The allogeneic effect: the mechanism of allosuppression by Lyt-1, Ia- T cells

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.     

Regulation of T cell growth factor production: arrest of TCGF production after 18 hours in normal lectin-stimulated mouse spleen cell cultures

The detailed kinetics of TCGF accumulation in Con A-stimulated spleen cell cultures shows a maximum at 24 hr, with a subsequent decrease in activity. This decrease is not due to the appearance of inhibitory substances "masking" TCGF activity. Pulse experiments show that the rate of TCGF production falls sharply after 18 hr and is completely arrested after 24 hr of Con A stimulation. The arrest in TCGF production is the result neither of culture depletion in medium components nor of limiting accessory cell function or inactivation of the lectin, and it thus seem to be the result of inactivation of TCGF-producing T cells. This regulation is not the result of a TCGF-mediated feedback mechanism but rather of lectin-induced suppressive cells that appear in culture after 24 hr and turn off de novo production of TCGF in fresh cultures.     

Mechanism of T cell activation. II. Antigen- and lectin-dependent acquisition of responsiveness to TCGF is a nonmitogenic, active response of resting T cells

Small resting T cells, which do not respond to T cell growth factor (TCGF), acquire responsiveness upon a short (4-hr) pulse of specific ligands by presenting growth receptors for TCGF. The results demonstrate that the same mechanisms operate in the specific induction of primary MLR in that a 5-hr MLR is sufficient to render the responder cells reactive to TCGF. Furthermore, the results demonstrate that an active "response" by the resting T cells is required for expression of functional growth receptors, as demonstrated by the fact that: 1) a 4-hr pulse of concanavalin A (Con A) at 4 degrees C did not result in gain of reactivity to TCGF, whereas a 4-hr pulse at 37 degrees C did; 2) this metabolic requirement for acquisition of responsiveness to TCGF was not due to a secondary requirement for cap-formation of Con A-binding membrane structures, as normal responses were observed in the presence of cytochalasin B (cyt B); 3) the process of Con A-induced acquisition of susceptibility to TCGF was puromycin sensitive.     

The effect of T cell growth factor on the generation of cytolytic T cells.

The development of cytotoxic effector cells through primary allogeneic mixed tumor-lymphocyte culture (MTLC) was found to be accompanied by the production of T cell growth factor (TCGF). Addition of supplemental TCGF to MTLC resulted in the generation of significantly greater quantities of effector cells, and these effector cells displayed augmented cytotoxic activity. The TCGF-induced effect could not by duplicated by the addition of fresh medium or a mitogenic concentration of concananvalin A. Although TCGF augmented the proliferation of antigen-nonreactive cells, antigen-reactive cells appeared to be preferentially stimulated by TCGF. Finally, it was shown that depletion of TCGF from MTLC resulted in an impairment of proliferation and differentiation of cytotoxic effector cells. These findings demonstrate that soluble factors are involved in the regulation of in vitro cell-mediated immune responses in an analogous manner to similar factors that have been shown to regulate humoral immune responses. Therefore, the forces affecting TCGF production may modulate the amplitude of a T cell-mediated cytolytic response.     

Ligand-activated T cell growth factor-induced proliferation: absorption of T cell growth factor by activated T cells.

The fate in culture of the T cell growth factor (TCGF), which is required for continued growth of human cultured T cells (CTC) in vitro, was studied. TCGF activity was stable for 7 days at 37 degrees C. However, it was no longer detectable after incubation with actively growing CTC at 37 degrees C for 3 days. This loss of TCGF activity also occurred quite rapidly and was detectable within 1 hr of incubation of 0.3 ml supernatant with 2 to 5 x 10(7) CTC at 23 degrees C. 2 x 10(8) mononuclear peripheral blood leukocytes were not effective in removing TCGF activity, and incubation with similar numbers of cells from B and T cell lines had no effect. Three-day-old concanavalin A and phytohemagglutinin blasts were very reactive with TCGF, so that 10(7) or 2 x 10(7) cells consistently removed TCGF activity. These experiments suggested specific absorption of TCGF by activated T cells, and led us to develop a model of ligand-activated TCGF-induced proliferation of T cells: Ligands induce production of TCGF by T-producer cells and deliver a first signal to the T-responder cells. This causes a receptor for TCGF to appear on T-responder cells. Only then does TCGF deliver the obligatory second signal that is needed to drive the T-responder cells into proliferation.     

