Comparison of allogeneic and self-restricted stimulation of lymphokine production by dual-reactive cloned T cells

Murine T cell clones were derived that proliferated in response to stimulation by alloantigen or by ovalbumin (OVA) in the presence of irradiated syngeneic spleen cells. Two cloned cell lines of strain B10.BR (H-2k) proliferated in response to alloantigen encoded by I-Ab, whereas the response to OVA was restricted by an element encoded by I-Ak. A cloned cell line of strain B10.A (H-2a) proliferated in response to alloantigen encoded by I-As, whereas the response to OVA was restricted by an element encoded by I-Ak. Cloned cells were stimulated by alloantigen or by OVA to produce lymphokines and to incorporate thymidine. Culture supernatants were collected 24 hr later and were assayed for interleukin 2, colony stimulating factor, interferon, Ia-inducing activity, and interleukin 3; thymidine incorporation was measured 72 hr after stimulation. For each clone tested, stimulation by alloantigen or by OVA led to the production of an identical array of lymphokines. Likewise, the strength of stimulation by alloantigen was approximately equal in magnitude to the strength of stimulation by a particular concentration of OVA. Lymphokine production and thymidine incorporation were co-variant measures of the intensity of stimulation. These data, in combination with data linking OVA reactivity and alloreactivity to identical regions of the major histocompatibility complex, suggest that dual reactivity represents a cross-reaction between alloantigen and self determinants associated with nominal antigen.     

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.     

Eosinophils and immune mechanisms. III. Production of the lymphokine eosinophil stimulation promoter by mouse T lymphocytes.

The lymphoid cell population responsible for production of eosinophil stimulation promoter (ESP), a lymphokine which increases migration of eosinophils, was investigated in murine Schistosoma mansoni infection. Con A challenge induced ESP production, whereas LPS did not. Prior treatment with anti-thetaC3H alloantiserum plus complement in vitro eliminated ESP production; in vivo treatment with rabbit anti-mouse thymocyte serum consistently reduced ESP production by splenic lymphoid cells, but affected lymph node cell ESP production only after exceptionally large doses. Thymocytes did not produce significant amounts of ESP; nor did lymphoid cells from congenitally athymic mice. Depletion of B lymphocytes and macrophages by nylon fiber adherence eliminated antigen-induced ESP production; this was partially restored by addition of non-immune, 72-hr peritoneal exudate cells. Con A-induced ESP production was not affected by nylon fiber treatment. These results demonstrate that ESP is produced by an ATS-sensitive, peripheralized T lymphocyte population, and suggest a macrophage requirement for antigen-induced production of this lymphokine.     

A quantitative analysis of antigen-triggered lymphokine production by activated T cells

We have developed a theoretical model to describe the triggering of lymphokine release from antigen-specific, activated T (T') cells, and we have used this model to define parameters that regulate this interaction. Under assay conditions of T' cell excess, the efficiency of triggering is a function of the target cell type. When various H-2k-bearing target cells were used to trigger B10.AQR T' cells activated against B10.A cells (anti-Kk), a hierarchy of triggering efficiency was observed with B10.A Con A blasts greater than R1.1 tumor cells greater than B10.A spleen cells greater than B10.A lymph node cells. There was a 10-fold difference in triggering efficiency between Con A blasts and lymph node cells. A similar pattern of reactivity was observed for various H-2d-bearing target cells used to trigger CBA T' cells specific for BALB/c antigens (anti-H-2d). Under assay conditions of T' cell excess, the order of reaction, i.e., the number of target cells per T' cell required to trigger lymphokine release, remained constant for the different target cell types. However, the order of reaction can vary with conditions of T cell activation. CBA T' cells activated against BALB/c spleen cells exhibited pseudo-first-order reactivity when triggered for lymphokine release with P815 target cells, whereas CBA T' cells activated against UV-irradiated P815 cells exhibited pseudo-second-order reactivity when triggered by the same tumor cells. Thus, T cells with the same apparent specificity can be qualitatively different in their reactivity with antigen. Under assay conditions of target cell excess, our analysis indicated that no T-T interaction was involved in the triggering reaction. Finally, our analysis was applied to study the nature of cross-reactivity. The results indicated that the triggering of lymphokine elicited by a cross-reactive antigen was due to the reactivity of a discrete subset of T' cells.     

Production of a cloned mouse by nuclear transfer from a fetal fibroblast cell of a mouse closed colony strain.

