Generation of cytotoxic T lymphocytes from thymocyte precursors to trinitrophenyl-modified self antigens. I. Requirement of both killer-helper factor(s) and interleukin 2 for CTL generation from a subpopulation of thymocytes

The requirement for signals in the induction of cytotoxic T lymphocytes (CTL) from thymocyte precursors has been investigated. Either unfractionated or peanut agglutinin-binding (PNA+) C3H/He thymocytes were stimulated with mitomycin C(MMC)-treated, 2,4,6-trinitrophenyl(TNP)-modified syngeneic spleen cells in the presence of a variety of lymphokine preparations. Cellfree supernatant (CFS) from purified protein derivatives(PPD-CFS) stimulated Mycobacterium tuberculosis (Tbc)-primed cells, or partially purified interleukin 2 (IL 2) mediated strong cytotoxic responses in unfractionated thymocytes, whereas only PPD-CFS at final concentrations beyond 30% was active for CTL generation in PNA+ thymocytes. Neither IL 2 at concentrations of below 60 U/ml nor a low concentration of PPD-CFS (at final below 10%) had such a capacity. The addition of monoclonal anti-IL 2 receptor antibody completely blocked CTL generation induced by PPD-CFS in PNA+ thymocytes. In contrast, anti-immune interferon (IFN-gamma) antibody showed a marginal effect. PPD-CFS (10%) and IL 2 (10 U/ml) could synergistically trigger PNA+ thymocytes to induce CTL generation. These results suggested that both IL 2 and "helper" factors other than IL 2 are required for CTL generation from PNA+ thymocytes. We refer to these kinds of helper factors as killer helper factors (KHF). Partially purified IL 2-free KHF show two peaks of activities at apparent m.w. 14,000 to 34,000 and 44,000 to 90,000, and are heterogeneous with respect to isoelectric point, which is between 4.5 and 5.1. Cultures that received TNP-modified syngeneic cells and KHF on day 0 and IL 2 on day 2 generated potent CTL responses, whereas the addition of IL 2 on day 0 followed by the addition of KHF on day 2 to the culture was ineffective, suggesting that KHF is required in the early phase of the culture to achieve optimal CTL responses.     

A human helper T cell clone secreting both killer helper factor(s) and T cell-replacing factor(s)

A human helper T cell clone (d4), which showed its helper effect on the differentiation of both T and B cells, was established by MLC reaction of normal T cells against a B lymphoblastoid cell line (CESS) followed by cloning in the presence of IL2 and x-irradiated CESS and autologous non-T cells. d4 cells helped the induction of cytotoxic T cells against UV-treated CESS cells. Antigen-stimulated d4 cells secreted helper factor(s) involved in the induction of cytotoxic T cells (killer helper factor(s), KHF), and KHF activity could be separated into two fractions, one with the m.w. of 15,000 to 20,000 and the other with the m.w. of 45,000 to 50,000. The factor with 15,000 to 20,000 m.w. showed IL 2 activity; the other factor showed gamma-interferon activity without IL 2 activity, suggesting that both IL 2 and gamma-interferon exerted KHF activity. d4 cells or their culture supernatant showed helper activity in the induction of IgG in a B cell line (CESS). The helper activity of the supernatant (TRF) was absorbed with CESS cells but not with IL 2-dependent CTLL, whereas KHF activity was absorbed with IL 2-dependent CTLL but not with CESS cells. The results showed that TRF and KHF were distinct molecules and a single helper T cell clone could secrete helper factors for both B and T cells.     

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.     

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.     

