In an attempt to facilitate the long-term proliferative growth and subsequent cloning of cytotoxic T lymphocytes (CTL's) against syngeneic murine 203-glioma (20-methylcholanthrene-induced ependymoblastoma of C57BL/6 mouse origin), sensitized T lymphocytes from tumor-bearing mice were cultured in the presence of T cell growth factor (TCGF). Of five clones established by a limiting dilution technique, two clones (G-CTLL 1 and 2) exhibited tumor-specific cytotoxicity. G-CTLL 1 cells, which possessed much higher cytotoxic activity than G-CTLL 2 cells, were further analyzed. G-CTLL 1 cells were maintained in a TCGF-dependent exponential proliferative culture for over 18 months and continued to mediate an extremely high cytotoxic activity with the target specificity (50- to 100-fold increases over the peak cytotoxic activity of sensitized T lymphocytes in tumor-bearing mice). Their phenotypes of surface antigens were Thy-1+ (weak positive), Lyt-1.-2.+3+, and asialo-GM1-, and their cytotoxicity was blocked by adding only anti-Lyt-2 monoclonal antibodies. These results indicated that the cloned cells originated from CTL's. The cloned cells were characterized by the production of immune interferon with the glioma antigen-stimulation, suggesting that the immune interferon could enhance the cytotoxic activity of the CTL clone at the site of a clone-target cell recognition event.

The mechanism responsible for the lymphocytotoxicity associated with congenital adenosine deaminase (ADA) deficiency has been ascribed to an accumulation of dATP. Elevated levels of dATP can then lead to inhibition of DNA synthesis by inhibiting ribonucleotide reductase and causing a depletion of the other deoxynucleotide triphosphates (dNTP). This hypothesis was derived principally from studies with murine and human lymphoblastoid cell lines (LCL) and apparently confirmed in a limited number of investigations with lectin-stimulated lymphocytes. Our biochemical studies of lectin-stimulated mouse and human lymphocytes were not consistent with the dATP model and suggested that AdR exerted effects on lymphocyte activation that preceded the initiation of DNA synthesis. In the current studies, we focused on the effects of AdR on the early events in T lymphocyte activation, because we found they were the most sensitive to AdR toxicity. AdR blocked neither the production of T cell growth factor (TCGF) by lectin-stimulated lymphocytes nor the expression of TCGF receptors as detected by the anti-Tac monoclonal antibody that recognizes the human TCGF receptor. AdR did, however, block the early TCGF-dependent events leading to the entry into the cell cycle. By using the metachromatic fluorescence stain acridine orange, we found that AdR blocked the increased synthesis of RNA that characterizes the entry into the G1 phase of the cell cycle from the G0, resting state. Because these early effects were caused by the lowest doses of AdR, and because they preceded the synthesis of DNA by 15 to 20 hr, it suggested that these effects may be principally responsible for the in vivo toxicity associated with ADA deficiency. Furthermore, none of the other proposed biochemical mechanisms, e.g., inhibition of methylation, diminution of ATP levels, or incorporation of AdR into polyadenylated RNA, appeared adequate to explain AdR toxicity during T lymphocyte activation.

Interleukin-2 (IL-2) is a type of the lymphokine which Morgan et al. found in 1976 to be a specific growth factor of T lymphocytes. Initially, it was called the T cell growth factor (TCGF), but it was renamed interleukin-2 (IL-2) in 1979. IL-2 is an essential factor for the differentiation and growth of T cell and NK cell, and it is involved in the adaptive immune reaction through such cells. Thus, IL-2 can be expected to play an important role in the improvement of the immunologic adaptive function in various immune system failure type disease and in malignant tumors among others. With the recent production of recombinant IL-2, the latter's biological and immunological functions are gradually coming to light, and its clinical applicability is also being seen in terms of effectiveness. Thus, administration of IL-2 alone, the induction of IL-2-dependent, tumor-specific CTL (cytotoxic T lymphocyte), or the induction and growth by IL-2 of lymphokine-activated killer (LAK) cell, have paved the way for adoptive immunotherapy. The clinical phase I study of IL-2 (TGP-3) has been completed, and the second phase study is now in progress. A pilot study is also under way using LAK cells.

