Skewed T-cell receptor repertoire: more than a marker of malignancy, a tool to dissect the immunopathology of inflammatory diseases.

The highly diverse heterodimeric surface T cell receptor (TCR) gives the T lymphocyte its specificity for MHC-bound peptides needed to initiate antigen-recognition. In normal peripheral blood, spleen and lymph nodes, the TCR repertoire of the T lymphocytes is usually polyclonal. However, in malignancies such as leukemias, as well as in lymphoproliferative diseases of mature T cells, the TCR is a reflection of the clonality of the malignant cells and is therefore monoclonal. Several clinical conditions (mainly solid tumors and autoimmune diseases) have been described where the TCR repertoire is restricted. The ability to demonstrate clonal TCR usage provides a useful tool to dissect the immunopathology of inflammatory diseases. In this review we discuss these findings and propose to sub-divide diseases with restricted TCR repertoire into a group of conditions in which there is a known TCR ligand, as opposed to diseases in which the restricted TCR repertoire is the result of impaired T-cell development. This classification sheds light on the pathogenesis of several inflammatory diseases.

Fibroblasts mediate T cell survival: a proposed mechanism for retention of primed T cells.

This report describes a salvage pathway whereby activated T lymphocytes revert to nonproliferating cells in the absence of antigen or mitogenic signals. After the removal of mitogenic cytokines, cultured T lymphocytes cease dividing and rapidly begin to undergo cell death. However, the addition of fibroblasts to interleukin 2 (IL-2)-propagated T cells results in prolonged survival of the previously activated T lymphocytes in the absence of proliferation. The prevention of cell death is also achieved by conditioned medium from the fibroblasts. T lymphocytes cultured with fibroblasts or the conditioned medium retain the ability to be restimulated if mitogenic stimuli are added to the culture. The activity is not accounted for by IL-1-7. The studies suggest a stromal cell-mediated, nonspecific mechanism for survival of primed T lymphocytes in a nonproliferating state.

Studies on the mode of action of diphtheria toxin. VII. Toxin-stimulated hydrolysis of nicotinamide adenine dinucleotide in mammalian cell extracts.

When diphtheria toxin and NAD are added to soluble fractions containing aminoacyl transfer enzymes isolated from rabbit reticulocytes or from HeLa cells, free nicotinamide is released and, simultaneously, an inactive ADP ribose derivative of transferase II is formed. The reaction is reversible, and in the presence of excess nicotinamide, toxin catalyzes the restoration of aminoacyl transfer activity in intoxicated preparations. In living cultures of HeLa cells, the internal NAD concentration is sufficiently high to account for the rapid conversion, catalyzed by a few toxin molecules located in the cell membrane, of the entire cell content of free transferase II to its inactive ADP ribose derivative. Completely inactive ammonium sulfate fractions containing soluble proteins isolated from cells that have been exposed for several hours to excess toxin, can be reactivated to full aminoacyl transfer activity by addition of nicotinamide together with diphtheria toxin. Transferase II appears to be a highly specific substrate for the toxin-stimulated splitting of NAD and thus far no other protein acceptor for the ADP ribose moiety has been found.

Longitudinal analysis of T cell receptor (TCR) gene usage by human immunodeficiency virus 1 envelope-specific cytotoxic T lymphocyte clones reveals a limited TCR repertoire.

Human immunodeficiency virus 1 (HIV-1) infection is associated with a vigorous cellular immune response that allows detection of cytotoxic T lymphocyte (CTL) activity using freshly isolated peripheral blood mononuclear cells (PBMC). Although restricting class I antigens and epitopes recognized by HIV-1-specific CTL have been defined, the effector cells mediating this vigorous response have been characterized less well. Specifically, no studies have addressed the breadth and duration of response to a defined epitope. In the present study, a longitudinal analysis of T cell receptor (TCR) gene usage by CTL clones was performed in a seropositive person using TCR gene sequences as a means of tracking responses to a well-defined epitope in the glycoprotein 41 transmembrane protein. 10 CTL clones specific for this human histocompatibility leukocyte antigen-B14-restricted epitope were isolated at multiple time points over a 31-mo period. All clones were derived from a single asymptomatic HIV-1-infected individual with a vigorous response to this epitope that was detectable using unstimulated PBMC. Polymerase chain reaction amplification using V alpha and V beta family-specific primers was performed on each clone, followed by DNA sequencing of the V-D-J regions. All 10 clones utilized V alpha 14 and V beta 4 genes. Sequence analysis of the TCR revealed the first nine clones isolated to also be identical at the nucleotide level. The TCR-alpha junctional region sequence of the tenth clone was identical to the junctional region sequences of the other nine, but this clone utilized distinct D beta and J beta gene segments. This study provides evidence that the observed high degree of HIV-1-specific CTL activity may be due to monoclonal or oligoclonal expansion of specific effector cells, and that progeny of a particular CTL clone may persist for prolonged periods in vivo in the presence of a chronic productive viral infection. The observed limited TCR diversity against an immunodominant epitope may limit recognition of virus variants with mutations in regions interacting with the TCR, thereby facilitating immune escape.

