The evidence that schizophrenia may involve infection by a virus (or viruses) has been indirect. The recent discovery, however, of the human retroviruses--human T-cell lymphoma-leukemia virus-I, and II (HTLV-I, -II) and human immunodeficiency virus (HIV)--now also known to affect the central nervous system (CNS), together with the development of new techniques in retrovirology, have made it possible to investigate more directly the role of this class of viruses as an etiology of schizophrenia. In our first effort to screen for the presence of a T-cell lymphotropic virus in schizophrenia, short-term tissue cultures of peripheral lymphocytes from 17 chronic schizophrenic patients and 10 normal controls were established. The cells were cultured in the presence of T-cell growth factor (TCGF, IL-2), and the culture supernatants were tested for the presence of the retroviral enzyme reverse transcriptase. No T-cell-associated reverse transcriptase activity was detected in cultures from patients or normal controls. Therefore, the data do not provide evidence for a role for T-cell lymphotropic retroviruses as an etiology of schizophrenia.

Cells with the ability to suppress cytotoxic T lymphocyte generation are found in the spleens of whole-body-irradiated (WBI) mixed allogeneic and syngeneic bone marrow transplant recipients in the early weeks after BMT. Previous studies have indicated that suppression is mediated by "null cells" similar to natural suppressor (NS) cells (1), and have ruled out several possible trivial explanations for the suppressive effect. We report here the results of additional experiments designed to assess possible mechanisms of suppression. We compared the cell populations after 5 days' incubation of cultures containing normal responding splenocytes plus irradiated allogeneic stimulator cells, with or without a cocultured suppressive chimeric splenocyte population. The data indicate that total viable cell yields are only slightly reduced, if at all, in suppressed cultures, but that the proportion of T cells is markedly reduced as measured at the end of the incubation period. Splenocytes from early BMT recipients do not appear to proliferate during the suppression of a mixed lymphocyte culture, and such populations represent only 15% of cells at the end of the 5-day incubation period. Suppression is strongest when the suppressive population is added at the initiation of MLC, and is lost if addition is delayed beyond day 3. Suppression can be overcome by T cell growth factor (TCGF)--and, to a lesser extent, by recombinant IL-2 (rIL-2), although resting suppressive populations do not consume appreciable amounts of these lymphokines. These results therefore suggest that suppression in MLC may occur primarily during the induction of helper T lymphocytes.

The long-term in vitro growth in T-cell growth factor (TCGF) of murine cytotoxic T-lymphoid cells directed against syngeneic tumor antigens was investigated. C57BL/6 mice were immunized to syngeneic FBL-3 lymphoma by two ip injections of irradiated FBL-3 lymphoma cells. Splenocytes from these animals were injected into mice with disseminated lethal FBL-3 tumor. The injection of cyclophosphamide (Cy) plus immunized lymphocytes significantly improved survival with cure of 53% of 38 animals. In comparison, treatment with Cy alone resulted in 0 of 31 cured and treatment with Cy plus unimmunized cells resulted in 0 of 40 cured (P less than 0.0005). These in vivo immunized lymphocytes were reexposed to FBL-3 tumor in vitro for 5 days in lectin-free TCGF (LF-TCGF). Although in vivo and in vitro sensitized lymphocytes exhibited no cytotoxicity toward fresh FBL-3 tumor cells in an 18-hour 51Cr release assay, expansion of appropriately sensitized cells in LF-TCGF resulted in significant lysis of fresh FBL-3 tumor cells. This significant lysis was specific and lysed syngeneic FBL-3 but not syngeneic MCA-103 fresh tumor targets. This maximal specific cytotoxicity was maintained for 2.5 months. A screening assay was developed that permitted rapid identification and isolation of low-frequency cytotoxic clones with reactivity specific for FBL-3 tumor. Several of these cloned cells were grown for almost 3 months with maintenance of high degrees of specific lysis (as much as 4,500 lytic U/10(6) cells). These cytotoxic lines and clones will be of value for the study of tumor-host immunologic interactions and perhaps for use in adoptive immunotherapy.