The immunoregulatory effects of TCGF (T-cell growth factor) on the generation and growth of syngeneic murine malignant glioma (20-methylcholanthrene-induced 203-glioma)-specific killer T-cell were investigated in C57BL/6 adult mice in order to clarify the immunopotential usefulness for anti-tumor local adoptive immunotherapy against malignant brain tumor. TCGF was prepared and assayed. Briefly, 5 x 10(6) ml mouse spleen cells were cultured with 2 microgram/ml concanavalin A in RPMI-1640 medium supplemented with 2% fetal calf serum for 24 hours. Culture supernatants were concentrated by ammonium sulphate precipitation (55 to 80% saturation) and purified by gel filtration (Sephadex G-100, a molecular weight from 30 to 36,000 daltons) and ion exchange chromatography (DEAE-cellulose, elution with 0.15 M in NaCl at ph 7.4). The purified TCGF had no IFN activity. Assays for TCGF was performed for quantitative analysis using 203-glioma-specific killer T cell clone (G-CTLL), which was obtained by limiting dilution method (0.3 cells/well in 96 well microtiter plate) and maintained for over 6 months in the presence of TCGF. Titer (U/ml) of TCGF was defined as the quantity of TCGF required to obtain one-half of the maximal stimulation of G-CTLL proliferation assay. It was confirmed that the specific killer T-cell against 203-glioma was generated in mice after intracranial as well as subcutaneous inoculation of the tumor cells. The killer T-cell activity of spleen cells, however, began to be severely impaired 2 weeks after intracranial inoculation concurrently with the increased intracranial pressure due to developing the tumor growth. Sensitized lymphocytes obtained from intracranial and subcutaneous tumor-bearing mice were assessed for CTL (cytotoxic T-lymphocyte) activity in MLTC (mixed lymphocyte-tumor cell culture) for 18 hours by microcytotoxicity assay. The specific cytotoxicity against 203-glioma cells was enhanced when sensitized lymphocytes from intracranial and subcutaneous tumor-bearing mice were pre-cultured with optimal TCGF (20 U/ml) for over 5 days. After the treatment of sensitized lymphocytes with anti-Thy-1 monoclonal antibody and complement, however, the specific cytotoxicity of sensitized lymphocytes was eliminated almost completely. Therefore, it was thought that TCGF possesses immunoregulatory effects of enhancement of killer T-cell activity. On the contrary, TCGF had no influence on normal T lymphocytes and the growth of 203-glioma cells in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

The current understanding in biology and function of 4 growth factors is reviewed. PDGF suggests functions for proto-oncogens in normal cells, which may interact in tightly linked hierachies to induce malignant growth. PDGF-requirement of normal fibroblast cell-lines is lost when the cells are infected with tumor viruses. TGF is able to stimulate growth of normally anchorage dependent cells in an anchorage independent manner in soft agar. This ability is thought to be the best in-vitro correlate of neoplastic transformation. The peptide hormones bombesin/gastrin releasing factor and EGF can act as autocrine growth factors in various lung cancer cell-lines and stimulate clonal tumor cell growth in-vitro. The potential clinical application of these types of growth factors may enable the in-vitro growth from any lung cancer patient and allow individual drug testing. TCGF produced by T-cells to activate T-cells, is central to immune stimulation and immune response. Models for potential indirect anticancer effects either by in-vivo administration or by in-vivo incubation plus passive transfer of T-cells are presented to be initiated in future clinical trials.

Red deer (Cervus elaphus) represent an appropriate large animal model to study the immunology of tuberculosis, being naturally susceptible to Mycobacterium bovis infection. Cell-mediated immune responses were investigated in deer displaying protective- or disease-type reactions, following immunization with M. bovis bacille Calmette-Guerin (BCG) or infection with virulent M. bovis, respectively. T cell responses were measured as antigen-dependent cell proliferation and production of T cell growth factor (TCGF) following in vitro stimulation with M. bovis antigens (live or heat-killed BCG, or PPD). T cells from immunized deer proliferated less in response to soluble denatured culture antigen (purified protein derivative, PPD) than to particulate BCG, although there were no differences in the magnitude of these responses between the two groups of animals. Cells derived from immunized deer produced less TCGF than cells from infected deer when stimulated with PPD in vitro, although responses to BCG antigens were similar between the two groups. The majority of TCGF activity was neutralized by anti-IL-2 antibodies, regardless of the animal group or source of antigen used for in vitro stimulation. After 7 days in vitro culture with antigen, blast cells staining positively for alpha beta (CD4, CD8) and gamma delta T cell receptors were recorded. The majority of blasts were CD4+, although in immunized deer fewer CD4+ blasts were produced following in vitro stimulation with PPD than with BCG antigens. These results, together with previous reports from our laboratory, represent the only detailed examinations of T cell responses to M. bovis in this naturally-susceptible ruminant species.

