Effector activity of OKT4+ and OKT8+ T-cell subsets in lectin-dependent cell-mediated cytotoxicity against adherent HEp-2 cells

The role of OKT4+ and OKT8+ T-cell subsets was studied in lectin-dependent cell-mediated cytotoxicity (LDCC) against adherent HEp-2 human epipharynx carcinoma target cells. LDCC was evaluated by detachment from the monolayer of [3H]thymidine prelabeled HEp-2 cells in a 24-hr assay with a concanavalin A (Con A) dose of 25 microgram/ml at effector:target cell ratios of 5:1, 25:1, and 50:1. Under these conditions but without Con A considerable natural cell-mediated cytotoxicity (NCMC) was not elicited; however, the cytotoxicity was significantly augmented in the presence of Con A (=LDCC) by sheep erythrocyte rosette-forming T lymphocytes and by both OKT4+ and OKT8+ T-cell fractions. LDCC activity by isolated OKT8+ T cells was superior to that by OKT4+ T cells and unfractionated T lymphocytes. By contrast, addition of either OKT4+ or OKT8+ T cells together with unfractionated T lymphocytes, or OKT4+ and OKT8+ T cells mixed at ratios of 1:1, 1:2, and 2:1, to target cells did not result in major differences in comparison of LDCC activities by these mixed effector cell populations with each other or with that by unfractionated T lymphocytes. Parallel studies were carried out to determine the effect of OKT4+ and OKT8+ T-cell subsets on the Con A-induced proliferation of peripheral blood mononuclear cells (PBMC). While OKT8+ T cells inhibited the mitogenic response to Con A, OKT4+ T lymphocytes had no major effect. A higher responsiveness of the OKT8+ to OKT4+ T-cell subset in LDCC to HEp-2 targets and in Con A-induced lymphocyte proliferation is suggested.     

Inability of immunocompetent thymocytes to produce T-cell growth factor under adenosine deaminase deficiency conditions

Five density-defined subpopulations of rat thymocytes were separated by isopycnic centrifugation on a discontinuous density Ficoll gradient and compared with respect to their response to Con A stimulation under normal and adenosine deaminase (ADA) deficiency conditions. This study shows that (a) immunocompetent (low-density) thymocytes and splenic T lymphocytes produce T-cell growth factor (TCGF) in response to mitogenic stimulation in normal culture conditions, but are unable to synthesize effective TCGF in the presence of an adenosine deaminase inhibitor and excess substrate (ADA deficiency conditions), and (b) high-density immunoincompetent thymocytes proliferate and differentiate into mature T cells in response to Con A if effective exogenous TCGF is added to the culture medium but are unable to do so under ADA deficiency conditions.     

Glucocorticoid receptors in subpopulations of human lymphocytes defined by monoclonal antibodies

Glucocorticoid receptors (GR) were investigated in subpopulations of lymphocytes identified by monoclonal antibodies. Purified T (OKT3+) and non-T lymphocyte subpopulations were isolated from human peripheral blood using Degalan bead columns coated with rabbit anti-human IgG. Purified subpopulations of OKT4+ and OKT8+ lymphocytes were obtained by coating the nonadherent population (T cells) from the first column with OKT4+ or OKT8+ and pouring it into a second Degalan column, coated with goat anti-mouse IgG. GR content and affinity were analyzed by a whole cell assay with [3H]dexamethasone as tracer. The numbers of GR in lymphocyte subpopulations (OKT3+ cells, non-T cells, OKT4+, and OKT8+ cells) were nearly equal. It is concluded that the differential effects of glucocorticoids on the circulatory kinetics of OKT4+ and OKT8+ cells probably are not related to differences in glucocorticoid receptors of these T-cell subpopulations.     