The effect of a panel of monoclonal antibodies and heteroantibodies on T-cell proliferation in various assay systems has been examined. The antibodies tested were directed against T-cell differentiation antigens, HLA-DR antigens, and structures defined by an anti-human VH antiserum. As the test cell system highly purified subpopulations of T-cell growth factor (TCGF)-dependent T-cell lines activated either by mitogen or antigen were used. A survey of the data indicates the following: (1) Mitogenic and antigenic triggering of T lymphocytes are mediated through partly different membrane structures. (2) Antigenic stimulation by purified protein derivative (PPD) as well as polyclonal activation induced by OKT3/anti-Leu 4 monoclonal antibodies can be inhibited by heteroantibodies raised against human immunoglobulin VH fragments thus pointing to a possible connection between the antigens detected by these antisera. (3) There does not seem to be differences between the two major subpopulations of T lymphocytes (i.e., helper/inducer and suppressor/cytotoxic cells) as to how they respond to antigens or mitogens in the investigated assay systems. (4) A clear distinction was found between T blasts specific for PPD and allogeneic cells as compared to cytotoxic T cells (CTL), as the T4 and T8 antigens seem to be functionally important for antigen recognition among CTL but not for the blasts proliferating in response to PPD and allogeneic cells. (5) An inhibitory effect of OKT3/anti-Leu 4, OKIa1, and anti-HLA-DR on TCGF-dependent growth was detected, possibly indicating a steric relationship between these antigens and TCGF receptors on mitogen-induced T blasts. (6) Soluble factors obtained after incubating adherent cells with OKIa1 and anti-HLA-DR antibodies seemed to have an inhibitory effect on overall T-cell proliferation stressing the importance of studying the T-cell activation process at different levels in these kinds of experiments. (7) The results further suggest a complexity in the build up of antigen receptors on the various T-effector cells, perhaps also involving receptors for growth factors, HLA-DR antigens, and receptors for the latter.

The culture supernatants of Con A-activated human peripheral blood mononuclear cells (PBM) contained at least two regulatory factors upon B cell proliferation. One was B cell growth factor (BCGF), which activated antigen-stimulated B cells to proliferation and clonal expansion, and the other was its inhibitory factor, arbitrarily named B cell growth inhibitory factor (BIF). This BIF inhibited the effect of BCGF on anti-mu-stimulated B cells or the monoclonal mature B cell line (CLL-T.H.) obtained from the peripheral blood lymphocytes of B cell-type chronic lymphocytic leukemia patients, which were activated only with BCGF and without adding other proliferating stimuli (e.g., anti-mu). BIF activity was detected in the 24 hr culture supernatants of Con A-activated human PBM in FCS containing medium and also in serum-free RPMI 1640 medium. This substance with BIF activity could not be derived from FCS. Con A-induced BIF (m.w. of 80,000 and an isoelectric point of pH 5.4) was analyzed by Sephadex G-200 gel filtration and chromatofocusing. BIF was stable at pH 2.0 and at 56 degrees C for 30 min. Partially purified BIF had no effect on cell viability and almost no interferon activity (less than 1 IU/ml). BIF with high titer had a slight but significant inhibition on TCGF-dependent T cell growth and on PHA or Con A responses, but the extent of these inhibitions was far less than that of BCGF-dependent B cell growth. Absorption of BIF with Con A blasts made its inhibition on T cell growth even less. On the other hand, BIF activity could not be absorbed with Con A blasts but was almost absorbed with large numbers of CLL-T.H. cells. BIF had almost no inhibitory effect on the proliferation of a mouse fibroblast cell line (NIH 3T3), a mouse myeloma cell line (NS-1), human lymphoid cell lines (MOLT-4, HSB-2, and Daudi), or a human myeloid cell line (K-562). BIF-producing cells were estimated to be T cells and were identified as T8+ T cells. On the other hand, Con A-induced BCGF was demonstrated to be produced predominantly by T4+ T cells. These results show that human B cell proliferation is regulated by interaction between T4+ and T8+ cells via soluble factors, namely BCGF and BIF, respectively.

The lymphocyte colony forming capacity of peripheral blood mononuclear cells from normal controls and from two patients with chronic OKT8+ lymphocytic leukaemia was determined in agar culture under PHA stimulation. The number and size of the colonies in patients were reduced compared to normal. The lymphocytic phenotype of colony cells was studied with monoclonal antibodies in colonies harvested from agar culture and in colonies expanded in liquid culture in the presence of TCGF. This study was performed in individual colonies and in pooled colonies. Colonies from normal controls contained a mixture of the OKT4+ and OKT8+ lymphocyte subsets. In contrast, colonies from the two patients contained essentially OKT4+ lymphocytes. The data indicate that, in the patients, progenitors of the OKT8+ subset are unresponsive to normal proliferative and/or differentiative stimuli under the present culture conditions.

