Immunoregulatory activity of recombinant human tumor necrosis factor (TNF)-alpha: induction of TNF receptors on human T cells and TNF-alpha-mediated enhancement of T cell responses

The expression of specific tumor necrosis factor (TNF) membrane receptors and biological effects of recombinant TNF (rTNF)-alpha on normal human T lymphocytes were studied. Although resting T cells lacked specific binding capacity for rTNF-alpha, high affinity (Kd 70 pM) TNF receptors were de novo induced upon primary activation of T cells. Comparison of TNF receptor expression with that of high affinity interleukin 2 (IL-2) and interferon-gamma (IFN-gamma) receptors, respectively, revealed similarities to IL 2-receptor expression with respect to kinetics of induction. However, maximum expression of TNF receptors (approximately equal to 5000/cell at day 6) and subsequent decline occurred approximately 3 days after the peak of IL 2-receptor expression. In contrast, no change in the expression of IFN-gamma receptors (Kd 10 pM, 300 to 400 receptors/cell) was found in the course of T cell activation. On activated TNF receptor positive T cells, TNF-alpha exerted multiple stimulatory activities. Thus TNF increased the expression of HLA-DR antigens and high affinity IL 2 receptors. As a consequence, TNF-treated T cells showed an enhanced proliferative response to IL 2. Moreover, TNF-alpha was effective as a co-stimulator of IL 2-dependent IFN-gamma production. These data indicate that TNF-alpha may regulate growth and functional activities of normal T cells.     

The released interleukin 2 receptor binds interleukin 2 efficiently

The released interleukin 2 receptor (IL 2R) molecule was characterized in order to clarify its biochemical structure and to determine its functional capacity. Enzymatic digestions demonstrated that the released IL 2R, like the cell surface IL 2R, is a complex glycoprotein, modified by the addition of both N- and O-linked carbohydrates and sialic acid residues. It has a peptide backbone that is approximately 10 Kd smaller than that of its membrane-associated counterpart. Affinity chromatography demonstrated that released IL 2R from either an HTLV-I-positive T cell line (HUT-102) or PHA-activated normal peripheral lymphocytes binds efficiently to purified recombinant IL 2. Furthermore, the interaction between the growth factor and the released receptor does not appear to require any accessory molecules. These observations suggest a potentially significant role for the released IL 2R in the regulation of IL 2-dependent lymphocyte functions.     

TCGF(IL 2)-receptor inducing factor(s). II. Possible role of ATL-derived factor (ADF) on constitutive IL 2 receptor expression of HTLV-I(+) T cell lines

Interleukin 2 (IL 2) receptor (IL 2-R) is constitutively expressed on T cell lines established from the patients with adult T cell leukemia (ATL), which is a human T cell leukemia lymphoma virus (HTLV-1)(+) T4(+)-leukemia endemic in Japan, the United States, and other countries. Many of these cell lines continuously produce an acidic lymphokine, ATL-derived factor (ADF), which preferentially induces the synthesis and expression of IL 2-R on a sensitive HTLV-1(-) non-T cell line (YT). The induced IL 2-R was characterized by the binding of 125I-IL 2 and flow cytometry by using fluoresceinated anti-human IL 2-R monoclonal antibodies (anti-Tac). Scatchard analysis with 125I-IL 2 showed ADF induced high-affinity receptor sites on YT cells. To test the possibility that ADF produced by HTLV-1(+) T cells is involved in the abnormal expression of IL 2-R, we studied the effect of ADF on an HTLV-1(+) IL 2-dependent T cell line (ED) in which the beta-chain gene of the T cell antigen receptor (T beta) was rearranged. Unlike IL 2-independent HTLV-1(+) cell lines that constitutively expressed Il 2-R, the IL 2-R expression on ED cells declined in the absence of crude IL 2 or recombinant IL 2. When either ADF or recombinant IL 2 was added to the culture of ED cells, there was a dose-dependent enhancement of IL 2-R expression in 24 hr. ADF and IL 2 showed a synergism in the IL 2-R induction, and both factors were needed to induce the maximal receptor expression in these T cells. The lack of IL 2 production by ADF-treated YT, as well as ED cell line suggested IL 2 may not be involved in the IL 2-R induction by ADF. Northern blot hybridization with human IL 2-R cDNA probe showed the increase of IL 2-R mRNA in YT cells after ADF-treatment. ADF also enhanced IL 2-R expression of a rat T cell line transformed by HTLV-1(TARS-1), as demonstrated with anti-rat IL 2 receptor monoclonal antibodies (ART-18). An ADF-like IL 2-R-inducing factor was also detected in the conditioned medium of two HTLV-1(+) rat T cell lines (TARL-2 and TART-1), which constitutively expressed a higher number of Il 2-R than TARS-1 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of interleukin 2 receptors on interleukin 3-dependent cell lines

