IL-2-dependent proliferation of murine T cells requires expression of both the p55 and p70 subunits of the IL-2 receptor

An IL-4-dependent T cell clone (LD8) was isolated from the murine IL-2-dependent cytotoxic T cell line C30.1. This clone has lost the capacity to proliferate in response to IL-2 after long-term culture in IL-4. LD8 cells express the p70, but not the p55, subunit of the IL-2R on their cell surface. The number of p70 IL-2R molecules on LD8 cells is comparable with the number of high-affinity IL-2R on the parental C30.1 cell line. LD8 cells can efficiently internalize IL-2 through the p70 IL-2R subunit. Following stimulation by IL-2, LD8 cells up-regulate p70 IL-2R mRNA, but do not express p55 IL-2R mRNA. IL-2-dependent proliferation of LD8 cells was reconstituted after introduction and expression of a human p55 IL-2R cDNA. To further investigate the role of p70 IL-2R, we have measured IL-2-induced proliferation of C30.1 cells in the presence of three anti-p55 IL-2R mAb (5A2, PC61, and 7D4) that recognize different epitopes. Under the experimental conditions used, the combination of anti-p55 IL-2R mAb prevents the formation of high-affinity IL-2R, but does not affect the binding of IL-2 to p70 IL-2R or IL-2 internalization. However, these three mAb inhibit proliferation of C30.1 cells even in the presence of IL-2 concentrations sufficient to saturate p70 IL-2R. Together these results demonstrate that p70 IL-2R alone is not sufficient to transmit IL-2-induced growth signals and that formation of p55-p70 IL-2R complex is required for IL-2-dependent proliferation of murine T cells.     

Cloning and expression of murine IL-12.

Human IL-12 (NK cell stimulatory factor, cytotoxic lymphocyte maturation factor) is a heterodimeric cytokine that can act as a growth factor for activated human T and NK cells, enhance the lytic activity of human NK/lymphokine-activated killer cells, and stimulate the production of IFN-gamma by resting human PBMC. Because in our hands, human IL-12 did not elicit similar responses in murine lymphocytes, we have cloned and expressed the murine IL-12 subunit cDNA in order to obtain recombinant protein for murine studies. Comparison of the predicted amino acid sequences of the murine subunits with their human counterparts revealed that the p40 subunits are more highly conserved than the p35 subunits (70% vs 60% identity, respectively). The sizes of the p35 and p40 subunit mRNA were estimated to be 1.5 kb and 2.6 kb, respectively. RNA blot analysis showed that p35 mRNA was expressed in lymphoid tissues (spleen, thymus) and nonlymphoid tissues (lung, brain), whereas p40 mRNA expression was only detected in lymphoid cells. Incubation of splenocytes with pokeweed mitogen did not significantly affect p35 mRNA levels, however, it resulted in a decrease of p40 mRNA. Coexpression of the murine p35 and p40 cDNA clones in COS cells resulted in the secretion of IL-12, which was active in human and mouse T cell proliferation, murine NK cell activation, and murine IFN-gamma induction assays. Transfection of each subunit cDNA alone did not result in measurable secreted IL-12 activity. A hybrid heterodimer consisting of murine p35 and human p40 subunits retained bioactivity on murine cells; however, the combination of human p35 and murine p40 was completely inactive on murine cells. These results indicate that the observed inability of human IL-12 to act on murine cells is largely determined by the p35 subunit.     

Production of lymphokine mRNA by CD45R+ and CD45R- helper T cells from human peripheral blood and by human CD4+ T cell clones

The expression of lymphokine mRNA by human CD4+CD45R+ and CD4+CD45R- Th cells was assessed after mitogen stimulation. These Ag have previously been shown to relate closely to virgin and primed T cells, respectively. CD4+CD45R+ (virgin) and CD4+CD45R- (primed) cell fractions were isolated by sorting double-labeled cells with a fluorescence-activated cell sorter. CD4+CD45R+ cells produced high levels of IL-2 mRNA when stimulated with either PMA together with calcium ionophore, or with PHA, but they expressed only trace quantities of mRNA for IL-4 or IFN-gamma. In contrast, CD4+CD45R- cells produced high levels of mRNA for IL-2, IL-4, and IFN-gamma. After 14 days of continuous culture, CD4+CD45R+ Th cells lost expression of the CD45R Ag, but gained high level expression of CDw29, such that they were indistinguishable from the cell population which originally expressed this Ag. At the same time, they acquired the ability to synthesize IL-4 mRNA. It seemed likely that the broad lymphokine profile of primed Th cells might mask clonal heterogeneity. Analysis of 122 CD4+ T cell clones showed that all of them synthesized IL-2 mRNA. One clone failed to express IL-4 mRNA, but did produce those for IL-2 and IFN-gamma. A total of 34 of the clones was investigated to determine expression of IFN-gamma mRNA; two of these clones were negative for IFN-gamma mRNA, and both expressed IL-2 and IL-4 message. These data suggest that while fresh virgin and primed peripheral blood T cells show a clear resolution of lymphokine production, a simple subdivision of human CD4+ T cell clones on the basis of their lymphokine production (such as that reported for mouse Th cell clones) is not possible.     