Strategies to overcome obstacles to successful immunotherapy of melanoma.

The immunogenicity of malignant melanomas has been recognized by the observed recruitment of tumor-specific cytotoxic T-cells (CTL), leading to the identification of several melanoma associated antigen (MAA). However, numerous strategies to treat melanoma with immunotherapy have resulted in only partial success. In this editorial, we discuss recent data related to the ability of tumors to elude immune responses. We therefore discuss different strategies to induce a clinically effective immune response. These approaches include 1) immunostimulation: including peptide/protein based vaccines, dendritic cell vaccines, and adoptive cell transfer; and 2) overcoming immunosuppression, including targeting of checkpoint molecules such as CTLA-4, circumventing the activity of Tregs, and assuring antigen expression by tumor cells (thwarting antigen silencing). Finally, we discuss recent advances in gene therapy, including adoptive therapy with engineered T cell receptors (TCRs). These issues lead to the conclusion that successful immunotherapy in malignant melanoma requires a combination of strategies aimed at both inducing immunostimulation and blocking immunosuppression.

1 alpha,25-dihydroxyvitamin D3 induces maturation of the human monocyte cell line U937, and, in association with a factor from human T lymphocytes, augments production of the monokine, mononuclear cell factor.

The monocyte factor, interleukin 1, or other factors homologous with interleukin 1, modulates functions of a variety of cells, including T and B lymphocytes, synovial cells, and chondrocytes. We have reported that a human monocyte cell line, U937, produces interleukin 1 when incubated with a soluble factor from lectin-stimulated T lymphocytes. We have also shown that U937 cells have a specific cytosolic receptor for 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25[OH]2D3). We now report that 1 alpha,25(OH)2D3(10(-11)-10(-10) M) induces maturational changes in the U937 cells similar to those produced by conditioned medium from lectin-stimulated T lymphocytes (increase in Fc receptors and OKM1 binding and decrease in proliferation), but does not induce monokine production as measured by mononuclear cell factor activity. 1 alpha,25(OH)2D3 is 200-300-fold more effective than 25-hydroxyvitamin D3, which is consistent with the known biological potency of these vitamin D3 metabolites. 1 alpha,25(OH)2D3 and the lymphokine together markedly augment maturational effects and, in addition, augment monokine production. The specificity of the interaction is further demonstrated by the lack of augmentation of monokine production with 1 beta,25-dihydroxyvitamin D3 in the presence of lymphokine. These interactions of a classical hormone and the hormonelike product(s) of the immune system with U937 cells serve as a model for human monocyte/macrophage differentiation and suggest a role for these interactions in some aspects of inflammation.

Expression of HLA-A2 antigen in human melanoma cell lines and its role in T-cell recognition.