Several mouse IL 3-dependent cell lines, IC2, LT4, FDC-P2, and PB-3C, derived from spleen or bone marrow cells were shown to express low affinity receptors for IL 2 (Kd; 0.5 to 8 X 10(-8) M). High affinity receptors for IL 2 were not detected on the IL 3-dependent cells within the experimental limitation of this study. The clones did not respond to IL 2 at all at the concentration as high as 25 micrograms/ml. The number of the receptors expressed on those clones was estimated to be 0.2 to 2 X 10(5)/cell, which is comparable with the number of those on IL 2-dependent T cell clones. Expression of IL 2 receptor was confirmed in mRNA levels for both IC2 and LT4 cells. A relatively low level expression of one (4.5 Kb) of four IL 2 receptor mRNA species was observed with those IL 3-dependent clones compared with IL 2-dependent T cells. It seems that these low affinity receptors may be expressed on IL 3-dependent cells that undergo differentiation or maturation in mast cell and some myeloid cell lineages.     

Demonstration of up-regulated IL 2 receptor expression on an in vitro cloned BCL1 subline

We have established BCL1 CL-3 cells capable of responding to B15-TRF and interleukin 2 (IL 2). This clone has both high affinity and low affinity receptors for IL 2 (IL 2R), but IL 2 by itself did not stimulate either proliferation or immunoglobulin (Ig) secretion. B15-TRF, which possesses both growth and differentiation activity, causes an increase in size of CL-3 cells and renders CL-3 cells responsive to IL 2, including an increased expression of IL 2R (eight-fold to 10-fold) and the differentiation of CL-3 cells into Ig secretion (60 to 80% of cultured cells). CL-3 cells pretreated with B15-TRF for 12 hr become competent to respond to IL 2 by up-regulation of IL 2R within 12 hr. In contrast CL-3 cells pretreated with IL 2 for 12 hr required 24 hr B15-TRF stimulation to result in IL 2R up-regulation. Thus the ordered action of B15-TRF and IL 2 is the most effective operational pathway for the up-regulation of IL 2R. This IL 2-mediated IL 2R up-regulation and induction of Ig synthesis depends upon the concentration of IL 2 in the culture. Both responses seem to be caused by IL 2 molecules bound to high affinity IL 2R. However, the possibility of involvement of low affinity IL 2R can not be vigorously excluded. In fact the level of IL 2 required for a response is far higher than that needed for activated T cell proliferation. This cloned BCL1 subline promises to be a useful tool for studying the regulation and mechanisms of B cell responses.     

Synergistic and antagonistic effects of IL-1 alpha and IL-4, respectively, on the IL-2-dependent growth of a T cell receptor-gamma delta+ human T leukemia cell line