Structure-function study of the p55 subunit of murine IL-2 receptor by epitope mapping.

Using a cell-free translation system we have expressed the Mr 55,000 subunit of the murine IL-2R (p55 IL-2R), which binds IL-2 with low affinity (Kd = 10 nM). Mutants and truncated forms of p55 IL-2R have been used to map the epitopes recognized by three anti-p55 IL-2R mAb: 135D5, 7D4, and 2E4. The mAb 135D5 inhibits IL-2 binding to p55 IL-2R and recognizes an epitope located between amino acids 64 to 125. This epitope can be mimicked by a synthetic peptide corresponding to the region defined by residues 72 to 88. However, the mAb 7D4 and 2E4 do not affect the IL-2 binding to p55 IL-2R. These mAb recognize an epitope of p55 IL-2R lying between residues 125 to 212 that can be mimicked with a peptide corresponding to amino acids 188 to 208. A strong correlation emerged between the experimental results on epitope mapping and predictions of potential antigenicity of murine p55 IL-2R. In addition, we described two internal initiation sites of p55 IL-2R mRNA under the in vitro conditions used leading to the production of significant amounts of N-terminal truncated p55 IL-2R proteins.     

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.     

The murine interleukin 1 beta gene: structure and evolution.

We have isolated from a genomic library a murine recombinant clone containing the gene coding for interleukin-1 beta m-RNA. A 7000 b.p. DNA fragment has been sequenced. Sequences homologous with human IL-1 beta cDNA have been found distributed within 7 exons. The translation of these sequences allows the prediction of a protein 269 aminoacids long. Hybridization of P388D1 RNA from cells stimulated with phorbol myristic acetate with a genomic DNA probe shows the existence of a 1.6 Kb murine IL-1 beta mRNA which is absent in the unstimulated cells. The comparative analysis between the murine IL-1 beta and the human IL-1 alpha genes shows extreme conservation of the aminoacids at the exon junctions. This observation together with the similarity in number and size of the exons suggests that these genes have diverged from a common ancestor.     

Functional heterogeneity among human inducer T cell clones

Analysis of mouse CD4+ inducer T cells at the clonal level has established that a dichotomy among CD4+ T cell clones exists with regard to types of lymphokines secreted. Mouse T cell clones designated Th1 have been shown to secrete IL-2 and IFN-gamma, whereas T cell clones designated Th2 have been shown to produce IL-4 but not IL-2 or IFN-gamma. To determine if such a dichotomy in the helper inducer T cell subset occurred in man, we examined a panel of human CD4+ helper/inducer T cell clones for patterns of lymphokine secretion and for functional activity. We identified human T cell clones which secrete IL-4 but not IL-2 or IFN-gamma, and which appeared to correspond to murine Th2 clones. In marked contrast to murine IL-2 secreting Th1 clones which do not produce IL-4 or IFN-gamma, we observed that some human T cell clones secrete IL-2, and IFN-gamma as well as IL-4. Southern blot analysis indicated that these multi-lymphokine-secreting clones represented the progeny of a single T cell. IL-4 secretion did not always correlated with enhanced ability to induce Ig synthesis. Although one T cell clone which secreted IL-2, IL-4, and IFN-gamma could efficiently induce Ig synthesis, another expressed potent cytolytic and growth inhibitory activity for B cells, and was ineffective or inhibitory in inducing Ig synthesis. These results indicate that although the equivalent of murine Th2 type cells appears to be present in man, the simple division of T cells into a Th1 and Th2 dichotomy may not hold true for human T cells.     

Molecular structure of the human argininosuccinate synthetase gene: occurrence of alternative mRNA splicing.

