We provide here a protocol for production of T cell growth factor (TCGF) using cells isolated from defibrinated blood. Whether combined in allogeneic pools or tested as single donors, these cells consistently yield high activity TCGF, following PHA stimulation. Protocols using cells isolated from heparinized blood have often included addition of indomethacin or removal of adherent cells, or both, to overcome the problem of frequent nonproducing cultures. Our failure to find nonproducing cultures using this source of cells suggests that inhibitory cells or factors are removed by the blood clot formed during defibrination. A distinct economic advantage is gained by using these cells, not only because of the reliability of obtaining active supernatants, but also because the same blood can be used for preparation of a serum pool for cell cultures and the erythrocytes are useful for absorption of contaminating PHA present in the TCGF-containing media. Not only can defibrinated blood leukocytes be stimulated by PHA to release TCGF, but when cultured in allogeneic mixtures containing no PHA, they also release active TCGF.

Lymph and supernatants derived from efferent lymphocytes leaving the popliteal lymph nodes of sheep responding to human red cells or dinitrophenylated bovine serum albumin were examined for the presence of T-cell growth factor (TCGF). Efferent cells from normal sheep, but not from antigen-stimulated sheep, were found to release low levels of TCGF when incubated in medium for 12 hr in the absence of any exogenous stimulus. High levels of TCGF were found in normal lymph and also in immune lymph collected from sheep during the first 6 hr of immune responses. There were no detectable levels of TCGF in lymph collected later in the response. The lymphokine appeared to be a single molecular species of 10,000-20,000 molecular weight as assessed by exclusion chromatography. Efferent cells expressing receptors for TCGF were found in efferent lymph during the first 12 hr of the response. The results demonstrate for the first time that TCGF is produced in vivo and that asynchrony exists between TCGF production and expression of receptors for TCGF on efferent cells released by the stimulated node. Based on the known kinetics of previously reported synergistic factors, mitogenic factors, and T-cell-replacing factors in sheep efferent lymph and their physical characteristics it was concluded that the TCGF detected in lymph is distinct from these factors.

Infants with congenital CMV infection have a specific defect in CMV-induced lymphocyte proliferation, providing a model for investigation of mechanisms of viral immune recognition and immune response. In the present study the possible role of a defect in lymphokine activation of CMV-specific T helper cells (Th) was examined. IL-1 activity was detected in supernatants of patient mononuclear cell (MNC) cultures stimulated with CMV. In contrast, no IL-2 activity could be detected in supernatants of CMV-stimulated MNC cultures, whereas PHA induced normal IL-2 production. Addition of low concentrations of either crude TCGF or recombinant IL-2 (rIL-2) resulted in 2-4 fold augmentation of CMV-specific lymphocyte proliferation; exogenous IL-2 had no effect on MNC responses to HSV. CMV-specific Th lines/clones were established from three congenital CMV patients by initial stimulation of MNCs with CMV antigen and 0.1 U/ml rIL-2, followed by repeated stimulation with CMV, HLA-matched allogeneic feeder cells and 10% TCGF. The resulting CMV Th lines/clones proliferated specifically in response to stimulation with CMV antigen and produced endogenous IL-2. Thus, the immune deficiency associated with congenital CMV may either be due to an intrinsic defect in CMV-Th activation or CMV-specific suppressor cell activity.

The PCl-6 T cell line, derived from mice sensitized by skin painting with picryl chloride (PCl), shows antigen dependence for DNA synthesis and for lymphotoxin (LT) production. These cells produce LT, but not interferon (IFN), when exposed to 2,4,6-trinitrobenzene sulfonic acid- (TNBS) coupled syngeneic spleen cells. Concanavalin A (Con A) induces IFN production by PCl-6 cells, and IFN levels, but not LT titers, are increased by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). These results support the noncoordinate regulation of these two lymphokines. Line 32, a T cell growth factor- (TCGF) dependent T cell line and its Ly-1.2+, 2.2- derivative clone, 32H1, produce both antiviral and antiproliferative activity after exposure to several different mitogens. Tests for acid lability, sensitivity to anti-mouse IFN-alpha, beta antisera, and antiproliferative activity on non-mouse target cells indicates that an Ly-1+ clone, in the absence of both TCGF and accessory cells, can produce at least three separate lymphokine activities after Con A exposure: IFN-gamma (Type II), IFN-alpha, beta (Type I), and LT.