Production of alpha-lymphotoxin by human T-cell subsets

Human T cells were isolated from peripheral blood lymphocytes (PBL) and sensitized to allogeneic PBL in a one-way mixed-lymphocyte culture. These sensitized T cells were fractionated on the basis of their possession of Fc receptors for IgG (TG+) or IgM (TM+), or the absence of both IgG and IgM receptors (TG-M-). When restimulated with alloantigen of the same derivation, TG+, TM+, and TG-M- cells yielded almost equal amounts of cytotoxin. Anti-alpha-lymphotoxin serum neutralized most of this cytotoxic activity indicating that alpha-lymphotoxin (alpha-LT) constituted most of this activity. Although TG-M- cells function as effectors in allogeneic cytotoxicity, TG+ cells lyse IgG-coated targets in an antibody-dependent cell-mediated cytotoxic (ADCC) reaction, which has been shown to be mediated in part by alpha-LT. Whether TM+ cells can be cytotoxic is not clear. In addition, freshly isolated human T-cell subsets were stimulated with phytohemagglutinin-P (PHA-P). After PHA stimulation, TG+, TM+, and TG-M- cells produced similar amounts of soluble cytotoxin, which was largely neutralized by anti-alpha-LT. The TG+ cells incorporated less thymidine than the TM+ or TG-M- cells. Likewise, OKT4+ and OKT8+ subsets, isolated with the aid of monoclonal OKT8 or OKT4 antibody and complement, yielded lymphotoxin after stimulation with PHA. It is shown that all T-cell subsets, as defined here, can produce lymphotoxin. Furthermore, depending on the assay system, cytotoxicity can be clearly demonstrated in all of these subsets, except in TM+ cells, where positive and negative results have been reported.     

Differentiation antigens in human T-cell activation: Evidence that anti-VH antibodies react with a membrane structure on human T Lymphocytes distinct from the antigens detected by monoclonal antibodies of the OKT and Leu series

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 V_H 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 V_H 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, OKIal, 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 OKIal 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.     

Release of hemopoietic factors by normal human T cell lines with either suppressor or helper activity

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.     

Study of DTH and resistance in Mycobacterium lepraemurium infection using a T-cell line isolated from mice infected with Mycobacterium bovis (BCG)

A T-cell line of mixed phenotype (60% L3T4+, 40% Lyt-2+) was isolated from mice infected with Mycobacterium bovis (BCG). This line responded to M. lepraemurium and BCG but not to M. leprae and produced TCGF spontaneously. It also produced factors which stimulated macrophages to secrete hydrogen peroxide and superoxide anion. In vivo studies showed that only L3T4+ cells were required to transfer DTH responses and that Lyt-2+ cells suppressed this response. Both L3T4+ and Lyt-2+ cells were required to inhibit M. lepraemurium multiplication in vivo.     

Impaired T- and B-cell functions in patients with Hodgkin's disease

Summary The mitogenic response of T-cell subsets, the production of interleukin-1 (Il-1) and interleukin-2 (Il-2) and in vitro immunoglobulin production was investigated in patients with Hodgkin's disease (HD). The mitogenic response of mononuclear cells (MNC) and the OKT4⁺ and OKT8⁺ subsets was greatly reduced in advanced disease stages and could only partially be restored with exogeneous Il-2. In untreated patients with HD — except those with highly advanced disease — the OKT4⁺ lymphocytes showed normal response to phytohemagglutinin in contrast to the MNC suggesting inhibiting agents or cells within the MNC. These findings corresponded to reduced Il-2 synthesis of MNC, whereas isolated OKT4⁺ — cells produced normal or elevated amounts of Il-2. MNC or monocytes produced normal or even higher amounts of lipopolysaccharide-induced Il-1 than controls. The results do not confirm a defect in this component of the interleukin system in HD. The immunological impairment was not limited to the T-cell system but involved B-cell activation and differentiation as well. The pokeweed mitogen-induced IgM, IgG and IgG production was highly suppressed in untreated HD, whereas the MNC of previously treated patients produced subnormal amounts of immunoglobulin in vitro. It is not yet clear whether this defect is T-cell-mediated or primarily a B-cell deficiency.     

