We analysed the phenotypic characteristics of mouse NK cells susceptible to boosting activity by T-cell-growth-factor (TCGF) and the characteristics of NK-cell progenitors able to grow in vitro in the presence of TCGF. Our data show that while both the above-mentioned cell populations are Asialo GM-1+, they differ in the expression of Thy 1 alloantigen. In addition, we also analysed the possible regulatory mechanisms capable of influencing the growth of TCGF-sensitive NK-cell progenitors. The data here reported suggest that the growth capability of NK cell progenitors is under the control of the thymus.

The rhesus monkey (Macaca mulatta) has long been an important model in biomedical and behavioral research. The biomedical importance of M. mulatta is due to its 93% genetic similarity with humans and its complex social behavior. The recent sequencing of the M. mulatta genome has enhanced its role in biological research. However, the use of the macaque as an experimental model in cytogenetic assays has been problematic due to difficulties in obtaining large numbers of well-spread cells in metaphase without the use of extremely toxic mitogens such as staphylococcal enterotoxin A (SEA). Here we describe a technique for culturing and producing sufficient numbers of cells in metaphase using the common mitogens phytohemagglutinin (PHA), concanavalin A (ConA), and T-cell growth factor (TCGF) which act synergistically to induce M. mulatta T-lymphocyte division. Using this method we have obtained a mitotic index in 48 h cultures of 12.0+/-2.2 metaphase cells/100 cells (n=5 animals). Fluorescence in situ hybridization with whole chromosome painting of M. mulatta cells was performed with human whole-chromosome probes that labeled the following chromosomes for human (M. mulatta): 1(1), 2q(12), 2p(13), 4(5) pairs in red, and 3(2), 5(6) and 6(4) pairs in green. In humans this probe combination simultaneously paints 3 chromosome pairs in red and 3 in green, whereas in M. mulatta 4 chromosome pairs are labeled in red and 3 pairs are labeled in green. Using this method we show a baseline frequency of 0.026 translocations per 100 whole-genome cell equivalents in peripheral blood lymphocytes obtained from unexposed adolescent non-human primates. This method will add to the usefulness of M. mulatta as an animal model in biomedical research.

Conditions for the production and assay of ovine T cell growth factor (TCGF) activity were evaluated. Peripheral blood leukocytes were stimulated with concanavalin A (Con A) in the presence of 2% autologous serum or serum-free media. Supernatants were harvested after 30 hr and concentrated for further characterization. A 28 hr proliferation assay with 2.5 X 10(4) 24 hr Con A blasts per well was optimal for detection of TCGF. Peak TCGF activity occurred with a 30-37 KD molecular weight fraction. Production and assay of TCGF were performed under autologous conditions to reduce background stimulation which occurred when fetal bovine serum was present. This methodology required no cell lines or inbred animals and should be adaptable to the study of immunostimulatory molecules of other outbred species.

We attempted to elucidate the mechanism of action of T cell growth factor (TCGF)-expanded cells after stimulation with concanavalin A (Con A), a process which reduces the severity of adjuvant arthritis (AA), as seen in transferred syngeneic rats. TCGF-expanded cells were fractionated by Percoll density gradient or by the panning method, before the transfer. Transfer of cells with a density of between 1070 and 1079 suppressed AA most effectively, compared with other fractions with a density of less than 1070. A large number of the cells with density 1070-1079 were of the W3/25 phenotype. The transfer of W3/25 positive cells obtained by the panning method from TCGF-expanded cells reduced AA but not W3/25 negative cells. The suppressor function of TCGF-expanded cells was examined in an assay system in vitro. The addition of Con A-stimulated cells to the assay culture (but not expanded with TCGF), suppressed both the proliferation of spleen cells stimulated with Con A and the IgG production stimulated with lipopolysaccharide. The addition of TCGF-expanded cells to the assay culture had no effect on these responses. Thus, the W3/25 cells in the TCGF-expanded cells seem to function as suppressor-inducer cells, affect the presuppressor cells of the recipient rat and differentiate to suppressor effector cells.

The study of bovine cytokines and lymphocyte surface markers has been restricted by the lack of stable, long-lived, easily maintained cell lines. To address this problem, bovine mononuclear leucocytes were fused with the mouse thymoma cell line BW5147 to produce interspecies hybrid cell lines and clones. Hybridomas were produced which contained more than 40 chromosomes and expressed one or more bovine leucocyte surface markers, they grew well and were cloned without the use of exogenous factors. Fusion rates were improved when exogenous factors, including recombinant human interleukin-2 (rhIL-2), were added. Some hybrids secreted immunoregulatory factors able to maintain the growth of either an IL-2-dependent bovine T-cell line (C625) and/or preactivated normal bovine B cells. Anti-human IL-2 partially blocked the T-cell growth factor (TCGF) activity produced by these hybrids. The release of growth factors by the hybrid clones 5/AA6/16 and 5/AA6/19 was augmented by concanavalin A stimulation.