Previous studies have suggested that, in human melanoma, expression of HLA-A2 antigen is important for tumor cell recognition by autologous T-lymphocytes. Because of the recent demonstration that expression of HLA Class I antigens may be selectively lost in several human tumors, including melanoma, we derived pairs of tumor infiltrating lymphocytes (TIL) and melanoma cell lines from 4 human lymphocytic antigen (HLA)-A2+ patients with metastatic melanoma. We observed that, although all 4 TIL cultures expressed HLA-A2 antigen, only 2 melanoma cell lines did so. Melanoma cells derived from the other 2 patients showed neither surface expression of the HLA-A2 antigen nor presence of the corresponding mRNA. We also observed some correlation between loss of HLA-A2 expression and level of c-myc transcription. TIL derived from patients whose melanoma cell lines had normal expression of HLA-A2 had a CD8 phenotype and were capable of lysing autologous melanoma cells. Melanoma cell killing was CD3 and major histocompatibility complex Class I restricted in both cases, but HLA-A2 restricted in only one case. On the other hand, TIL derived from the 2 patients whose melanoma cell lines had lost expression of HLA-A2 had a predominant CD4 phenotype and virtually no cytotoxic activity. Preincubation of the HLA-A2 negative melanoma cell lines with alpha- or gamma-interferon did not induce the re-expression of the HLA-A2 antigen. In an attempt to restore HLA-A2 antigen expression in one of the melanoma cell lines that were HLA-A2 negative, we transfected these cells with the HLA-A2 gene subcloned in the pSV2-neo vector. Four transfected clones, with high levels of HLA-A2 antigen expression, were expanded and characterized. Proliferative and cytotoxic activities of TIL against the autologous transfected clones as well as the untransfected parental melanoma cell line were measured and compared. CD4+ TIL showed no difference in the proliferative response to autologous parental and HLA-A2 transfected clones. However, we observed selective recognition of the HLA-A2 expressing clones by autologous cultured peripheral blood lymphocytes (which contained CD8 cells) as well as allogeneic CD8+ TIL with a HLA-A2 restricted pattern of recognition. In contrast, virtually no cytotoxic activity was detected against either parental or HLA-A2 transfected clones. Overall, our data suggest that selective down-regulation of HLA-A2 antigen expression in melanoma cells may represent one of the mechanisms by which tumor cells escape immunological recognition.

Characteristics of lymphocytes cultured from murine viral myocarditis specimens: a preliminary and technical report.

Long-term culture of lymphocytes from myocardial biopsy specimens has been reported. To test the usefulness of this technique in experimental models of murine myocarditis, the culture and characterization of lymphocytes from mice infected with encephalomyocarditis virus or coxsackievirus B3 were studied. Lymphocytes were successfully cultured from three hearts of encephalomyocarditis virus-infected mice in interleukin 2-containing culture. After approximately 10 days, lymphocytes migrated out of myocardium in the chronic stage of myocarditis and gradually increased in numbers. More than half of the exuding cells were Thy 1.2-positive. None of the myocardial samples from coxsackievirus B3-infected mice grew out lymphocytes. Pathologic examination of all specimens showed myocardial necrosis with lymphoid infiltration. Although further studies are needed, this preliminary study suggests that the technique of lymphocyte culture may promote new insights into the pathogenesis of experimental myocarditis.

Fibroblast-derived factors preserve viability in vitro of mononuclear cells isolated from subjects with HIV-1 infection.

OBJECTIVE: Peripheral blood mononuclear cells (PBMC) from HIV-infected subjects have an increased mortality rate (MR) when incubated in vitro for 3 days in a culture medium. We have previously shown that fibroblast-conditioned medium (FCM) can preserve viability, without significant activation, of human lymphocytes in vitro. We therefore tested the ability of two FCM and other factors to reduce spontaneous MR in HIV-positive PBMC. METHODS: PBMC were cultured for 3 days in control medium and medium supplemented with FCM or recombinant cytokines [interleukin (IL)-2, IL-6, IL-7, granulocyte macrophage colony-stimulating factor]. Cells viable at day 3 were counted in a cytofluorimeter after staining with ethidium bromide. DNA was extracted from the cultures and evaluated for the presence of low molecular weight fragmentation. RESULTS: The MR of PBMC from 51 HIV-positive subjects and from 21 healthy controls were 30.1 and 9.5%, respectively (P < 0.0001). The MR was higher in 40 patients with a CD4+ lymphocyte count < 400 x 10(6)/l than in subjects with a count > 400 x 10(6)/l (32.84 versus 20.96%; P = 0.047). IL-2 and FCM significantly reduced MR in HIV-positive subjects (MR: 17.8 and 20.4%; P: < 0.001 and 0.005, respectively). This effect was more evident in subjects with a CD4+ lymphocyte count < 400 x 10(6)/l and in subjects with negative p24 antigenaemia. Cellular proliferation accounts for increased survival in IL-2-supplemented cultures but not in those with FCM. DNA was extracted from fresh PBMC and cells cultured for 3 days for 22 HIV-positive cases. DNA degradation was documented and bands related to an apoptotic mechanism of death observed, especially in subjects with more advanced disease. CONCLUSIONS: Our data suggest that FCM inhibits accelerated cell death in vitro of PBMC isolated from HIV-positive patients.