The TALL-103/2 cell line was derived from an immature acute T lymphocytic leukemia with T-myeloid differentiating capacity. The leukemic cells were first expanded in recombinant human IL-3 in which they acquired a myeloid phenotype, and subsequently were adapted to grow in human rIL-2 in which they became lymphoid committed. The TALL-103/2 cell line expresses only T cell-specific differentiation Ag (CD2, CD3, CD7, and CD8) but has retained the CD33 myeloid Ag originally present on the IL-3 expanded population. By using mAb directed at the TCR-alpha beta or specific for framework determinants on human TCR-gamma and -delta chains, the TALL-103/2 cells were shown to be WT31-, TCR delta 1+, TCS-1+, and Ti gamma A-, thus representing a T cell subset expressing the nondisulfide-linked form of the TCR-gamma delta. The TALL-103/2 cells have been maintained for more than 1 y in the presence of human rIL-2 on which they are strictly dependent. Chemical cross-linking and immunofluorescence studies indicate the presence of both high and intermediate affinity IL-2R on the TALL-103/2 cells. Whereas mAb antiTac and H-31 with reactivity to the IL-2R alpha-chain (p55) compete only partially for the IL-2-induced proliferation of these cells, mAb TU27, specific to the IL-2R beta-subunit (p75), inhibits such growth completely even at high concentrations of IL-2. The interactions of the two T cell-stimulating factors IL-1 and IL-4 on the IL-2-dependent growth of TALL-103/2 cells were investigated. IL-1 alpha synergizes with IL-2 in supporting the short and long term growth of this cell line, whereas IL-4 abrogates its growth. These effects are, at least in part, due to the modulation of IL-2R expression induced by the two lymphokines. Functionally, the TALL-103/2 cells display MHC-nonrestricted cytotoxic activity that is significantly enhanced by addition of either IL-4, IL-6, or IFN-gamma. Because of its properties and its stable requirement for IL-2 for continuous growth, this T lymphocytic leukemia-derived cell line represents an interesting model to analyze ontogeny and function of leukemic T cells.     

Activation by IL-2, but not IL-4, up-regulates the expression of the p55 subunit of the IL-2 receptor on IL-2- and IL-4-dependent T cell lines

We have investigated the effects of IL-2 and IL-4 on different parameters of T cell activation using three T cell lines. The Th cell line L14 and the cytotoxic T cell line C30.1, both grown in IL-2-containing medium, and a line derived from C30.1 cells (line 1) cultured in IL-4 for a prolonged period were studied. All three cell lines could be activated with IL-2 or IL-4. T cell stimulation by either IL-2- or IL-4-induced identical patterns of cell size enlargement and transferrin receptor expression. However, only IL-2 up-regulated cell-surface expression of the p55 subunit of the IL-2R (p55 IL-2R) as measured by flow cytometry and RIA. This difference was also reflected by the accumulation of soluble p55 IL-2R in the culture medium. No significant increase in expression of membrane or soluble forms of p55 IL-2R was detected after IL-4 stimulation. mAb specific for p55 IL-2R which block IL-2-induced T cell growth did not affect IL-4-mediated T cell proliferation indicating that p55 IL-2R is not involved in IL-4-mediated T cell growth. Analysis of IL-4R expression performed on line 1 using biotinylated IL-4 revealed that IL-4, but not IL-2, is capable of increasing IL-4R expression. Together these results suggest that during IL-2- or IL-4-induced T cell proliferation, each lymphokine specifically up-regulates its own receptor.     

Receptor-mediated endocytosis of interleukin 2 in a human tumor T cell line. Degradation of interleukin 2 and evidence for the absence of recycling of interleukin receptors

We have previously described a human tumor T cell line, IARC 301, which constitutively expresses biologically functional interleukin 2 (IL2) receptors. The fate of IL2 after binding to surface high affinity receptors was investigated. After a few minutes, IL2 is internalized at 37 degrees C and is subsequently degraded and released in the medium. The half-life for surface high affinity IL2 receptors, as measured in the presence of cycloheximide, is about 1 h and does not depend upon the presence of IL2. After trypsin digestion of cell surface high affinity receptors in the absence of protein biosynthesis, we could not detect any receptor reappearance on the cell membrane, whether IL2 was added or not. Taken together these results show that IL2 receptors are constantly internalized in these cells in the presence or absence of IL2 and that they do not recycle to the plasma membrane after receptor-mediated endocytosis.     