Peripheral blood lymphocytes from a patient with chronic lymphocytic leukaemia of T cell origin (T-CLL PBL), were found to lack PHA and Con A responsiveness and to have a very poor responder activity in mixed lymphocyte cultures. In spite of the presence of Ia-like antigens on 36% of the T-CLL PBL, negligible stimulator capacity in MLC was observed. Proliferation of the T-CLL PBL could be induced and maintained both by exogenous T cell growth factor (TCGF) containing PHA, and mitogen-free TCGF. In addition, restoration of the responder and stimulator capacity in MLC was obtained in the presence of mitogen-free TCGF. These results indicate that the lack of response to mitogens and alloantigens of the T-CLL PBL has to be attributed to the failure of these cells to produce TCGF upon activation.

Mononuclear blood cells from patients with different types of leukemia, and from controls as well as cells from established lymphoblastic cell lines were analyzed with respect to terminal transferase (TdT) activity and T-cell growth factor (TCGF; Interleukin 2, IL-2), to determine the significance of TCGF production and response as functional markers for human leukemias. The data obtained so far suggest that the aberrant proliferation and lack of maturation observed in these leukemias may be associated with or be the result of a break-down in cellular-mediated control of proliferation.

No significant differences were found between hyperplastic tonsillitis and recurrent tonsillitis in the intensity of cell-mediated immune response of tonsillar T cells, the ratio of T cell subsets, the ratio of Tac-positive cells, and the stimulation index determined after incubation in the presence of TCGF alone. However, the proportion of the cells in phase S+G2+M, the ratio of OKT9-positive cells to total tonsillar lymphocytes, and the amount of polyamines (especially putrescine) were greater in hypertrophied tonsils than in the tonsils with recurrent tonsillitis. Some characteristics of recurrent tonsillitis are discussed from these differences. Comparison of tonsillar lymphocytes from children vs. adults produced similar results. The proportion of cells in phase S+G2+M, the ratio of OKT9, and the amount of polyamines were greater in tonsils of children. In addition, differences in the function of tonsillar and peripheral lymphocytes from the patient are also discussed.

The T lymphocytes, OKT4-depleted T lymphocytes, monocytes, macrophages and the bone marrow early erythroid progenitor (BFU-E) interactions have been evaluated in normal subjects and in uremic patients in predialysis phase (PDP), on hemodialysis (HD) or continuous ambulatory peritoneal dialysis (CAPD). In the same subjects the T-cell growth factor (TCGF) activity and the T-cell subset identification have also been assessed. The results show in uremic patients, particularly in the presence of reduced OKT4 subset and TCGF activity, a blockage of the interactions between immunocompetent and bone marrow cells which normally could stimulate early erythroid progenitor growth. Since this blockage is partially removed by CAPD, this may suggest a more efficient removal of an interaction inhibitor with CAPD than with other dialysis techniques. The mechanism by which such a material may act appears unclear, but it is possible to suppose an inhibiting activity on the production of TCGF by lympho-monocyte cells which under normal conditions could be considered as an early erythropoiesis regulatory factor.

This brief review of our experiments concerning the in vivo activity of crude Il-2 led us to the following conclusion: The first is the existence, in vivo, of a cyclophosphamide-sensitive T-cell controlling the activity of a serum born Il-2 inhibitor in thymus-bearing normal mice. Under in vivo conditions which are characterized by high Il-2 inhibitor activities, locally applied Il-2 administered along with antigen amplified in vivo CTL-responsiveness, yet the effect observed was poor. Crude Il-2 proved to be a potent immuno-enhancing agent in the athymic (nu/nu) mouse, which lacks Il-2 inhibitor activity. It was found that together with antigen administration of Il-2 to nude mice results in the generation of highly reactive T-helper cells, as well as in the generation of alloreactive CTL.