Clonal dominance among synovial tissue-infiltrating lymphocytes in arthritis.

T-cell receptor gene rearrangement using a C beta probe was evaluated in 12 patients with rheumatoid arthritis, 2 with juvenile rheumatoid arthritis, and 1 with systemic lupus erythematosus, and in all the samples a dominant T-cell receptor gene rearrangement was noted. In rheumatoid arthritis identical T-cell receptor gene rearrangement was noted in freshly isolated synovial tissue-infiltrating lymphocytes (TIL) and the corresponding interleukin 2-propagated culture. TIL from five different joints obtained from one rheumatoid arthritis patient shared one dominant band, and TIL from three joints had an identical rearrangement. Limiting dilution experiments showed that 10% of T-cell clones had rearrangements matching the corresponding bulk in one rheumatoid arthritis patient. These findings lend further support to the suggestion that the clonal dominance noted among synovial tumor-infiltrating lymphocytes is the result of an in vivo process reflecting a selective T-cell receptor gene usage.

Cytotoxic activity of graft-infiltrating lymphocytes correlates with cellular rejection in cardiac transplant patients.

Lymphocytes propagated from allografts have shown a wide spectrum of activity during rejection including cytotoxicity, proliferation, and lymphokine production. It is necessary to correlate these activities to the rejection process to understand the in vivo immune response. The frequent need to obtain a biopsy of human cardiac allografts permits the evaluation of the function of the graft-infiltrating lymphocytes (GIL) as related to development of the rejection process. Lymphocyte cultures established from biopsies taken before, during, and after rejection episodes of grade 1.0 or greater were assayed for surface antigen expression using flow cytometry, proliferative activity using a primed lymphocyte test (PLT), and cytotoxicity using a cell-mediated lympholysis assay. Fifteen rejection episodes were followed from 10 patients. Two patients were followed through two different rejection episodes and one patient through four rejection episodes. CD8+ cells usually predominated during the rejection episode. Following the rejection episodes the GIL showed a shift toward higher proportion of CD4+ cells. Most cultures taken prior to and during rejection episodes (8/9 and 12/13 assayed, respectively) demonstrated greater than 30% killing of targets bearing donor-related HLA antigens. Seven of 15 cultures remained cytotoxic after a rejection episode whereas 8 of 15 lost cytotoxicity. The patients whose cultures remained cytotoxic after a rejection episode went on to further rejection episodes at 6, 7, 11, 20, 37, or 118 days later. Those patients whose cultures were no longer cytotoxic did not experience any subsequent rejection episode until at least 257 days later.(ABSTRACT TRUNCATED AT 250 WORDS)

Clonal analysis of graft-infiltrating lymphocytes from renal and cardiac biopsies. Dominant rearrangements of TcR beta genes and persistence of dominant rearrangements in serial biopsies.

Graft-infiltrating lymphocytes from both human renal and cardiac allografts were propagated in interleukin 2 in order to evaluate rearrangements in the T-cell receptor (TcR) beta-chain genes. Individual biopsies from renal allografts during episodes of cellular rejection were examined as well as multiple biopsies of heart transplant patients from whom endomyocardial samples were taken prior to, during, and after episodes of rejection. TcR beta-chain rearrangements were evaluated in Southern blots using DNA extracted from interleukin 2-propagated cells and digested with restriction endonucleases permitting assessment of rearrangements to both C beta 1 and C beta 2. Rearrangements shared among greater than 5% of the "bulk" culture appear as nongermline bands when hybridized with a C beta probe. Single-cell progeny were generated from limiting dilution, and the rearrangements among the cloned progeny compared to the "bulk" of the cultured progeny of graft-infiltrating lymphocytes. The results indicate that "dominant" rearrangements are a common feature of renal allograft-infiltrating lymphocytes (14 of 15 cases examined). Since the number of cells which can be recovered from a given cardiac biopsy may be limiting, evaluation of clonal dominance from these cultures is more difficult to evaluate. However, sharing of "dominant" rearrangements among multiple biopsies from the same cardiac allograft patient indicates an in vivo selection for T cells with the same receptor rearrangement. Analysis of individual clones showed 3/33 clones from a renal allograft sharing the "dominant" rearrangement noted in the bulk culture, but none of these "dominant" clones showed antidonor specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

T-cell receptor V-gene usage in synovial fluid lymphocytes of patients with chronic arthritis.

