Functional IL-2 Receptors Research Articles

Upregulation by Retinoic Acid of Interleukin-2-Receptor mRNA in Human T Lymphocytes

It was previously demonstrated that retinoic acid (RA) can enhance the functional responses of human T lymphocytes by increasing surface IL-2Rα chain protein expression on proliferating T blasts, resulting in augmented IL-2-dependent growth. In the present study, we used IL-2- maintained lymphoblasts generated from human thymocytes to show that RA enhancement of IL-2Rα is accompanied by an increase in steady-state levels of IL-2Rα mRNA. This increase occurred within 1 hr after the addition of RA to the culture and was inhibited by the presence of the protein synthesis inhibitor cycloheximide. RA did not alter the stability of either IL-2Rα on the cell surface or IL-2Rα mRNA, suggesting regulation by RA at the level of transcription. This contention was supported by the demonstrated ability of RA to activate the IL-2Rα promoter in a chloramphenicol acetlytransferase plasmid construct that was transfected into the blast cells. In addition to inducing IL-2Rα expression. RA also increased the surface expression and mRNA levels of IL-2Rβ, the 75-kDa component of the IL-2 receptor that mediates IL-2 signal transduction. These new findings showing regulation by RA of both IL-2Rα and IL-2Rβ suggest multiple parthways by which this retinoid can modulate functional IL-2 receptors.

Expression of a functional p75 interleukin-2 receptor on lung lymphocytes from patients with human immunodeficiency virus type 1 (HIV-1) infection.

Lung involvement in patients affected by HIV-1 infection is characterized by an alveolitis sustained by the accumulation of CD8+ T lymphocytes. To investigate whether in situ T cell growth plays a relevant role in the pooling of CD8+ lymphocytes, we have analyzed the activity of two lymphokines involved in the mechanisms of T cell proliferation, i.e., interleukin-2 (IL-2) and interleukin-4. To this aim, following appropriate triggering and blocking, the expression and the functional role of IL-2 receptors (IL-2R) (both p55 and p75 chains) and IL-4 receptors have been analyzed on T lymphocytes obtained from the bronchoalveolar lavage (BAL) of 16 HIV-1+ patients. Molecular and phenotypic studies we performed demonstrated that CD8+ lymphocytes from the BAL of HIV-1 + patients strongly expressed the p75 chain of IL-2 receptor, while neither p55 mRNA nor its surface membrane product (Tac antigen) was detectable; in addition, there was no expression of IL-4 receptors. IL-2 stimulation was able to induce T cell growth in a dose-dependent manner, whereas IL-4 did not. Finally, using mAbs which specifically block the p55 or p75 IL-2R, we showed that both subunits of IL-2R were involved in the proliferative activity of lung lymphocytes. The results obtained in the present study directly demonstrate that BAL T lymphocytes of HIV-1 + patients express a fully functional IL-2 receptor apparatus, pointing to the role for this lymphokine in maintaining the alveolitis taking place in the lungs of AIDS patients.

Characterization of the interleukin 2 receptors (IL-2R) expressed on human natural killer cells activated in vivo by IL-2: association of the p64 IL-2R gamma chain with the IL-2R beta chain in functional intermediate-affinity IL-2R.

Interleukin 2 (IL-2) receptors expressed on the surface of activated T cells and natural killer (NK) cells exhibit a variety of affinity states depending on their subunit composition. Low-affinity binding is associated with a 55-kDa alpha chain, intermediate-affinity binding with a 70-75-kD beta chain, and high-affinity binding with a bimolecular complex of the alpha and beta subunits. In a previous study of the IL-2 receptors expressed on NK cells obtained from cancer patients after in vivo IL-2 therapy, we documented a discrepancy between the level of beta chain and the level of intermediate-affinity IL-2 binding sites expressed on the cell surface. Based on this result, we postulated that formation of intermediate-affinity receptor sites required a component in addition to the beta chain, and that this component was present at limiting levels in the patient NK cells. In the present study we have examined the structure of the intermediate-affinity receptor complex using monoclonal antibodies that recognize the beta chain, but that do not interfere with its ability to bind IL-2. Evidence is presented establishing the physical association of a novel protein of 64 kD with the beta chain in intermediate-affinity IL-2 binding sites. This molecule, termed IL-2R gamma chain, coprecipitated with beta chains prepared from cells that had been incubated with IL-2, but was undetectable in immunoprecipitates prepared in the absence of IL-2. Examination of gamma chain expression in post-IL-2 therapy NK cells, where only low levels of intermediate-affinity IL-2 binding were detectable, revealed that the gamma chain was associated with, on average, only 10-12% of the beta chains expressed on such cells. This contrasted with approximately equal levels of beta and gamma chain expression on YT cells, a cell line that has both high levels of cell surface beta chain expression and high levels of IL-2 binding. Thus, the ratio of gamma chain to beta chain present in the immunoprecipitates roughly correlated with the proportion of beta chain involved in intermediate-affinity receptor sites. This result suggests that the 64-kD gamma chain is the component responsible for regulating the affinity of IL-2 association with the beta subunit. By further defining the structural components necessary for IL-2 receptor formation, these studies provide additional insight into mechanisms whereby lymphocytes might regulate their responsiveness to IL-2.

