Isolation and partial characterization of human T-cell growth factor

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).     

Effects of cytokines on in vitro growth of tumor-infiltrating lymphocytes obtained from human primary and metastatic liver tumors

Summary Tumor-infiltrating lymphocytes (TIL) were isolated from 22 human primary and metastatic liver tumors, and expanded in vitro in the presence of either interleukin-2 (IL-2, 100 U/ml) plus tumor necrosis factor (TNF, 1000 U/ml), IL-2 (1000 U/ml) plus IL-4 (1000 U/ml) or IL-2 (1000 U/ml) alone. TIL proliferated in culture in 20/22 cases. Among different cytokine combinations, TNF and IL-2 were most effective in promoting the outgrowth of CD3⁺ CD8⁺ T lymphocytes (mean ± SEM: 90%±5) in the cultures of TIL from primary liver tumors. Cytotoxicity against autologous tumor cells was demonstrated in all early cultures of TIL from primary liver cancers in the presence of IL-2 plus TNF. In contrast, cultures of TIL derived from colon cancer metastatic to liver had significantly lower levels of autotumor cytotoxicity and proportions of CD3⁺ CD8⁺ cells (40%±13) than those of TIL from primary liver tumors. The addition on day 0 of interferons ( or ) to TIL cultured in the presence of TNF and IL-2, significantly augmented cytotoxicity against autologous tumor. In contrast, incubation of TIL in the presence of IL-4 and IL-2 did not result in increased autotumor responses in the cultures of TIL from primary liver tumors. The expansion (-fold) of TIL (day 30) cultured in the presence of IL-2 alone compared to that in the presence of TNF and IL-2 was significantly greater for hepatocellular carcinoma (median, 280 vs 260) than for autologous peripheral blood lymphocytes (36 vs 27), cholangiocarcinoma (42 vs 51) or TIL from metastatic colon cancer (39 vs 30). Outgrowth of TIL in IL-2 plus TNF offers an opportunity for in vitro enrichment in cells with autotumor cytotoxicity in primary liver tumors. However, this cytokine combination was unable to promote and sustain growth of autotumor effectors from TIL in metastatic liver cancer.Supported by ACS grants IM27 077 and IM588 A (TLW) and Organ Transplant Program Project 1P01-CA-4744501 AZ     

Cytochalasins distinguish by their action resting human T lymphocytes from activated T-cell blasts

In the majority of resting human peripheral T lymphocytes obtained from separate individuals cytochalasin B (CB) and D (CD) cause a disappearance of microvilli and induce a rapid formation of prominent sac and bleb-like projections with a length of 1–10 μm randomly distributed over the cell surface. During mitogen stimulation the cells lose the tendency to develop such projections when subsequently exposed to CB and CD. By contrast, in activated T lymphocytes the cytochalasins provoke an asymmetric localization of microvilli including cell surface antigens and actin to a prominent protuberance often separated from the cell body by a constriction. This protuberance is distinct from conventional spontaneous uropods formed by conA-stimulated lymphocytes in relation to contact with other cells and with non-cellular surfaces. The cytochalasins therefore in their action distinguish resting small lymphocytes from activated T-cell blasts.     

Adjuvant properties of transfer factor preparations from human tonsillar lymphocytes]

Low molecular components (Mol. m below 10 000 dalton) of the extract prepared from the human tonsils lymphocytes produced an adjuvant effect on th formation of tuberculin sensitivity in guinea pigs immunized with BCG. This interspecies adjuvant effect was proportional to the dose of the "transfer-factor" preparations administered, depended on the method of their preparation, was expressed in administration of the preparations simultaneously with the immunization, before it or after the tuberculin tests, and under definite conditions was replaced by the immunodepressive effect.     

