The thymic preparations thymosin fraction 5 (TF5) and synthetic thymosin alpha 1 (T alpha 1) were examined for their ability to enhance growth factor production by human peripheral blood mononuclear cells (PBMC). The results showed that both TF5 and T alpha 1 were capable of enhancing the production of a B cell growth factor (BCGF-12kD) and T cell growth factor (TCGF; IL-2). Enhancement by T alpha 1 could be obtained at 100-200-fold lower concentrations than that seen with TF5. In contrast, no enhancement of growth factor production was obtained with control preparations of non-thymic tissue extracts at any concentrations used. It was observed that stimulation of BCGF-12kD and IL-2 was most significantly obtained when the PBMC were activated with lectin. Furthermore, no direct effect of thymic hormones on test B and T cells was observed. These observations provide the first direct evidence that production of B cell growth factors can be enhanced by thymic hormones. In addition, these studies suggest that thymic hormones may regulate B cell responses by acting on mature activated T lymphocytes.

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.

The role of clinical status and chemotherapeutic intervention on native and inducible natural killer cell (NK) activity in breast cancer was ascertained by determining the K562 cytotoxicity capacity of peripheral blood lymphocytes. The level of NK activity in breast cancer patients receiving chemotherapy (n = 62) was significantly lower than that observed in patients currently receiving no treatment (n = 56) (at effector: target [E:T] ratios of 20:1, 10:1, and 5:1, 23.8%, 17.9%, and 12.1% versus 34.9%, 25.6%, and 15.9%, respectively; P less than 0.01, two-way analysis of variance). The absolute level of NK activity in peripheral blood of cancer patients on therapy was further reduced when compared with untreated patients and healthy controls when reductions in lymphocyte counts concomitant with chemotherapeutic intervention were included in calculations of NK activity. T-cell growth factor (TCGF) increased NK activity in all breast cancer patients and healthy controls with maximal stimulation of basal activity at a concentration of 10% (volume/volume [v/v]) TCGF. The percent stimulation of basal NK activity by TCGF was significantly greater in patients receiving chemotherapy (26.4%, 24.3%, and 19.0% at an E:T of 20:1, 10:1, and 5:1, respectively; n = 23) than in untreated patients (16.6%, 18.5%, and 18.9%; n = 21) and healthy controls (23.5, 18.6, and 8.1; n = 8) (P less than 0.05 and P less than 0.01, respectively, two-way ANOVA). The influence of soluble factors and agents in serum on peripheral blood NK activity was assessed by monitoring the effects of autologous plasma on basal and TCGF-stimulated NK activity. Autologous plasma at concentrations less than or equal to 10% (v/v) enhanced basal NK activity. Levels of inducible NK activity in the presence of either 10% TCGF, 5% plasma, or a combination of both were not significantly different in statistical comparisons of both the effects of inducer and therapeutic modality. At concentrations of plasma greater than 10% (v/v), progressively decreasing NK activities were observed. T-cell growth factor could partially reverse the inhibitions of NK activity by 25% autologous plasma. In 13 experiments, basal NK activity and NK activity in the presence of 10% TCGF, 25% autologous plasma, and a combination of TCGF and plasma were 27.6%, 46.0%, 16.3%, and 28.1%, respectively (E:T = 20:1). This study indicates that NK function is compromised in breast cancer patients receiving cytotoxic drug-therapy. The potential use of TCGF in adjuvant immunotherapy as a modulator of NK function has been demonstrated.(ABSTRACT TRUNCATED AT 400 WORDS)