The tumor promoter teleocidin induces IL 2 receptor expression and IL 2-independent proliferation of human peripheral blood T cells

In testing the recently discovered tumor promoter teleocidin (TCD), we found that like phorbol esters, TCD was mitogenic to human peripheral blood lymphocytes (PBL) and preferentially stimulated sheep erythrocyte-rosetted (ER) T cell-enriched populations. Stimulation of PBL with TCD induced synthesis and expression of receptors for interleukin 2 (IL 2), as shown by dot-blot analysis with the use of a synthetic oligonucleotide probe, cell surface staining with anti-Tac antibody followed by fluorescence-activated cell sorter analysis, and a functional proliferation assay in which TCD-stimulated cells were washed free of TCD and were recultured with human recombinant IL 2 (rIL 2). Increased expression of cell surface markers after TCD stimulation of PBL is not general, because TCD did not affect the expression of Leu-2a antigen, and it also reduced the density of Leu-3a and Leu-4 antigens. Stimulation of cultured, IL 2 receptor-positive PBL with rIL 2, but not TCD, was blocked by anti-rIL 2 antibodies. Furthermore, IL 2-specific mRNA was not detected in TCD-stimulated PBL, demonstrating that IL 2 was not required for TCD-induced T cell proliferation. In addition, TCD replaced IL 2 in inducing short-term proliferation of IL 2-dependent murine cytotoxic T cell lines. The findings that TCD induced IL 2-independent proliferation of T cells, and TCD and IL 2 synergized in inducing T cell proliferation, suggest that they initiate T cell proliferation via different mechanisms. The IL 2-independent activation of T cells, and the induction of IL 2 receptor expression by TCD, may be related to its ability to activate protein kinase C in cell membrane.     

Soluble interleukin 2 receptors are released by long term-cultured insulin-specific T cells transiently after contact with antigen

An enzyme-linked immunosorbent assay was used to quantitate soluble interleukin 2 receptors (IL 2R) released by antigen-dependent, insulin-specific murine T cells into the culture supernatant, as well as cell-associated IL 2R present in cell lysates. IL 2R were released solely after T cell activation by antigen. The release of IL 2R was transient, reaching optimal levels within 72 hr after antigen challenge and gradually declining to background levels thereafter, when the cells were subcultured in IL 2-enriched medium. The decrease in the amount of IL 2R released during culture in IL 2-containing medium paralleled the decrement in cellular IL 2R detected in cell lysates, in cell surface-expressed IL 2R as determined by cytofluorometry, as well as in high-affinity IL 2R. In contrast, IL 2R were constitutively released by an IL 2-dependent T cell clone. Soluble IL 2R might exert an immunoregulatory function by competing with cellular IL 2R for IL 2 binding.     

Stimulation of immunoglobulin secretion in human B lymphocytes as a direct effect of high concentrations of IL 2

In certain human IgM and IgG cell lines, immunoglobulin (Ig) secretion is highly stimulated by a B cell inducing factor (BIF) that is free of interleukin 2 (IL 2). BIF also induces Ig secretion in purified peripheral blood B cell populations that have been mitogenically stimulated by Staphylococcus aureus bacteria. Low concentrations of IL 2 (less than 20 U/ml) are not active in these systems. We now show that IL 2 at concentrations above 100 U/ml can induce Ig secretion in these blood B cells and B cell lines. Both conventional IL 2, purified from the human JURKAT and gibbon MLA-144 cell lines, and recombinant IL 2 are active. Very high concentrations approaching 10(4) U/ml are optimal for Ig secretion. Antibody to the T cell IL 2 receptor, anti-Tac, did not inhibit stimulation of the IgM cell line SKW6.4 by IL 2, and no Tac antigen was detected on the cells. The 9B11 monoclonal anti-IL 2 antibody that neutralizes T cell growth activity also abrogates stimulation of Ig secretion by conventional and recombinant IL 2 in the SKW6.4 cell line. However, the 1H11 monoclonal anti-(conventional thr3-glycosylated IL 2), which does not neutralize T cell growth activity, does inhibit induction of Ig secretion by the corresponding IL 2 in the B cell line. These results suggest that IL 2 stimulates B cells via a low-affinity interaction with a receptor different from the Tac receptor identified on T cells, and that the active site on the IL 2 molecule for B cells differs from that for T cell targets. If IL 2 promotes Ig secretion by binding with a low affinity to the B cell BIF receptor, IL 2 and BIF could be homologous proteins.     