Type I and II insulin-like growth factor receptors on human phytohemagglutinin-activated T lymphocytes

Human T cells activated with mitogens, antigens, or antibodies to the T-cell receptor complex acquire a cascade of new receptors, including the receptors for interleukin-2, transferrin, and insulin. We investigated whether receptors for insulin-like growth factors (IGF) also were expressed on activated T cells. Based on competitive binding studies, immunoprecipitation of labeled cell surface receptors and blocking of radiolabeled peptide binding by a specific monoclonal antibody (alpha IR-3) to the type I IGF receptor, as well as affinity crosslinking of radiolabeled peptides to their receptors, we concluded that both type I and type II IGF receptors are expressed on activated T cells. A specific binding site for IGF-II also was observed on the type I IGF receptor which was not inhibited by alpha IR-3. Receptors for IGF were more numerous on activated T cells than on resting T cells, and their peak expression appeared by the peak of DNA synthesis. Thus, human activated T cells were shown to express both type I and II IGF receptors which could potentially play a role in the regulation of T-cell proliferation, differentiation, and function.     

Proliferation of human malignant lymphocytes induced by anti-IgM independent of B cell growth factor

Human malignant B lymphocytes were identified that proliferate in response to small doses of anti-immunoglobulin. Proliferation was induced by monoclonal mouse anti-HIgM, polyclonal goat anti-HIgM, and F(ab')2 fragments thereof, in vitro, and was not accompanied by immunoglobulin secretion. Proliferation was found to be unaffected by T cell depletion and was not enhanced by supplementation with B cell growth factor. Culture fluids from unstimulated malignant lymphocytes as well as from malignant lymphocytes stimulated with anti-HIgM contained no measurable B cell growth factor activity. Thus, proliferation of these malignant lymphocytes was not dependent on the presence of T lymphocytes and was independent of the presence of B cell growth factor. These results imply that B cell stimulatory factors may not be required for proliferation of all human B lymphocytes. Moreover, these results imply that treatment with anti-immunoglobulin reagents may be inappropriate for some B lymphocyte malignancies.     

Mitogenic and mitogenically defective phytohemagglutinin isolectins stimulate T-cell growth factor (interleukin 2) production and response in fresh and cultured human T lymphocytes

L4-PHA (L4) and E4-PHA (E4) lectins isolated from Phaseolus vulgaris have different mitogenic properties. The mechanisms of the differences in mitogenic behavior were sought in the interaction of lectin, lymphocyte subsets, and T-cell growt factor (TCGF) also known as interleukin 2 (IL-2). TCGF activity in culture supernatants ( L4S ; E4S ) from L4- and E4-stimulated, freshly isolated lymphocytes was assayed as stimulation of DNA synthesis in TCGF-dependent continuous T-cell cultures (CTC). E4S contained less TCGF than did L4S . Addition of partially purified TCGF does not increase the stimulation of fresh lymphocytes by L4 or E4. L4 and E4 equally stimulate both helper (OKT4+) and suppressor (OKT8+) cells. The ability of L4 to further stimulate CTC is slowly lost (15 greater than 30 greater than 45 days). It is concluded that production of TCGF is not rate limiting in E4 and L4 stimulation of lymphocytes. The growth of CTC, which requires the presence of TCGF, remains sensitive to, but not dependent on, L4 for at least 30 days.     

Transforming growth factor beta is an important immunomodulatory protein for human B lymphocytes