Colony growth of T lymphocytes in a soft agar culture system, following stimulation with phytohaemagglutinin (PHA), is enhanced by T cell-derived conditioned medium (CM). The putative enhancing factor present in this CM has been termed lymphocyte colony-enhancing factor (LCEF). The purification of LCEF from CM has been complicated by the fact that active CM could be obtained only in the presence of 7-8 mg/ml of serum proteins. In order to circumvent this problem, CM which was shown to be active in the colony formation assay, was produced in the presence of a serum fraction precipitated by 1.6 M ammonium sulphate (AS). The first step of the purification of this CM was fractionation by 1.6 M AS, whereby the active material was fully recovered in the supernatant. The 20-fold purified material was applied on a DEAE-cellulose column. Two peaks of activity were obtained using the colony formation assay. The purification achieved by this second step was 5-10 fold for the first peak, eluting around 0.06 M NaCl, and two- to three-fold for the second peak, eluting around 0.19 M NaCl. Two peaks of activity were also obtained upon hydrophobic chromatography on a phenyl-Sepharose column, with enhancement factors of 5-8 for the first peak and 25-60 for the second peak. These results imply the presence of more than one LCEF molecule in the original CM. None of the LCEF-active fractions contained any T cell-growth factor (TCGF) activity; LCEF seems therefore to be a molecular entity distinct from TCGF.

Many different types of virus particles are able to non-specifically impede the ability of human peripheral blood lymphocytes to respond to mitogenic ro alloantigenic stimuli. This result is not obtained if ultra-purified virus is employed, although virus which has been banded only once through sucrose generally retains inhibitory potential. Ultra-pure virus is relatively unable to bind to cell surfaces, suggesting the importance of physical contact between viruses and cells in order for the observed inhibition to occur. Addition of exogenous T-cell growth factor (TCGF) to cultures containing virus, cells and stimulus causes a dose-dependent reversal of the usual inhibitory effect.

We have investigated the use of T-cell growth factor (TCGF) to isolate and grow, in long-term culture, lymphoid cells with immunologic reactivity directed against syngeneic murine and autologous human tumors. Splenocytes from mice immune to a methylcholanthrene-induced sarcoma were expanded in TCGF, both before and after in vitro mixed lymphocyte-tumor cultures, and expressed high levels of cytotoxicity for fresh syngeneic solid tumor cells. Cloned lines have been isolated with a high level of specific lysis for the immunizing tumor. Similar studies of cytotoxic reactivity to a syngeneic FBL-3 lymphoma have given rise to long-term cytotoxic cell lines growing in TCGF capable of curing mice with disseminated lymphoma in adoptive transfer studies. Exposure to TCGF, of human peripheral lymphoid cells from cancer-bearing patients, results in the development of cytotoxicity to autologous fresh tumor. We have used clonal analysis by limiting dilution techniques to isolate individual cloned cells with this autologous antitumor reactivity. Infusion to autologous cytotoxic cells expanded 10,000-fold in TCGF and labeled with 111In into three cancer patients resulted in cell localization initially to the lung and subsequently to the liver and spleen. The application of these techniques for the cloning and expansion of antitumor T-lymphoid cells in TCGF has offered a new approach to adoptive immunotherapy.

Cyclosporin A (CyA) interferes with immune responses and prevents growth factor release by stimulated T cells. However, it is not known whether this is due to an effect on the accessory cells, required for T-cell responses, whether antigen recognition cannot occur, or whether later steps, leading to lymphokine production, are blocked. For this reason, the effect of CyA on homogeneous populations of T tumour cells was investigated. The immunosuppressive compound efficiently prevented T-cell growth factor (TCGF) (interleukin 2) release by stimulated tumour cells. Still, the cells retained the surface antigen T3, known to be involved in T-cell stimulation, after treatment with CyA. Furthermore, CyA failed to affect the inhibition of proliferation, observed in a T-cell tumour in response to stimulation, indicating that the cells had received the stimulatory impetus, TCGF release, induced by treatment with a phorbol ester, was only partly sensitive to inhibition by CyA, demonstrating that CyA will interfere with discrete aspects of the stimulation of a T-cell.

Lymphocytes from athymic and normal mice were tested and compared for cytotoxic activity after in vitro cultures supplemented with concanavalin A or T-cell growth factor (TCGF). Our results indicate that, in addition to cytotoxic T cells, natural killer (NK) activity can be recovered from lymphocytes cultured in the presence of TCGF and that this is not the result of contaminating interferon in the TCGF preparations. The NK nature of the effector cells was established by means of a panel of target cells, including a teratocarcinoma tumour cell, which enabled the distinction of T-cell- and NK-cell-mediated lysis.

