Antiproliferative Effect Of IFN-gamma Research Articles

IRF-1 expression in normal human epidermal keratinocytes.

Interferon gamma (IFN-gamma) is a potent inducer of cell growth arrest in human epidermal keratinocytes. Interferon regulatory factor-1 (IRF-1), an interferon-inducible gene, is a mediator of interferon action. It has also been suggested that IRF-1 may have a functional role as a tumor suppressor gene and may be associated with the antiproliferative effect of IFN-gamma. We examined the expression of IRF-1 mRNA in normal human epidermal keratinocytes (NHEK). Northern blot analysis demonstrated an increased expression of IRF-1 mRNA by IFN-gamma in NHEKs. This increased expression of IRF-1 mRNA by IFN-gamma was not blocked by treatment with cycloheximide, but was abolished by treatment with actinomycin D. In addition, neither pretreatment with TPA, a protein kinase C (PKC) activator, nor treatment with H7, a PKC inhibitor, affected the induction of IRF-1 mRNA by IFN-gamma. These results indicate that IFN-gamma-induced IRF-1 mRNA in NHEKs is transcriptional and PKC-independent.

Control of cell cycle regulatory proteins and modulation of STAT1 proteins by IFN-gamma in human prostatic JCA-1 cells

The addition of human recombinant IFN-gamma (10(2) and 10(3) IU/ml) inhibited human prostatic JCA-1 cell growth by 27% and 64%, respectively. Since the high dose of IFN-gamma elicited an increase in G(1) concomitant with a decrease in G(2)/M phases of the cell cycle, changes in the expression of cell cycle regulatory protein molecules were analyzed by Western blots. Results of these experiments show that IFN-gamma down regulated the G(1)/S transition molecules, e.g., cyclin D1, the cyclin-dependent protein kinase Cdk4, and the retinoblastoma gene product (pRB), but increased the G(2)/M transition molecules, e.g., cyclin B1 and p34(cdc2) (Cdk1). Possible modulation of Cdk-inhibitors (CDKIs), e.g., p53 and p21(WAF1), which have checkpoint functions in the cell cycle, by IFN-gamma, was also studied. The p53 was induced by both 10(2) and 10(3) IU/ml IFN-gamma. At 10(3) IU/ml, IFN-gamma inhibited p21(WAF1), increased the expression of STAT1 alpha, and sustained the elevated STAT1 alpha for up to 96 h. Thus several mechanisms may be involved in the antiproliferative effects of IFN-gamma.

Differences of reactivity to interferon gamma in HeLa and CaSki cells: a combined immunocytochemical and flow-cytometric study.

We characterized the changes induced by treatment for 48 h with 100 U/ ml interferon gamma (IFN-gamma) on HeLa and CaSki cells, derived from human uterine carcinomas and containing human papillomavirus (HPV) type 16 and HPV type 18 respectively, by studying cell growth, cell morphology, the cell cycle and expression of epidermal growth factor (EGF) receptor, filaggrin-profilaggrin and MHC class II antigen, HLA-DR. The response of the two cell lines to IFN gamma differed in some cases. In both cell lines, the cells remained viable; cell growth was similarly inhibited as shown by cell counts. Signs of morphological changes were essentially observed in HeLa cells. The cell cycle phases, analyzed by flow cytometry were more disturbed in CaSki than in HeLa cells; the proportion of CaSki cells in S phase increased and those in G2 + M decreased. Expression of EGF receptors related to proliferation increased only in CaSki cells while expression of filaggrin-profilaggrin, a marker of differentiation, and HLA-DR, a marker of epithelial cell immune response, was enhanced in both cell lines. The presence of filaggrin-profilaggrin being unexpected in these cells, the specificity of the reaction with the monoclonal antibody AKH1 was confirmed by immunoblotting. In conclusion, our results show that the two cell lines reacted differently to IFN gamma although they are of similar origin and the different antigens studied may be useful to predict the progression of lesions infected with HPV towards malignancy or the reactivity to IFN gamma of such lesions. However, enhanced synthesis of EGF receptors is probably independent of the antiproliferative effect of IFN gamma but an increase in HLA-DR antigen expression by epithelial cells, which corresponds to an immune response favored by IFN gamma, could act synergistically with cell growth inhibition and differentiation to exclude tumoral and/or HPV-infected cells.

