We have developed a rapid limiting dilution analysis technique for quantitating alloantigen-specific TCGF-secretory T lymphocytes (operationally defined as helper T lymphocytes or HTL) in murine lymphoid populations. A simple permutation of this technique allows the distinction between HTL that have encountered antigen in vivo and the large population of naive precursor HTL (pHTL) with the same alloantigen specificity. We present evidence to validate these LDA techniques, and show that lymphoid tissue from mice bearing sponge matrix allografts, but not isografts, contain HTL that have been activated in vivo. In addition, we demonstrate a requirement for restimulation with alloantigen for secretion of lymphokine by the in vivo-activated cells. We suggest that this differential LDA technique could serve as a valuable tool in evaluating in vivo immunity and/or the in vivo efficacy of immunosuppressive, as well as immunopotentiating, therapies.

The ability to grow antigen-spectfic human T-cell clones in vitro has been instrumental in understanding T-cell function. A major breakthrough in T-cell culture in vitro was the discovery of the T-cell growth-inducing properties of interleukin-2 (IL-2), originally called T-cell growth factor or TCGF, by Morgan et al (1), who for the first time were able to grow human T-lymphocytes, isolated from human bone marrow. Shortly thereafter, Gillis and Smith (2) reported the cloning and long-term culture of mouse cytotoxic T-cells, using TCGF. In spite of the success of growing human T-cells in vitro, the cloning of these cells, however, turned out to be more difficult in contrast to mouse cell lines that could be maintained in culture in the presence of TCGF-containing supernatant only, long-term cultures of cloned human allo-antigen-specific T-cell lines (3, 4) needed repetitive stimulation with specific allo-antigen for then growth (3) This led to the general assumption that antigen-specific IL-2-dependent T-cell lines and T-cell clones would lose then antigen responsiveness when propagated with IL-2 in the absence of specific antigen.

We previously reported that incubation of human peripheral blood mononuclear cells (PBMC) for 5 days with T-cell growth factor (TCGF) resulted in lymphokine-activated killer activity against endothelial cells (EC). In this paper we report on the effects of short-term incubation of PBMC with lymphokines. We show that incubation of PBMC with lymphokines during an 18-hr period is sufficient to generate a strong cytolytic response against EC. The cytolytic capacity of the effector cells was directly dependent on the dose of lymphokine added during the induction phase. When PBMC were separated into adherent and non-adherent cells, the non-adherent fraction could be induced to lytic activity against EC, whereas the adherent cells could not. When PBMC were separated, using 2-amino-ethylisothiouronium bromide hydrobromide-treated sheep red blood cells (AET-SRBC), into T- and non-T-cell fractions, the latter fraction could be induced to lyse EC. TCGF-induced cell-mediated EC lysis could not be inhibited using anti-T3 nor anti-LFA-1 antibodies. Lysis of EC by TCGF-stimulated effector cells was strongly inhibited by the addition of unlabelled K562 target cells, whereas cold OKT3 hybridoma cells did not exert such an effect.

CONCLUSIONS

the kinetics of the induction of lytic activity against EC, as well as the cell separation experiments, suggest that short-term-activated NK cells may lyse EC. This hypothesis was confirmed using monoclonal antibody and cold target cell analysis.

Both TGF beta 2 and 5 have been described in the South African clawed frog Xenopus laevis and have been cloned from the tadpole-derived fibroblast cell line, XTC. Because TGF beta has such a profound inhibitory effect on the mammalian immune system, this study was performed to determine whether TGF beta: (a) has any in vitro effects on the growth of Xenopus lymphoblasts, and (b) is produced by mitogen-activated Xenopus lymphocytes. Following stimulation with mitogen or alloantigen, T lymphocytes from Xenopus secrete a T-cell growth factor (TCGF) that is functionally homologous to mammalian interleukin-2 (IL-2). Both recombinant human TGF beta 1 and Xenopus TGF beta 5 inhibit TCGF-induced proliferation of Xenopus splenic blasts and this inhibition can be reversed with anti-pan TGF beta antiserum. The Xenopus mitogen-induced saturated ammonium sulfate precipitated TCGF-containing supernatant (SAS TCGF SN) also contains latent TGF beta as assayed on mink lung fibroblasts and Xenopus splenic blasts, and experiments utilizing anti-TGF beta antiserum showed that only TGF beta 5 is present in this supernatant.