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.

T cell growth factor (TCGF) resulting from the incubation of murine splenocytes with concanavalin A (Con A) has been partially purified and separated from Con A. Sequential application of 50-70% saturated ammonium sulfate precipitation and G-100 gel filtration chromatography has resulted in a 300-fold purification of TCGF with a 60% yield. 99.99% of Con A has been removed from the TCGF by these steps. TCGF exists in two molecular weight forms of about 50,000 and 25,000 daltons. TCGF activity is degradable by trypsin digestion and is stable at 56 degrees C for 30 min, but is inactivated by heating at 80 degrees C. Lymphoid cells activated by either Con A or allogeneic in vitro sensitization will grow in TCGF free of Con A but fresh splenocytes will not grow in the absence of Con A, implying a need for prior activation before TCGF sustained growth of T cells can be achieved.

BACKGROUND

Interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15, all T-cell growth factors (TCGFs), utilize the common IL-2 receptor gammac chain as a critical signaling component in their receptor complexes. We have bred IL-2-/- and IL-4-/- double knockout (DKO) mice and showed vigorous islet allograft rejection by DKO hosts. The identity of TCGFs that support the IL-2- and IL-4-independent allograft rejection is unclear.

METHODS

We analyzed IL-9 gene expression in rejecting islet allografts in wild-type and in DKO mice, as well as in human renal transplant biopsy specimens, by reverse transcriptase polymerase chain reaction and compared the expression of IL-9 with that of other TCGFs.

RESULTS

IL-9 gene expression was not detected in rejecting murine islet allografts in either wild-type or DKO recipient mice despite robust expression of other TCGFs, including IL-7 and IL-15. IL-9 transcripts were also not expressed in any of the human renal transplant biopsies obtained 4 to 251 days after transplantation, regardless of the presence or absence of histological evidence of rejection. Despite expression of IL-9 by DKO splenic cells upon in vitro mitogenic stimulation, IL-9 alone was unable to stimulate the proliferation of concanavalin A-activated splenic leukocytes harvested from DKO mice.

CONCLUSIONS

IL-9 is conspicuously absent despite vigorous expression of IL-2, IL-4, IL-7, and IL-15 genes during acute allograft rejection.

The discovery, characterization, and purification of human T-cell growth factor (TCGF) has led to the establishment of continuously growing T-lymphoblast cell lines from normal people and from patients with certain T-cell neoplasias. In contrast to normal T-cells, neoplastic mature T-cells respond directly to TCGF, requiring no prior lectin or antigen in vitro activation. The transformed T-cell lines have phenotypic characteristics consistent with the neoplastic cells of their disease of origin. A novel retrovirus, human T-cell lymphoma-leukemia virus (HTLV), has been isolated from the fresh and cultured cells of two of these patients. Subsequent characterization of this virus has shown that it is not significantly related to any known animal retrovirus, is not an endogenous (genetically transmitted) virus of man, and so far has been associated only with fresh or cultured T-cells from patients with T-cell neoplasia. These results suggest that HTLV infected some mature T-cells of some people and that it might be involved in some neoplasias involving these cells.

The effect of cimetidine, an H-2 receptor antagonist, on activation of PBL from both normal individuals and melanoma patients was studied. It has been shown that cimetidine enhanced, though moderately, the production of TCGF from normal PBL after PHA-P stimulation. In addition, cimetidine significantly augmented TCGF-induced proliferation of normal PBL, as well as proliferation induced by allogeneic cells (MLC) by PPD, Con A, and PHA. In PBL samples where coincubation with cimetidine had limited or no effect, preincubation of PBL with cimetidine prior to the addition of IL-2 and other T cell activators showed a significant enhancement effect. This effect mediated by cimetidine was further demonstrated on PBL from melanoma patients whose T cell responses were initially low. The possibilities are discussed that: (a) cimetidine treatment inactivates suppressor cell activity, thus enhancing T cell mediated responses; or (b) cimetidine may act directly at effector cell level.