In vitro cultures of human peripheral blood lymphocytes, both unstimulated G0 cells as well as phytohemagglutinin (PHA) stimulated cells, have been used in the investigation of DNA repair following different types of damage, including that induced by ultraviolet light, ionizing radiation, and chemical agents. We report here repair of DNA damage in cultured normal human T-lymphocytes after treatment with the alkylating agents, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or methanesulfonate (MMS), as measured by the alkaline elution technique. T-lymphocytes cultured with different sources of T-cell growth factors (TCGFs) were shown to have similar repair levels, as measured by loss of single-strand breaks. However, diminished repair was observed with in vitro culture age when T-cells were cultured with PHA and TCGF for a 3-week period. Cell-cycle analysis performed on asynchronously growing cultures indicated that differential repair with in vitro aging was not cell-cycle-related. Fluorescence Activated Cell Sorting (FACS) was used to determine the percentages of CD4+ and CD8+ T-cell subsets present during the in vitro culture periods. Cultures consisted primarily of CD4+ cells until day 20 when the percentage of CD8+ cells increased to approximately 50% of the T-cell population. Neither the absolute percentages of CD4+ and CD8+ cells not the CD4+/CD8+ ratios correlated with repair rates of cultured T-cells. Therefore, the observed decreases in the repair of alkylating agent-induced damage are not due solely to the change in the CD4+/CD8+ ratio.

Expression of alien histocompatibility antigens on Epstein-Barr virus transformed, cultured lymphoblastoid cell lines (LCL), which were established from normal peripheral blood lymphocytes (PBL), was studied by means of mixed lymphocyte reaction (MLR), cell mediated lysis (CML) and primed lymphocyte typing (PLT). Stimulation of PBL by autologous LCL resulted in some MLR responses and generation of cytotoxic effector cells against autologous LCL. Restimulation of PBL by 16 individual allogeneic PBL failed to prime an individual lymphocytes against autologous LCL in PLT tests. However, stimulation of PBL by a pooled normal PBL resulted in generation of cytotoxic cells against autologous LCL. Culturing of stimulated PBL in the presence of T cell growth factor (TCGF) for 30 days was shown to maintain cytotoxic effector cells in the cell population.

Thy.1lowCD3- cells obtained from nylon wool-passed murine bone marrow (NW-BM) cells by cell sorting did not express CD4, CD8, or T cell receptor-alpha/beta and -gamma/delta on their cell surfaces. An extremely limited number of B10.BR (H-2k) responder lymph node (LN) cells were stimulated with B10.D2 (H-2d) stimulator spleen cells in cultures containing the minimum required dose of rat T cell growth factor (TCGF). In these cultures, the generation of cytotoxic T lymphocytes (CTL) was very low. B10.BR Thy.1lowCD3- NW-BM cells, added to these cultures, could augment the CTL generation vigorously, but neither B10 (H-2b) nor B10.D2 cells could. When B10 LN cells were used as responder cells in these cultures, B10 Thy.1lowCD3- NW-BM cells could augment the CTL generation, but neither B10.BR nor B10.D2 cells could. Similar findings were obtained when Lyt-2+ cells or Thy.1+L3T4- (CTL precursor) cells sorted from spleen cells were used as responder cells. Both elements, rat-TCGF and Thy.1low CD3- NW-BM cells, were essential for this augmentation of the CTL generation in this culture system because neither one alone could augment generation, and rat-TCGF could be replaced by Thy.1+ Lyt-2- helper T (Th) cells sorted from spleen cells. These findings showed that NW-BM cells could augment CTL precursors in a self-major histocompatibility complex (self-MHC)-antigen restricted manner, and further that both NW-BM cells and Th cells had different and independent functions to induce CTL.