Stimulation of mouse lymphocytes by a mitogen derived from Mycoplasma arthritidis. IV. Murine T hybridoma cells exhibit differential accessory cell requirements for activation by M. arthritidis T cell mitogen, concanavalin A, or hen egg-white lysozyme

A T-cell mitogen present in culture supernatants of Mycoplasma arthritidis (MAS) is known to exhibit an absolute dependence on E alpha-bearing accessory cells (AC), which appear to function by binding the mitogen. We therefore compared the specificity and nature of the AC requirements for MAS and antigen-induced production of IL 2 in T hybridoma cell lines originating from a fusion by using hen egg-white lysozyme (HEL)-specific, H-2d-restricted T blasts. A marked specificity was noted in the ability of the hybridoma lines to become activated by Con A, MAS, or HEL antigen. Thus all three lines produced IL 2 in response to Con A without the addition of B lymphoma AC. Two lines responded to MAS, but only in the presence of AC, and only one line responded to HEL antigen in the presence of AC. Using the HEL responsive T hybridoma line, we demonstrated that disrupted AC and AC membranes could present MAS but not HEL. MAS rapidly associated with AC at 4 degrees C, whereas HEL failed to do so. Paraformaldehyde-fixed AC could absorb the mitogen in MAS and present it to T hybridoma cells within several minutes, whereas HEL antigen could only be presented by fixed AC if there was a prolonged period of incubation (greater than 30 min) at 37 degrees C before fixation. The combined data indicate that metabolically active cells are not required for the association of MAS with AC or for presentation of MAS to T hybridomas. In contrast, HEL antigen requires metabolically active cells for both of these processes. Thus, the mitogen in MAS can bind to AC without any processing requirements, and it is likely that the resulting complex of mitogen and Ia molecules can directly activate T hybridoma cells.     

Natural killer (NK) cell activating factor released from murine thymocytes stimulated with an anti-tumor streptococcal preparation, OK-432

Natural killer (NK) activity of mouse splenocytes was significantly augmented when the splenocytes were incubated for 3 to 4 hr with culture supernatants of mouse thymocytes stimulated by OK-432, an antitumor preparation from the Streptococcus pyogenes SU-strain. Antiviral activity was also detected in the culture supernatants, but IL 2 activity was not. When the culture supernatants of thymocytes stimulated by OK-432 were fractionated on a column of Blue Sepharose CL-6B, NK enhancing activity and antiviral activity were observed in partly overlapping fractions that bound to the column. However, the antiviral activity in the Blue Sepharose-bound fraction was neutralized completely by treatment with anti-IFN (alpha, beta) antiserum, whereas significant NK cell enhancing activity was still observed after treatment with anti-IFN (alpha, beta) antiserum. When the Blue Sepharose-bound fraction was subjected to gel filtration, the NK cell enhancing activity was detected in the 25,000 to 35,000 and 40,000 to 67,000 m.w. regions, but antiviral activity was observed in the over 67,000 m.w. region. These results indicate that a new kind of lymphokine, called natural killer cell activating factor (NKAF), distinct from IFN and IL 2, was found. The NKAF was found to have the following properties: its pI value is between pH 5.5 and 6.5, it binds to concanavalin A- and lentil agglutinin-Sepharose, and it is stable with pH 2-24 hr treatment. In addition, NKAF-producing cells were peanut agglutinin (PNA)-thymocytes when thymocytes were fractionated by the agglutination-sedimentation method with the use of PNA.     

Intrathymic differentiation of cytotoxic T lymphocyte (CTL) precursors. I. The CTL immunocompetence of peanut agglutinin-positive (cortical) and negative (medullary) Lyt 123 thymocytes

To analyze the developmental and functional interrelationship between cortical and medullary thymocytes, the peanut agglutinin-(PNA) binding capacity was used to separate thymocytes into PNA+ (cortical) and PNA- (medullary) thymocytes. Virtually, all positively selected PNA+ thymocytes (90% of the overall thymocyte population) expressed the Lyt 123 phenotype, whereas 90% of negatively selected PNA- thymocytes expressed Lyt 1 alloantigens, about 10% being Lyt 123 thymocytes. Provided, the requirement of Lyt 1 T helper cells was bypassed by Interleukin 2, a nonspecific mediator of T help, PNA+ Lyt 123 thymocytes mounted cytotoxic T cell responses comparable in magnitude to that of peripheral T cells. Their repertoire included antigenic disparities coded for by the complete MHC complex, H-2K, I-A, H-2D, mutational events at H-2K, as well as antigenic disparities expressed on TNP conjugated- and Sendai virus-infected syngeneic cells. PNA- Lyt 123 thymocytes represent a highly reactive pool of primary cytotoxic T lymphocyte (CTL) precursors for both alloreactive and H-2-restricted CTL responses. Since PNA- thymocytes include also Lyt 1 T helper cells, PNA- responder thymocytes are able to mount autonomously (CTL responses. Our data are first to provide direct evidence that Lyt 123 cells represent a common source of alloreactive and H-2-restricted CTL precursors in unprimed lymphocyte populations. Moreover, the apparent immunocompetence of cortical PNA+ thymocytes is now explained by their lack of T helper cells.     