Involvement of Two Regulatory Elements in Interferon-γ-Regulated Expression of Human Indoleamine 2,3-Dioxygenase Gene

Interferon (IFN)-gamma-induced expression of indoleamine 2,3-dioxygenase (IDO) gene is implicated in the antimicrobial and antiproliferative effects of IFN-gamma in cell cultures. Earlier studies identified a 96 base pair (bp) regulatory region upstream of the IDO gene that conferred IFN-gamma response to the chloroamphenicol acetyltransferase (CAT) gene linked to herpesvirus thymidine kinase promoter. The IFN-gamma-responsive region was further narrowed to a 67 bp fragment by 3' deletion. This 67 bp fragment contains several sequence elements of potential interest, including a 14 bp sequence homologous to the ISRE sequence found in IFN-alpha-inducible genes and two palindromic sequences (PE I and PE II) homologous to the GAS sequence identified in IFN-gamma-inducible genes. Site-directed mutagenesis studies showed that IFN-gamma-induced expression of IDO-CAT constructs involved cooperation between two elements: the ISRE homolog and the PE II (but not PE I). Either element alone with its flanking sequence was inadequate in conferring an IFN-gamma response to CAT reporter gene. Two IFN-gamma-regulated protein factors interacting with these two elements were identified. The factor binding to the ISRE region was induced with a slower kinetics, required new protein synthesis, and reacted with antibodies to IRF-1. The factor interacting with the PE II region appeared rapidly after treatment with IFN-gamma independently of new protein synthesis, and its binding to DNA probe was blocked by antibodies to p91 factor, reported to bind to GAS element.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell death suffers a TKO.

The cytokine interferon-gamma (IFN-gamma), initiates both cell cycle arrest and cell death in certain cell lines. Through a novel strategy of cell transfection with episomal vectors expressing antisense cDNAs, Deiss et al. have demonstrated that it is possible to isolate genes that are required for the initiation of cell death by the cytokine IFN-gamma. This approach, referred to as TKO, for Technical Knock Out, has identified several genes whose activity appears to be essential for the induction of apoptosis by IFN-gamma in HeLa cells. Interestingly, these genes appear to mediate IFN-gamma-induced apoptosis in HeLa cells, but their inhibition by antisense does not ameliorate the antiproliferative effects of IFN-gamma in these cells. The clever strategy employed by these authors holds promise for others who wish to isolate genes required for other differentiative processes in cultured cell lines.

Interferon-gamma in multiple myeloma.

Biological heterogeneity is a characteristic of multiple myeloma. A dysregulated cytokine network underlies the various phases of the disease. Numerous cytokines, either promoting or inhibiting plasma cell growth, are involved in tumor control. Interferon-gamma (IFN-gamma) showed the most powerful inhibiting activity on myeloma cell proliferation. This effect was demonstrated on IL-6 dependent myeloma cell lines, but not on IL-6 independent ones. It was also evident on fresh explanted bone marrow myeloma cells. The antiproliferative effect of IFN-gamma seems mainly due to the inhibition of IL-6, the central myeloma growth factor. IL-6 inhibition may occur at various levels: a downregulation of IL-6 receptor has been reported, and also a block of the IL-6 signal transduction pathway via interaction with cytoplasmic proteins such as p91 has been suggested. Our findings showed that IFN-gamma strongly inhibited myeloma cell proliferation to the same extent as dexamethasone (DEX), whereas interferon-alpha (IFN-alpha) inhibited Ig secretion. The combined use of interferons (IFNs) showed inhibitory activities both on proliferation and Ig synthesis that paralleled the effects of DEX. In some cases, IFN-gamma was also shown to augment monoclonal immunoglobulin secretion suggesting a possible differentiating activity on plasma cells. The in vitro data encouraged pilot studies to evaluate the in vivo antitumor effects of IFN-gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiproliferative effects of interferon gamma in combination with alpha-difluoromethylornithine on human carcinoma cell cultures.