The focus of this study was to elucidate the influence of lymphocytes on phagocytic cell-dependent chemiluminescence (CL). CL was induced by in vitro addition of mitogens, particulate substances and Keyhole Limpet Hemocyanin (KLH)-antigen. CL was measured in spleen cells of immunologically naive mice and in mice immunized with KLH. Mice immunized with KLH and Freund's adjuvant incomplete (FA) showed a lower CL-response than mice injected with FA only. No influence on CL-activity by lymphocytes derived from immunologically naive mice could be found. Adoptive transfer of T cells from KLH-sensitized donor mice did not influence CL-activity elicited with Zymosan in spleen cells derived from KLH-unsensitized recipient mice. Preincubation of spleen cells with supernatants of T cell cultures or with T cell growth factor (TCGF) stimulated with KLH or Con A could not trigger CL-reactivity. Therefore, the lower CL-response of spleen cells derived from KLH-sensitized mice appears to be not a consequence of a suppressive cellular or lymphokine effect, but is due to direct influence of KLH on accessory cells themselves.

OBJECTIVE

Interleukin-2 (alias: IL-2, TCGF, Lymphokine), a type of interleukin, is also a potent signalling molecule in the signalling cascade of the immune-mediated activation of T Lymphocytes leading to the destruction of haematopoietic stem cell (HSC) which is the basis of acquired aplastic anaemia (AAA). The objective was to study the association of IL-2 in the bone marrow plasma (BMP) and peripheral blood plasma (PBP) in AAA patients.

METHODS

A total of 52 BMP and PBP-paired samples (both from the same patients) was collected from the confirmed AAA patients and 10 healthy individuals. The level of IL-2 was measured by the quantitative enzyme-linked immunosorbent assay (ELISA). The Mann-Whitney U test was used for statistical analysis.

RESULTS

Significantly increased level of IL-2 was observed in the BMP than PBP of AAA patients. The level of IL-2 in PBP and BMP was found to be very low in the control cases. Considerably increased levels of IL-2 were found in the PBP and BMP of AAA patients as compared to controls (48.54 ± 21.89 vs. 1.99 ± 1.25 p-value < 0.00001) and (75.33 ± 41.9 vs. 3.12 ± 1.82; p-value < 0.00001) respectively. Among these patients, the IL-2 levels were higher in patients with Very Severe Aplastic Anaemia (VSAA) and Severe Aplastic Anaemia (SAA) than those with Non-severe Aplastic Anaemia (NSAA) in the PBP (65.6 ± 23.61 vs. 31.72 ± 7.64; p-value 0.00338) and (45.37 ± 16.25 vs. 31.72 ± 7.64; p-value 0.01468) respectively. Again the IL-2 levels were higher in patients with VSAA and SAA than those with NSAA in the BMP (115.01 ± 38.91 vs. 38.32 ± 19.49; p-value < 0.00001) and (66.44 ± 23.34 vs. 38.32 ± 19.49; p-value 0.0006). The IL-2 level was higher in VSAA than SAA in PBP (65.6 ± 23.61vs. 45.37 ± 16.25; p-value 0.0114) and BMP (115.01 ± 38.91 vs. 66.44 ± 23.34; p-value 0.00044).

CONCLUSIONS

This study emphasized on the bone marrow and blood plasma levels of IL-2 in aplastic anaemia and their relationship with disease severity. The results indicate towards the fact that IL-2 may have an important association with the marrow failure of AAA patients and thus can help in disease development. Further study is necessary for better understanding.

We have examined the identity of the T colony progenitor cell (TCPC) in T cell depleted peripheral blood mononuclear cells (PBM-E-). PBM were cultured in a limiting dilution assay utilizing PHA, T cell growth factor (TCGF) and irradiated feeder cells in semi-solid medium. The TCPC frequency was 0.10 in PBM-E+ and 0.0006 in PBM-E-. The observed TCPC frequency in PBM-E- was directly proportional to the number of contaminating PBM-E+; all of the T colonies which arose from PBM-E- could be accounted for by contaminating T cells. In other experiments, E+ cells were generated from PBM-E-, incubated in the presence of PHA and TCGF. This appearance of E+ cells could be totally abrogated by incubation with hydroxyurea. Similarly, the TCPC frequency in PBM-E- was increased by pre-incubation with PHA and TCGF, and this increase was blocked by hydroxyurea. These experiments suggest that a small number of contaminating T cells can rapidly expand in culture and that these residual T cells, not a population of pre-T cells, are the source of TCPC within the PBM-E- population.