In this study we analyzed the usage frequencies of the TCR V-gene segments by alpha beta+ T cells present in synovial fluid of 17 patients with chronic arthritis, including rheumatoid arthritis. The results of this study, obtained from semiquantitative PCR analyses, showed that in all patients most of the TCR V alpha- and V beta-gene segments could be detected both in fresh PBMCs and in fresh SFMCs. The relative frequencies of use of these V-region genes were variable between the different patients. Although there was some skewing of increased usage frequencies of particular TCR V alpha and V beta genes among SFMC-derived TCRs when compared with PBMCs, we could not correlate such increased TCR V-gene usage with the inflammation in the joints as a disease-specific marker.

Phenotypic characteristics of lymphoid populations of middle gestation human fetal liver, spleen and thymus.

Mononuclear cells isolated from liver, spleen and thymus of fetuses between 18 and 24 weeks gestational age were stained for a number of lymphoid cell markers by indirect immunofluorescence and analyzed by flow cytometry. Studies were carried out on freshly isolated mononuclear cell preparations and on cultured cells after selective expansion in interleukin 2 (IL2). Many mononuclear cells in fresh isolates of liver and spleen could not be identified with antibodies to mature T- and B-cell markers. An average of 3% of isolated liver cells and 34% of isolated spleen cells stained positively for CD3, and 19% of liver cells and 37% of spleen cells stained positively for CD20. Lymphoid cells of the fetal thymus were an average 67% CD3+, 76% CD4+, 84% CD8+, and showed greater CD45RO staining (93%) than mononuclear cells of other tissues. Propagation of liver and spleen cell populations in culture favored CD3 phenotypes and CD8 phenotypes. Propagated T cell populations of liver and spleen were primarily TCR alpha/beta+ (81% in liver, 85% in spleen), suggesting a selective advantage in IL2 expansion of alpha/beta T cells over gamma/delta T cells. Propagated gamma/delta T cells of liver and spleen were predominantly TCR gamma/delta 2+. Whereas propagated cells of liver and spleen consisted of approximately 10% gamma/delta+ cells, thymus-derived cells expanded in culture were only an average of 2% TCR gamma/delta+, demonstrating a rarity of IL2-responsive gamma/delta T cells in middle gestation fetal thymus.

Phenotypic characteristics of lymphocyte populations isolated from middle gestation human placenta.

In order to characterize the phenotypic composition of populations of lymphoid cells in maternal and fetal tissues during the period of middle gestation, mononuclear cells were isolated from maternal peripheral blood, fetal spleen, fetal thymus and placenta of 18-24 week pregnancies. Peripheral blood and placental isolates were stained for a number of lymphoid cell markers by indirect immunofluorescence and analyzed by flow cytometry. Studies were performed on both freshly isolated mononuclear cell preparations and in vitro cultured cells after selective expansion in interleukin 2 (IL2). Fresh placental mononuclear cell isolates were an average 20% CD3+; their CD4/CD8 ratios varied among individuals. An average of 68% of the lymphocytes isolated from maternal peripheral blood were CD3+. Placental and maternal peripheral blood isolates had comparable percentages of CD16+ and CD20+ cells, while CD56+ cells were present at significantly greater numbers in the lymphocyte compartment of placenta (17%) than in peripheral blood (3%; P < 0.01). Lymphocyte isolates were expanded by culture with IL2 and PHA and stained to determine if propagated lymphocyte populations are representative of initial isolates. Expansion of all lymphocyte isolates favored CD3 phenotypes and CD8 phenotypes. Compared to expanded placenta-derived populations, expanded peripheral blood lymphocytes were similar with regard to percentages of all phenotypes except gamma/delta T cells which represented more of placental lymphocytes (10%) than peripheral lymphocytes (5%; P < 0.01). Surface HLA typing determined propagated placenta-derived lymphocytes to be of maternal and not fetal origin. In vitro propagation of placental mononuclear cell isolates may therefore provide populations of maternal CD3+ lymphocytes for assessment of function and specificity.

T cell receptor usage by HLA-DR3-specific T cell clones isolated from a renal allograft.