We have tested a closed colony mouse strain as a source for nuclei donors to determine differences in cloning efficiency. When donor nuclei were isolated from fetal fibroblast cells and injected into recipient oocytes from closed colony mice (ICR), reconstructed oocytes developed to full term and the success rate of cloning was 0.1%. This result indicates that cloning efficiency does not depend on the cell type. The body weight of the cloned mouse was lighter than controls, and the lifetime of the cloned mouse was the average for a mouse. These results contradict commonly-held views on cloning.     

Antigen-reactive cloned helper T cells. II. Exposure of murine cloned helper T cells to IL 2-containing supernatant induces unresponsiveness to antigenic restimulation and inhibits lymphokine production after antigenic stimulation

Cloned murine helper T lymphocytes (HTL) reactive to alloantigen or to ovalbumin (OVA) become unresponsive to antigenic restimulation after exposure to antigen or to culture supernatant fluids (SF) containing multiple lymphokine activities. Unresponsiveness is manifest by a failure of antigen-stimulated cells to incorporate thymidine or to produce lymphokines after antigenic challenge. Antigen-unresponsive HTL, however, will incorporate thymidine when exposed to an exogenous source of interleukin 2 (IL 2). The duration of unresponsiveness to antigen is correlated with the concentration of IL 2 in SF to which the cloned HTL had been exposed. Chromatographic fractionation of IL 2-containing supernatant from EL-4 thymoma cells (EL-4 SF) yielded a pool of SF that was enriched for IL 2 activity. Exposure of HTL to lymphokines contained in this pool induced unresponsiveness to antigen that was comparable to that observed when HTL were exposed to unfractionated EL-4 SF. Unresponsiveness to antigen also developed after cloned HTL were stimulated with concanavalin A (Con A) or with OVA and syngeneic splenic filler cells. We have used monoclonal antibody (mAb) GK1.5 (anti-L3T4) to investigate the role of lymphokine production in the induction of unresponsiveness. This antibody did not inhibit IL 2-induced thymidine incorporation by cloned HTL, and did not inhibit the induction of unresponsiveness after exposure of cloned HTL to EL-4 SF. In the presence of mAb GK1.5, however, HTL that were stimulated with Con A or OVA did not become unresponsive to antigenic restimulation, an effect that was overcome by the addition of EL-4 SF. These results suggest that HTL become unresponsive to antigen after exposure to IL 2-containing SF, and that stimulation by antigen or Con A can induce the unresponsive state by virtue of stimulating lymphokine production.     

Activation of MHC-restricted rat T cells by cloned syngeneic thyrocytes

We have previously demonstrated that rat thyrocytes express MHC class II Ag (RT1.B&D) in response to IFN-gamma. To determine whether MHC class II-positive thyrocytes can be recognized by MHC-restricted T cells, we used our clone of rat thyroid cells (1B-6) derived from the Fisher rat thyroid cell line (FRTL-5) and known to express MHC class II Ag in response to recombinant rat IFN-gamma. CD4+ and CD8+ normal syngeneic Fisher rat spleen T cells were selected by flow cytometry and averaged greater than 96% purity. We demonstrated that irradiated MHC class II-positive but not class II-negative 1B-6 thyrocytes stimulated CD4+ T cells in a primary sensitization reaction over 4 days. In contrast, CD8+ T cells had no response in similar experiments. This stimulation of CD4+ T cells was dose dependent for 1B-6 thyrocytes and was abrogated by anti-rat MHC class II mAb (MRC OX-6). Autoreactive (Fisher) and alloreactive (Buffalo) T cell lines and isolated CD4+ T cells derived from these lines, which were developed against Fisher rat spleen cells, similarly recognized MHC class II Ag expressed on 1B-6 cells but had no detectable response to 1B-6 MHC class II-negative thyrocytes or MHC class II-positive human thyroid cells. The CD4+ T cell recognition of 1B-6 cells via MHC class II Ag supports our previous data with autologous human thyroid T cell co-cultures and is indicative of an autospecific role for thyrocytes in the development of autoimmune thyroiditis.     