Supernatants from Con A-stimulated rat spleen cell cultures containing TCGF were able to increase the sensitivity of the microcytotoxicity assay. Spleen cells from mice immunized with irradiated syngeneic tumour were precultivated for 24 h in a TCGF-containing medium. The precultivated immune spleen cells, when allowed to react with syngeneic tumour target cells, displayed a higher cytotoxic effect than the same immune spleen cells precultivated in a medium without TCGF. Similarly, the addition of TCGF-containing medium to the effector cells during the microcytotoxicity assay increased the cytotoxic effect. Combination of both schedules, i.e., precultivation of the immune effector cells in a TCGF-containing medium and addition of TCGF-containing medium to the effector cells during the microcytotoxicity assay produced the highest increase in sensitivity of the microcytotoxicity assay.

Normal mouse spleen cells were cultured in different conditioned media (CM). Mixed lymphocyte culture supernatant (MLC SN) was shown to promote the proliferation of cytotoxic, Thy-1+, Lyt-1+, Lyt-2+, asialo-GM-1+, weakly adherent cells with numerous vacuoles and lysosome-like cytoplasmic granules. In contrast, the Con A SN induced the proliferation of non-cytotoxic, Thy-1-, Lyt-1-, Lyt-2-, asialo-GM-1-, non-adherent cells with numerous cytoplasmic granules. The ultrastructural morphology of these cells and the cytochemical characteristics of their granules enable us to identify them as mast cells. The different effects of both CM could be related to their T-cell growth factor (TCGF) content. When the amount of TCGF of these two CM was determined (by assaying growth-stimulating activity for T-cell colonies), it appeared that the MLC SN contained larger amounts of TCGF than the Con A SN used in these experiments.

By two different fusions between a cytotoxic T-lymphocyte (CTL) clone and a polyoma virus (Py)-transformed fibroblast line, 40 hybrid clones have been generated. It has been demonstrated that they were all TCGF independent for multiplication. Moreover, some of these hybrids were functional for cytolytic expression, whether or not TCGF was present either at the time of fusion or in the selective media. Two clones generated from the same fusion were markedly cytolytic and were able to remove TCGF from their culture medium, suggesting that they possessed TCGF receptors. These clones also secreted discrete amounts of a TCGF-like factor. The effect of TCGF on hybrid cell proliferation is discussed.

Murine T cell lines that help induction and generation of allospecific cytotoxic T cells (CTL) from thymocytes cultured together with allogeneic stimulator cells have been established. It was done by successively culturing T cell blasts obtained from mixed lymphocyte cultures (MLC) in the medium supplemented with T cell growth factor (TCGF). The helper function of the long-term cultured T cells was antigen specific, radiation resistant, and not H-2 restricted. The helper function has been retained for more than 8 mo by the cells continually proliferating in the TCGF-supplemented medium. The T stimulated responsiveness of thymocytes to T cell mitogens. CTL activity seen in the original MLC waned rapidly during successive culture of this T cell line in TCGF medium T cell blasts from MLC successively cultured in continual presence of TCGF and the stimulator alloantigens retained strong CTL activity against the allogeneic stimulator cells for about 3 mo. All these T cell lines proliferating in TCGF medium expressed Thy 1 antigen and showed the appearance of a large T cell blast. These T cell lines could not proliferate in the absence of TCGF and lost responsiveness to T cell mitogens intrinsically.

The rat natural killer (NK) cell, defined here by spontaneous lysis of H2-negative embryonal carcinoma (EC) cells, was investigated with respect to its antigenic phenotype. Rat spleen cells were separated by panning into adherent and non-adherent populations after incubation with monoclonal antibodies defining differentiation antigens of rat lymphoid cells. This method achieved considerable NK enrichment in some of the panned populations (for example, W3/13 non-adherent cells, up to 20-fold), but emphasized the heterogeneous surface antigen expression of the NK cell type. These enrichment procedures were used to establish bulk cultures of rat NK cells which increased in specific activity over several weeks when grown in T cell-growth factor (TCGF) and could be cloned in soft agarose.

