rosetting and Ficoll-Hypaque gradients, were seeded into microculture wells by limiting dilution or micromanipulation techniques and were incubated under culture conditions that allow clonal expansion of essentially all T cells. After 15 to 20 days, microcultures were stimulated with PHA and CSF activity was assayed in culture supernatants 24 hr thereafter. About 45% (1/2.3) of peripheral blood T cells were found to give rise to CSF-producing progenies. Moreover, when fluorescence-activated cell sorter-purified T4+ and T4- (or T8- and T8+) were analyzed, the frequency of the precursors of CSF-producing cells was 1/1.5 in the T4+ subset, whereas approximately one-third of the T8+ cells had this functional potential. To additionally characterize T cells responsible for CSF production, unfractionated T cells as well as T4+ and T4- cells were cloned by single cell micromanipulation. The resulting clones were analyzed simultaneously for the production of IL 2, production of CSF and for cytolytic activity in a lectin-dependent assay. It was found that 25/48 clones obtained from unfractionated T cells produced CSF, whereas 23/48 and 19/48 produced IL 2 or had cytolytic activity respectively. Six of the 25 CSF-producing clones had only this functional capability, whereas the remaining clones in addition displayed cytolytic activity (4/25), IL 2 production (10/25), or both (5/25). A similar functional heterogeneity was observed among T4+ and T4- clones, thus indicating that T cells producing CSF are functionally heterogeneous within both the T4+ and T8+ subsets.     

Inhibition of transformed T cell growth in vitro by monoclonal antibodies directed against distinct activating molecules

Stimulatory of antigen-specific murine T cell hybridomas with the appropriate antigen has been shown to cause lymphokine secretion and inhibition of spontaneous cell growth. In this study, the effect of cellular activation on the growth of transformed T cells, of known or unknown antigen specificity, was explored with stimulatory monoclonal antibodies (mAb) that recognize nonclonally distributed T cell surface molecules. Anti-CD3 antibodies stimulated interleukin 2 (IL-2) secretion while they inhibited murine and human T cell tumor growth in vitro. Both responses required external cross-linking of the anti-CD3 antibodies. Activation via two molecules that are not physically associated with the T cell antigen receptor, Thy-1 and Ly-6, also inhibited transformed T cell growth while inducing IL-2 secretion. Notably, an anti-Thy-1 mAb that did not cause the transformed T cells to secrete lymphokines failed to affect their growth, and in fact blocked the growth inhibition induced by the stimulatory mAb. Regardless of which stimulating mAb was used, IL-2 production and cell growth were inversely proportional, manifesting similar antibody dose-response curves. Activation of a T cell hybridoma with stimulatory mAb resulted in rapid lysis, as evidenced by the release of 51Cr and lactate dehydrogenase. Cell cycle analysis demonstrated that cellular activation was accompanied by a cell cycle block between the G1 and S phases, and probably a slowing of the transit of cells already in S. These results demonstrate that the growth of a spectrum of neoplastic T cells, murine and human, can be inhibited by what are normally growth-promoting signals for non-transformed T cells.