Interleukin-2 (IL-2) prepared from Con A-activated rat spleen cells was partially purified using hydroxylapatite chromatography (HTP) and chromatofocusing on Mono P. IL-2 eluted in a major peak between 0.1 and 0.25 M NaCl in PBS (purification factor 36-fold) and in a second peak in the high salt elution (purification factor 5-fold). When analysed on Mono P, the major peak was found to resolve into four components with apparent pI values in the range of 7.05-5.80; further activity eluted in the high salt fraction. Similar patterns were observed using high salt eluted activity with minor variations in the apparent pI values. Neuraminidase treatment caused a shift in IL-2 charge towards more basic pI values. This analysis of the multiple species of IL-2 suggests that part of the heterogeneity may be due to variation in the degree of sialylation of the peptide chain.

Rat thymocytes were separated into five density layers by isopycnic centrifugation on a discontinuous-density Ficoll gradient and each fraction was tested for metabolic functions at different times during a 96-h culture period in various conditions. Immediately after separation the rate of oxygen consumption per unit of cellular volume is virtually the same in all fractions. However, the capacity to synthesize nucleic acids, proteins, purine and pyrimidine nucleotides by the de novo pathway and the activity of the salvage pathway of purine nucleotides is much greater in the low density fractions (fractions 1 and 2). In in vitro cell culture conditions (1) in the absence of mitogens this metabolic activity (which demonstrates a high spontaneous mitotic capacity of cells in the low-density fractions) decays rapidly to a level which is practically negligible after 24 h of culture; (2) in the presence of mitogens alone: PHA, Con A, PWM or PNA, with or without prior neuraminidase treatment, after 24 h an induction of the synthesis of nucleic acids and protein is observed concomitant to the apparition of blast cells. This blastogenesis peaks at 72 h and occurs in fractions 1 and 2 only; (3) in the presence of Con A and 2-mercaptoethanol the blast transformation corresponding to the response of the thymocytes to TCGF is observed in all of the thymocyte fractions and extends beyond 72 h of culture. It remains much more intense in the low-density fractions 1 and 2.

Transplantation tolerance was induced in cyclophosphamide-treated, B-cell-depleted chickens by transfer of allogeneic bursal cells. To study the presence of specific suppressor cells in tolerant birds, mitomycin-C-treated peripheral blood lymphocytes (PBL) from tolerant recipients were cocultured in mixed lymphocyte cultures of normal syngeneic responder and allogeneic stimulator cells. No evidence of suppression was detected, since responses in cultures with tolerant cocultured cells were on the same level as the mixed lymphocyte reaction (MLR) of normal responders without cocultured cells. However, responses in cultures with normal cocultured cells were significantly higher. If cocultured cells were not treated with mitomycin C, responses to tolerizing alloantigen were equally high in cultures with tolerant cocultured cells as in cultures with normal cocultured cells. Likewise, when lymphocytes from tolerant chickens were mixed with normal syngeneic cells in graft-versus-host (GVH) splenomegaly assay, no suppression was detected, but the GVH reaction was even stronger than the reaction induced by the mixture of cells from two normal chickens. Furthermore, administration of chicken T cell growth factor (TCGF) into the cultures enhanced considerably the MLR of tolerant cells against the tolerizing alloantigen, but not against syngeneic or third-party stimulator cells. These results indicate that the transplantation tolerance in B-cell-chimeric chickens is due to lack of alloantigen-specific helper cells. When exogenous help is offered to tolerant cells either by normal syngeneic cells or by exogenous TCGF, the reactivity of tolerant cells against the tolerogen is reestablished.