The possible functional involvement of the T4 molecule in T-cell recognition of and/or activation by the class II HLA antigens SB was investigated. SB antigens are encoded by the SB gene that (1) maps between GLO and HLA-DR; and (2) codes for Ia-like molecules that are similar to but distinct from HLA-DR molecules. Both cytotoxic T lymphocyte (CTL) and proliferative responses to SB antigens were found to be mediated by OKT4+, OKT8- cells. SB-specific cytotoxicity and proliferation were analyzed in the presence of a series of monoclonal antibodies (OKT4, 4A, 4B, 4C, and 4D) that react with distinct epitopes on the OKT4 molecule. SB1-, SB3-, and SB4-specific CTL were partially inhibited by OKT4A and 4B, but not by OKT4, 4C, and 4D. SB2-specific CTL were not substantially inhibited by any of the OKT4-related antibodies. SB-specific proliferative responses to SB1, 2, 3, and 4 were strongly blocked by OKT4A and 4B but not by OKT4, 4C, and 4D. Proliferative responses of SB-primed cells stimulated with TCGF-containing supernatants alone were not inhibited by any of the OKT4-related antibodies, but were completely inhibited by the anti-Tac monoclonal, which reacts with the TCGF receptor. These results indicate that: (1) the T4 marker may be expressed on most T cells, regardless of their function, that recognize allogeneic Ia or self-Ia plus foreign antigens; and (2) the T4 molecule may be involved in T-cell recognition of and/or activation by class II HLA antigens.

We investigated the effect of both partially purified (TCGF) and recombinant interleukin-2 (rIL-2) on the tumor-directed cytotoxic activity of canine peripheral blood mononuclear cells (PBMC) using three normal canines. No cytotoxic activity was displayed by unstimulated effector cells at 3 h of incubation; however, the cytotoxic effect was observed in a 16-h assay. PBMC of all canines displayed significant levels of lytic activity after stimulation for 4 to 7 days with both types of IL-2 against a variety of allogeneic and xenogeneic neoplastic cells in 3-h 51Cr release assay. The cytotoxic activity of cultured cells increased proportionally in the 16-h assays. Morphological examination of the May-Grünwald and Giemsa stained cytocentrifuged slides of cultured cells on each day of assay showed an increase in large granular lymphocytes (LGLs) beginning on day 4 and reaching a peak on day 7 of culture.

A two-color flow cytometric analyses of the PBL of 51 patients (24 resectable, and 27 nonresectable), and of 30 healthy controls has been completed. The proportion of antigens, on the PBLs, defined by combinations of anti-Leu 2a and anti-Leu 15, anti-Leu 3a and anti-Leu 8, anti-Leu 3a and anti-HLA-DR, anti-HLA-DR and anti-Leu 2a and, anti-HLA-DR and anti-IL 2R, was not significantly different among the three groups. TCGF increased the proportion of Leu 2a+ Leu 15-, Leu 3a+ Leu 8-, HLA-DR+ Leu 2a+, Leu 3a+ HLA-DR+ and, HLA-DR+ IL 2R+ cells and reduced the proportion of Leu 2a+ Leu 15+ cells in gastric cancer patients, while the cultivation by IL 2 (2 weeks) increased the proportion of Leu 3a+ Leu 8-, HLA-DR+ IL 2R+ and Leu 3a+ HLA-DR+ cells, and did not change the proportion of Leu 2a+ Leu 15- and Leu 3a+ Leu 8+ cells, and decreased the proportion of Leu 2a+ Leu 15+ cells. These results, taken together with our previously published studies, suggest that the TCGF-activated Leu 2a+ Leu 15- subset but not the Leu 2a+ Leu 15+ subset serves as a phenotypic marker of suppressor-effector T cells, and that the IL 2-activated Leu 3a+ Leu 8- subset contained killer T cells against tumor cells in our experimental systems.