Mice were injected in the foot pad with either 5 x 10(5) syngeneic plasmacytoma (MOPC104E) or fibrosarcoma cells (Meth A). Lymph nodes containing tumor cells were harvested 14 days later and cultured. In the presence of recombinant interleukin-2 (r-IL-2) predominantly tumor cells proliferated. Culture with T cell growth factor (TCGF) resulted in the growth of lymphoid cells. Concanavalin A (Con A) had only a modest effect on elimination of tumor cells in the culture. Tumor-infiltrating lymphocytes (TIL) prepared from the lymph nodes showed specific tumor-neutralizing activity when grown in the presence of TCGF. In vitro examination revealed that Meth A cells could not be lysed by TIL, while TIL from MOPC tumors showed tumor specific activity. This study may explain negative results in human trials with TIL induced by IL-2 alone.

Burst promoting activity (BPA) and colony stimulating activity (CSA) were measured in the supernatants from six T-cell lines which are IL-2 independent and do not excrete human T-cell leukaemia virus. Two cell lines were shown to produce high levels of BPA, namely MLA-144 and the Jurkat cell line. MLA-144 is a gibbon T-cell line which can proliferate and produce BPA in serum free HB101 medium. The BPA from this line was shown to act directly on BFU-E by studies using highly purified peripheral blood derived BFU-E. The Jurkat CM had far less activity against purified progenitors. These cells lines will be of value in the study of erythropoietic regulation.

The production of T-cell growth factor (TCGF) by acute lymphoblastic leukaemia (ALL) cells was determined in seven children and in three adults. A significant production of TCGF by adult, but not childhood, ALL cells was observed. The adult ALL cells were classified as "non-T-non-B" by surface marker analysis. It is suggested that TCGF production may not be confined to the cells of T-lineage.

A patient with T-cell prolymphocytic leukemia (T-PLL) is described. The malignant T-cells from the patient were predominantly Leu-2-positive, indicating a suppressor phenotype. The cells were then tested to determine their functional capabilities. The patient's Leu-2-positive cells initially suppressed B-cell proliferation, as predicted by their phenotype but later functioned as T helper cells in the pokeweed mitogen system without a change in phenotype. The cells also responded inadequately to alloantigen and mitogen despite addition of exogenous T-cell growth factor (TCGF). Leu-2-positive prolymphocytes from the spleen of the patient were constitutive producers of TCGF. Surface phenotype using monoclonal antibody was inadequate to predict T-cell function of the cells from this patient with T-PLL. In addition, these data suggest there may be functional subpopulations within the OKT8+ phenotype. Constitutive TCGF production by malignant post-thymic T-cells may represent a mechanism by which these cells sustain their own growth.

ATL-cell lines producing C-type retroviruses were established by culture of the peripheral blood mononuclear cells from a patient with chronic ATL. These cell lines had the surface feature of inducer/helper T-cells with electron microscopic finding of C-type retrovirus on the cell surface. Chromosome analysis revealed the presence of a marker chromosome, but did not identify 7 trisomy nor 14q+. Of the cell lines, the line established by culture with TCGF was named "Yana-I" and the line established by culture of Yana-I without adding TCGF was named "Yana-II". Serum ATLA-antibody was positive in 1.5% of the healty individuals examined (4/257). Among malignant lymphoma patients, ATLA antibody was positive in none of the patients with B-cell type lymphoma (0/10) and Hodgkin's disease (0/2). In contrast, ATLA antibody was found in half of the patients with T-cell type lymphoma (3/6) and all with ATL (4/4). This suggested that C-type retrovirus in closely associated with ATLL.

Culture of blood T lymphocytes collected from normal individuals and cancer patients were carried out in presence of T cell growth factor (TCGF); these cultures presented cytotoxic activity directed against different targets (lectin activated cells, autologous cancer cells, antibody coated cells and K 562). In order to study separately the different effector subpopulations, isolation of single cultured cells were performed with the help of a micropipette under microscope and monoclonal cultures were carried out in presence of TCGF. In the preliminary cytotoxic assays performed in the clones: (1) a marked activity directed against lectin targets was observed in many clones and (2) an important N K activity was exhibited by the clone 45 B9 (65% of the tested cells lysed human lymphoma K 562 cells).