The antiproliferative effects of human recombinant interferon gamma (IFN gamma) in combination with alpha-difluoromethylornithine (DFMO) or as single agents were assessed on human cell cultures derived from carcinomas of the breast (MCF-7), the ovary (EFO-27) or the kidneys (EGI-4). Results were obtained in proliferation assays by direct cell counting. The cell lines differed considerably in their sensitivities to the antiproliferative effect of IFN gamma as compared by the 50% inhibition doses of the growth (ID50). In contrast to the findings with IFN gamma, similar antiproliferative effects resulted from the application of comparable doses of DFMO. While IFN gamma induced cytotoxic effects in EGI-4 cells, DFMO produced only cytostatic actions in the cell lines analyzed. Synergistic growth inhibition resulted from the combined application of IFN gamma and DFMO in EFO-27 cell cultures. This finding was most pronounced after treatment with IFN gamma or DFMO doses below the respective ID50 values. However, antagonistic effects occurred in cells of the line EGI-4 after DFMO had been combined with IFN gamma at concentrations below the cytotoxic dose range. Within the sensitivity of our proliferation assay, no synergistic interactions were found in MCF-7 cell cultures. In the cell lines tested, no relation between the sensitivity for the single agents and the effectivity of the drug combination was identified. Despite promising synergistic effects in the moderately IFN gamma-sensitive ovarian carcinoma cell line EFO-27, the efficacy of the IFN gamma/DFMO combination was restrained by possible antagonistic effects as demonstrated in the highly IFN gamma-sensitive EGI-4 renal carcinoma cell cultures. We conclude that the differential interaction patterns in the cell cultures analyzed preclude general suggestions for clinical studies using IFN gamma and DFMO.

Effect of tumor necrosis factor‐α and interferon‐γ on the growth of a human salivary gland cell line

Interferon-gamma (IFN-gamma) is a product of activated T-lymphocytes, and tumor necrosis factor-alpha (TNF-alpha) is a product of both lymphocytes and macrophages. These cell types are often present at sites of tissue damage secondary to chronic infection or autoimmune disease. The purpose of this study was to characterize the effects of TNF-alpha and IFN-gamma on a human submandibular gland epithelial cell line (HSG). IFN-gamma caused a concentration-dependent decrease in HSG cell growth (approximately 70% in 6 days). Conversely, TNF-alpha alone had little effect on the growth of these cells. When these cytokines were added in combination (20 units/ml TNF-alpha and 1,000 units/ml of IFN-gamma), there was a synergistic antiproliferative effect; no apparent cell growth was observed. The cytokine-induced antiproliferative effect was reversible. After the apparent cessation of cell growth for 3-6 days, removal of the cytokines permitted complete growth recovery. Further, cells that recovered and exhibited growth patterns that were similar to control cells remained susceptible to the antiproliferative effects of the cytokines. Flow cytometry revealed that the percentage of cells in G0/G1 with the combination of cytokines was significantly increased by 24 h. The antiproliferative effect of IFN-gamma alone and that of IFN-gamma and TNF-alpha in combination were blocked completely using an antibody to the IFN-gamma receptor. A hypothesized mechanism of tissue damage in autoimmune inflammatory disorders is via up-regulation of cell surface markers such as intercellular adhesion molecule type I (ICAM-1) and histocompatibility antigen HLA-DR which can exacerbate the inflammatory process. Treatment of HSG cells with IFN-gamma, with or without TNF-alpha, resulted in increased levels of ICAM-1 and the acquisition of HLA-DR expression. These aggregate data suggest that IFN-gamma alone can regulate the expression of cell surface markers involved in the inflammatory process as well as cause a potent yet reversible inhibition of HSG cell growth that is modulated by the presence of TNF-alpha.

Antiparasitic and antiproliferative effects of indoleamine 2,3-dioxygenase enzyme expression in human fibroblasts.

Studies were carried out to evaluate the proposed role of indoleamine 2,3-dioxygenase (INDO) induction in the antimicrobial and antiproliferative effects of gamma interferon (IFN-gamma) in human fibroblasts. The INDO cDNA coding region was cloned in the pMEP4 expression vector, containing the metallothionein (MTII) promoter in the sense (+ve) or the antisense (-ve) orientation. Human fibroblasts (GM637) stably transfected with the sense construct expressed INDO activity after treatment with CdCl2 or ZnSO4, but cells transfected with the antisense construct did not. The growth of Chlamydia psittaci was strongly inhibited in INDO +ve cells but not in INDO -ve cells after treatment with Cd2+ or Zn2+. The inhibition correlated with the level of INDO activity induced and could be reversed by the addition of excess tryptophan to the medium. The growth of Toxoplasma gondii was also strongly inhibited in INDO +ve cells but not in INDO -ve cells after treatment with Cd2+. Expression of Cd(2+)-induced INDO activity also inhibited thymidine incorporation and led to cytotoxicity in INDO +ve cells but not in INDO -ve cells. Thus, the induction of INDO activity by IFN-gamma may be an important factor in the antimicrobial and antiproliferative effects of IFN-gamma in human fibroblasts.

Hematopoietic progenitors and synergism of interferon-gamma and stem cell factor.