Taenia taeniaeformis: cellular reconstruction of athymic mice and role of L3T4+ helper T lymphocytes in the early infection

The role of T helper lymphocytes (L3T4+) in the early response to Taenia taeniaeformis metacestodes was investigated. Athymic BALB/c-nu/nu mice (susceptible) were inoculated intraperitoneally with the following cell populations from congenic BALB/c-nu+ + mice (resistant): (a) whole spleen single cells, (b) thymus single cell suspensions, or (c) spleen cells pretreated with anti-L3T4 monoclonal antibody before the injection. The mice were given 3 weekly injections of cells and then infected orally with 300 eggs 7 days after the last injection. Cryostat sections of the liver from the infected mice were examined at 6 days postinfection (PI) for parasite viability, the numbers of eosinophils, and L3T4+ T lymphocytes present within 100 micron of the parasite and for the presence of biotin in hepatocytes (involved in biosynthesis of fatty acids) around the parasite. The success of the cellular reconstitution of athymic mice with the lymphoid cells was measured by a T-cell mitogenic assay with concanavalin A (ConA). The cellular reconstitution of athymic mice with a mixture of lymphoid cells from the spleen and thymus of BALB/c-nu/ + mice resulted in both parasite death and eosinophil infiltration. Reconstitution with mature splenic cells alone resulted in a greater parasite killing and eosinophil infiltration as compared to reconstitution with thymic cells. The better reconstitution with splenic cells was reflected in a greater mitogenic response to ConA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Separation and biological properties of Phaseolus vulgaris isolectins

Two isolectins (L₄E₀-PHA and L₀E₄-PHA) from red kidney beans (Phaseolus vulgaris) were isolated by affinity chromatography on immobilized thyroglobulin and by chromatography on hydroxyapatite. The L₄E₀-PHA siolectin was not retarded on the affinity column and was eluted from the hydroxyapatite column with a 0.1 M Phosphate buffer. This isolectin had no erythroagglutinating activity but had high lymphoagglutinating and lymphocyte stimulating activities. The L₀E₄-PHA isolectin was adsorbed on the affinity column and was eluted from the hydroxyapatite column with a 0.25 M Phosphate buffer. This isolectin has a high erythroagglutinating activity, a very low lymphoagglutinating activity and no lymphocyte stimulating activity. These two isolectins are shown however to be closely related with respect to their oligomeric structure and reactivity towards anti-PHA antibodies. The lack of mitogenic activity of the L₀E₄-PHA isolectin suggests that in the other isolectins, the E monomer is not responsible for their mitogenic activity and that the membrane glycoproteins, which contain the E monomer — specific oligosaccharide, are not involved in the process inducing mitosis.     

Mitogen activation induces the enhanced synthesis of two heat-shock proteins in human lymphocytes

We have used mitogenic lectin (PHA) and a monoclonal antibody (OKT3) to stimulate human peripheral blood (G0) lymphocytes, in the presence of monocytes, and have found two major preferentially synthesized proteins, 73 and 95 kD, which are induced by the mitogens. The elevated synthesis of both proteins begins approximately 4-6 h after mitogen addition (early to mid G0/G1) before entry into first S phase. Maximum synthesis of both proteins is reached by 12 h after mitogen addition when P95 synthesis represents approximately 4%, and P73 approximately 2%, of the total protein synthesis, compared with less than 0.5% for each protein in cells cultured without mitogen. Thus, the proteins appear to be major components of activated cells. We find that both P73 and P95 are induced by heat stress as well as mitogenic stimulation. The induction of the proteins is not affected by either deleting glucose from the culture media or, alternatively, by supplementing it. Using polyclonal antibodies prepared to each of the proteins isolated from mitogen activated cells and monoclonal antibodies that were raised to heat shock proteins, we are able to show that P95 is electrophoretically and immunologically identical to the HSP 90 induced by heat stress. P73 is one of the 70 kD HSPs, (termed HSC 70; Pelham, H. R. B. 1986. Cell. 46: 959-961), but is different from the most strongly heat inducible form of HSP 70 (72 kD). The distribution of both proteins in subcellular fractions of mitogen activated lymphocytes is similar to the reported localization of the respective HSP's in other cell types. The results suggest that HSP 90 and HSC 70 may have functional roles in stress response and growth processes of human lymphocytes.     

Determination of the T-cell subset producing gamma-interferon in tuberculous pleural effusion

The percentage of cells of different T-cell subsets and their functions in tuberculous pleural effusion were investigated. The percentage of OKT4-positive cells was 65 +/- 2% (mean +/- SEM, n = 8) and that of OKT8-positive cells was 19 +/- 3% (n = 8). Pleural T lymphocytes of patients with tuberculous pleurisy responded well to stimulation with purified protein derivative of tuberculin, and deoxyribonucleic acid synthesis was observed along with gamma interferon (IFN-gamma) production. When pleural T lymphocytes of patients with tuberculous pleurisy were treated with OKT4 monoclonal antibody and complement, a significant decrease in IFN-gamma production was observed in all eight patients (P less than 0.005), whereas no definite decrease in IFN-gamma production was found after treatment with OKT8 monoclonal antibody and complement. These results suggest that at least the OKT4+/OKT8- T-cell subset is responsible for the antigen-specific IFN-gamma production in pleural T lymphocytes of patients with tuberculous pleurisy.     