Interleukin 2 mediates an alteration in the T200 antigen expressed on activated B lymphocytes

We have examined the effect of exogenous IL 2 on cell surface antigen expression in LPS/dextran sulfate-activated murine B cells with the use of a panel of fluorescein-conjugated lectins. Elevated binding of the lectins PNA and SBA to activated B cells was found to be mediated by IL 2-containing supernatants from stimulated EL4 cells as well as by recombinant IL 2. These lectins have specificity for terminal beta-(1-3)-N-acetylgalactosaminyl residues; thus, the quantity or accessibility of these moieties is mediated by IL 2 in activated B lymphocytes. PNA binding in all strains tested, regardless of MHC or background genes, was found to be elevated fivefold to 15-fold by exogenous IL 2. To observe this effect, IL 2 must be added during the first 24 hr of culture. Based on anti-Thy-1 + complement depletion studies, T cells were not required, suggesting a direct effect of IL 2 on B cells. The glycoprotein responsible for this elevated binding of PNA has an Mr of approximately 220K and by immunodepletion was shown to belong to the T200 (Ly-5) family of cell surface antigens. These data demonstrate that exogenous IL 2 can mediate alterations in T200 expression on activated B cells that may be related to IL 2-driven modulation of B cell proliferation and/or differentiation.     

Functional analysis of cloned macrophage hybridomas. IV. Induction and inhibition of mixed lymphocyte responses

A series of macrophage (M phi) hybridomas were generated by fusion of drug-marked P388D1 (H-2d) tumor cells with CKB (H-2k) splenic adherent cells. The ability of this panel of cloned M phi hybridomas expressing various levels of surface Ia antigens to induce allogeneic mixed lymphocytes responses (MLR) was examined. All MLR stimulatory M phi hybridomas expressed surface Ia antigens. However, some Ia+ and all Ia- M phi hybridomas were unable to induce vigorous MLR responses. Furthermore, even after induction of surface Ia antigen expression with Con A supernatants (Con A Sn) or purified interferon-gamma, the nonstimulatory M phi hybridomas remained ineffective at inducing strong MLR proliferative responses. Furthermore, addition of the latter M phi hybridoma clones (both with and without Con A Sn treatment) to conventional MLR cultures resulted in inhibition of MLR responses. The series of inhibitory M phi hybridomas secreted normal levels of IL 1 upon stimulation with lipopolysaccharide. After surface Ia induction with Con A Sn, the inhibitory M phi hybridomas could stimulate secretion of IL 2 and expression of IL 2 receptors. Moreover, although they inhibited conventional MLR responses, IL 2 production and IL 2 receptor expression were not significantly inhibited. Addition of these M phi hybridomas 24 to 48 hr after initiation of MLR response also inhibited MLR proliferation. The results indicated that the group of inhibitory M phi hybridomas can inhibit MLR responses after IL 2 secretion and acquisition of IL 2 receptors. Finally, this inhibitory activity has been maintained during 1 yr of continuous in vitro culture, and the hybridomas represent a stable "homogeneous" subpopulation of inhibitory macrophages. Thus, the inhibitory phenotype appears to reflect arrest at a distinct differentiation stage.     