We have previously shown that the proportion of CD8-expressing T cells (CD8bright+ and CD4+ CD8dim+ cells) in the peripheral blood of chickens increases around 8 days after a primary infection with Eimeria tenella oocysts. The increase in the CD8+ eight cells coincides with enhanced responses after in vitro stimulation with parasite antigen. In the study described here, the responsiveness of these day 8 PBL was further characterized by determining their capacity to proliferate and to produce cytokine (IFN-gamma) upon stimulation with E. tenella sporozoite antigen, or non-specific stimuli like T cell growth factor (TCGF) and anti-CD3 monoclonal antibody (MoAb). Comparing the responsiveness of infected responder (day 8) and control chickens, non-specific triggering induced cytokine production in cells from infected animals and proliferation in cells from control animals. When triggered with E. tenella sporozoite antigen, lymphocytes from infected chickens responded with proliferation and cytokine production, in contrast to lymphocytes from control animals that did not respond. The phenotype of the lymphocytes involved in the parasite-specific proliferation and cytokine production, was characterized in a blocking assay using MoAb directed against the CD4 or CD8 molecule. The results suggest that CD8bright+ as well as CD4+ (CD4+ CD8dim+ and possible CD4+, single positive) lymphocytes are responsible for the IFN-gamma production measured after stimulation with parasite antigen, whereas the specific proliferative response appears to be caused by CD4+ (CD4+ CD8dim+ and possibly CD4+ single positive) lymphocytes. We speculate that the CD8bright+ cells, present in the circulation around 8 days after a primary E. tenella infection, act as effector cells in protective immune responses, whereas CD4+ cells play an important helper function in these responses.

Human PBL cells, sensitized in vitro to TNP or FITC-modified autologous stimulators, have been successfully grown in long-term culture by using TCGF derived from PHA-activated tonsil cells. Long-term cultures consist almost exclusively of T lymphocytes as defined by the formation of spontaneous rosettes with sheep erythrocytes and C-mediated lysis with heteroantibodies to human T cells (R alpha T H). The majority, but not all of these T cells, bear surface Ia antigens as detected by C-mediated lysis in the presence of alpha p23,30. Functionally, these cultures maintain high levels of hapten-specific cytolytic activity during many weeks of culture. In addition, a number of these T cell lines exhibit hapten specific proliferation when co-cultured with x-irradiated hapten modified autologous cells. During this proliferative response, helper factor(s) are released that trigger B cell differentiation into AFC. Data are presented that demonstrate that helper factor(s) production by T cell line cells is preferentially triggered by autologous, but nt allogeneic stimulator cells, bearing the relevant hapten.

In recent years investigators from a number of laboratories have described antigen nonspecific, lymphocyte-mediated cytotoxicity generated by TCGF alone, in the absence of antigen or mitogen. The exact origin of the cells mediating this cytotoxicity, in either mouse or humans, is unknown. We found that when mouse spleen cells are incubated with higher than normal concentrations of TCGF, good levels of cytotoxicity toward allogeneic, NK, and untransformed self target cells are generated by day 5 or 6 in culture. We were unable to block the lysis of any of these target cells with antibodies to target cell class I antigens. However, generation of this cytotoxicity from naive spleen cells was very strongly blocked by anti-class I MHC antibodies. When T cells from spleen were extensively purified, they did not respond to TCGF at any concentration unless adherent cells were added back. Generation of cytotoxicity under these conditions was also blocked by class I antibodies. Generation of promiscuous killing activity by PMA and ionomycin, on the other hand, was class I independent. Our data suggest that pre-CTL, under the influence of TCGF, can be activated to CTL and that under the continued influence of TCGF can be driven into a so-called "promiscuous" state of cytotoxicity. Possible roles for class I antigens in this process are discussed.

The amount of T-cell growth factor (TCGF) produced in vitro in antigen-stimulated lymphocyte cultures was measured by induction of growth in cell lines dependent on the factor. The method distinguished eight herpes-simplex-seronegative subjects from ten seropositive ones, and these results correlated with those of the lymphocyte blast transformation (LBT) test. The LBT responses and TCGF amounts produced in cultures with purified tuberculin (PPD) were also in good correlation (r = 0.833, P less than 0.001). Considerable levels of the factor were already present 12 h after the beginning of the culture. The method offers rapid quantification of cell-mediated immunity to specific antigens.

We have developed a simple assay for human peripheral-blood suppressor cells induced by soluble protein antigens. Suppressor cells were obtained from 6-day cultures in the presence of tetanus toxoid or PPD and were assayed in co-cultures with fresh autologous PBLs. The proliferative response in this second culture showed significant suppression varying from 50% to 90%. The suppressor cells are OKT8-positive, resistant to X irradiation and non-specific in their function. They do not impede either the function or production of TCGF in the secondary culture but we are unable to detect a non-specific suppressor factor made by these cells.