To better characterize the inflammatory response that occurs in the nervous system in multiple sclerosis (MS), T-cell receptor (TCR) gene expression was quantified from cerebrospinal fluid (CSF) cells of 21 patients with active disease. Unstimulated CSF cells expressed each of 22 different TCR beta chain variable region (V beta) gene families in proportion to their expression in simultaneously sampled peripheral blood. When CSF cells from individuals with MS were expanded by in vitro culture in T-cell growth factor/interleukin 2 and 4-containing medium (TCGF/IL2/IL4), restricted numbers of V beta genes were expressed. In many subjects, expanded CSF cells expressed predominantly V beta 2. In contrast to CSF, expansion of corresponding peripheral blood mononuclear cells (PBMC) in TCGF/IL2/IL4 resulted in persistent expression of all V beta gene families. Within individuals, different V beta genes were overexpressed by PBMC compared with CSF cells. No effect of the HLA haplotype of the individual on CSF V beta gene expression was observed. Expanded CSF cells retained their capacity to respond to mitogen stimulation, but the proliferative response to myelin basic protein (MBP) was not enhanced. Finally, freshly obtained CSF cells stimulated directly with MBP also expressed a limited number of V beta genes, although these were generally different from patterns observed following stimulation with TCGF/IL2/IL4. Thus, restricted populations of T cells capable of responding to TCGF/IL2/IL4, presumably reflecting in vivo activated cells, are compartmentalized in the nervous system in MS.

The common gammac-chain is an essential signaling component shared by all known T cell growth factor (TCGF) receptors (i.e., IL-2, IL-4, IL-7, IL-9, and IL-15). In the present study, we have studied the effect of gammac-chain blockade on T cell activation and allograft rejection. Treatment of B6AF1 (H-2b/d.k) recipient mice with anti-gammac mAbs induced long-term survival of DBA/2 (H-2d) islet allografts (>150 days, n = 8), whereas control Ab-treated mice rejected the islet allografts within 17 days (n = 6). The state of engraftment induced by the anti-gammac mAbs was remarkably stable, as recipient mice bearing the primary islet allografts accepted a second DBA/2 islet allograft without further immunosuppression and systemic administration of high doses of IL-2Ig fusion protein failed to provoke rejection. Blocking the gammac-chain inhibited T cell proliferation and induced T cell apoptosis by repressing expression of Bcl-2. Our data suggest that one means of inducing T cell apoptosis and stable allograft survival can be achieved via gammac-chain blockade.

Fourteen days' culture of human peripheral blood lymphocytes (PBL) with recombinant interleukin 2 (rIL 2) or T cell growth factor (TCGF) results in the generation of lymphokine-activated killer (LAK) effector cells which have the unique property of lysing natural killer (NK)-resistant human tumor cells, Daudi, as well as NK-sensitive, K562 cells. LAK cells were generated from both normal and gastric cancer patients' PBL. However, LAK cell activities induced by rIL 2 or TCGF decreased with the progress of the tumor growth. In addition, TCGF-induced LAK cell activities were found to be lower than the rIL 2-induced LAK cell activities. Different mechanisms may be involved in the decreases of the rIL 2-induced and TCGF-induced LAK cell activities. This study further demonstrates that the cell types involved are also heterogeneous, as determined by phenotypic characteristics. The LAK-effector cell type was analyzed by two-color flow cytometry. RIL 2-induced LAK cells showed increased proportions of CD4+Leu 8- and Leu 7+CD16-, and a decreased proportion of CD8+CD11- cells, which are believed to be associated with killer T cell functions. In contrast, TCGF-induced LAK cells revealed significantly increased proportions of CD8+CD11- and CD4+Leu8- cells, and a decreased proportion of Leu 7+CD16- cells. Thus, LAK cells with different surface phenotypes were induced by the cultivations with rIL 2 and with TCGF.