Biological transduction can be defined as the triggering of a cellular response by the binding of molecules of effector substances to specific cellular sites. An example of biological transduction, analyzed in this report, is the triggering of T-cell proliferation by the binding of T-cell growth factor (TCGF) to specific TCGF-binding sites on responsive T-cells. Sigmoidal or S-shaped curves often result when measurements of biological response are plotted as a function of concentration of effector substance. Such curves suggest that effector molecules must bind a critical number of cellular sites, and this critical number of bound complexes must undergo secondary events (cross-linking, association, internalization, second messenger release, etc.) in order to initiate the biological response. The method described here estimates the critical number of cellular sites (R) and the probability of these secondary events (PS/B) as follows: (1) The total number of cellular sites (N) is estimated from binding data, and the probabilities (PB) of effector molecules binding to a site are estimated from response data. (2) The response data are assumed to follow the summed binomial distribution function, which is equated to the incomplete beta function. (3) R and PS/B are estimated by applying nonlinear regression to the incomplete beta function. The T-cell data to which the method was applied gave N = 15,000, R = 5, and PS/B = 7.22 x 10(-4). These results show that the binding of very few TCGF molecules is required for activation of T-cells and that the probability of the secondary events leading to cell proliferation is much smaller than the probability of TCGF binding to T-cells. The method described can be used to analyze any biological transduction experiments where both binding and biological response data are available.

Flow cytometry was used to evaluate nucleic acid synthesis in irradiated mixed lymphocyte cultures (MLC) compared to nonirradiated control cultures. Two staining methods were used (propidium iodide and acridine orange). We showed that RNA and DNA synthesis are retarded in MLC receiving 0.2 Gy. This effect was reversed by lymphocyte growth factor.

Following secondary stimulation of mixed leucocyte culture (MLC) populations with a class I plus II HLA difference, antigen-specific suppressor-effector cells of the OKT8+4- phenotype are generated. On the other hand, stimulation with a class II HLA genetic difference only gave rise to OKT4+ suppressor T cells. These effector cell populations which were maintained as "lines" by the addition of exogenous T cell growth factor (TCGF) and feeder cells, mediated antigen-specific suppression over a 6-8 week period during which they were assayed. The data reported here demonstrate that the phenotype of suppressor cells, generated in allogenic MLC, depends on the nature of the class of HLA antigen recognized as disparities in the suppressor-generating MLC. Moreover, both OKT8+4- and OKT4+ suppressors, obtained following stimulation with class I plus II or class II genetic disparities, respectively, appear to be specific for DR (or related class II product) antigens.

The ontogeny of IL 2-responsive cells in the thymus of CBA/J mice was examined in neonatal animals and in fetuses at 14, 16, and 18 to 20 days gestation. The thymocytes were tested for responsiveness to 2 micrograms/ml Con A, TCGF, IL 2, and co-stimulation by Con A plus TCGF or IL 2. These responses were compared with those of thymocytes of 6- to 8-wk-old CBA/J. Thymocytes (1 X 10(5)) were cultured, and the reaction was measured at maximum response (96 hr). Neonatal animals gave an unusually high response to TCGF or partially purified IL 2 alone, approximately five times greater than the adult. A low but significantly enhanced proliferation, stimulated by partially purified IL 2 alone, was observed with 14-day fetal thymocytes, even though cultures of these cells in medium alone had higher background proliferation than any other age tested. In the co-stimulator reaction, proliferation significantly above background was measured at 16 days of gestation with Con A plus TCGF. The magnitude of the co-stimulator reaction increased with age, especially between the 16th and 18th day of gestation and immediately after birth.

Prior studies have shown that intraperitoneal (ip) injection of 25 IU of human rIL-2 can effectively modulate in vivo immune reactivity to thymus-dependent and thymus-independent type 2 immunogens in Xenopus laevis, the South African clawed toad, but is less successful at affecting toad cells in vitro. Here we compare the capacities of human rIL-2 and autologous TCGF to modulate Xenopus splenocytes in vitro and find that autologous TCGF (1) is more effective at stimulating mitogenesis, (2) can serve as a ligand for inducible receptors that will also bind rIL-2 and an F1*-mouse anti-human p55 antibody, and (3) will regulate the expression these receptors.