In-vitro infection of normal human lymphocytes with HTLV-I (human T-cell lymphotropic retrovirus type I) has been carried out to study the target cell specificity and the kinetics of infection. Cord blood (CBL) and adult peripheral blood lymphocytes (PBL) have been co-cultivated with irradiated HTLV-I donor cells (MT2 and C91PL lines). Established ('immortalized') HTLV-I positive cell lines were obtained only from CBL: in comparison with PBL, a less mature phenotype of T-cell subsets and a lower interferon-gamma production was evidenced in CBL. A progressive variation of differentiation antigen representation and of exogenous T-cell growth factor (TCGF, interleukin-2, IL-2) medium concentration was observed with increasing time from infection. The four established lines obtained showed a predominant T3+, T4+, T8-, Tac+ phenotype and a reduced TCGF requirement. Studies on kinetics of HTLV-I infection showed that p19 and p24 viral antigens became expressed after a lag phase of 5 weeks. DNA Southern blot analysis indicated that a shift from polyclonal to monoclonal pattern of proviral integration occurred with time of culture, both complete and defective copies being transmitted from donor to recipient cells.

Optimized production conditions and a functional assay of avian T cell growth factor (TCGF) or interleukin 2 (IL-2) are described. Treatment of lymphocytes with mitogen (Con A)-coated chicken red blood cells (MRC) resulted in markedly enhanced mitogenic response and IL-2 secretion compared to stimulation with free Con A. A positive correlation (r = 0.89) was found between mitogenic response and IL-2 activity of conditioned media. Enrichment of target cells, i.e., Con A lymphoblasts, by Percoll consistently improved the sensitivity of the IL-2 assay. The half-life time of chicken IL-2 at 40 degrees C was 9.7 +/- 1.7 h, which was considerably shorter than the value obtained for murine IL-2, i.e., 53.1 +/- 8.5 h. High concentrations of conditioned media were found to contain a dialysable factor that suppressed IL-2 promoted blast proliferation. The relevance of the data for in vitro analysis of T cell function as well as for establishing T cell lines in the chicken system are discussed.

It has been suggested that allospecific T-cell clones lose specific reactivity after approximately 30 cell doublings and subsequently acquire suppressor and NK-like characteristics. We have tested this hypothesis by assaying paired functional and nonfunctional TLCs for suppressor activity in PLT and MLC cocultures. Two sets of clones were initially studied: the first pair consisted of clone S5.2B, a functional TLC, and S5.14A, a nonfunctional TLC; the second pair of clones tested was comprised of two different expansions of the same clone S5.5A (nonfunctional) and S5.5B (functional). These experiments yielded no evidence for suppressive activity by nonfunctional clones toward functional clones, furthermore, the addition of nonfunctional clones to primary MLC assays had no effect on the level of responsiveness. Eight clones were subcloned and 89 subclones were retested for function after approximately 50 cell doublings. Generally, the subclones failed to suppress MLC proliferation. A minority of TLCs could suppress MLC responses, but this "suppression" was reversible with the addition of 2% exogenous TCGF. However, eight subclones and two parental TLC lines did suppress MLC responses in the presence or absence of TCGF, but the suppressive effects in such cocultures were reversible in the presence of tylocine, an anti-mycoplasma antibiotic. Therefore, human T-cells, cultured for extended periods, do not inexorably and universally lose specific alloreactivity and gain suppressive characteristics due to some presumed differentiative event.

Somatic cell fusion between cytolytically active, T cell growth factor- (TCGF) dependent murine T cell lines (CTL lines) and noncytolytic, TCGF-independent murine T lymphoma lines has yielded two types of somatic cell hybrids (5): cytolytic hybrids, growth of which is dependent on TCGF, and hybrids with very weak or undetectable cytolytic activity which grow at the same rate with or without TCGF. Here we report that the former can produce stable variants that resemble the latter type. Some of these TCGF-independent variants still have TCGF receptors. High susceptibility to the cytotoxic effects of Vicia villosa lectin, a marker distinguishing the parental CTL lines from T lymphomas, is expressed by the TCGF-dependent hybrids but not by the TCGF-independent variants. The two types of hybrids also differ in the expression of surface glycoproteins. We propose that there exists a genetic element in the CTL line that represses the TCGF-independent replication mechanism of the T lymphoma parent in the TCGF-dependent hybrids and that this genetic element is lost or switched off in the TCGF-independent variants.