Interferon-gamma (IFN-gamma), an immunoregulatory cytokine produced by activated T cells and natural killer cells in response to viral infection or other stimuli, is generally recognized as a suppressor of hematopoiesis. IFN-gamma inhibited in vitro colony formation by granulocyte-macrophage (GM), erythroid and multipotential progenitors. This cytokine exerted direct suppression on the proliferation process, but not on the commitment, of GM progenitors. The antiproliferative effects of IFN-gamma may, in part, result from the prolongation of the doubling time of GM progenitors. Clinically, IFN-gamma may play an important role in the pathogenesis of pancytopenia in aplastic anemia and in the hemophagocytic syndrome. However, as well as showing inhibitory effects, IFN-gamma increased the number of pure and mixed megakaryocyte colonies formed by post-5-fluorouracil treated bone marrow cells and, moreover, the addition of IFN-gamma to culture containing stem cell factor resulted in a synergistic effect on the development of both primitive hematopoietic progenitors and mature populations. These findings suggest that IFN-gamma has bifunctional activity in hematopoiesis.

Regulation of indoleamine 2,3-dioxygenase gene expression in human fibroblasts by interferon-gamma. Upstream control region discriminates between interferon-gamma and interferon-alpha.

The interferon (IFN)-gamma-mediated induction of indoleamine 2,3-dioxygenase (IDO) enzyme, which converts tryptophan into N-formylkynurenine, has been implicated in the inhibition of intracellular pathogens, e.g. Toxoplasma gondii and Chlamydia psittaci, and in the antiproliferative effect of IFN-gamma on tumor cells. The IDO activity is induced strongly in many cell types by IFN-gamma but rather poorly by IFN-alpha or -beta. A genomic DNA clone containing part of the transcribed region of the IDO gene and approximately 13 kilobases (kb) of the 5'-upstream DNA sequence was isolated and analyzed. An approximately 1.4-kb fragment of this clone, containing 329 nucleotides of the transcribed sequence and approximately 1.1 kb of the 5'-upstream sequence, when ligated to chloramphenicol acetyltransferase (CAT) structural gene made its expression inducible by IFN-gamma, but this construct responded poorly, if at all, to IFN-alpha 2. Deletion constructs derived from this plasmid narrowed down the IFN-gamma-responsive region to a 151-nucleotide segment (-495/-344) which also contained a 14-nucleotide sequence (GGTTTCAGTTTTCC) highly homologous to the IFN(alpha)-stimulated response element (ISRE) that has been found so far in all cellular genes inducible with IFN-alpha or -beta. Expression of CAT activity was stimulated by IFN-gamma more effectively than by IFN-alpha 2 when a 155-nucleotide fragment (-495/-340) containing the 151-nucleotide segment required for IFN-gamma response was inserted before herpes simplex virus thymidine kinase promoter linked to CAT structural gene. The results indicate that despite the presence of an ISRE, the control region of the IDO gene can distinguish between IFN-gamma and IFN-alpha. This may account for the differential activation of IDO gene expression by IFN-gamma as against IFN-alpha or -beta in intact cells, and suggests that the response of ISRE to IFN-alpha or -beta may be governed by other features in the upstream control region of this gene.

Anti-proliferative effect of IFN-gamma in immune regulation. IV. Murine CTL clones produce IL-3 and GM-CSF, the activity of which is masked by the inhibitory action of secreted IFN-gamma.

We have demonstrated recently that rIFN-gamma inhibits the proliferation of murine bone marrow cells stimulated with rIL-3 or recombinant granulocyte-macrophage (GM)-CSF. In light of this finding, three murine CD8+ CTL clones whose supernatants had been shown previously not to contain detectable levels of CSF activity but which contained a relatively high level of IFN-gamma were reassessed for their ability to secrete IL-3 and GM-CSF. Supernatants from CTL clones activated with anti-CD3 mAb failed to stimulate the IL-3-dependent cell line FDCP1. However, these supernatants were indeed able to stimulate the proliferation of FDCP1 cells if anti-IFN-gamma mAb was present. This stimulatory activity was specifically neutralized by anti-IL-3 mAb. Supernatants from two of the three clones stimulated the proliferation of a GM-CSF-responsive HT-2 cell line, and this activity was neutralized by anti-GM-CSF antibody. The otherwise modest ability of CTL supernatants to stimulate the proliferation of fresh bone marrow cells was augmented considerably in the presence of anti-IFN-gamma mAb, and this activity was appropriately blocked by anti-IL-3 and anti-GM-CSF antibodies. mRNA for IL-3 and GM-CSF, as well as for IFN-gamma and TNF-alpha, was detected in cells that secreted those lymphokines, and the time course of appearance of each mRNA correlated with secretion of the appropriate lymphokine activity. However, the time course of mRNA accumulation for each lymphokine was distinct, the order of expression of these genes apparently being TNF-alpha, then IFN-gamma and GM-CSF, and finally IL-3. Our results emphasize that potential interactions among lymphokines must be considered when interpreting data obtained from lymphokine bioassays and suggest an immunoregulatory role for CTL through the secretion of several of the same lymphokines produced by HTL.