T (E+) lymphocytes purified from lymphoepithelial thymoma surgically removed from an asymptomatic 12 yrs old girl were cultured in presence of PHA-P; culture supernatant contained a T cell growth factor (IL-2) biochemically different from that purifiable from supernatants of normal T cells. Differences in molecular weight, isoelectric point and ion exchange elution pattern would allow to compare this abnormal IL-2, (named Thy-IL-2 from Thymus) to that already characterized from the HUT 102 T cell line (L-TCGF peak II). A comparison of biochemical properties of Thy-IL-2 and normal IL-2 (N-IL-2) is also presented; the biological significance of such differences are discussed.

An athymic mouse-derived immature T-cell clone, N-9F, was not maintained by interleukin-2 alone but required another soluble factor, contained in concanavalin A-stimulated rat splenocyte culture supernatant, namely T cell growth factor (TCGF), for its proliferation. An N-9F-proliferation factor (NPF) was isolated in a pure form from TCGF. N-9F cells and immature thymocytes proliferated in the presence of NPF at 10-11-10-8 g/ml in a dose-dependent manner, but adult thymocytes were not stimulated by NPF. NPF increased DNA synthesis of N-9F. NPF increased CD4 and CD8 double negative thymocytes and CD8 single positive thymocytes in fetal thymus organ culture. A hamster anti-NPF antiserum possessing the capacity to neutralize N-9F proliferation activity of NPF decreased double negative thymocytes. The amino-terminal amino acid sequence of NPF was identified to be Ser-Leu-Pro-Cys-Asp-Ile-Cys-Lys-Thr-Val-Val-Thr-Glu-Ala-Cys-Asn-Leu-Leu-Lys-Asp- and was identical to that of rat saposin A. The apparent molecular weight of NPF, 16000, was comparable to that of saposin A. A rabbit anti-mouse recombinant His-tag (mrH)-saposin A antibody recognized a 16000 MW molecule in TCGF. A Hitrap-saposin A antibody column bound NPF and pulled down the NPF activity in TCGF. Thus, NPF in TCGF was a saposin A-like protein possessing the capacity for growth and differentiation of immature thymocytes.

An athymic mouse-derived immature T-cell clone, N-9F, was not maintained by interleukin-2 alone but required another soluble factor, contained in concanavalin A-stimulated rat splenocyte culture supernatant, namely T cell growth factor (TCGF), for its proliferation. An N-9F-proliferation factor (NPF) was isolated in a pure form from TCGF. N-9F cells and immature thymocytes proliferated in the presence of N-9F at 10(-12)-10(-9)M in a dose-dependent manner, but adult thymocytes were not stimulated by NPF. NPF increased DNA synthesis of N-9F. NPF increased CD4 and CD8 double negative, single positive and double positive thymocytes in fetal thymus organ culture. A hamster anti-NPF antiserum possessing the capacity to neutralize N-9F proliferation activity of NPF neutralized the increasing effect of NPF on immature thymocytes. All effects of NPF was inhibited by mAb QR6.6 to recognize a 100 kDa surface molecule of N-9F. The amino-terminal 20 amino acid sequence of NPF was identified and identical to that of rat saposin A. The apparent molecular weight of NPF, 16000, was comparable to that of saposin A. A Hitrap-mouse recombinant His-tag-saposin A antibody column bound NPF, pulled down the NPF activity in TCGF, and the antibody recognized a 16kDa molecule in western-blotting of TCGF. Thus, NPF in TCGF was a saposin A-like protein possessing the capacity for growth and differentiation of immature thymocytes. The physiological significance of NPF in the growth and differentiation of immature thymocytes was discussed in view of the characteristic distributions of NPF and the molecule recognized by its mAb QR6.6 in fetal thymi.