Production of interleukin 1 by adult T cell leukemia (ATL) cell lines

The accessory function for T cell activation and the production of interleukin 1 (IL 1) of adult T cell leukemia (ATL) cell lines were studied in vitro. ATL cell lines such as Hut-102, MT-1, and MT-2 functioned as accessory cells for the stimulation of human T cell proliferative response induced with concanavalin A (Con A) and induced allogeneic mixed lymphocyte reaction. Cell lysates of three ATL cell lines and the culture supernatant of MT-2 cells had activities to stimulate murine thymocyte proliferative response. Then we studied physicochemical properties of the factors produced by MT-2 cells. The m.w. of the factors were approximately 15,000 by Sephacryl S-200 column chromatography, and their isoelectric point values were 5.4 and 4.8 by chromatofocussing technique. No fraction contained interleukin 2 (IL 2) activities to stimulate IL 2-dependent murine cytotoxic T cell line. The thymocyte-stimulating activities of the factors were absorbed with rabbit anti-IL 1 alpha antiserum, but not with anti-IL 1 beta antiserum. Furthermore, messenger RNA extracted from MT-2 cells hybridized to complementary DNA of IL 1 alpha, but not of IL 1 beta, by Northern blot hybridization analysis. The factors from MT-2 cells could stimulate the production of IL 2 and the expression of IL 2 receptors of human T cells in the presence of Con A as well as recombinant IL 1 alpha and IL 1 beta did, and these activities were also blocked by rabbit anti-IL 1 alpha antiserum, but not by anti-IL 1 beta antiserum. These results suggest that the factors produced by MT-2 cells correspond to IL 1 alpha. However, the accessory function of MT-2 cells for T cell activation was not blocked by rabbit anti-IL 1 antiserum. These results suggest that ATL cell lines produce IL 1-like factors, but the accessory function of ATL cell lines for T cell activation is mediated by some other mechanisms rather than by secreted IL 1-like factors.     

Down-regulation of high affinity interleukin 2 receptors in a human tumor T cell line. Interleukin 2 increases the rate of surface receptor decay

We have described a human tumor T cell line, IARC 301, which constitutively expresses high affinity interleukin 2 (IL2) receptors, and showed that after binding to its receptors, IL2 is endocytosed and degraded. Here we present evidence that IL2 down-regulates its own high affinity receptors. Within 1 h, IL2 induces a 60% decrease in surface receptor expression. In order to maintain this down-regulation, IL2 concentration must be high enough for the receptors to be saturated throughout the incubation. The effect of IL2 on the kinetics of receptor internalization was investigated with two approaches. First, the initial rate of IL2 internalization was measured, and no difference could be detected whether the receptors were saturated with IL2 or only partially occupied. Second, the initial rate of surface receptor decay was followed and found to be significantly decreased in the presence of IL2. Although the half-life of IL2 receptors is very short in the absence of IL2, t 1/2 approximately 65 min, suggesting that these receptors are constantly endocytosed, it can still be reduced to t 1/2 approximately 25 min when the receptors are saturated with ligand. This suggests that occupied receptors are internalized faster than and independently from free receptors. The difference in internalization rates can explain the observed receptor down-regulation.     

Human interleukin‐23 receptor antagonists derived from an albumin‐binding domain scaffold inhibit IL‐23‐dependent ex vivo expansion of IL‐17‐producing T‐cells

Engineered combinatorial libraries derived from small protein scaffolds represent a powerful tool for generating novel binders with high affinity, required specificity and designed inhibitory function. This work was aimed to generate a collection of recombinant binders of human interleukin‐23 receptor (IL‐23R), which is a key element of proinflammatory IL‐23‐mediated signaling. A library of variants derived from the three‐helix bundle scaffold of the albumin‐binding domain (ABD) of streptococcal protein G and ribosome display were used to select for high‐affinity binders of recombinant extracellular IL‐23R. A collection of 34 IL‐23R‐binding proteins (called REX binders), corresponding to 18 different sequence variants, was used to identify a group of ligands that inhibited binding of the recombinant p19 subunit of IL‐23, or the biologically active human IL‐23 cytokine, to the recombinant IL‐23R or soluble IL‐23R‐IgG chimera. The strongest competitors for IL‐23R binding in ELISA were confirmed to recognize human IL‐23R‐IgG in surface plasmon resonance experiments, estimating the binding affinity in the sub‐ to nanomolar range. We further demonstrated that several REX variants bind to human leukemic cell lines K‐562, THP‐1 and Jurkat, and this binding correlated with IL‐23R cell‐surface expression. The REX125, REX009 and REX128 variants competed with the p19 protein for binding to THP‐1 cells. Moreover, the presence of REX125, REX009 and REX115 variants significantly inhibited the IL‐23‐driven expansion of IL‐17‐producing primary human CD4⁺ T‐cells. Thus, we conclude that unique IL‐23R antagonists derived from the ABD scaffold were generated that might be useful in designing novel anti‐inflammatory biologicals. Proteins 2014; 82:975–989. © 2013 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