Mechanisms controlling TCGF production by cloned sublines of EL-4 azgr in response to stimulation by anti-Thy-1 antibody

The effect of xenogeneic anti-Thy-1 antibody on T cell growth factor (TCGF) production by T lymphoma cell lines has been examined as a model system for T cell activation. EL-4 G12 (a cloned subline of the producer EL-4 azgr cell line) produced TCGF when stimulated by a high concentration of anti-Thy-1, but none was induced by low concentrations of anti-Thy-1. Large amounts of TCGF were produced when these cells were cultured with Fc-receptor positive (FcR+) accessory cells. TCGF production by EL-4 G12 showed dose response kinetics similar to TCGF production by anti-Thy-1-stimulated, purified normal spleen T cells. Goat anti-rabbit IgG (GaRIG) and protein A substituted for this accessory helper effect, but neither FcR+ cells nor Protein A worked when (Fab')2 anti-Thy-1 was used instead of IgG anti-Thy-1. Anti-T-200 monoclonal antibody inhibited anti-Thy-1-induced TCGF production by EL-4 G12 and accessory cells. Phorbol myristic acetate and lymphocyte-activating factor also substituted in part for the accessory cell help. The data suggest there are at least 2 different accessory cell help mechanisms in anti-Thy-1-induced TCGF production, anti-Thy-1-bound membrane aggregation either by GaRIG, Protein A or FcR, and a LAF-dependent mechanism.     

Regulation of T-cell functions by L-lactate

Lactate is a product of glycolytically active macrophages. After stimulation with concanavalin A accessory cell-depleted splenic T-cell populations were found to produce only minute amounts of T-cell growth factor (TCGF); but substantial amounts of TCGF were produced if the cultures were supplemented either with splenic adherent cells or with lactate but not with interleukin-1 (IL-1). IL-1 was capable, however, of supporting TCGF production by the thymoma subline EL4-6.1. TCGF production in cultures of accessory cell-depleted splenic T-cell populations was demonstrable with 10(-3) M L-lactate, and optimal responses (plateau level) were obtained with 4-6 X 10(-2) M L-lactate. Cultures of macrophages were found to accumulate up to 5 X 10(-2) M lactate. Our experiments indicate, therefore, that lactate serves as a regulatory signal by which macrophage-like accessory cells enhance helper-T-cell functions. Lactate is apparently not the only mediator of accessory cell function since plateau levels of TCGF production were markedly lower with lactate than with splenic accessory cells; but L-lactate was found also to determine the magnitude of T-cell-mediated immune responses in vivo and in cultures of unfractionated lymphocyte populations. The production of interferon in accessory cell-depleted and concanavalin A-treated T-cell cultures, however, was not significantly affected by lactate. Concanavalin A-stimulated splenic T-cell populations were found to consume glucose rapidly and to release lactate into the supernatant. This indicates that the cells contain more lactate and pyruvate than they can utilize by their respiratory metabolism. The administration of external lactate or pyruvate was found to inhibit the utilization of glucose by the mitogenically stimulated T cells.     

T cell growth factor from human splenic cell cultures: conditions for its production, and its utilization for maintenance of cytotoxic T cell lines

We prepared the T cell growth factor (TCGF) from human spleen cell cultures stimulated with phytohemagglutinin (PHA). Various cell culture conditions and agents supporting the active TCGF production of the spleen cells were examined. The highest TCGF activity was obtained in the supernatants under the conditions that 2 x 10(6)/ml spleen cells were stimulated with PHA for 48 hr. Production of TCGF from spleen cells depended markedly on their individual sources. Addition of indomethacin to the culture or irradiation of the responding spleen cells increased TCGF activity in the supernatant of the culture. Further, addition of irradiated cells of an Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line (LCL) to spleen cell cultures stimulated with PHA greatly enhanced TCGF production. Human splenic TCGF facilitated the establishment of human cytotoxic T cell (Tc) lines specific for EBV-transformed LCL cells when those Tc line cells were stimulated periodically with irradiated autologous LCL cells but not with the other two types (K-562 or Molt-4) of cells. Allogeneic LCL stimulators allowed the Tc line cells to proliferate. However, Tc line cells cocultured once with allogeneic LCL stimulators no longer exhibited EBV-specificity in their cytotoxicities.     