The kinetics of allohelp mediated by diffusable factors revealed that help by nonirradiated T cells (TOR) peaked at 48 to 72 hr, followed by a sharp decline if the T cells remained in the cultures. The temporal decrease in help after 72 hr was not mediated by suppressor lymphokines because mixtures of early (24 to 48 hr) and late (120-hr) allogeneic supernatants enhanced help synergistically. Lyt-1, Ia- T cells mediated the temporal decline in help and suppressed allogeneic B cell activation in co-cultures, and this "down-regulatory" activity (allosuppression) was radiosensitive. Help by irradiated T cells (T1000R) increased gradually until it plateaued between 96 and 120 hr. The helper activities of the allogeneic supernatants were directly proportional to their T cell growth factor (TCGF) activities. In addition, their kinetics were identical, and the removal of TCGF from 48-hr allogeneic supernatants by adsorption with TCGF-dependent HT-2 cells depleted both helper and TCGF activities. Help was restored to depleted 48-hr and 120-hr allogeneic supernatants by preparations of TCGF obtained from concanavalin A (Con A)-stimulated FS6-14.13 hybridoma cells that were adsorbed with lipopolysaccharide (LPS)-activated B cells or normal spleen cells (NS), but not with HT-2 cells. These results indicate that allohelp is dependent on TCGF. Moreover, help was dependent on at least one factor in addition to TCGF, because a high level of synergy occurred between TCGF and the "help-deficient" 120-hr allogeneic supernatant. In conclusion, the mechanism whereby Lyt-1, Ia- T cells regulated B cell activation with positive and negative allogeneic effects was through the production and subsequent exhaustion of TCGF, respectively. The production of TCGF and help was radioresistant, but exhaustion of TCGF and suppression was radiosensitive.

We cultured immunosuppressor T cells from gastric cancer patients using T-cell growth factor (TCGF) prepared from human tonsil or spleen. Peripheral blood lymphocytes cultured for 3-4 weeks with TCGF strongly inhibited the lymphocyte-proliferative response to alloantigen or PHA. Quantitative fluorescence measurement for immunological analysis of phenotypic characterization of the cells was made on a FACS-IV, using monoclonal antibodies (anti Leu-I, anti Leu-2a, anti Leu-3a, anti Leu-4, anti Leu-5, anti Leu-7, anti HLA-DR) and goat anti-human immunoglobulin. Immunosuppressor T cells grown in the presence of TCGF showed phenotype Leu-1+, 2a+, 3a-, 4+, 5+, 7-, HLA-DR+, human Ig-. Culture of immunosuppressor T cells activated by tumor cell antigen in vitro was successful only when the cells derived from patients with disseminated, nonresectable type of gastric carcinoma. Our findings suggest that TCGF-dependent immunosuppressor T cells are the result of a large tumor burden; this may explain the depression of in vitro or in vivo cell-mediated immune responses frequently found in such cancer patients.

Autologous mixed lymphocyte tumor cell cultures (MLTC) generated cytotoxicity against autologous tumors in 5 out of 15 (33%) patients with head and neck cancer. Further cultivation of MLTC-activated lymphocytes with T cell growth factor (TCGF) resulted not only in the propagation of cytotoxic T lymphocytes (CTL) and an increase of cytotoxicity against autologous tumors, but also in the induction of killer cells against allogeneic tumor cells. The results of crisscross tests using fresh tumor cells from 17 donors and lymphocytes from 10 donors indicate that cultivation of MLTC-activated lymphocytes with TCGF generated killer cells cytotoxic for allogeneic tumors in most cases, but not for autologous or allogeneic phytohemagglutinin-induced lymphoblasts. Further, the results of a cold target inhibition test undertaken in an autologous tumor killing system suggest that at least 2 different subsets, specifically cytotoxic for autologous tumors and cytotoxic for both autologous and allogeneic tumors, were developed in the CTL induced by autologous MLTC followed by cultivation with TCGF. The phenotypes of the effector cells were shown to be OKT3+, Leu2a+, OKT8+, OKM1-, and Leu7- by blocking assay using complement and monoclonal antibody against the T cell surface marker.

Estimation of a content of secondary structures from amino acid composition of IL-2 (TCGF), PDGF and transforming protein p28sis of simian sarcoma virus by means of the previously suggested nomograms (V.P. Zav'yalov and A.I. Denesyuk (1982) Immunol. Lett. 4, 7-14) allowed us to conclude that a dominant secondary structure of these proteins should be alpha-helix. Its content in PDGF and p28sis was estimated to be approximately equal to 50%, and that in IL-2 to be 50-65%. The content of beta-pleated structure in PDGF and p28sis does not exceed 10%, and that in IL-2 is found to be 20-30%. In all the proteins investigated five alpha-helical segments can be distinguished, of which one of the surfaces contains clusters of bulky hydrophobic side chains. As the number of alpha-helical segments satisfying the principles of packing into a hydrophobic core as well as the overall length of a polypeptide chain of IL-2 and PDGF coincided with those for IFNs, it was decided to arrange alpha-helices of these proteins into a globular structure by analogy with IFNs (V.P. Zav'yalov and A.I. Denesyuk (1984) Doklady Akad Nauk S.S.S.R. 275, 242-246). In consequence, identical side chains of amino acid residues were found at 9 out of 29 positions in hydrophobic cores of IFN-beta and IL-2, p28sis (PDGF), respectively. Thus the homology of hydrophobic cores of the proteins compared is greater than 30%. Probability of chance coincidences of amino acid residues in the hydrophobic cores of p28sis (PDGF) and IFN-beta is found to be 0.03, and that deduced from comparison of IL-2 and IFN-beta is 0.05.(ABSTRACT TRUNCATED AT 250 WORDS)