Production of human/mouse hybridomas secreting a human lymphokine,osteoclast activating factor

A human/mouse hybridoma was developed which has the property of secreting a human bone resorbing factor similar or identical to the osteoclast activating factor (OAF) isolated from human tonsil lymphocytes. Mouse plasmacytoma cells negative for OAF production were fused with an enriched subpopulation of human tonsil lymphocytes that had been activated with phytohemagglutinin (PHA) to produce OAF (G. E. Nedwin, M. A. Mohler, and R. A. Luben, submitted for publication). Culture supernatants from mixed hybridomas contained a bone resorbing protein shown to cause the release of ⁴⁵Ca from previously labeled mouse calvaria. The bone resorbing activity from these hybridomas was inhibited by the presence of OAF-specific monoclonal antibodies. Several hybridomas retained OAF production following limited dilution cloning. One clone, CD6.20, showed a biphasic dose-response curve for bone resorption similar to that of purified OAF from PHA-activated human tonsil lymphocytes. OAF production in the CD6.20 cell line has been retained for over 100 passages. Karyotype analysis of this cell shows the presence of human chromosomes 10 and 18 and the X chromosome.     

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.     

Activation of B cells by autoreactive T cells: cloned autoreactive T cells activate B cells by two distinct pathways

Although the existence of autoreactive T cells has been widely reported, the functional capacities of these populations have been less well defined. Studies were therefore carried out to characterize the relationship of autoreactive T cells to antigen-specific major histocompatibility complex (MHC)-restricted T cells in their ability to act as helper cells for the induction of immunoglobulin synthesis by B cells. A number of autoreactive T cell lines and clones were isolated from antigen-primed spleen and lymph node cell populations. Autoreactive T cells were found to proliferate in response to direct recognition of syngeneic I-A or I-E subregion-encoded antigens in the absence of any apparent foreign antigen. It was shown that cloned autoreactive T cells were capable of activating B cell responses through two distinct pathways. After appropriate stimulation by syngeneic cells, autoreactive T cells polyclonally activated primed or unprimed B cells to synthesize IgM antibodies. These activated T cells functioned in these responses through an MHC-unrestricted pathway in which polyclonal responses were induced in both syngeneic and allogeneic B cells. These cloned autoreactive T cells were also able to activate IgG responses by primed B cells through a different activation pathway. In contrast to the polyclonal activation of IgM responses, the induction of IgG antibodies by the same cloned T cells required primed B cells and stimulation with the priming antigen. The activation of B cells to produce IgG was strongly MHC restricted and required the direct recognition by the autoreactive T cells of self MHC determinants expressed on the B cell surface, with no bystander activation of allogeneic B cells. These results indicate that cloned autoreactive T cells resemble antigen-specific MHC-restricted T cells in their ability to function as T helper cells through distinct MHC-restricted and MHC-unrestricted pathways.     

Regulation of lymphokine response during reinfection by influenza virus. Production of a factor that inhibits lymphokine activity

Mononuclear cells, obtained from the spleens and lungs of influenza virus-seropositive C57BL/6 mice at 2 to 4 days after re-infection with homologous virus (strain A/Bangkok/1/79), produced a low m.w. factor in vitro that prevents the biologic expression, but not production, of the lymphokine, leukocyte migration inhibition factor (LIF). The low m.w. factor inhibited LIF activity without destroying the LIF molecule inasmuch as simple dialysis restored lymphokine activity to culture supernatants. Production of the low m.w. factor was observed from 2 to 4 days after re-infection, at which time the delayed-type hypersensitivity response to viral Ag was suppressed. In contrast, LIF was produced by splenocytes and lung mononuclear cells obtained at all times tested after re-infection (from 2 to 30 days). Production of the low m.w. factor required re-infection of influenza A virus-seropositive mice with type A virus; re-infection with influenza B virus failed to induce production. Ag specificity was also required in vitro for splenocytes to produce the factor; cells from type A virus-re-infected mice required type A Ag stimulation. Cell depletion studies with mAb plus C revealed that macrophages and T cells along with Ag stimulation were required for factor production by spleen cells. However, mononuclear cells obtained within 4 days from the lungs of re-infected mice did not require in vitro Ag stimulation for production of the low m.w. factor, and factor production was dependent upon the presence of CD4+ (L3T4) cells in the culture. Fractionation of culture supernatants over a Sephadex G-50 column indicated that the factor had a molecular mass of 2 to 3 kDa, and by FPLC chromatofocusing over a Mono P column, the factor eluted at a pH of approximately 8.2. Thus, re-exposure of influenza virus-seropositive mice to homologous virus resulted in the production of a low m.w. factor that prevented the biologic expression of LIF, but not its production. Lymphokines are an important component of the delayed-type hypersensitivity response; the presence of mononuclear cells secreting a low m.w. factor and LIF concomitantly at the site of virus replication (lungs) and the capacity of the factor to block the biologic expression of LIF in vitro suggest that the factor may have a role in the regulation of a delayed-type hypersensitivity response in vivo during re-infection.     