Cloning of the gamma chain of the human IL-2 receptor.

A third subunit, the gamma chain, of the human interleukin-2 receptor (IL-2R) was identified, and a complementary DNA clone encoding this member of the cytokine receptor family was isolated. The gamma chain is necessary for the formation of the high- and intermediate-affinity receptors, which consists of alpha beta gamma heterotrimers and beta gamma heterodimers, respectively. The IL-2R on murine fibroblastoid cells can be internalized after binding IL-2 only if the gamma chain is present; alpha and beta are insufficient for internalization. Thus, the gamma chain is an indispensable component of the functional IL-2R.

Tunicamycin inhibits function and expression of the high-affinity IL-2 receptor in a murine IL-2-dependent cell line

Murine interleukin-2-dependent T-lymphocytes (CT6) were treated with tunicamycin, an inhibitor of both glycoprotein and ganglioside synthesis, to study the involvement of glycosylation in the IL-2 proliferative response. Tunicamycin inhibited proliferation in a dose-dependent manner at concentrations which did not inhibit protein synthesis (10–50 ng/ml). Swainsonine, a glycoprotein processing inhibitor, had no effect on proliferation. Inhibition of proliferation by tunicamycn was accompanied by an inhibition of binding of 125I-IL-2 to its high-affinity receptor. Scatchard analysis showed that receptor number was decreased by tunicamycin treatment. On the other hand, tunicamycin did not affect either the binding of the monoclonal antibody 7D4, specific for the 55 kDa low-affinity protein subunit of the IL-2 receptor, or the recycling of the IL-2 receptor. To determine the specific effects of tunicamycin on the biosynthesis of particular CT6 glycoconjugates, cells were radiolabeled with 3H-glucosamine and incorporation into ganglioside, neutral glycolipid and glycoprotein fractions was measured. Low doses of tunicamycin inhibited ganglioside synthesis and glycoprotein glycosylation to the same extent, whereas no effect on neutral glycolipid synthesis was observed. These results suggest that glycosylation of glycoprotein and/or gangliosides might play an important role in the formation of a functional high-affinity IL-2 receptor complex in CT6 cells.

An associated molecule, p64, with IL-2 receptor beta chain. Its possible involvement in the formation of the functional intermediate-affinity IL-2 receptor complex.

We identified previously a membrane molecule, p64, which co-precipitates with the IL-2R beta-chain in human T cells. We have now investigated the biologic significance of p64 in the formation of the functional IL-2R complex with cell lines transfected with cDNA of IL-2R alpha- and/or beta-chains. Two functional parameters associated with IL-2R, IL-2 binding ability and association of p64 with the beta-chain, were examined. Two subclones, MOLT beta-11 and MOLT beta-12, of an IL-2R beta cDNA-transfected MOLT4 clone expressed similar numbers of IL-2R beta molecules on cell surfaces and bound to IL-2 with intermediate affinity. However, the numbers of IL-2 binding sites were significantly lower than those of IL-2R beta molecules and considerably different between the two subclones. The amount of p64 co-precipitated with IL-2R beta was proportional to numbers of the IL-2 binding sites in the two subclones. In addition, neither p64 co-precipitation nor IL-2 binding was detected in HeLa and COS7 cells transfected with IL-2R beta, and no p64 precipitation was seen even in those transfectants with both IL-2R alpha and beta cDNAs, which bind to IL-2 with high affinity but are not able to transduce intracellular signals. These results suggest the possibility that p64 associates with IL-2R beta and has an important role in formation of the functional IL-2R complex.