Regulation of macrophage growth and antiviral activity by interferon-gamma.

Interferons, in addition to their antiviral activity, induce a multiplicity of effects on different cell types. Interferon (IFN)-gamma exerts a unique regulatory effect on cells of the mononuclear phagocyte lineage. To investigate whether the antiviral and antiproliferative effects of IFN-gamma in macrophages can be genetically dissociated, and whether IFN-alpha and IFN-gamma use the same cellular signals and/or effector mechanisms to achieve their biologic effects, we have derived a series of somatic cell genetic variants resistant to the antiproliferative and/or antiviral activities of IFN-gamma. Two different classes of variants were found: those resistant to the antiproliferative and antiviral effects of IFN-gamma against vesicular stomatitis virus (VSV) and those resistant to the antiproliferative effect, but protected against VSV and encephalomyocarditis virus (EMCV) lysis by IFN-gamma. In addition, a third class of mutants was obtained that was susceptible to the growth inhibitory activity, but resistant to the antiviral activity of IFN-gamma. Analysis of these mutants has provided several insights regarding the regulatory mechanisms of IFN-gamma and IFN-alpha on the murine macrophage cell lines. The antiproliferative activity of IFN-gamma on these cells, in contrast to that of IFN-alpha, is mediated by a cAMP-independent pathway. The antiproliferative and antiviral activities of IFN-gamma were genetically dissociated. Variants were obtained that are growth resistant but antivirally protected, or are growth inhibited but not antivirally protected against VSV or EMCV. The genetic analysis indicated that IFN-alpha and IFN-gamma regulate the induction of the dsRNA-dependent P1/eIF-2 alpha protein kinase and 2',5'-oligoadenylate synthetase enzymatic activities via different pathways. Finally, a unique macrophage mutant was obtained that was protected by IFN-gamma against infection by VSV, but not EMCV, suggesting that antiviral mechanisms involved in protection against these different types of RNA viruses must be distinct at some level.

Antiproliferative effect of IFN-gamma in immune regulation. III. Differential selection of TH1 and TH2 murine helper T lymphocyte clones using recombinant IL-2 and recombinant IFN-gamma.

Supernatants collected after primary or secondary stimulation of spleen cells contain different arrays of lymphokines. Primary supernatants from spleen cells stimulated with Con A or allogeneic spleen cells (MLC-SF) contain IL-2 but little IL-4 or IGN-gamma; in contrast, secondary MLC-SF contains IL-2 as well as substantial IL-4 and IFN-gamma. Our laboratory previously had always used secondary MLC-SF for cloning T cells, and had routinely obtained TH1 helper T lymphocyte clones. In the present study, when primary Con A-SF was used as source of growth factors, TH2 and not TH1 clones were preferentially derived. Considering the possibility that IFN-gamma may be one important factor in determining whether TH1 or TH2 clones are preferentially obtained, clone derivation was then performed either in the presence of rIL-2 or rIL-2 plus rIFN-gamma. The majority of clones derived using rIL-2 alone were TH2 cells, whereas the majority of clones derived using rIL-2 plus rIFN-gamma were TH1 cells. Using either procedure, some clones were obtained that produced IL-2, IL-4, and IFN-gamma. These data are consistent with our previous observations that IFN-gamma inhibits the proliferation of TH2 but not TH1 clones, and suggest that the presence of IFN-gamma during an immune response would result in the preferential expansion of helper T lymphocytes of the TH1 phenotype. Our procedure for the differential selection of TH1 and TH2 clones reactive with the same Ag should be useful for designing in vitro systems for studying the function of these cell subsets in specific immune responses.