Five T-cell clones reactive with HBsAg were obtained from peripheral blood lymphocytes of a HBV vaccine recipient by means of in vitro stimulation of lymphocytes for 5 days by HBsAg and subsequent re-culture of lymphocytes with HBsAg, feeder cells and T cell growth factor (TCGF) for 6 days, followed by the limiting dilution method. Clones, and subclones derived from one original clone showed a specific proliferative response to HBsAg in the presence of autologous feeder cells but not to unrelated antigens, such as influenza virus antigens, herpes simplex antigens, toxoplasma antigens, and PPD. All clones were found to have a surface marker of helper/inducer phenotype, Leu 1+, Leu 2a-, and Leu 3a+, detected by an indirect immunofluorescence method. These clones, however, did not show a substantial helper effect for in vitro anti-HBs production by autologous B cells. This suggests that the mechanism of the helper effect by specific T-cell clones is implicated, and functions of such clones other than specific helper effect on antibody-producing B cells must be considered.

The long-term maintenance of T cells "cloned" by limiting dilution in TCGF was enhanced by the use of irradiated autologous lymphoblastoid cell line (LCL) cells as well as irradiated LCL cells of the individual to which the T cells were originally primed. It was possible to obtain more than 1 X 10(12) cells from a "clone" seeded at one cell per well. Some of the clones tested express primed LD-typing activity.

Human T lymphocytes (CCTC) have been maintained in continuous culture by a new method. The ability of CCTC to produce three types of T cell response has been determined and compared to the published responses of T cells grown in 'T cell growth factor' (TCGF) medium. Unlike TCGF cells, CCTC will not give proliferative responses when stimulated with PHA, Con A, or allogeneic lymphocytes even when supplemented with autologous PBL as a source of accessory cells. Instead, CCTC are potent inhibitors of the proliferative response of normal cells in MLC. This suppressive activity is not specific for histocompatible cells. Finally, CCTC express cytotoxic activity to a number of human lymphoid cell lines and this activity appears to be different from the polyclonal cytotoxicity reported for TCGF-maintained cells. Our method of long-term T cell culture therefore appears to grow cells with different properties from TCGF cells.

The effects of transfer of T cell growth factor (TCGF)-expanded spleen cells after concanavalin A (Con A) stimulation into syngeneic Lewis rats were studied. The recipient rats were immunized with complete Freund's adjuvant for induction of adjuvant arthritis (AA) or chick type II collagen in incomplete Freund's adjuvant for induction of collagen-induced arthritis (CIA) on day 0. Each of 5 X 10(7) cultured cells without mitogenic stimulation, 2 X 10(7) Con A-stimulated cells, or 1 X 10(7) TCGF-expanded cells cultured for 8 days (4 days X 2 culture cycles) after Con A stimulation was given on days 0 and 7. Both transfers of the cultured cells without stimulation and TCGF-expanded cells markedly diminished the severity of AA and CIA. On the contrary, transfer of Con A-stimulated cells led to no suppressive activity. In addition, transfer to TCGF-expanded cells significantly lowered the titre of anti-type II collagen antibody compared to that of control rats. The transfer of 1 X 10(7) TCGF-expanded cells was optimal for suppressing AA, in terms of cell number. This observation suggests that these cells were much more effective than were the unstimulated cultured cells, for which more than five times the number was required for the same suppressive activity. As far as the phenotypic proportion of helper (W3/13) and suppressor (OX-8) cells is concerned, we found no significant differences between the cultured cell groups and the freshly separated spleen cell group. The precise mechanism of these suppressive effects is the subject of further study. The transfer of TCGF-expanded cells appears to have a potent immunomodulatory effect.