A total of 13 cancer patients were treated with Adoptive Chemoimmunotherapy (ACIT) using alloactivated HLA haploidentical lymphocytes. Donor lymphocytes were activated in vitro using a pool of irradiated allogeneic lymphocytes (MLC-cells) and some further expanded by culturing in T-cell growth factor (TCGF-cells). The first 6 patients received i.v. cyclophosphamide (CPM) followed 24 h later by escalating doses of MLC-cells, then 7 days later they received an infusion of TCGF-cells. Minimal toxicity was seen. The next 7 patients received CPM (800 mg/m2) and a combined MLC and TCGF-cell infusion (total cell dose ranged from 0.79 x 10(10) to 2.26 x 10(10)). Of these 7 patients, 3 developed mild graft-versus-host reaction (GVHR) which resolved without treatment, and 2 patients had progressive GVHR which was arrested by methylprednisolone (2 mg/kg). Peripheral blood lymphocytes from these 2 patients, during the GVHR, had increased activated T-cells (OKT-10+ and OK-Ia+). In vitro expansion, in TCGF, of these activated T-cells enabled HLA typing to prove they were of donor origin. Only 1 clinical antitumor response was observed in the first 6 patients. The results of this study indicate that this form of ACIT can be given to patients with acceptable toxicity. Self-limited or easily controlled GVHR may be induced and primed donor cells persisting in the circulation are probably responsible. Further testing is required to determine whether the immune response induced by this form of ACIT may be therapeutically effective.

Human antigenic determinants present on activated T and B lymphocytes and not on resting B and T cells defined by serological studies are reported. The reactivity of 13 sera against human PBM activated by various agents was tested using microlymphocytotoxicity technique. Results showed that generally similar reactions are present on B and T blasts whatever the technique of activation (PHA, ConA, PWM, TCGF, EBV and allo activation). Absorption elution studies with 5 sera on 10 alloactivated cells clearly demonstrated their independence from DR determinants. The analysis of results obtained by these 15 sera on blasts from 50 donors showed no correlation with HLA-A, -B, -C, DR and -MB antigens, neither can it be HT (Qa-like) antigens. Furthermore, 5 clusters of sera are reported. The weak correlation coefficients cannot affirm the existence of a system with an allelic distribution although each cell except one out of a panel of 50 individuals carries at most 2 specificities. The segregation of 3 of these specificities within 5 informative families showed no linkage with HLA genes.

A method for the facile removal of mitogens or inducers of lymphokine production from cell culture medium of stimulated cells is described. The technique is based on the covalent attachment of biotin to mitogen or inducer and the removal of the biotinylated products from stimulated cell culture medium using immobilized avidin. Using this procedure, biotin-labeled staphylococcal enterotoxin A (SEA-B) was shown not to differ significantly from unmodified SEA in its capacity to stimulate mitogenesis and induce production of immune interferon (IFN) and T-cell growth factor (TCGF) in cultures of human mononuclear cells from peripheral blood. SEA-B was also shown not to differ from SEA in its binding to SEA antibodies. Results of mitogenicity studies and competitive radioimmune assay (RIA) measurements indicate that SEA-B is essentially completely removed from stimulated cell culture medium by absorption with avidin coupled to Sepharose 4B.

A cDNA clone coding for the murine IgG1 induction factor has been isolated. The translation products directed by this clone were analyzed in different biological assays. The data obtained show that the IgG1 induction factor: Is involved in the regulation of IgG responses, by increasing IgG1 and decreasing IgG3 and IgG2b secretion; Induces hyper-Ia expression on resting B lymphocytes; Synergizes with anti-Ig in inducing DNA synthesis in resting B lymphocytes; Synergizes with DxS in inducing DNA synthesis by B lymphocytes; It induces DNA synthesis by either the T cell line CTL-L or Con-A blasts. Thus, this lymphokine in addition to IgG1 inducing activity has also BSF-1, BCGF-II and TCGF like activities. The fact that a single molecule can perform all the above listed functions has implications for our view of lymphocyte activation. It indicates that considering the B cell response as an ordered series of independently controlled events, is an oversimplified view of the dynamic process through which B cells are activated and also indicate the functional interconnection of the different elements of the immune system.

Crude T cell growth factor was prepared from pig blood cells in mixed lymphocyte culture together with Concanavalin A. The TCGF was recovered from the crude supernatant by ammonium sulphate precipitation and fractionated by gel exclusion chromatography to yield active fractions corresponding to an apparent molecular weight of 23,000d. The TCGF was further purified by isoelectric focussing and was found to migrate as a single peak of pI 5 - 5.5. The crude preparation was found to support the growth of mouse and sheep activated cells but had no effect on human activated cells. Human TCGF supported the continued growth of activated pig cells whereas mouse and sheep TCGF had no such effect.