Phytohemagglutinin isolectin stimulation of human lympocytes cultured in serum free medium.

The ability of the red kidney bean (Phaseolus vulgaris) Phytohemagglutinin (PHA) isolectins L4 and E4 to transform human lymphocytes cultured in serum free or serum supplemented medium was studied. Previous similar studies done in fetal bovine serum (FBS) supplemented medium have shown L4 to be 30–60 times more potent a mitogen than E4. In serum free conditions, this difference was much less, L4 being only 3–9 times more potent than E4. In serum free medium, optimal mitogenic concentrations of L4 and E4 were 1.1.–3.3 ug/culture and 3.3–10 ug/culture respectively as compared to 3.3–10 ug/culture and 90–270 ug/culture for L4 and E4, respectively, in FBS suplemented medium. L4 stimulated lymphocytes in serum containing medium transform more rapidly than do L4 treated cells cultured in serum free conditions. Fetuin added to serum free cultures of lymphocytes more effectively inhibited transformation induced by E4 than by L4. Although the binding of 1251 E4 and L4 to lymphocytes was greatly reduced by the addition of FBS to the medium, the reduction in E4 binding was much greater than that in L4 binding. Neither L4 or E4 caused the death of lymphocytes cultured in serum free or FBS supplemented medium. These results confirm the previously described difference in mitogenic potential between L4 and E4. However, the results of earlier studies done in FBS supplemented medium include several artifacts related to the differential interaction of PHA isolections with fetuin and other serum glycoproteins.     

Characterization of a long-term T-helper-cell line which produces IL-2 and induces immunoglobulin secretion

Six OKT4+ human T-cell lines that require continuing PHA stimulation and TCGF for continuous growth were established. The cells from all six of these T-cell lines became smaller in size and lost the cell surface Ia antigen when they were grown in phytohemagglutinin (PHA)-depleted growth factor. These cells were unable to survive in the absence of PHA even if exogenous factor was present in great abundance. One of the cell lines (FL) was capable of providing helper functions. In the presence of PHA and phorbol myristate acetate, FL cells produced a growth factor, tentatively identified as Interleukin 2 (IL-2) by its ability to promote the proliferation of an IL-2-dependent murine T-cell line. Moreover, when FL cells were cocultured with B cells, pokeweed mitogen-induced immunoglobulin production was enhanced.     

Effect of monoclonal antibodies (MoAb) to class I and class II HLA antigens on lectin- and MoAb OKT3-induced lymphocyte proliferation

We have examined the effect of several monoclonal antibodies (MoAb) to monomorphic determinants of class II HLA antigens, and MoAb to monomorphic determinants of class I HLA antigens and to beta-2-microglobulin (beta 2-mu) on lectin- and MoAb OKT3-induced proliferation of human peripheral blood mononuclear cells (PBMNC) and cultured T cells (CTC). Some, but not all, anti-class II HLA MoAb inhibited the proliferative response of PBMNC to MoAb OKT3 and pokeweed mitogen (PWM). The degree of inhibitory effect varied considerably. This effect was not limited to anti-class II HLA MoAb since anti-class I HLA MoAb and anti-beta 2-mu MoAb also inhibited MoAb OKT3- or PWM-induced proliferative responses. In contrast, the response of PBMNC to phytohemagglutinin (PHA) and concanavalin A (Con A) was not blocked by any anti-class II HLA MoAb. However, some anti-class II HLA MoAb also inhibited the proliferative response of CTC plus allogeneic peripheral blood adherent accessory cells (AC) to PHA or Con A as well as to MoAb OKT3 or PWM. This may be attributable to the substantially greater class II HLA antigen expression by CTC than by fresh lymphocytes. Pretreatment of either CTC or AC with anti-class II HLA MoAb inhibited OKT3-induced proliferation. In contrast, pretreatment of CTC, but not AC, with anti-class I HLA MoAb inhibited the proliferative response of CTC to OKT3. Pretreatment of CTC with anti-class I HLA MoAb inhibited PHA-, Con A and PWM-induced proliferation, to a greater degree than the anti-class II HLA MoAb. It appears as if lymphocyte activation by different mitogens exhibits variable requirements for the presence of cells expressing major histocompatibility determinants. Binding of Ab to membrane markers may interfere with lymphocyte-AC cooperation, perhaps by inhibiting binding of mitogens to their receptors or by interfering with lymphocyte and AC function. We also have examined the role of class II HLA antigens on CTC by depleting class II HLA-positive cells. As expected, elimination of class II HLA-positive AC with anti-class II HLA MoAb plus complement caused a decrease in proliferation of CTC in response to all the mitogens tested. In contrast, elimination of class II HLA-positive CTC was shown to clearly increase proliferation of CTC, perhaps because this may deplete class II HLA-positive suppressor cells.     