The role of polyamines in interferon and retinoic acid mediated synergistic antiproliferative action

Retinoic acid alone has no effect on the human breast cancer cell line BT-20 but can amplify the antiproliferative action of interferon-gamma (IFN-gamma). In our system ornithine decarboxylase (ODC) activity correlates well with growth rate; it was investigated whether the antiproliferative effects of IFN-gamma and IFN-gamma plus retinoic acid could be attributed to suppression of ODC activity. The ODC inhibitor difluoromethyl-ornithine (DFMO), which is active as a single agent did not enhance growth inhibition induced by the biological response modifiers. The substitution of the BT-20 cells with putrescine, the product of the enzymatic reaction mediated by ODC, reversed DFMO induced antiproliferative action. On the other hand putrescine did not affect the proliferation of BT-20 cells treated with interferon alone or in combination with retinoic acid.

Anti-proliferative effect of IFN-gamma in immune regulation. II. IFN-gamma inhibits the proliferation of murine bone marrow cells stimulated with IL-3, IL-4, or granulocyte-macrophage colony-stimulating factor.

A biphasic dose response curve was observed when the bone marrow-derived cell line FDCP1, used as an indicator line for IL-3 bioassays, was exposed to supernatants from some activated T cell clones but not others. The active component which inhibited proliferation at the higher supernatant concentrations appeared to be IFN-gamma, based on the following observations. 1) Only those culture supernatants which contained IFN-gamma gave a biphasic dose response curve; 2) with these supernatants, an anti-IFN-gamma mAb augmented the proliferation of FDCP1 cells at the higher supernatant concentrations; and 3) rIFN-gamma profoundly inhibited the proliferation of FDCP1 cells stimulated with rIL-3 or rIL-4. rTNF-alpha inhibited FDCP1 proliferation only to a modest extent, yet the combination of rTNF-alpha + rIFN-gamma provided greater inhibition than each agent alone. The proliferation of a second bone marrow-derived cell line, DA1, was not inhibited by rIFN-gamma or rIFN-gamma + rTNF-alpha when stimulated with rIL-3 or recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF). Fresh bone marrow cells also showed a suboptimal proliferative response when stimulated with T cell supernatants containing IFN-gamma, and this response was augmented considerably upon the addition of anti-IFN-gamma mAb. Bone marrow cell proliferation was observed upon exposure to rIL-3, rIL-4, or rGM-CSF, and these responses were inhibited by rIFN-gamma; rTNF-alpha also produced a synergistic effect with these cells. Bone marrow cell colony formation stimulated by rIL-3 or rGM-CSF also was inhibited by rIFN-gamma. Colony formation in bone marrow cell cultures was not observed in response to rIL-4. Collectively, these results suggest that Th1 cells, which in addition to IL-3 and GM-CSF also produce IFN-gamma, may regulate hemopoietic cell proliferation and colony formation differently from the way Th2 cells do, which do not produce IFN-gamma.

Anti-proliferative effect of IFN-gamma in immune regulation. I. IFN-gamma inhibits the proliferation of Th2 but not Th1 murine helper T lymphocyte clones.

A biphasic dose-response curve was observed when the IL-1-dependent HTL clone D10 was exposed to IL-1 plus supernatants from some activated T cell clones but not others. The active component that inhibited proliferation at high concentrations of these supernatants appeared to be IFN-gamma based on the following findings: 1) the biphasic pattern of responsiveness correlated with the presence of IFN-gamma in the supernatants; 2) an anti-IFN-gamma mAb augmented the proliferation of D10 cells to these supernatants; 3) rIFN-gamma inhibited profoundly the response of D10 cells stimulated with rIL-1 plus supernatant from activated D10 cells or with rIL-1 plus rIL-4; 4) the response of D10 cells to rIL-1 plus rIL-2 also was inhibited by rIFN-gamma, although to a lesser extent. The proliferation of an additional Th2 clone stimulated with rIL-1 plus rIL-4 or rIL-2 also was inhibited by rIFN-gamma, implicating IFN-gamma as an inhibitory lymphokine for Th2 cells in general. rIFN-gamma did not affect the proliferation of two Th1 clones, nor did it affect the proliferation of an unconventional HTL clone which produces both IL-4 and IFN-gamma and proliferates in response to IL-2 or IL-4 in an IL-1-independent fashion. The proliferation of D10 cells stimulated by Ag or by immobilized anti-CD3 antibody also was blocked by rIFN-gamma, whereas IL-4 production in response to these stimuli was unaffected, indicating that proliferation and not general cell function was specifically inhibited. Collectively, these data implicate IFN-gamma as a suppressive factor for the proliferation of the subset of HTL designated Th2, and suggest that the relative amounts of the various lymphokines present during an immune response may direct which T cell types increase in number.