Interleukin 2 inhibits antigen-stimulated lymphokine synthesis in helper T cells by inhibiting calcium-dependent signalling

Certain L3T4+, Lyt-2- cloned murine helper T lymphocytes (HTL), when cultured with a high concentration of interleukin 2 (IL 2), become temporarily unresponsive to antigenic stimulation, as indicated by failure to proliferate and by reduced secretion of lymphokines when challenged with antigen. Exposure of cloned HTL to IL 2 also renders these cells less responsive to concanavalin A (Con A). Here we demonstrate that antigen-unresponsive HTL also accumulate reduced levels of lymphokine mRNA, thus indicating a pretranslational block of the response to antigen. However, HTL which had been pretreated with IL 2 and were unresponsive to antigen responded strongly to antigen + A23187 or to A23187 + PMA but failed to respond to antigen + PMA. With HTL made unresponsive to antigen or to Con A by exposure to IL 2, increases in intracellular calcium ion levels stimulated by Con A also were reduced. Thus, for mouse HTL clones, the IL 2-induced state of unresponsiveness to antigen or Con A appears to reflect an inability of such HTL to increase intracellular free calcium to a level sufficient for activation of lymphokine genes.     

Characterization of effector-target conjugates for cloned human natural killer and human lymphokine activated killer cells by flow cytometry.

The present studies demonstrate that the intracellular fluorochromes calcein and hydroethidine can be used for quantification of effector-target conjugates involving cloned human natural killer (NK) or interleukin-2 (IL-2) activated human lymphokine activated killer (LAK) cells by dual color flow cytometry without potential artifacts that might result from extensive modification of effector and/or target cell membranes. Cloned NK cells and LAK cells form conjugates with cultured cell lines regardless of susceptibility to lysis. The strength of the interactions in these conjugates was investigated using a variable speed vortexer. Even relatively gentle vortexing disrupted most conjugates involving fresh human peripheral blood lymphocytes (PBL) but only about one-fourth of conjugates between K-562 cells and human PBL that had been cultured with or without IL-2 by this treatment. The rate of conjugate formation for LAK cells was determined to be about 3 times faster than for cloned NK cells, and both rates are considerably faster than the reported rate of formation of cytotoxic T lymphocyte (CTL) target conjugates. The differences in the rate of conjugate formation are apparently not related to target cell specificity, since LAK cells form conjugates with susceptible and resistant cell lines at comparable rates. When effector-target conjugates are incubated at 37 degrees C in the absence of calcium--thereby precluding lysis--the percentage of conjugated LAK or cloned NK cells decreases logarithmically with time. These results suggest that an initial equilibrium between free and conjugated lymphocytes gradually shifts in favor of unconjugated cells.     

Antigen-reactive cloned helper T cells. I. Unresponsiveness to antigenic restimulation develops after stimulation of cloned helper T cells

Murine helper T lymphocyte (HTL) clones reactive to ovalbumin (OVA) were maintained in continuous culture in vitro. Clones were propagated by weekly stimulation in the presence of irradiated splenic filler cells, antigen, and supernatant fluid (SF) containing IL 2. By varying the quantity of these reagents in cultures of HTL cells, the reactivity to antigen of the cloned cells was altered markedly. After stimulation by antigen or SF, HTL clones became profoundly unresponsive to antigenic restimulation. Cells remained unresponsive for 2 to 9 days after stimulation, depending on the culture conditions that were chosen for their maintenance. The addition of SF containing a high concentration of IL 2 prolonged the duration of unresponsiveness by 3 days, and the presence of a non-T splenic filler cell increased the period of unresponsiveness by an additional 4 days. The use of a high concentration of OVA in cell cultures also prolonged the time of unresponsiveness. The results described here demonstrate that the response to antigen of HTL cells is down-regulated after stimulation, and appears to be correlated with exposure to SF that contains IL 2.     