The production of T-cell growth factor (TCGF) in vivo in sheep

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.     

Regulation of T-cell activation and T-cell growth factor (TCGF) production by hydrogen peroxide

Activated macrophages are known to release a variety of immunoregulatory substances including the low-molecular-weight substances hydrogen peroxide and lactate. We report here that lactate but not hydrogen peroxide is capable of supporting a substantial production of T-cell growth factor (TCGF) in cultures of accessory cell-depleted splenic T-cell populations after stimulation with concanavalin A. Hydrogen peroxide and its biosynthetic precursor superoxide anion (O2-) mediate, however, a strong augmentation of the TCGF production by accessory cell-depleted T-cell populations in the presence of lactate. Lactate inhibits the incorporation of [3H]thymidine in short-term cultures (18-26 hr) of accessory cell-depleted T cells. This confirms the rule that (optimal) production of T-cell growth factor requires a growth inhibitory signal. Concentrations of hydrogen peroxide which augment TCGF production most effectively (i.e., 1 X 10(-5) M) do not inhibit the incorporation of [3H]thymidine; and higher concentrations (3 X 10(-5)-1 X 10(-4) M) of hydrogen peroxide inhibit both the production of TCGF and the incorporation of [3H]thymidine. In agreement with the augmenting effect of hydrogen peroxide on TCGF production, it was observed that the proliferative response in mixed lymphocyte cultures is suppressed by catalase and augmented by 1 X 10(-5) M H2O2. Proliferative and cytotoxic responses in mixed lymphocyte cultures with an external source of interleukin 2 (IL-2) in contrast, are not augmented by 1 X 10(-5) M H2O2. The relatively high concentration of 1 X 10(-4) M hydrogen peroxide was found to inhibit the proliferative responses in mixed lymphocyte cultures with or without external IL-2 but not the cytotoxic response in the presence of IL-2. This indicates that CTL precursor cells may be relatively resistant against H2O2.     

Effect of cyclosporin A on human lymphocyte responses in vitro. III. CsA inhibits the production of T lymphocyte growth factors in secondary mixed lymphocyte responses but does not inhibit the response of primed lymphocytes to TCGF

The mechanism whereby Cyclosporin A (CsA) inhibits secondary mixed lymphocyte responses was assessed. CsA added to secondary MLR cultures inhibited proliferation and induction of cytolytic lymphocyte activity. This inhibition was found to be associated with the inhibition of T lymphocyte stimulating growth factor(s) (TCGF) production in the supernatants of secondary MLR cultures. As little as 1.0 micrograms/ml of CsA added to secondary MLR cultures resulted in no measurable TCGF activity. In contrast, moderate doses of CsA (1.0, 2.5 micrograms/ml), which completely inhibited the secondary MLR response to alloantigen, did not inhibit the proliferative and CML response of alloantigen-primed lymphocytes to these stimulating growth factors. Even at high doses of CsA (20 micrograms/ml), substantial levels of proliferation (50% of control response) and CML induction (60% of control response) were observed when the primed cells were exposed to secondary MLR supernatants containing TCGF activity. It was concluded that inhibition of secondary mixed lymphocyte responses by CsA may be due in part to the inhibition of TCGF production rather than the inhibition of the effect of TCGF on mature cytotoxic T lymphocytes.     