The mitogenic response to Con A and the production of T cell growth factor or interleukin 2 (IL 2) by splenic and peripheral blood lymphocytes of obese strain (OS) chickens with spontaneous autoimmune thyroiditis have been investigated. By using an optimized method with Con A-coated chicken erythrocytes (MRC), lymphocytes of OS chickens were found to exhibit significantly elevated mitogenic responses as compared with cells from either Normal White Leghorn chickens (NWL) or animals of the Cornell C-Strain (CS), from which the OS has originally been developed. This difference was observed throughout ontogeny up to 15 mo of age, and was associated with increased levels of IL 2 activity in the culture supernatants. The elevated responsiveness of OS T lymphocytes was also found to be manifested in the expression of receptors for IL 2, because Con A-stimulated lymphocytes of OS birds were significantly more effective than those from normal controls in absorbing IL 2 activity from conditioned media (CM) of stimulated spleen cells. High concentrations of CM were suppressive in IL 2 assays, signaling the presence of an inhibitory factor(s) in addition to IL 2. An additional indication for defective immunoregulation was that CM from OS lymphocyte cultures showed significantly less of this suppressive activity in comparison with CM of normal (NWL and CS) lymphocyte cultures. Finally, the spontaneous uptake of 125IUdR of embryonic and early post hatching OS spleen lymphocytes was consistently and significantly enhanced. This difference, however, in contrast to the one observed in Con A responses, was found to decrease with age. The data are discussed in view of the contradictory results concerning T cell functions reported for several autoimmune states in mammals.

The supernatant of TE+ lymphocytes isolated by density gradient from a lympho-epithelial thymoma contained a biochemical variant of T cell growth factor (IL-2). This protein showed two peaks with an apparent molecular weight of 18 and 28 kd by Sephadex G 100 chromatography. Further purification was achieved by DEAE Sephacel and CM Sepharose ion-exchange chromatography. By isoelectric focusing the isoelectric point was found to differ from that of normal. IL-2. Based on these results, the IL-2 variant appears to be a new kind of lymphokine distinguishable from normal IL-2 and similar to a TCGF (L-TCGF peak II) purified from the malignant T cell clone HUT 102.

It would be of great interest to obtain permanent T-cell lines retaining specific activity without either allogeneic or xenogeneic stimulation. Functionally active hybrids between cytolytic T cells and thymoma were previously reported, but they had to be selected in a TCGF-containing medium. This study contains new results and reports the preparation of a hybrid cell from a cytolytic T cell and a polyoma virus-infected fibroblast, in which the T-cell characteristics dominate over the polyoma-transformed characteristics. A differentiated T-cell function (i.e., cytolysis) persists and the differentiated line does not require TCGF. The loss of cytolytic activity during in vitro evolution may be due to a selection favouring transformed cells, as suggested by concomitant enhancement of the transformed phenotype and chromosome loss.

This study shows that anti-Tac antibody does not bind to human thymocytes unless they are activated. Human thymocytes could be induced to express Tac antigen (TCGF receptor) on their cell surface by Concanavalin A. B lymphoblastoid cells or 12-O-tetradecanoylphorbol 13-acetate alone did not induce TCGF receptors, but they did exert a very marked synergistic effect with Concanavalin A. This observation is consistent with our earlier finding that B lymphoblastoid cells secrete a factor which exerts a synergistic effect on the induction of lymphokine secretion by thymocytes and T-cells. The early expression of Tac antigen was independent of thymocyte proliferation. Anti-Tac antibody (10(-3)) inhibited the expression of TCGF receptors and late proliferation of thymocytes. TCGF did not reverse this inhibition nor did it prevent the binding of Tac antibody, but it enhanced the expression of Tac antigen.

A simple two-step method involving ammonium sulfate precipitation followed by hydrophobic chromatography is described for the separation of T cell growth factor (TCGF) from a number of other factors contained in medium conditioned by concanavalin A-stimulated spleen cells. Thus, granulocyte-macrophage colony-stimulating factor, P cell-stimulating activity, pluripotential stem cell-supporting activity and interferon activity were not detected in TCGF partially purified by these steps. T cell-replacing factor co-purified with TCGF. Macrophage activity factor (MAF) co-purified with TCGF, but the ratio of MAF to TCGF activities was reduced more than 20-fold relative to that in crude conditioned medium. All of the factors were present in the 50-80% saturated ammonium sulfate fraction, however, levels of concanavalin A were reduced by 98% in this step. TCGF, separated in this way from these other regulatory factors will be useful in experiments analyzing the actions of TCGF on mixed populations of cells.