The growth and differentiation of B cells to immunoglobulin (Ig)-secreting cells is regulated by a variety of soluble factors. This study presents data that support a role for transforming growth factor (TGF)-beta in this regulatory process. B lymphocytes were shown to have high-affinity receptors for TGF-beta that were increased fivefold to sixfold after in vitro activation. The addition of picogram quantities of TGF-beta to B cell cultures suppressed factor-dependent, interleukin 2 (IL 2) B cell proliferation and markedly suppressed factor-dependent (IL 2 or B cell differentiation factor) B cell Ig secretion. In contrast, the constitutive IgG production by an Epstein Barr virus-transformed B cell line was not modified by the presence of TGF-beta in culture. This cell line was found to lack high-affinity TGF-beta receptors. The degree of inhibition of B cell proliferation observed in in vitro cultures was found to be dependent not only on the concentration of TGF-beta added but also on the concentration of the growth stimulatory substance (IL 2) present. By increasing the IL 2 concentrations in culture, the inhibition of proliferation induced by TGF-beta could be partially overcome. In contrast, the inhibition of Ig secretion induced by TGF-beta could not be overcome by a higher concentration of stimulatory factor, demonstrating that the suppression of B cell differentiation by TGF-beta is not due solely to its effects on proliferation. Furthermore, it was demonstrated that B lymphocytes secrete TGF-beta. Unactivated tonsillar B cells had detectable amounts of TGF-beta mRNA on Northern blot analysis, and B cell activation with Staphylococcus aureus Cowan (SAC) resulted in a twofold to threefold increase in TGF-beta mRNA. Supernatants conditioned by unactivated B cells had small amounts of TGF-beta, SAC activation of the B cells resulted in a sixfold to sevenfold increase in the amount of TGF-beta present in the supernatants. Thus, B lymphocytes synthesize and secrete TGF-beta and express receptors for TGF-beta. The addition of exogenous TGF-beta to cultures of stimulated B cells inhibits subsequent proliferation and Ig secretion. TGF-beta may function as an autocrine growth inhibitor that limits B lymphocyte proliferation and ultimate differentiation.     

Characterization of structural domains of the human epidermal growth factor receptor obtained by partial proteolysis

Partial cleavage with trypsin has been used to study the structure of the epidermal growth factor (EGF) receptor purified from human carcinoma cells. Following affinity labeling of the receptor with 125I-EGF or the ATP analogue 5'-p-fluorosulfonyl benzoyl[14C]adenosine, metabolic labeling with [35S]methionine, [3H]glucosamine, or [32P]orthophosphate, or in vitro autophosphorylation with [gamma-32P]ATP, tryptic cleavage defines the following three regions of the 180-kDa receptor protein: 1) a 125-kDa trypsin-resistant domain which contains sites of glycosylation, EGF binding, and an EGF-specific threonine phosphorylation site; 2) an adjacent 40-kDa fragment which contains serine and threonine phosphorylation sites and is further cleaved to a 30-kDa trypsin-resistant domain; and 3) a terminal 15-kDa portion of the receptor that contains the sites of tyrosine phosphorylation and is degraded to small fragments in the presence of trypsin. Both the 125- and 40-kDa regions of the EGF receptor appear to be required for receptor-associated protein kinase activity since separation of these regions by tryptic cleavage abolishes this activity, and both regions are specifically labeled with an ATP affinity analogue, suggesting that both are involved in ATP binding. Additional 63- and 48-kDa phosphorylated fragments are generated upon trypsin treatment of EGF receptor from EGF-treated cells. The potential usefulness of partial tryptic cleavage in studying the EGF receptor and the possible biological function of the 30-kDa trypsin-resistant fragment of the receptor are discussed.     

Functional characterization of human hepatocyte growth factor mutants obtained by deletion of structural domains.

Human hepatocyte growth factor (hHGF) consists of characteristic structural domains. In this study, we prepared mutant proteins lacking each of these domains and examined their biological activities for stimulation of hepatocyte DNA synthesis, inhibition of Meth A cell growth, and induction of MDCK cell dissociation. We also examined their interactions with the c-met/HGF receptor by competition analysis and by analysis of levels of tyrosine phosphorylation. The mutant proteins lacking the N-terminal, the first kringle, or the second kringle domain were not biologically effective and could not compete with hHGF bound to the c-met/HGF receptor. The results indicate that these domains are necessary for the biological activities of hHGF mediated by binding to the c-met/HGF receptor. The mutant proteins lacking the third or fourth kringle domain moderately retained biological activities and the receptor binding. The relative levels of the tyrosine phosphorylation of the c-met/HGF receptor by these mutant proteins correlated well with the relative potencies of the biological activities when compared with that of the wild-type hHGF. The mutant protein lacking the light chain was not effective in the biological activities and tyrosine phosphorylation of the c-met/HGF receptor, but competed with hHGF bound to the c-met/HGF receptor. These results suggest that the heavy chain plays an important role in the interaction of hHGF with the c-met/HGF receptor and that the light chain is further required for the tyrosine phosphorylation of the c-met/HGF receptor.     