In vitro induction and expression of interleukin 2 receptor in a clonal T helper cell differentiation model

Induction and expression of interleukin 2 (IL 2) receptor have been studied using a poly( Glu60 Ala30 Tyr10 ) (GAT)-specific T cell clone of mouse origin. This clone (52-3) has been characterized and it exhibits functional properties of T helper (TH) cells: it leads to a specific anti-DNP response in the presence of DNP-GAT and DNP-primed B cells and it secretes biological activities which can induce polyclonal B cell proliferation and IgM secretion. In vitro this clone mimics the activation stages of normal T lymphocytes and can be obtained under two states of differentiation. depending on the antigen-specific signal provided by antigen-presenting cells (APC). The expression of IL 2 receptor by this clone has been studied by (i) its response to IL 2, (ii) its capacity to absorb IL 2 bioactivity, and (iii) its reactivity with monoclonal antibody 7D4 specific for mouse IL 2 receptor. All the results indicate that the unstimulated state does not express the IL 2 receptor while the activated state does. Clone 52-3 has been compared with clone 14-1.6 that derives from a TH cell line and expresses the IL 2 receptor constitutively. 52-3 offers a good experimental model for studying in vitro, in a clonal TH cell population, the detailed mechanism of IL 2 receptor induction.     

Effect of interleukin 1 on the expression of interleukin 2 receptor (Tac antigen) on human natural killer cells and natural killer-like cell line (YT cells)

The effect of interleukin 1 (IL 1) on the expression of interleukin 2 receptor (IL 2R/Tac antigen) on human natural killer (NK) cells and the NK-like cell line, YT was studied with the use of a fluoresceinated anti-IL 2R monoclonal antibody and a Spectrum III flow cytofluorometer. IL 2R was expressed on approximately 10% of NK cells. The expression of IL 2R on NK cells was increased to approximately 25% by the in vitro culture with monocytes or IL 1 and to a less extent by the culture with IL 2 or interferon-gamma (IFN-gamma). IL 2R was expressed on approximately 50% of YT cells without any stimulations. The expression of IL 2R on YT cells was increased up to almost 100% by the culture with IL 1 or monocytes, but not with IL 2, IFN-alpha, IFN-beta, IFN-gamma, or lectins such as concanavalin A and phytohemagglutinin-P. IL 1 absorbed with YT cells or murine thymocytes lost both IL 1 activity detected by the stimulation of murine thymocyte proliferative response and enhancing activity of IL 2R expression on YT cells, suggesting that IL 1 has both activities. However, the assay system of the expression of IL 2R on YT cells is much more sensitive than the stimulation of murine thymocyte proliferative response. By the kinetic study, the enhancement of IL 2R expression was induced by only a 2-hr incubation of YT cells with IL 1. This enhancement did not proceed at 4 degrees C or by the treatment of YT cells with actinomycin D or cycloheximide, suggesting that this enhancement is energy dependent and requires the synthesis of RNA and protein but not DNA. Thus IL 1 plays an important role for the regulation of the expression of IL 2R on NK cells, and IL 1-dependent IL 2R expression on YT cells may give us a good model for the study of the molecular mechanism of the regulation of IL 2R expression.     