A medullary carcinoma with lymphocytic infiltration is associated with relatively good prognosis following radical mastectomy in breast cancer. This suggests that tumor infiltrating lymphocytes (TIL) play an important role in the immunological resistance of the host against cancer. The cytotoxic activities of TIL in murine mammary carcinoma SC42 and SC115 of syngeneic DS mice were assessed with 51Cr release assay in this study. The tumor masses were minced and trypsinized, and lymphoid cell rich fraction was separated with Percoll discontinuous gradient centrifugation. During 10 days culture with T cell growth factor (TCGF) obtained from Con A stimulated rat spleen cells, the tumor cells were collapsed and lymphocytes were expanded. The cultured TIL from SC42 tumor had stronger cytotoxicity against SC42 tumor cells than the cultured TIL from SC115 tumor and the cultured spleen cells of SC42 tumor bearer, and the cultured TIL from SC115 tumor showed predominant activity against SC115 tumor cells. Nonspecific cytotoxic activity and natural killer (NK) activity of these TIL were weaker than the cultured spleen cells. The culture of the spleen cells with the tumor cells and TCGF failed to induce tumor-specific cytotoxic lymphocytes. Moreover, the tumor-specific cytotoxicity was reduced by the treatment of anti thy-1, 2 and complement, and NK activity was reduced by the treatment of anti-asialo GM1 and complement. These results indicate that the tumor-specific cytotoxicity is latent in the T cell population of TIL and is induced by the culture with TCGF.

We analyzed the release of activities capable of stimulating the in vitro growth of human hemopoietic progenitor cells by long-term cultured T cell growth factor (TCGF)-dependent human T lymphocytes. Seven cell lines tested produced colony-stimulating activity (CSA) as well as burst-promoting activity (BPA). The CSA stimulated primarily the growth of the cells forming colonies after 14 days of incubation. In addition the supernatants from these seven T-cell lines showed the ability to induce the in vitro growth of mixed granulocyte, erythroid, megakaryocyte, macrophage colonies (CFU-GEMM). The release of hemopoietic factors did not depend on the presence of accessory cells or phytohemagglutinin or serum during the incubation for factor production. In six of the T cell lines the majority of the cells were reactive to the OKT 8 monoclonal antibody (MoAb), whereas one cell line contained mostly OKT 4+ cells. Suppressor activity was detected in three tested OKT 8+ cell lines, while the one OKT 4+ displayed helper activity. All cell lines produced hemopoietic factors with equal efficiency. These results indicate that factors affecting human hematopoiesis are produced by normal T lymphocytes in long-term culture and this property is not related to the helper or suppressor activity of the cultured cells.

Mitogenic factor was produced by sensitized guinea pig lymph node cells stimulated with a specific antigen. Both T lymphocytes and macrophages were required for the production of this factor. The culture supernatant of lymphocytes containing the mitogenic factor exhibited a strong helping effect on the proliferative response of T lymphocytes to phytohemagglutinin (PHA). Mitogenic factor and the factor with the helping activity coeluted in the molecular weight range of 25,000-35,000 daltons in gel filtration. Furthermore the fraction containing mitogenic factor was found to support the proliferation of lymphoblasts induced by PHA or antigen, suggesting that the mitogenic factor may be the guinea pig equivalent of T cell growth factor (TCGF) reported in the mouse, rat, and human. On the other hand, the T cell-activating monokine of the guinea pig, possessing the helping activity for the proliferative response of T lymphocytes to PHA, never exhibited TCGF-like activity.

The E-rosette receptor (CD2, T11) is a differentiation antigen expressed on immature and mature human T lymphocytes. Activation of T cells from human peripheral blood with phytohemagglutinin (PHA) or with monoclonal antibody to the CD3-Ti complex (anti-Leu-4) caused the expression of CD2 to increase 10- to 20-fold. Dual parameter correlated analyses with antibody to the T cell growth factor (TCGF) receptor (anti-Tac) and anti-CD2 antibody demonstrated that the increase in CD2 expression occurred at the same time and on the same cells that expressed the TCGF receptor after stimulation with PHA. The increased expression of CD2 and the initial expression of Tac were totally inhibited by cycloheximide, but were not affected by sufficient actinomycin-D to block the T cell proliferative response. The expression of CD2 was compared with the expression of CD4 and CD8, i.e., T cell differentiation antigens on cytotoxic/suppressor or helper T cells, respectively. Although virtually all of the small percentage of freshly isolated Tac+ peripheral blood cells belonged to the CD4+, CD8- subset, both CD4+ and CD8+ T cells were equivalently activated by PHA to express Tac. By 20-30 hr after activation, the expression of CD4 or CD8 was initially decreased 10-50%. Subsequently, the expression of CD4 and CD8 returned to the levels on resting T cells but did not increase further. Therefore, the increase in CD2 expression does not reflect a universal property of cell surface antigens on activated T lymphocytes.