Normal mouse spleen cells were activated by Con A and suppressor T cells were enriched by the use of peanut agglutinin (PNA). Cells of the enriched suppressor T cell fraction were further cultured in a medium containing T cell growth factor (TCGF) and the suppressor T cell lines were established. These cell lines have been growing in culture for more than one year and retained surface characteristics of Thy-1+, Lyt-1-, 2- and bear receptor sites for PNA. The suppressor clones exerted an inhibitory effect on in vitro antigen specific and antigen nonspecific PFC responses. The culture supernatant of these clones had a suppressive effect on the in vitro humoral responses, indicating that a soluble factor mediates at least partly the suppressive activity of the antigen nonspecific suppressor T cells. Moreover, it was revealed that the suppressor cells must exist at an early stage of PFC culture to exert their activity.

The in vitro effects of hydrocortisone on T cell activation and tolerance induction were investigated using human influenza virus immune T cell lines and clones. Hydrocortisone at 10(-9) to 10(-6) molar concentrations was able to inhibit the antigen induced but not the T cell growth factor (TCGF) mediated proliferative response of both the lines and clones. However, hydrocortisone was able to inhibit TCGF production by cloned T cells. The proliferative response of cloned T cells to intact influenza virus A/Texas/1/77 was more markedly inhibited by equivalent concentrations of hydrocortisone than was the response of that clone to a 24 amino acid sequence (p20) of the haemagglutinin molecule implying that hydrocortisone may also act at the level of antigen processing. Furthermore hydrocortisone was able neither to induce T cell tolerance alone nor to inhibit antigen specific tolerance induction. However, hydrocortisone did lower the antigen threshold for tolerance induction. The possible mechanisms of hydrocortisone activity in the modulation of T cell regulation in autoimmune disease are discussed.

Squirrel monkeys are experimental hosts useful for studies on human malaria. In the present work, in vitro lymphocyte reactivity was measured by proliferation in the presence of Plasmodium falciparum-derived products, and found to depend on the previous malarial status of the animals and upon the source and/or the nature of the P. falciparum-derived material. Special attention was given to the IL-2/IL-2R pathway of lymphocyte activation. Culture supernatant from P. falciparum-parasitized erythrocytes exerted an inhibitory effect towards T lymphocytes obtained from P. falciparum-non-immune squirrel monkeys, when activated, for instance, by PHA. These lymphocytes did not incorporate tritiated thymidine (neither they did proliferate) although they expressed IL-2 alpha and beta binding chains and secreted IL-2 (or at least TCGF). This inhibitory effect could not be rescued by the addition of rhIL-2, although the assayed lymphocytes could retain the ability to continue their cell cycle progression and divide after removal of the P. falciparum-derived inhibitory product(s). The incidence of anti-mitogenic molecules which impair the IL-2/IL-2R pathway of lymphocyte activation in malaria related processes is discussed.

Human T-cell growth factor (TCGF) has been isolated from conditioned media of the Jurkat T-leukemia cell line. Using a high-efficiency isolation procedure involving hollow fiber concentration, gel filtration and 3 steps of reverse-phase HPLC we obtained 100 to 600 pmol TCGF per liter of conditioned medium. Jurkat cell-derived TCGF (jTCGF) has a molecular weight of 15,750. The amino acid composition of jTCGF agrees well with that derived from the cDNA sequence coding for this protein (Taniguchi et al, Nature 302, 305, 1983). jTCGF is highly active in vitro in stimulating the proliferation of T-cells as measured by 3H-thymidine incorporation into DNA (half-maximal stimulation with 3 fmol/100 microliters well).

OKT 3 monoclonal antibodies strongly inhibit the proliferation of T-cell growth factor (TCGF)-dependent continuously-growing T-lymphoblasts. The inhibition was shown to be dose-dependent reaching close to 70% in a concentration of 10 micrograms/ml. By using a purified radiolabelled TCGF preparation the inhibition was shown to take place at the level of growth factor binding. These results are consistent with OKT 3 monoclonal antibodies reacting with a membrane structure on or close to the TCGF receptor on activated human T-lymphocytes.