TCGF (IL 2)-receptor inducing factor(s). I. Regulation of IL 2 receptor on a natural killer-like cell line (YT cells)

A continuous cell line (YT cells) with inducible receptor for T cell growth factor (TCGF)/interleukin 2 (IL 2) was established from a 15-yr-old boy with acute lymphoblastic lymphoma and thymoma. YT cells were tetraploid, having 4q+ chromosomal markers, and proliferated continuously in vitro without conditioned medium (CM) or IL 2. They were weakly positive for OKT9, OKT11, and Tac antigen (Ag), a determinant closely associated with the receptor for IL 2 (IL 2-R), and were negative for OKT1, OKT3, OKT4, and OKT8 Ag. YT cells also expressed HNK-1 Ag and Fc receptors for IgG, which are expressed on natural killer (NK) cells. They retained a killing activity against human cell lines, including K562 (myeloid), T, and B cell lines. Unlike Tac Ag/IL 2-R(+) cell lines derived from adult T cell leukemia (ATL), YT cells were negative for HTLV, as proved by Southern blotting with cDNA for viral DNA. The expression of Tac Ag was markedly enhanced in 18 hr, when YT cells were incubated with CM from PHA-stimulated peripheral blood leukocytes (PBL) or spleen cells, as determined by immunofluorescence by using flow cytometry and binding assay with 125I-anti-Tac antibody (Ab). The binding study with 125I-labeled recombinant IL 2 showed 3.2 X 10(4) IL 2 receptor sites on YT cells precultured with CM. PHA-P and Con A neither agglutinate nor enhance the expression of IL 2-R/Tac antigen on these non-T cell line cells. Furthermore, neither recombinant IL 2 nor gamma-interferon could induce IL 2-R on YT cells, suggesting the presence of a unique IL 2-R inducing factor in PBL or spleen CM. Unlike Tac Ag on HTLV(+), ATL-derived cell lines (Hut-102, MT-1, ATL-2), the expression of Tac Ag on YT cells was down-regulated by anti-Tac Ab. The induction of Tac Ag/IL 2-R on YT cells seemed specific, because the enhancement of Tac Ag expression was not associated with that of Ia Ag and T9/transferrin receptor.     

Expression of interleukin 1 receptors on human peripheral T cells

The expression of interleukin 1 receptors (IL 1R) on human peripheral T cells was studied by the binding assay with 125I-labeled recombinant human interleukin 1 (IL 1) alpha and IL 1 beta and by the flow cytofluorometry with the fluorescein isothiocyanate (FITC)-conjugated IL 1 alpha. Peripheral blood lymphocytes expressed only few IL 1R without any stimulations. When they were stimulated with concanavalin A (Con A), IL 1R-positive cells began to increase by 4 hr, reached the maximum level at 48 hr, and then gradually decreased. The kinetics of the expression of IL 1 alpha R and IL 1 beta R showed the same pattern. Furthermore the binding of 125I-labeled IL 1 alpha to IL 1R on T cells was inhibited by the addition of either cold IL 1 alpha or IL beta, but not by interleukin 2 or interferons. The similar results were observed in the binding of 125I-labeled IL 1 beta. These results suggest that IL 1R on human peripheral T cells reactive for IL 1 alpha and IL 1 beta were identical. By Scatchard plot analysis, the numbers of IL 1R were estimated as 40 and 350 molecules per cell before and after Con A stimulation, respectively, and their Kd values were 3.1 X 10(-10) M and 2.8 X 10(-10) M. When purified T cells alone were stimulated with Con A, IL 1R were only marginally expressed. However, by the addition of monocytes, IL 1R were expressed on T cells in a dose-dependent manner. The maximum response was induced in the presence of 10% monocytes. The maximum IL 1R-positive T cells were approximately 30% by the detection of the flow cytofluorometry with FITC-conjugated IL 1 alpha. This enhancing activity of IL 1R expression on T cells by monocytes was inhibited by the addition of an anti-HLA-DR antibody or by the treatment of monocytes with the anti-HLA-DR antibody and complement. Furthermore T cell proliferative responses induced with IL 1 and Con A were also enhanced by the addition of HLA-DR-positive monocytes. These results suggest that IL 1R are expressed as the result of monocyte-T cell interaction in the early stage of T cell activation, and the expression of IL 1R on T cells and the responsiveness of T cells for IL 1 require the accessory function of HLA-DR-positive monocytes.