In order to evaluate the T cell receptor (TCR) usage by clones of human allograft infiltrating lymphocytes, this study utilized polymerase chain reaction (PCR) amplification of TCR transcripts from five clones which were previously shown to react with a human leucocyte antigen (HLA)-DR3 mismatch between a living related kidney donor and recipient. The five CD4+ (CD8-) clones, which were selected for TCR analysis, proliferated in response to HLA-DR3 and three of the clones were also cytotoxic against the same target cells. After identification of the TCRAV and TCRBV usage of the clones, the sequence of the TCR alpha and beta were determined by direct sequencing of the PCR product. The results indicate that several different TCRAV and TCRBV gene segments are used among the different clones, but the two clones that were both cytotoxic and proliferative in response to HLA-DR3 shared identical TCRAV27-J42-C and TCRBV13-D1-J1S2-C1 transcripts. The additional three clones showed various TCRAV and TCRBV transcripts, but evaluation of the CDR3 region of the TCR beta chain, corresponding to the peptide antigen binding sites, demonstrated shared amino acid motifs which resulted both from germline sequences and combinations of n-region and germline-derived codons. These results suggest that the repertoire for anti-HLA-DR3-reactive clones can include a diverse expression of TCR, but there may be selection for some clones, as well as conserved motifs in the CDR3 region of anti-DR3 specific clones.

Adoptive immunotherapy of murine pulmonary metastases with interleukin 2 and interferon-gamma.

We examined the activities of activated lymphocytes, interferon-gamma (IFN-gamma), and interleukin 2 (IL-2) in adoptive immunotherapy of pulmonary metastases. Pulmonary metastases produced in Balb/c mice by a single tail-vein injection of 5 X 10(5) murine sarcoma (MCB8) tumor cells on day 0 were treated with combinations of Con A-activated lymphocytes (CAL) (3 X 10(7) cells on days 3 and 7), IL-2 (5 X 10(4) U three times a day on days 3 to 8), and IFN-gamma (5 X 10(4) U/mouse on days 1 to 8). Treated tumors contained increased numbers of infiltrating Thy-1.2+ lymphocytes and a predominance of L3T4+(CD4+) lymphocytes. The level of expression of class I and class II MHC antigens by tumor cells in the lung was increased after treatment. Mice that received CAL + IL-2 + IFN-gamma showed approximately 80% reduction in tumor burden as compared to controls (P = 0.001). Mice treated with IL-2 + CAL, or IL-2 + IFN-gamma, displayed approximately 50% reduction (both P less than 0.02 as compared to triple therapy), whereas IL-2, IFN-gamma, or CAL administered as single agents had little effect on pulmonary metastases. We conclude that adoptive immunotherapy with activated lymphocytes and IL-2 is enhanced by IFN-gamma.

Interleukin 1 production by the human monocyte cell line U937 requires a lymphokine induction signal distinct from interleukin 2 or interferons.

A soluble product from cloned human T lymphocytes is capable of stimulating U937 cells, a line of human monocytes, to produce interleukin 1 (IL 1). We previously reported that U937 cells exposed to T lymphocyte-conditioned medium secrete mononuclear cell factor (MCF), which increases collagenase and prostaglandin E2 production by adherent rheumatoid synovial cells. Whereas structural and functional homologies between lymphocyte-activating factor (LAF, or IL 1) and MCF were described, previous attempts to measure LAF secretion by lymphokine-stimulated U937 cells were unsuccessful. Although the crude supernatants of cultured U937 cells exposed to medium from lectin-stimulated peripheral blood or cloned T lymphocytes contained MCF activity, no LAF activity was detected. After these crude supernatants were chromatographed on Ultrogel AcA54, however, and the fractions were individually assayed for IL 1, MCF and LAF activities were coeluted with apparent m.w. approximately 14,000 to 23,000. The inability to detect LAF activity in the unfractionated medium was accounted for by an inhibitor of lymphocyte proliferation present in fractions of higher m.w. The T lymphocyte product that stimulated U937 cell maturation and monokine production was secreted in response to lectin-stimulation in a dose-dependent fashion. Although we have previously demonstrated that the hormone 1,25-dihydroxyvitamin D3 caused maturational changes in U937 cells, and other investigators have reported effects of alpha and gamma interferon, these changes are dissociable from IL 1 production. Thus, a distinct lymphocyte-derived signal, necessary for the production of IL 1 by U937 cells, can be identified and dissociated from other biologic products that cause "maturational" changes. The detection of LAF activity in U937 cell supernatants requires the removal of an inhibitor of lymphocyte proliferation.