Antitumor and therapeutic effects of spleen cells from tumor-bearing mice cultured with T cell growth factor and soluble tumor extract

Summary Spleen cells of BALB/c mice that had been inoculated with syngeneic plasmacytoma MOPC 104E were cultured for 11 days in T-cell growth factor (TCGF) and ultrasonicated tumor extract (USE). Cultured lymphocytes (MOPC-CL) possessed three-fold more lytic units than normal spleen cells cultured in TCGF without USE (N-CL). Moreover, the in vivo neutralization assay suggested that MOPC-CL were composed of at least two populations, one possessing tumor-specific and the other nonspecific antitumor activity. When 2×10⁷ of MOPC-CL were administered IP to mice that had been inoculated IP with 10⁵ MOPC 104E cells 5 days previously marginal prolongation of survival was observed. This effect was not augmented by the single injection of a larger number (5×10⁷) of CL, but was augmented by the repeated daily administration for 4 days (from day 5 to day 8 after the inoculation) of the same total number (5×10⁷) of CL. In addition, IP injection of the streptococcal preparation OK432 before the transfer of CL significantly enhanced the therapeutic efficacy, and resulted in a cure rate of 20%. The mechanism of this combined effect appears to involve the effect of OK432 on interleukin 2 (IL-2) regulation systems in vivo. Our culture system with TCGF and USE and our therapy system with OK432 and CL allow the clinical application of adoptive immunotherapy for the many types of solid cancers.     

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.     

The high specificities of Phaseolus vulgaris erythro- and leukoagglutinating lectins for bisecting GlcNAc or beta 1-6-linked branch structures, respectively, are attributable to loop B

Despite very similar tertiary structures based upon a common framework, legume lectins exhibit an amazing variety of sugar binding specificities. While most of these lectins recognize rather discrete sugar linkages, Phaseolus vulgaris erythroagglutinating and leukoagglutinating lectins (E(4)- and L(4)-PHA) are unique in recognizing larger structures. E(4)- and L(4)-PHA are known to recognize complex type N-glycans containing bisecting GlcNAc or a beta1,6-linked branch, respectively. However, the detailed mechanisms of molecular recognition are poorly understood. In order to dissect the contributions of different portions of each lectin, we carried out region-swapping mutagenesis between E(4)- and L(4)-PHA. We prepared six chimeric lectins by exchanging different combinations of loop B and the central portion of loop C, two of four loops thought to be important for the recognition of monosaccharides (Sharma, V., and Surolia, A. (1997) J. Mol. Biol. 267, 433-445). The chimeric lectins' sugar binding activities were evaluated quantitatively by surface plasmon resonance. These comparisons indicate that the high specificities of E(4)- and L(4)-PHA toward bisecting GlcNAc and beta1,6-linked branch structures are almost solely attributable to loop B. The contribution of the central portion of loop C to the recognition of those structural motifs was found to be negligible. Instead, it modulates affinity toward LacNAc residues present at the nonreducing terminus. Moreover, some of the chimeric lectins prepared in this study showed even higher specificities/affinities than native E(4)- and L(4)-PHA toward complex sugar chains containing either a bisecting GlcNAc residue or a beta1,6-linked branch.     

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.