Constitutive and mitogen-induced production of T cell growth factor by stable T cell hybridoma lines

Stable T cell growth factor- (TCGF; IL 2) producing cloned T cell hybridoma lines were constructed by fusing murine alloantigen-activated T cells with the 8-azaguanine-resistant lymphoma line, BW5147. Many, but not all, clones of one of these hybridomas, i.e., hybridoma 24, secreted TCGF constitutively, but production was markedly enhanced by stimulation with T cell mitogens. Large numbers of TCGF-secreting hybridoma cells in a stable functional state could be obtained from histocompatible mice inoculated with cloned T cell hybridomas. Moreover, such in vivo-derived hybridoma cells could be stimulated sequentially with mitogen at least twice to secrete their biologically-active product, resulting in larger TCGF yields from the same cells. The secreted product of these T cell hybridoma lines resembled TCGF isolated from other cellular sources in that it: a) supported the growth of a TCGF-dependent T cell line; b) provided help for the induction of alloantigen-reactive cytotoxic T lymphocytes from thymocyte precursors; c) facilitated concanavalin A-induced mitogenic responses of low thymocyte numbers; d) had an apparent m.w. of 30,000 to 40,000 by gel filtration chromatography; and e) was eluted from DEAE-Sephacel ion-exchange chromatography columns by salt concentrations of 30 to 150 mM NaCl. The ability of these T cell hybridomas to grow in vivo and retain their functional characteristics in a stable form should prove useful in terms of providing large numbers of TCGF-secreting cells and studying in vivo aspects of the production of TCGF as well as other immunoregulatory mediators.     

Growth of an interleukin 2/interleukin 4-dependent T cell line induced by granulocyte-macrophage colony-stimulating factor (GM-CSF)

Lymphokine activities in conditioned medium from activated helper T cell lines are most commonly defined by the proliferation of "specific" lymphokine-dependent cell lines. Various sublines of IL 2-dependent (and ostensibly specific) HT-2 and CTLL cells have now been shown to proliferate in response to BSF-1/IL 4 as well. After activation with antigen or mitogen, D10.G4.1, an antigen-specific cloned T helper cell that has recently been shown to produce IL 4 but not IL 2, secretes two distinct cytokines that induce the growth of HT-2 cells. These "T cell growth factors" (TCGF) can be separated by reversed phase high-performance liquid chromatography (RP-HPLC). The TCGF activity of one of these factors can be blocked by 11B11, an antibody specific for IL 4. The second TCGF activity is not affected by 11B11 or by antibodies specific for IL 2. This TCGF activity can be neutralized by a goat polyclonal antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF), and has a RP-HPLC elution profile identical to that of recombinant GM-CSF. Recombinant GM-CSF induces both proliferation and long-term growth of HT-2 but not CTLL cells, and this activity can be neutralized by the same antibody to GM-CSF. GM-CSF is best known as a factor that induces the maturation and growth of granulocytes and macrophages from bone marrow-derived hematopoietic precursor cells. The ability of GM-CSF to induce the growth of certain T cell lines indicates that this molecule may play a role in T cell-mediated immune responses, either as an autocrine growth factor or a paracrine stimulus from both lymphoid and nonlymphoid tissues that produce this cytokine.     

Detection of autologous mixed lymphocyte reaction responding cells and their precursor frequency in NZB mice

The autologous mixed lymphocyte reaction (AMLR) can be detected in older NZB mice after treatment of the responding cell population with monoclonal anti-I-A^d and complement and supplementation of the culture medium with T-cell growth factor (TCGF) from young animals. The addition of TCGF to cultures containing responding cells alone that had not been pretreated with anti-I-A plus complement resulted in high levels of background proliferation. This is indicative of a high number of preexisting I-A-positive, activated, TCGF-responsive T cells in these mice. These activated cells could also be removed by treatment with anti-I-A antibody and panning on anti-mouse Ig plates, or by BUdR and light killing of those cells proliferating in the presence of TCGF or purified IL-2. Prior treatment of the responding cells with anti-Lyt 2 and complement did not effect the AMLR. An NZB AMLR responding cell line was established using these methods. This line retained haplotype specificity in a proliferation assay. Limiting dilution analysis of the precursor frequency of AMLR responding cells in the nonautoimmune C58 and BALB/C strains in culture medium with TCGF gave a frequency of between 1 in 35,000 and 1 in 88,000. In young, AMLR-positive, NZB mice, supplementation with TCGF yielded precursor frequencies within the normal range. In older NZB mice, the addition of TCGF resulted in increased background proliferation of preactivated, IA⁺ T cells. After removal of these cells with anti-I-A plus complement, AMLR responding cells were found at normal frequency levels when stimulated in the presence of TCGF. In the oldest animals tested (greater than 18–20 weeks), normal precursor frequencies could not be demonstrated even after this treatment, representing a true decline in the AMLR responding cell number. AMLR deficiency in NZB mice appears therefore to be the result of the combined effects of decreased lymphokine production, excessive T-cell activation, and finally decreased numbers of AMLR responding cells.     