Functional status of interleukin 2 receptors expressed by immature (Lyt-2-/L3T4-) thymocytes

A subpopulation of phenotypically immature (Lyt-2-/L3T4-) thymocytes express receptors for the polypeptide hormone interleukin 2 (IL 2); however, these cells do not proliferate in vitro in response to IL 2. In investigating this phenomenon in greater detail, we observed that the IL 2 receptors (IL 2-R) on freshly isolated immature thymocytes bound IL 2 with about fivefold lower affinity (Kd approximately 100 pM) than IL 2-R on activated mature T cells and T cell lines (Kd approximately 20 pM). Furthermore, in contrast to activated T cells, Lyt-2-/L3T4- thymocytes did not endocytose bound IL 2. When stimulated in short-term culture with a combination of phorbol ester (PMA) and calcium ionophore, Lyt-2-/L3T4- thymocytes proliferated in a largely IL 2-dependent fashion. IL 2-R expression on these activated cells initially disappeared (at 24 hr) and subsequently reappeared (at 48 to 72 hr). Reexpressed IL 2-R on activated thymocytes resembled those on mature T lymphocytes in that they bound IL 2 with high affinity (Kd = 15 to 25 pM) and were capable of endocytosing IL 2. Taken together, these data place certain constraints on the putative physiologic role of IL 2 in intrathymic growth regulation.     

Regulatory effects of human IgE-binding factors on the IgE response of rat lymphocytes

Normal human peripheral blood T cells were propagated in the presence of human interleukin 2, and activated cells were incubated with human IgE-dimer to induce IgE binding factor formation. The cells were then fused with a mutant of the human T cell line CEM. Five of the T cell hybridomas formed IgE binding factors upon incubation with human IgE-dimer. Because IgE binding factors formed by the human T cell hybridomas had affinity not only for human IgE but also for rat IgE, the biologic activities of the factors were evaluated by using antigen-primed rat mesenteric lymph node (MLN) cells. When parent T cells were propagated with crude IL 2, which contained glycosylation enhancing factor (GEF), IgE binding factors formed by all of the five hybridomas had affinity for Con A, but only a fraction of the factors bound to lentil lectin. The 60,000 and 15,000 IgE binding factors formed by two representative hybridomas, i.e., 166A2 and 166G11, selectively potentiated the IgE-forming cell response of rat MLN cells. When parent T cells were obtained by propagation with purified IL 2, which did not contain GEF, and the cells were incubated with IgE-dimer in the presence of glycosylation inhibiting factor (GIF), T cell hybridomas constructed from the cells formed IgE binding factors that lacked affinity for Con A but bound to peanut agglutinin (PNA). The 30,000 IgE binding factors formed by two of such hybridomas, 398A3 and 400G2, selectively suppressed the IgE response of rat MLN cells. It was also found that the biologic activities and carbohydrate moieties of human IgE binding factors could be switched by changing the culture conditions of the hybridomas. When the 166A2 hybridoma was cultured with human IgE in the presence of bradykinin, essentially all of the IgE binding factors that were formed by the cells bound to lentil lectin, and the factors that were formed in the presence of bradykinin exerted higher potentiating activity than those obtained in the absence of bradykinin. On the other hand, IgE binding factors formed by the same cells in the presence of GIF had affinity for PNA, and selectively suppressed the IgE response of rat MLN cells.     

Induction of high affinity IL 2 receptors on B cells responding to anti-Ig and T cell-derived helper factors

We have reported that IL 2 is one of the essential helper factors in culture supernatants from concanavalin A-activated spleen cells or T cell hybridomas that support proliferation and immunoglobulin secretion in B cell cultures responding to anti-immunoglobulin. Here we show that cells in such cultures consume IL 2 and bear high affinity IL 2 receptors detected by binding of purified, radiolabeled IL 2. Induction of high affinity IL 2 receptors depends on addition of both anti-immunoglobulin and helper factors, and does not occur in cultures given only anti-Ig, only helper factors, concanavalin A plus helper factors, or LPS. The majority of IL 2 receptors are on cells that also bear endogenous membrane immunoglobulin, because they are found in the membrane immunoglobulin-positive fraction when cultured cells are separated by fluorescence sorting after overnight culture in the absence of anti-immunoglobulin to allow reexpression of membrane immunoglobulin.     

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.     