The T cell antigen receptor complex (CD3/Ti) plays a role in specific antigen recognition as well as in signal transduction, with its surface expression required for the function of several other structurally distinct receptor systems, including CD2, Ly-6(TAP), and Thy-1. In this communication, evidence is presented suggesting an association between the surface expression of CD3/Ti and that of the type 1 interferon (IFN) receptor in a CD4+ murine T cell clone. We tested the proliferative responses and their capacity to be inhibited by type 1 IFN with the wild-type, CD3/Ti-positive T cell clone and its CD3/Ti-negative variants did not respond to specific antigen or anti-CD3 antibody stimulation but they did respond to T cell growth factor (TCGF), stimulation as did the wild-type parental cells. Therefore, the type 1 IFN inhibition of TCGF-stimulated proliferative responses of wild-type and variant cells were compared. Both natural and recombinant type 1 IFNs inhibited TCGF-induced tritiated thymidine (3H-TdR) incorporation in the wild-type T cell clone, with a ID50 of 60-80 U/ml. By contrast, the variants required much higher doses of type 1 IFN. The ID50 with natural murine IFN-beta was 10,000 U/ml, but this same dose of human IFN-alpha A/D gave only a marginal inhibitory effect. Accompanying the loss of IFN responsiveness, these variants also exhibited a loss of high-affinity type 1 IFN receptors. Taken together, these data suggest that the CD3/Ti complex plays a role in the surface expression of the type 1 IFN receptor in a CD4+ T cell clone.(ABSTRACT TRUNCATED AT 250 WORDS)

This unit describes protocols employing cell lines or bioassays that can be used for the quantitation of murine total T cell growth factor (TCGF) activity, interleukin 2 (IL-2), and interleukin 4 (IL-4), and of human IL-2 and IL-4. The ability to distinguish between different growth factors is crucial to understanding the regulation of the immune response. The Basic Protocol describes the use of the CTLL-2 line to detect murine IL-2 and IL-4 in supernatants. One alternate protocol describes the detection of IL-2 in samples of human serum or supernatants using CTLL-2 cells, while other alternate procedures describe the detection and quantitation of murine IL-4 using a mutagenized subline of CTLL-2, CT.4S, and the detection of human IL-4 using a derivative of the CT.4S mouse cell line, CT.h4S. Support protocols are provided for the quantitation of CTLL-2, CT4.S, or CT.h4S proliferation using a standard [3H]thymidine incorporation method or by using the 3-(4,5-dimenthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Support Protocol 1). Support protocols also describe the calculation of cytokine units from samples based on DNA synthesis data and procedures for the maintenance of the CTLL-2, CT.4S, and CT.h4S cell lines.

Well-defined clones of human T-lymphocytes were produced and monoclonal T-cell cultures were maintained for long periods of time. Single T-lymphocytes were isolated with the help of a micropipette from PBL cultures prior to any cellular stimulation (MLC), collected separately at the bottom of a 200 microliter tissue culture microwell under the control of stereomicroscopic observation, and cultured with irradiated lymphoid cells in the presence of TCGF. After 12 days, 20-50% of the seeded wells exhibited clones of 3 x 104-105 T-lymphocytes, which were transferred to larger tissue culture wells (2 ml, LINBRO) for long-term culture. Recloning of the growing cloned cells under the same conditions as the primary culture was carried out successfully. In the preliminary cytotoxic assays performed in 11 clones (a) a marked activity directed against lectin-coated targets was observed in may clones; (b) an important NK-like activity was exhibited by the clone 45B9 (65% of the tested cells lysed K562 cell targets); (c) 2 clones did not demonstrate cytotoxic activity against either PHA-coated L-1210 cells or K562 cell targets. These results could be explained hypothetically by the difference of functional maturation of T-cells within each clone.