To maximize expression of a eukaryotic gene in Escherichia coli, a series of plasmids were constructed containing various synthetic ribosome-binding sites (RBS). These sites consist of a Shine-Dalgarno (SD) region (with translation stop codons in all three reading frames) positioned at distances 5-9 nucleotides (nt) from the AUG initiator codon of the gene coding for human T-cell growth factor (TCGF or IL-2). The region encompassing the RBS through the TCGF structural gene from each of these plasmids was inserted as a 'cassette' into seven different E. coli expression vectors, and TCGF production was measured. Our results demonstrate a greater than 2000-fold range of TCGF synthesis dependent upon the promoter and the synthetic RBS used. The translational efficiency of the TCGF gene was found to be influenced by the quality of the RBS, which is in part determined by the external sequence context of this site. The synthetic RBS, containing the necessary information for the translation initiation process, readily accessible by restriction sites, should be of general usefulness in obtaining maximum expression of eukaryotic genes in E. coli.

OBJECTIVE

The lysis of chondrocytes, the parenchymal cells of cartilage, by lymphocytes may provide a potent mechanism by which the immune system participates in sustaining joint damage in rheumatoid arthritis (RA). We studied the capability of lymphocytes from healthy individuals and patients with arthritis to lyse chondrocytes.

METHODS

Peripheral blood mononuclear cells (PMBC) were tested for their ability to lyse chondrocytes in a 51Cr-release assay. Enhancement of the chondrolytic activity was determined by preincubating the cells with T cell growth factor (TCGF) or recombinant interleukin-2 (rIL-2) before cytotoxic testing.

RESULTS

PBMC from healthy individuals possessed a low ability to lyse chondrocytes, whereas cells from the synovial fluid of patients with RA displayed higher chondrolytic activity. In RA, modulating factors must come into play because not all synovial fluid sample cells showed high chondrolytic activity and cells from synovial tissue had little or no lytic action on chondrocytes. Chondrolytic activities of cells from all sources, including PBMC from healthy subjects and patients with arthritis and cells isolated from synovial fluid or from the synovial tissue of RA patients, were greatly increased by incubating the cells with TCGF or rIL-2. In contrast, treatment of chondrocytes with interferon-gamma, which enhances major histocompatibility complex gene expression, decreased the susceptibility of chondrocytes to lysis.

CONCLUSIONS

These observations suggest a mechanism for joint damage in which the destruction of chondrocytes by lymphocytes is controlled by cytokines released during the inflammatory process in arthritic diseases.

We have previously shown that greater than 90% of B6.1 cells, a murine cytolytic T lymphocyte (CTL) cloned line which is solely dependent on T cell growth factor (TCGF) for continuous growth in vitro, accumulates in the G1 phase of the cell cycle after transfer into culture medium containing no TCGF. Moreover, when such quiescent cells are exposed again to TCGF, greater than 85% reenter the S phase and subsequently divide in a relatively synchronous fashion. In this study, the regulation of the rate of cell cycle progression of quiescent B6.1 cells after exposure to TCGF was analyzed using two complementary DNA staining techniques, namely, the propodium iodide method (to enumerate cells entering the S phase) and the Hoechst 33342-bromodeoxyuridine substitution technique (to enumerate cells which have gone through mitosis). After TCGF addition, quiescent B6.1 cells resumed DNA synthesis and divided after a lag phase of 10 and 20 h, respectively. The duration of the lag phase was found to be dependent on the length of time during which quiescent B6.1 cells had been deprived of TCGF, but was independent of the concentration of TCGF used for restimulation. In contrast, the proportion of cells responding to TCGF as well as the rate of their first passage through mitosis was dependent on TCGF concentration. The presence of TCGF for at least 6 h was required for a maximal response. Moreover, direct evidence was obtained that TCGF by itself was able to stimulate proliferation of quiescent B6.1 cells in the absence of other growth factors and serum constituents other than bovine serum albumin, transferrin, and lipids.