Sheep T-cell growth factor (TCGF) was prepared from concanavalin A-activated sheep peripheral blood cells and subsequently characterized by ammonium sulfate precipitation, gel exclusion chromatography, and isoelectric focussing. The TCGF was found in the 60-80% ammonium sulfate fraction and was shown to have an apparent molecular weight of 32,500 and an isoelectric point in the range pI 5.2-5.5. The ability of the sheep TCGF to promote proliferation of activated human, sheep, mouse, and rat cells was compared with that of human TCGF prepared by phytohemagglutinin stimulation of lymphocytes from multiple donors and TCGF prepared from concanavalin A-stimulated rat and mouse spleen cells. Human TCGF was found to act across all species barriers, rat TCGF supported the growth of cells of all species except human, and mouse only promoted the growth of activated mouse and rat cells. Sheep TCGF was unique in being unable to support the growth of any cells except autologous cells.

In the elderly, an impairment of the immune system could lead to increased incidence of infectious, neoplastic and autoimmune diseases; on the other hand, depression, which is the most common psychiatric problem in aged people, seems to be linked with alterations in immunological function. Thirteen institutionalized elderly subjects were studied to investigate the relationship between depression and immunological parameters. These subjects were selected as "healthy" according to the SENIEUR-EURAGE protocol and they belonged to a population already evaluated by our group--one year before--for psychological, endocrinological and immunological parameters. The lymphocyte mitotic response to PHA was greatly diminished in aged subjects, when compared to the adult controls. Depressed elderly showed impaired immunological function as compared with nondepressed ones, either "in vitro" or "in vivo". Lymphocyte stimulation with phytohemagglutinin (PHA), T cell growth factor (TCGF) production (induced by stimulation with PHA) and cutaneous delayed hypersensitivity (CDH) were reduced in depressed aged subjects. As far as lymphocyte proliferation with PHA in the whole group were concerned, no differences were found comparing the present results with those obtained in a former study. Although it is difficult to understand the significance of the immune imbalance associated with depression in the elderly, our results suggest that psychological status could influence the immunological functions in old people.

Immune system activation correlated with a naturally occurring infection has been found in the South African clawed frog Xenopus laevis. The microorganism thought to be the cause of this infection is coccobacilloid and approximately 1 micron in diameter. Since this microorganism does not grow on conventional bacterial media and it has been observed intracellularly, it may be an obligate intracellular bacterium. It has been found in Xenopus peripheral blood and in highly vascularized organs such as the spleen and liver. Splenomegaly is the only pathology thus far described for infected frogs; infection is not associated with increased morbidity or mortality. This infection has been found in all outbred frogs examined in shipments from one South African and three separate North American vendors, and has been transmitted to animals bred and raised in our laboratory. This infection has a profound effect on the immune system of Xenopus. Significant numbers of splenocytes from infected individuals exhibit morphology commonly associated with activated T lymphocytes. There is constitutive production of T-cell growth factor (TCGF) and both IgM and IgY. Freshly harvested splenocytes from infected animals proliferate in response to a TCGF-containing supernatant, indicating that they express receptors for TCGF, a trait exclusively exhibited by activated lymphocytes. These splenocytes also show an increase in the activation marker recognized by the monoclonal antibody FJ17.

Recently the authors established a method for culturing mouse splenic T-lymphocytes with T-cell growth factor (TCGF) and feeder cells in vitro. Using this method, T-lymphocytes grow for approximately 14 days with population doubling times of 27-29 hr; cloning efficiencies (CEs) of mouse spleen cells ranged from three to twelve percent. Using this colony forming assay, in vivo and in vitro radiosensitivity of mouse splenic T-lymphocytes in the G0 phase and in vitro radiosensitivity of proliferating T-lymphocytes (cycling T-lymphocytes) were examined. For in vitro irradiation, the dose-survival curve of T-lymphocytes in G0 phase gave a D0 value of 0.99 Gy and a Dq value of 0.87 Gy and that of cycling T-lymphocytes gave a D0 value of 1.04 Gy and a Dq value of 0.19 Gy. For in vivo irradiation, the dose-survival curve of T-lymphocytes gave a D0 value of 1.01 Gy and a Dq value of 0.73 Gy. These results suggest that the recovering activity from sublethal damage of G0 T-lymphocytes was more effective than that of cycling T-lymphocytes. Furthermore, this colony forming assay system appears to be very useful for screening the effects of in vivo exposure to toxic and/or mutagenic agents and for comparing the effects of in vivo exposure with those of in vitro exposure to toxic agents as well as radiation.