Signal requirements for lymphocyte activation: Role of a T-cell growth factor produced by guinea pig peritoneal exudate lymphocytes

Peritoneal exudate lymphocytes (PEL) from immunized guinea pigs, when pulsed with antigen, rapidly release a T-cell stimulatory factor (TSF). TSF nonspecifically enhances the proliferation of purified guinea pig T cells in the presence of another signal such as PMA, PHA, or Con A. On a per cell basis, antigen-pulsed PEL produce about 14 times more activity than similarly stimulated lymph node lymphocytes.Several lines of evidence support the view that TSF is the guinea pig equivalent of TCGF (IL-2). TSF containing supernatants have IL-2 activity when assayed on the IL-2-dependent CT6 cell line. When TSF containing supernatants were absorbed with CT6 cells, there was a significant decrease of both TSF activity as assessed on guinea pig T cells as well as IL-2 activity as assessed by the CT6 assay. Additionally, partially purified human and mouse IL-2 have TSF activity, while the macrophage product, IL-1, has no TSF activity. After chromatography on a S-200 column, TSF activity and IL-2 activity coelute at an apparent molecular weight of 19,000.     

Isolation of human nerve growth factor from placental tissue

Nerve growth factor (NGF) has been isolated from human placental tissue. Using the chicken embryo dorsal root ganglia assay, we determined levels of NGF activity for the amnion, placental cotyledons, cord serum, fetal serum, and maternal serum. The highest levels of NGF activity were measured in placental cotyledons. After homogenization and centrifugation of the placental cotyledons, the supernatant was sequentially chromatographed, at neutral pH, on Sephadex G-100, DEAE-11, and Sephadex G-150. A high-molecular-weight protein fraction (150,000), which contained all the biological activity, was isolated in this fashion. Analytical isoelectric focusing of this fraction revealed a basic protein component (pI 9.5) of the high-molecular-weight species. Assays for NGF activity of all protein components separated by analytical isoelectric focusing showed that NGF activity was associated only with the basic protein component. Correspondingly, preparative isoelectric focusing of the high-molecular-weight species yielded a basic protein with very high biological activity (1–3 ng per biological unit) that was immunochemically active against rabbit IgG made against mouse -NGF.To whom reprint requests should be addressed.     

Purification of a skeletal growth factor from human bone

A skeletal growth factor was isolated and purified from demineralized human bone matrix. A dose of 6 micrograms/mL of the purified factor significantly increased the proliferation rate of embryonic chick bone cells in serum-free culture (292% of controls, p less than 0.0001) but had no effect on embryonic chick skin cells plated at the same initial density. The factor is sensitive to inactivation by trypsin and urea, but not by collagenase, 20% butanol, or 1% mercaptoethanol. It is also resistant to inactivation by heat (stable for 15 min at 75 degrees C) and extremes of pH (stable for 30 min at 4 degrees C from pH 2.5 to 10.0). Purification of the active factor by selective heat and acid precipitations, molecular sieve column chromatography, and preparative polyacrylamide gel electrophoresis provided a material that was homogeneous by the criteria of high-pressure liquid chromatography, polyacrylamide gel electrophoresis, and isoelectric focusing. The apparent molecular weight is 83 000. The purified factor increases bone cell proliferation at doses comparable to other mitogens: 0.3 microgram/mL (3.6 nM) significantly increases DNA synthesis to 231% of controls (p less than 0.001). The purified factor was also active on cultured embryonic chick bones, enhancing the growth rate of tibiae and femurs, as measured by increased dry weight (185% of controls, p less than 0.025) and [3H]proline incorporation (164% of control, p less than 0.001), respectively.