We examined the role of accessory cells that cooperated with the cloned cytolytic T cell line, L3. Accessory cells and T cell growth factors (TCGF) acted synergistically to stimulate cloned L3 CTL to grow and produce cytolytic activity. Accessory cells were non-T, radioresistant, plastic adherent M phi-like cells that interacted with cloned L3 CTL via soluble factors. Peritoneal M phi, at low density, also cooperated with L3 cells but at high density, inhibited L3 cells. Both accessory and inhibitory M phi acted upon L3 CTL without the intervention of other T lymphocytes or nonadherent cells. Other published reports indicate that M phi may promote cytolytic activity indirectly, amplifying TCGF production by helper T lymphocytes. This report indicates that M phi may also promote cytolytic activity directly, amplifying the response of CTL to TCGF.

Peripheral blood lymphocytes from seven patients with adult T-cell leukemia (ATL) were found to lack PHA-responsiveness. However, in most of the cases, minute but distinct proliferation could be induced and maintained by human spleen cell conditioned medium containing PHA or by a combination of PHA and conditioned medium of gibbon cell line, MLA-144 (MLA-144 CM). These results indicate that the lack of response to mitogens of ATL cells might be attributed not only to the failure of these cells to produce T-cell growth factor (TCGF) upon activation, but also to their poor responsiveness to TCGF. Furthermore, a direct proliferative response to mitogen-free MLA-144 CM was shown in two out of seven patients; these two patients experienced rapidly progressive clinical courses. This observation raises the possibility that TCGF promotes the growth of ATL cells in vivo, and is related to the clinical course of the disease.

Human normal T cells were selected for in vitro cloning according to the expression of T4, T8 or T10 antigens on individual cells. Clones were produced from each of these cells irrespective of the antigenic phenotype of the parental cell. The cloned progeny manifested, in many cases, shifts in antigen expression. Thus, T4+T8- cells gave clones expressing predominantly T4-T8+ and vice versa. The clonal expression of T4 and T8 seemed to be mutually exclusive. Antigenic shifts were recorded also in clones derived from T4-T8-T10- cells, resulting in T10+ clones which were also either T4+ or T8+ and from T4+T8-T10+ cloned cells yielding clones of either T4+ or T8+ cells. Testing functional properties we found that NK activity was mediated not only by T10+ cells but also, in some cases, by T4+ and T8+ cells. Moreover, TCGF production, which may reflect helper activity, was mediated not only by T4+ cells. Only the cytotoxic (CTL) activity seems to be confined to the T8 phenotype. Thus, it appears that T antigens, which seemed to be molecular markers of differentiation, are not markers for terminal differentiation and do not always reflect defined functional properties. These conclusions are drawn from cloning of normal T cells which manifest properties different from those of T-cell lines or T hybridomas.

The in vitro transformation of normal T-lymphocytes by human T-cell leukemia/lymphoma virus (HTLV-I) is possible utilizing cocultivation techniques. We now report on a quantitative assay for HTLV-I transformation. Transformed cell lines were produced by cocultivation of either preactivated (phytohemagglutinin and T-cell growth factor) or nonactivated peripheral blood mononuclear cells with an equal number of lethally irradiated HTLV-I-positive donor cells (MT-2). After 14 days in liquid culture, transformed cells were plated in a 2-layer soft agarose system with or without T-cell growth factor (TCGF). Colony formation among 50 normal controls was observed at varying efficiencies with a mean number of 179 colonies (range, 6-599) in the presence of TCGF (up to a 2-log difference). The day 14 T-cell cultures demonstrated relatively low colony-forming efficiencies (less than or equal to 0.1%) and enhanced colony formation in the presence of TCGF. Day 14 after cocultivation was chosen for this assay based on a dose-response relationship between colony formation and the virus-positive donor cell inoculum and the known kinetics of colony growth of normal activated T-cells. An analysis of individual colonies indicated that they were of target cell origin and HTLV-I positive. Recombinant beta-interferon in increasing concentrations caused a decrease in colony formation as measured in this assay. Long-term cell cultures (2-18 months) showed higher colony-forming efficiencies (up to 1.0%) which were not enhanced by TCGF. The ability to quantitatively evaluate transformation via colony counts will provide an opportunity to study differences in transforming efficiencies attributable to varying target cells, donor cells, or blocking factors such as interferons, drugs, or anti-HTLV-I antibodies.