The human genome contains one expressed argininosuccinate synthetase gene and ca. 14 pseudogenes that are dispersed to at least 11 human chromosomes. Eleven clones isolated from a human genomic DNA library were characterized extensively by restriction mapping, Southern blotting, and nucleotide sequencing. These 11 clones represent the entire expressed argininosuccinate synthetase gene that spans 63 kilobases and contains at least 13 exons. The expressed gene codes for two mRNAs that differ in their 5' untranslated sequences and arise by alternative splicing involving the inclusion or deletion of an entire exon. In normal human liver and cultured fibroblasts, the predominant mature argininosuccinate synthetase mRNA lacks sequences encoded by exon 2 in the expressed gene. In contrast, the predominant argininosuccinate synthetase mRNA in baboon liver contains exon 2 sequences. A transformed canavanine-resistant human cell line in which argininosuccinate synthetase activity is 180-fold higher than that in wild-type cells contains abundant amounts of both forms of the argininosuccinate synthetase mRNA. The mRNA lacking exon 2 sequences is the more abundant mRNA species in the canavanine-resistant cells. These observations show that splicing of the argininosuccinate synthetase mRNA is species specific in primates and varies among different human cell types.     

Murine B lymphoma cell lines release functionally active interleukin 2 after stimulation with Staphylococcus aureus

In this study it is shown that an IL-2-like functional lymphokine activity derived from the murine B cell lines 2PK-3 and L10A2J after stimulation with Staphylococcus aureus can be blocked with anti-IL-2 mAb such as 9B11, DMS-1, and S4B6. Experiments demonstrate that the stimulation of the IL-2-sensitive cell line CTLL-2 by the IL-2-like functional activity derived from murine B cell tumors can also be blocked with the anti-IL-2R mAb PC61. Additionally, in RNA-RNA hybridization experiments with radiolabeled SP6-derived ssRNA probes developed from murine IL-2 genomic DNA sequences and specific for IL-2 mRNA, quantitatively significant amounts of IL-2-specific mRNA in both 2PK-3 and L10A2J are shown subsequent to stimulating the cells with S. aureus. These results suggest the murine B cell tumor lines 2PK-3 and L10A2J synthesize and release IL-2 after stimulation with selected polyclonal activators such as S. aureus.     

Nucleotide sequence of mouse IL-2 receptor cDNA and its comparison with the human IL-2 receptor sequence.

We have cloned cDNA encoding the mouse interleukin-2 (IL-2) receptor from a murine T cell line, CTLL using human IL-2 receptor cDNA as probe. COS 7 cells transfected with the cDNA expressed the antigen recognized by the monoclonal antibody against the murine IL-2 receptor. The cDNA identified 4 species of mRNA (4.5, 3.5, 2.2 and 1.5 kb) of the mouse IL-2 receptor in CTLL cells. Difference in the length of mRNA seems to be ascribed to the variable length of the 3' untranslated sequence. Total nucleotide sequence (approximately 1400 bp) of this cDNA was determined and compared with the human receptor. The nucleotide and amino acid sequences of the IL-2 receptor are 70% and 60%, respectively, homologous in average between the two species. The comparison has revealed several conserved regions localized to particular exons such as transmembrane and cytoplasmic portions, suggesting that these regions are important for receptor function and its regulation.     

Distribution and ontogeny of CD2 expression by murine T cells

We have raised a polyclonal antiserum to murine CD2 by immunization of a rabbit with a synthetic peptide corresponding to a hydrophilic sequence in the extracellular domain of the murine CD2 gene. The antiserum immunoprecipitates a 55 kDa protein, consistent with the size predicted by the cDNA sequence. Flow microfluorometric analysis of a panel of T cell tumors and clones demonstrated concordance of reactivity of intact cells with the anti-CD2 serum and the presence of CD2 mRNA. Surprisingly, although splenic T cells were found to uniformly express high levels of CD2, several of a panel of functional T cell clones were found to lack CD2 expression. This suggests that the clones lost CD2 upon in vitro cultivation, and may not be required for activation or maintenance in culture. Adult thymocytes exhibited heterogeneous expression of CD2. The majority of CD4-8- thymocytes expressed low levels and CD4+8+ thymocytes intermediate levels, whereas all CD4+8- and the majority of CD4-8+ thymocytes expressed high levels of CD2. Multiparameter analysis of CD2 expression and that of CD3, CD5, JIId, and IL-2R p55 chain showed that expression of CD2 correlates with the maturational state of thymocytes. Finally, analysis of fetal thymuses from timed pregnancies revealed that expression of CD2 is preceded by that of IL-2R p55 chain.