Constitutive and mitogen-induced production of T cell growth factor by stable T cell hybridoma lines

Stable T cell growth factor- (TCGF; IL 2) producing cloned T cell hybridoma lines were constructed by fusing murine alloantigen-activated T cells with the 8-azaguanine-resistant lymphoma line, BW5147. Many, but not all, clones of one of these hybridomas, i.e., hybridoma 24, secreted TCGF constitutively, but production was markedly enhanced by stimulation with T cell mitogens. Large numbers of TCGF-secreting hybridoma cells in a stable functional state could be obtained from histocompatible mice inoculated with cloned T cell hybridomas. Moreover, such in vivo-derived hybridoma cells could be stimulated sequentially with mitogen at least twice to secrete their biologically-active product, resulting in larger TCGF yields from the same cells. The secreted product of these T cell hybridoma lines resembled TCGF isolated from other cellular sources in that it: a) supported the growth of a TCGF-dependent T cell line; b) provided help for the induction of alloantigen-reactive cytotoxic T lymphocytes from thymocyte precursors; c) facilitated concanavalin A-induced mitogenic responses of low thymocyte numbers; d) had an apparent m.w. of 30,000 to 40,000 by gel filtration chromatography; and e) was eluted from DEAE-Sephacel ion-exchange chromatography columns by salt concentrations of 30 to 150 mM NaCl. The ability of these T cell hybridomas to grow in vivo and retain their functional characteristics in a stable form should prove useful in terms of providing large numbers of TCGF-secreting cells and studying in vivo aspects of the production of TCGF as well as other immunoregulatory mediators.     

T cell-induced expression of membrane IgG by 70Z/3 B cells

To study T cell regulation of B cell isotype differentiation, we determined the capacity of clonal T cell populations (hybridomas derived by fusing BW5147 with Con A-activated Peyer's patch (PP) and spleen T cells) to induce "downstream" isotype expression by the pre-B cell lymphoma 70Z/3. In initial studies, we found that 70Z/3 B cells cultured in the presence of LPS (1 microgram/ml) expressed membrane IgM (mIgM) but not membrane IgG (mIgG). In contrast, 70Z/3 B cells cultured with HAJ-3 T cells, a PP-derived T cell hybridoma (as well as other similarly derived PP and spleen hybridomas), or with HAJ-3 T cells plus LPS do express mIgG. Such expression occurred in spite of mitomycin C-induced blockage of cell proliferation, and is observed in 70Z/3 B cell subclones cultured with HAJ-3 T cells. For these reasons, it is not due to selective expansion of a small pre-switched mIgG-bearing 70Z/3 B cell subpopulation. In other studies it was shown that 70Z/3 B cells expressing mIgG after induction by HAJ-3 T cells continue to express mIgM and do not secrete IgG. Finally, exposure of 70Z/3 B cells to the macrophage factor IL 1 and the T cell factors IL 2, BSF-pl, and BCGF-II present in EL-4 cell supernatants did not result in mIgG expression. On the basis of these studies, we conclude that a clonal B cell population expressing mIgM can be induced by T cells to co-express mIgG. Because the B cells do not express mIgG unless exposed to T cells, this represents a T cell-induced isotype switch.     

T cell replacing factor for steroids (TRF-S): a 40,000 dalton protein produced by a T4+ T cell

The induction of polyclonal immunoglobulin (Ig) synthesis by glucocorticosteroids (GCS) in human peripheral blood lymphocytes is dependent on both T cells and monocytes. T cells can be replaced by a cytokine, T cell replacing factor for steroids (TRF-S), which promotes GCS-induced Ig production. T cells produce the cytokine when cultured with intact monocytes, with 24 hr monocyte supernatants, or with small quantities (0.1 U/ml or more) of highly purified interleukin 1 (IL 1). TRF-S was produced by isolated T4+ cells, whereas isolated T8+ cells were unable to help GCS-induced Ig synthesis. High pressure liquid chromatography with a gel permeation column revealed a single locus of activity that corresponded to an apparent m.w. of 40,000. At the dilutions utilized in culture, supernatants containing optimal TRF-S activity (3 U/ml final concentration in culture) were found to have less than 0.2 U/ml (final concentration) of interleukin 2 (IL 2) activity. Neither recombinant IL 2 nor recombinant interferon-gamma (IFN-gamma) over a broad range of concentrations was able to reproduce the capacity of TRF-S to induce the development of Ig-secreting cells with GCS. Thus, we report that TRF-S is synthesized primarily by T4+ T cells, and that its production is stimulated by small concentrations of IL 1. The apparent m.w. of TRF-S is 40,000, and its biological activity is distinct from that of IL 1, IL 2, and IFN-gamma.     