Splenocytes of normal (NS) and tumor-bearing (TBS) mice (Balb/c, C57BL/6, C3H) were stimulated in vitro for 24 h with concanavalin A and the amount of T cell growth factor (TCGF) generated was measured. TBS of mice carrying subcutaneous implants (greater than 1 cm tumor diameter) of several T lymphomas, pulmonary and mammary carcinomas, and a melanoma, or intraperitoneal implants (greater than 10(8) cells) of ascitic lymphomas produced (per culture) 40-90% less TCGF than that generated by NS. TBS also contained a greater proportion of phagocytic cells and a higher ratio of Lyt 2+/Lyt 1+ T cells as compared with NS. In cocultures consisting of NS and TBS (1:1), TCGF production by NS was markedly suppressed. In contrast, addition of up to 30% tumor cells to NS decreased production only slightly. Removal from TBS of either phagocytes or Lyt 2+ T cells, or treatment in vitro with indomethacin [IND; an agent inhibiting prostaglandin (PG) synthesis], appreciably reduced their capacity to inhibit NS and improved TCGF production in TBS cultured alone. TCGF production by TBS could be completely restored by depletion of both phagocytes and Lyt 2+ T cells. Elevated quantities of TCGF (up to threefold) were generated by TBS of mice pretreated in vivo 1-4 days previously with low doses of either cyclophosphamide or X-irradiation, with or without IND. It is concluded that suppression of TCGF production in vitro by TBS is mediated by phagocyte-released PG and by Lyt 2+ T lymphocytes.

The present study was designed to examine the cellular requirements for the generation of the suppressor T cells induced in the presence of fetal calf serum in culture. When C57Bl/6 mouse spleen cells were cultured for 4-5 days, these precultured cells were shown in mixing experiments to suppress the generation of cytotoxic effector cells (CTL) against allogeneic P815 cells or the generation of anti-SRBC humoral response by freshly explanted C57Bl/6 spleen cells. Spleen cells cultured in the presence of silica (0.5 mg) for 4 days, did not develop suppressor activity. However, when silica was added 3 days after the start of the suppressor generation culture, the development of suppressor cells was only slightly affected, although the phagocytic activity of these spleen cells was still totally abolished. When plastic or G-10 Sephadex column nonadherent spleen cells were cultured alone for 4-5 days, these cells did not suppress the generation of CTL or anti-SRBC humoral response. When the nonadherent spleen cells were cultured with plastic adherent spleen cells, however, suppressor cells developed and the suppressor activity of these cells was dependent on the number of adherent spleen cells co-cultured with the non-adherent spleen cells. This activity of the adherent spleen cells was insensitive to treatment with anti-Thy 1.2 serum plus complement and to X-irradiation. Furthermore, adherent PEC could not substitute for adherent spleen cells, indicating a possible tissue specificity for the macrophages in the adherent cell fraction which can function in supporting and/or accelerating the differentiation of "immature" suppressor T cells. Finally, culture-induced suppressor T cells were sensitive to X-irradiation and their activity was refractory to IL2 (TCGF), whereas the activity of alloantigen-induced suppressor cells was sensitive to IL2.

A method of establishing human T-cell lines in tissue culture media containing TCGF but not PHA is described. PBL, initially stimulated by PHA to produce TCGF, continue production for at least 48 hrs after the lectin has been washed off. The TCGF-conditioned medium initiates blast formation of T-cells from freshly isolated PBL and supports indefinite growth of the T-cell blasts from the primary PBL cultures and also from blast-cells obtained from clonally derived T-cell colonies grown in soft agar culture. The established cell lines are identified by cytochemical and biological means as belonging to the T-cell compartment and express spontaneous cytotoxicity against HeLa-cells. The continuously growing T-cells are unable to produce TCGF and depend strictly on external supply of the growth factor. PHA by itself does not support T-cell growth in spite of its ability to elicit a mitogenic response in PBL-cultures. Thus two types of cells must be involved in the mitogenic event: (i) a TCGF-producer and (ii) a TCGF-responder. PHA elicits in the former TCGF production and then TCGF in the later the mitogenic T-cell response. Therefore, not PHA but TCGF might be considered to be the T-cell mitogen.