Detection of HSV-1-induced lymphokine production in human cells by a direct indicator cell addition assay

These studies present an efficient and sensitive method for detection of T cell growth factor (TCGF) activity in human lymphocyte cultures and illustrate that T cell growth factors are associated with T lymphocyte-mediated anti-HSV-1 responses. Secretion of TCGF is induced after stimulation of human peripheral blood mononuclear cells ( PBMNC ) with herpes simplex virus type 1 (HSV-1). Lymphokine activity is detected in a simple, sensitive method by studying [3H]thymidine incorporation after the addition of murine CTLL -20 cells to cultures of gamma-irradiated (4000 R), virus-stimulated PBMNC . By using this assay, we find that PBMNC from seropositive but not seronegative individuals produce detectable TCGF activity in a dose-dependent manner after incubation with HSV-1. Maximum activity is detected between 24 to 48 hr of incubation and correlates with in vitro proliferation of nonirradiated PBMNC in response to the virus. In addition, gamma-irradiated (1000 to 3000 R) PBMNC , which are frequently used as a source of antigen-presenting cells (APC), can secrete TCGF after contact with HSV-1. Lymphokine production by the APC-containing population is eliminated by gamma-irradiation (5000 R); such APC can still present UV-inactivated HSV-1 to HSV-1-responsive lymphoblasts, indicating that lymphokine production by T cells residing in the APC population is not essential for antigen presentation.     

Selective effects of interferon on distinct sites of the T lymphocyte triggering process

Lectin- and antigen-induced proliferation of murine T cells consists of two major events, namely, a rapid induction of susceptibility to growth factors and a later-occurring, accessory cell-dependent production of T cell growth factors (TCGF). The mechanism by which interferon (IFN) inhibits T cell responses was studied accordingly. A decrease of Con A-induced proliferation was observed in the presence of increasing amounts of IFN. The reduced proliferative response in such cultures was found to be due to an accumulation of cells in the G0/G1 phase of the cell cycle. Furthermore, the results show that IFN did not inhibit the early events in T cell triggering, because the acquisition of responsiveness of resting T cells to TCGF was unaltered in the presence of IFN, nor did it interfere with production of TCGF. In contrast, IFN was found to interfere with the TCGF-dependent T cell blast growth. Cytofluorometric analysis of the proliferative phase revealed that IFN exerts its effect on T cells, which have entered the proliferative cycle, by a postmitotic accumulation in G0/G1, thus reducing the proliferating population. The results demonstrate that IFN primarily affects the later phase of proliferative activity after T cell triggering, leaving the helper cell functions untouched.     

Class I MHC antigens in the generation and expression of promiscuous cytotoxic cell function

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 induction of the human T-cell growth factor receptor precedes the production of RNA and occurs in the presence of inhibitors of RNA synthesis

The receptor for T-cell growth factor (TCGF) is an activation antigen that is present in low amounts on a small fraction of resting T lymphocytes. The TCGF receptor on human T cells can be detected with the anti-Tac monoclonal antibody within 7-12 h of stimulating the cells with phytohemagglutinin (PHA). In the current studies, we examined human lymphocytes cultured alone, with PHA, or with PHA plus sufficient actinomycin-D to inhibit RNA synthesis. After varying intervals, aliquots of the lymphocytes were stained with acridine orange (AO) or pyronin-Y(PY) to measure RNA and/or with anti-Tac plus FITC goat anti-mouse Ig. Tac expression began to increase after 6-8 h incubation with PHA, whereas increases in PY or AO staining were not detected until 12 h or later. Furthermore, the initial increase in Tac expression was not affected by sufficient actinomycin-D to block all detectable nucleic acid synthesis. Therefore, it appears that the initial expression of TCGF receptors detected after lymphocyte activation does not require de novo production of RNA.     

Initial characterization of sheep T-cell growth factor and its species-restricted activity on human, rat, and mouse cells

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