Cultures of murine T lymphocytes with cytotoxic activity towards syngeneic RBL-5 lymphoma cells were obtained from spleen cells of immunized animals after co-culture in vivo with irradiated RBL-5 cells. At different times after initiation, these mixed tumour-lymphocyte cultures (MTLC) were multiplied by transfer to conditioned medium (CM) containing T cell growth factor (TCGF) activity, produced by the stimulation of rat spleen cells with Con A. The effect of residual Con A was investigated by the addition of specific blocking sugar, alpha-methyl mannoside (alpha MM), to the CM in some experiments. This procedure did not reduce the growth potential of the cells, and resulted in a dramatic increase in the cytotoxic activity of the cultures as measured by a 4-hr 51Cr-release assay. The cultures multiplied 1 X 10(3)-fold over a 3-week period with retention of cytotoxicity for RBL-5 cells at levels up to 70-fold greater than those of the MTLCs from which they were derived. The cultured cells, when injected i.p. together with RBL-5 cells into normal mice, were shown to mediate a significant prolongation of the survival of the treated animals. This effect was, however, less dramatic than had been expected from the in vitro results. It would therefore appear that, while cells grown in tissue culture using Con A-conditioned medium may fulfill some theoretical requirements for the immunotherapy of experimental tumours, other factor(s) are required for full protection.

To isolate a stable tumor cell line source of IL-2 (TCGF), 19 murine T-cell lines and their derivatives were screened for both constitutive and mitogen-stimulated IL-2 production. The cloned subline of a mouse thymic lymphoma EL-4 designated as EL-4TF could be stimulated with PMA to produce 80 U/ml of IL-2. A TK- EL-4TFR mutant line has been selected from the EL-4TF cell population by treatment with 5-BrdUrd. The EL-4-TFR cells were stimulated with PMA and fused with the cells of thymic lymphoma BW5147. The resulting BH3 hybrid cell population was repeatedly cloned and tested for constitutive IL-2 production: two of the BH3 hybridoma clones were found to produce IL-2 constitutively. The IL-2 of EL-4TF origin was found to support permanent in vitro growth of IL-2 dependent, tumor-specific T killer cell line CTLL when present in culture medium at a concentration of at least 0.1 U/ml. Since the EL-4TF-derived IL-2 preparations were contaminated with PMA, it was of interest whether PMA alone has a growth-promoting activity in CTLL cell cultures. Permanent cultivation of CTLL cells in an IL-2-free medium containing PMA was not possible. However, both mitogenic and co-mitogenic effects of PMA on CTLL cells were observed.

We investigated whether concanavalin A-stimulated, TCGF-reactive, and TCGF-producing T cells could be separated with regard to their Lyt phenotypes. Normal spleen cells, pretreated with monoclonal anti-Lyt-1 or anti-Lyt-2 antibodies and complement were assayed for their capacity to respond to TCGF, as well as for their ability to produce TCGF after Con A stimulation. The results demonstrate that reactivity to TCGF is predominantly induced in Lyt-2+ lymphocytes, because it is reduced by more than 95% in Lyt-1+23- T cell populations as compared with cultures containing Lyt-1-23+ cells. Limiting dilution analysis revealed that up to 20% of the cells in Lyt-2-containing populations were induced by Con A to TCGF-reactivity, whereas 20-fold fewer clones were induced in the Lyt-2-depleted cell populations. On the other hand, the study of TCGF production in Lyt-1-23+ populations revealed that the former population was considerably enriched in TCGF-producing T cells, whereas the latter was substantially depleted. We conclude that TCGF-producing and TCGF-reactive T cells are largely distinct lymphocyte populations that can be separated on the basis of their Lyt phenotypes.

We describe the properties of two Ly-1+2- T cell clones (Ly-1.14 and Ly-1.21), which are maintained in long-term culture in the absence of other cell types. The clones require media containing a source of interleukin 1 as well as interleukin 2. They retain physiologic responses to interleukin 1, which is required for optimal production of T cell lymphokines by these clones in response to concanavalin A (Con A). The two Ly-1+2- T cell clones differ in their production of lymphokines after stimulation by Con A. The supernatant of clone Ly-1.21 promotes the proliferation of T cells maintained in long-term culture, induces antibody synthesis in cultures of B cells and antigen, and induces the differentiation of cytolytic cells in cultures of thymocytes and antigen; these assays define the properties of T cell growth factor (TCGF), T cell-replacing factor for B cells (TRF-B), and T cell-replacing factor for cytolytic cells (TRF-C), respectively. In contrast, the supernatant of clone Ly-1.14 contains only TCGF activity and does not promote antibody synthesis by B cells or differentiation of cytolytic cells from thymocytes. The results indicates that TCGF and TRF activities reside on independent, although perhaps related, molecules.