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.     

Immature and advanced patterns of T cell receptor gene rearrangement among lymphocytes in splenic culture

Bulk populations and 39 hybridomas from splenic Con A cultures were analyzed for rearrangements among TCR genes: alpha, beta, gamma, and delta. Patterns were categorized to reveal general rules governing gene rearrangement within the activated adult peripheral population. Many patterns of gene rearrangement were consistent with previous studies of T cell lines. Additional points of interest were the following: 1) A large proportion of Con A-stimulated splenic cells bore no TCR gene rearrangements. 2) One splenic hybridoma exhibited an unusual gene pattern, with rearrangements, at alpha and beta, but not J gamma 1 or J gamma 2 loci. 3) Multiple gamma rearrangements were noted other than V1.2-J2 and V2-J1. 4) One hybridoma exhibited TCR gene rearrangements typical of day 14 to 15 fetal thymocytes, as well as rearrangements at immunoglobulin gene loci. 5) Among hybridomas with J alpha rearrangements, homologous chromosomes exhibited rearrangements at similar positions along the J alpha locus.     

Mechanism of action of Cyclosporin A: inhibition of lymphokine secretion studied with antigen-stimulated T cell hybridomas

We have employed bifunctional T cell hybridomas, which can be stimulated to secrete lymphokine(s) and lyse specific target cells, to analyze the effect of Cyclosporin A (CsA) on T cell helper and effector functions. We report here the effects of CsA on antigen- and lectin-induced lymphokine secretion. We have found that a pharmacologic level of CsA (10 ng/ml) blocks antigen- and lectin-driven interleukin 2 (IL 2) secretion without affecting cell proliferation. In addition, one monoclonal hybridoma that is induced by concanavalin A to secrete colony stimulating factors (CSF) as well as IL 2 is concomitantly blocked by CsA for production of IL 2 and CSF. Because the hybridomas grow constitutively and are devoid of functional IL 2 receptors, they permit analysis of the kinetics of the inhibitory response. We have shown that CsA blocks not only stimulation of lymphokine secretion but also ongoing IL 2 production, probably by interfering with the effective interaction of receptor and antigen. Thus, blocking of IL 2 secretion from preactivated cells by CsA occurs by 1 to 2 hr, the time required to stop IL 2 production by removal of Ag/Lectin stimulator. The results are consistent with a mechanism of action of CsA on T cells that involves a direct interference of CsA with binding of Ag to Ag-receptor and results in blocking of induction and active secretion of multiple lymphokines.     

Lymphokine production by human T cell hybridomas

Human T cell hybridomas were generated by hybridization of SH9 cells, the 6-thioguanine-resistant variant of human T lymphoma Hut102-B2, with concanavalin A-stimulated human peripheral blood lymphocytes. The hybrid nature of the established cell lines was documented by the difference in D14S1 restriction fragment length polymorphism between SH9 and the hybridoma cell DNA, and by the expression of OKT11 antigen on hybrid cells. T cell growth factor, macrophage growth factor (MGF), and interferon (IFN) activities have been demonstrated in the supernatants of different hybrid cultures, but not in SH9 cell cultures. Substantial quantities of MGF were secreted by several hybrids including the L23 line. MGF activity was dose-dependent, heat-labile, and synergistic with indomethacin. High titers of IFN activity were found in the cultures of hybridoma L415 and its subclones. Neutralization with specific antisera showed the IFN synthesized by L415 clones was immune interferon (IFN-gamma). Like the parental SH9 line, all of the hybridomas producing these lymphokines exhibited a cell surface phenotype typical for helper T cells. The hybridoma system therefore shows potential for the study of various lymphokines produced by human helper T lymphocytes.