T-cell cultures derived from the blood of 14 patients with solid tumors were propagated with T-cell growth factor (TCGF). The cultures were initiated from lymphocytes exposed to autologous tumor-biopsy cells. TCGF was added either immediately or 3-10 days later. In the former culture type the cell yield on day 7 was considerably higher. The cytotoxic potential of the cultured cells was assayed on two occasions, between days 7 and 10 and between weeks 5 and 8. Cells of all but two cultures had the potential to lyse autologous tumor-biopsy cells. On the population level, cytotoxicity was specific for autologous tumor in those cultures that were driven to growth with TCGF after the 3rd day. These lymphocytes did not lyse allogeneic tumor-biopsy cells. In contrast, all five cultures initiated in the presence of TCGF exhibited a broader cytotoxic potential, i.e., in addition to the stimulator autologous-tumor cells, they also lysed other targets. Another difference between the two culture types was their behavior toward K562. Tested on the 7th day they all lysed K562; however, this function declined in strength or disappeared later in the cultures exposed to TCGF after the 3rd day. Reexposure of the lymphocytes to autologous tumor-biopsy cells after 2 weeks of culture period, but not on the 7th day, induced DNA synthesis. This secondary response was specific inasmuch as allogeneic tumor cells had little or no effect. One of the autotumor restimulated cultures was tested for cytotoxic potential. It increased against the autologous but not against other tumors or K562 cells.

It is often difficult to obtain sufficient quantities of leukocytes from bone marrow (BM) transplant recipients for post-transplant immunological monitoring and evaluation of engraftment. We have developed a cell culture expansion technique that allows HLA-A,B, and DR typing as well as karyotyping on limited numbers (less than 10(7)) of lymphocytes. Lymphocytes from healthy control donors were allo-activated in vitro and further expanded by culturing in T-cell growth factor (TCGF). After 14 days of culture, these cells were sent for blind HLA-A,B and DR typing and for karyotyping. Surface marker studies showed that 90% of the T-cells expanded in growth factor are OK-la positive. HLA typing with these DR-bearing in vitro expanded T-cells showed excellent correlation with HLA typing on fresh unexpanded lymphocytes. Karyotyping of expanded T-cells showed normal male or female cells as expected. This technique may be especially valuable when testing pediatric or adult patients with very low lymphocyte counts.

This report examines optimal culture conditions necessary for accurate and sensitive quantification of chicken T Cell Growth Factor (TCGF) activity. With this bioassay, TCGF is quantified by measuring its ability to cause proliferation of splenocytes prestimulated with mitogen. Proliferation is quantified by determining the optical density (OD) or "signal" of test samples in microtiter wells by measuring the incorporation of tetrazolium salt by live cells. To optimize assay conditions, systematic evaluation of the effects of cell culture variables was carried out with the constant aim of increasing signal to noise ratio in the assay. Higher signal to noise ratios were found when using Dulbecco's Modified Eagle's Medium (DMEM) rather than Roswell Park Memorial Institute Medium (RPMI) for basal tissue culture media containing the same supplements. The addition of lipid supplement to the assay system not only increased the proliferation signal, but also decreased the background OD. Incubation temperatures of 41 C rather than 37 C for both the mitogen prestimulation and proliferation phases of the assay also resulted in a higher signal to noise ratio. While incorporating the optimal experimental conditions, a finalized assay procedure employing test sample normalization with an internal assay standard was tested for accuracy. The assay can accurately detect 2 to 15 U/mL of TCGF activity. The within-assay variation ranged from 2 to 13% and the between-assay variation ranged from 11 to 22% depending upon the TCGF preparation being tested. The excellent reproducibility of this assay has facilitated investigations of TCGF production, processing, and purification.