Production of pyruvate by isolated mouse cumulus cells

Cumulus cells were isolated by hyaluronidase treatment of whole cumulus masses from superovulated, non-mated mice. The cells, in groups of approximately 200, were incubated for up to 4 h in 50 nl medium M2 at 37 degrees C, and serial 3-nl samples assayed for pyruvate using an ultramicrofluorescence technique. With 5.55 mM glucose, 23.3 mM lactate, or a mixture of the two substrates, the cumulus cells formed pyruvate at rates of 10.2, 9.6, and 8.9 fmol/cell/h, respectively. The concentrations of glucose, pyruvate, and lactate, as measured in 3-nl aliquots of rabbit oviduct fluid were 1.5 mM, 0.3 mM, and 3.7 mM, respectively. When incubated with 1 mM glucose and 3 mM lactate, mouse cumulus cells formed 7.5 fmol pyruvate/cell/h. The mean number of cumulus cells per ovum within a cumulus mass was 2,060. Intact cumulus masses from mated and non-mated superovulated mice, incubated with 1 mM glucose and 3 mM lactate, formed 22.6 and 23.3 pmol pyruvate/ovum/h, respectively. The results suggest that pyruvate production by cumulus cells may be important in supporting the nutrition of unfertilized and fertilized ova, and of spermatozoa, within the oviduct lumen.     

Production of the hyporeactivity factor by mouse cells

The mouse cells L 929 experiencing the state of hyporeactivity to repeated interferon induction with the aid of poly (I) X poly (C) secrete to the incubation medium a factor which suppresses interferon production. The factor is not neutralized by antiserum against murine interferon. Apart from hyporeactivity factor, interferon itself may inhibit its own production.     

Production of lymphokine mRNA by CD45R+ and CD45R- helper T cells from human peripheral blood and by human CD4+ T cell clones

The expression of lymphokine mRNA by human CD4+CD45R+ and CD4+CD45R- Th cells was assessed after mitogen stimulation. These Ag have previously been shown to relate closely to virgin and primed T cells, respectively. CD4+CD45R+ (virgin) and CD4+CD45R- (primed) cell fractions were isolated by sorting double-labeled cells with a fluorescence-activated cell sorter. CD4+CD45R+ cells produced high levels of IL-2 mRNA when stimulated with either PMA together with calcium ionophore, or with PHA, but they expressed only trace quantities of mRNA for IL-4 or IFN-gamma. In contrast, CD4+CD45R- cells produced high levels of mRNA for IL-2, IL-4, and IFN-gamma. After 14 days of continuous culture, CD4+CD45R+ Th cells lost expression of the CD45R Ag, but gained high level expression of CDw29, such that they were indistinguishable from the cell population which originally expressed this Ag. At the same time, they acquired the ability to synthesize IL-4 mRNA. It seemed likely that the broad lymphokine profile of primed Th cells might mask clonal heterogeneity. Analysis of 122 CD4+ T cell clones showed that all of them synthesized IL-2 mRNA. One clone failed to express IL-4 mRNA, but did produce those for IL-2 and IFN-gamma. A total of 34 of the clones was investigated to determine expression of IFN-gamma mRNA; two of these clones were negative for IFN-gamma mRNA, and both expressed IL-2 and IL-4 message. These data suggest that while fresh virgin and primed peripheral blood T cells show a clear resolution of lymphokine production, a simple subdivision of human CD4+ T cell clones on the basis of their lymphokine production (such as that reported for mouse Th cell clones) is not possible.     

A helper factor needed for the generation of mouse cytolytic T lymphocytes is made by tumor cell lines, cloned T cells, and spleen cells exposed to a variety of stimuli

A helper factor termed cytolytic T lymphocyte helper factor (CHF) that is needed for the generation of allospecific mouse cytolytic T lymphocytes (CTL) in vitro was produced by mouse spleen cells 3 to 4 days after the time when interleukin 2 (IL 2) had reached its maximal production. These kinetics were observed by stimulation of immune spleen cells with allogeneic tumor or spleen cells, with Sendai or influenza viral peptides, with virus infected cells, or with concanavalin A (Con A). CHF produced by rat spleen cells was able to help in the generation of mouse CTL, indicating that this cytokine was not restricted genetically. CHF could also be made by WEHI-3 and EL4 cell lines, as well as cloned cytolytic and helper T cells. The production of CHF by WEHI-3 cells argues that CHF is not IL 2. In addition, if CHF was not present early in the in vitro stimulation no CTL were generated, suggesting that CHF participated in the activation of CTL precursors. The addition of IL 2-containing conditioned medium to the CHF assay resulted in no substantial CTL generation, although significant cellular proliferation was observed. In contrast, CHF-containing conditioned medium allowed the generation of CTL in the absence of the same level of proliferation.