Phenotype and function of lamina propria T lymphocytes

Lamina propria T cells have a low expression of the CD45RA antigen and a high expression of the CD45RO antigen. This phenotype is characteristic for memory T cells (table 2). In addition, T cells in the effector compartment of the mucosa bear surface antigens which are very rarely found in other sites of the immune system. Intestinal T cells also express functional IL-2 receptors and IL-2 receptor alpha chain mRNA, and are able to synthesize high amounts of IL-2. However, another marker of memory T cells, CD29, is not expressed in high density in the lamina propria indicating that lamina propria T cells differ from 'classical' memory T cells. This is supported by functional studies in nonhuman primates infected rectally with C. trachomatis which show that lamina propria T cells do not proliferate after stimulation with antigen but rather provide helper function for immunoglobulin synthesis (table 2). The intestinal lamina propria therefore contains highly specialized T cells which have a distinctive phenotype and are activated. Functionally these T cells can be characterized as differentiated effector lymphocytes which respond to triggering the antigen-specific T cell receptor by secreting helper factors for B cells. This concept is supported by recent studies showing that the pattern of lymphokines produced by lamina propria T cells and the responsiveness to certain lymphokines differ from those of other lymphocyte populations [25]. Lamina propria T cells thus represent a subset of memory T cells with a unique maturational state.

Role of interferon-? on the in vivo expression of functional interleukin-2 receptors by murine macrophages

Interferon-gamma activates both in vitro and in vivo macrophage functions. Injection of rat recombinant interferon-gamma (rR-IFN-gamma) induced the expression of interleukin-2 receptors (IL-2R) by peritoneal macrophages from normal BALB/c and MRL-+/+ mice. Moreover, rR-IFN-gamma stimulated in a dose-dependent manner the oxidative burst of cells as revealed by luminol-dependent chemiluminescene (LDCL). Resident peritoneal macrophages from MRL-lpr/lpr (mice that develop a systemic lupus-like syndrome) showed a higher PMA-triggered LDCL response. This enhanced activity was accompanied by an increase in IL-2R expression (30% vs. less than 1%). The "activated" macrophages from rR-IFN-gamma-treated normal mice as well as MRL-lpr/lpr mice did not respond to the addition of recombinant interleukin-2 (rHu-IL-2) by an increase in LDCL. However, rHu-IL-2 triggering became efficient when cells enriched in IL-2R-bearing macrophages were preincubated overnight with rHu-IL-2R. This response may point out a functional role for IL-2R and provide a role for IL-2 in certain macrophage functions.

Functional p75 interleukin‐2 receptor expression on the fresh blast cells in childhood acute lymphoblastic leukemia with natural killer cell properties

Neoplastic cells of childhood acute lymphoblastic leukemia (ALL) with natural killer (NK) cell properties were studied for the expression of p75 interleukin-2 receptors (IL-2R) and the receptor functions. Freshly prepared blast cells from a patient with ALL had NK cell properties: (1) the phenotype such as CD56+, CD2+, E-rosette+, CD3-, and CD19-; and (2) the presence of spontaneous cytotoxicity against NK-sensitive K562 target cells. Although p55 Tac antigen was not detectable, there was the expression of p75 IL-2R on the freshly prepared blast cells: 70% of the cells reacted with Mik-beta 1 monoclonal antibody against p75 IL-2R as determined by flow cytometry. Two-color flow cytometry revealed that the blast cells expressed both p75 IL-2R and NKH-1. NK activity of the blast cells was augmented by their treatment with 1,000 U/ml recombinant IL-2 (rIL-2): the cytotoxicity level as percentage lysis increased to 38.7% from 22.0% when the normal lymphocyte value increased to 62.1% from 46.2%. Although the blast cells possessed no apparent level of proliferative capacity, the addition of 1,000 U/ml rIL-2 yielded a 2.7-fold increase in their thymidine uptake. These results demonstrate the expression of functional p75 IL-2R on the patient's blast cells with NK cell properties.

Murine thymic nurse cell clone supports the growth of fetal thymocytes in the presence of interleukin 2.

To investigate the role of thymic nurse cells (TNC) in activation and differentiation of fetal CD4-CD8- (double-negative) thymocytes, we have co-cultured murine fetal thymocytes (14-15 days of gestation) with an established murine TNC clone. We show here that TNC induced the growth of the fetal double-negative thymocytes in the presence of recombinant interleukin 2 (rIL2). Activated fetal thymocytes markedly formed lymphocyte-TNC complexes and proliferated extensively after 5 days in the co-culture. The activated fetal thymocytes in this co-culture condition remained double negative after 10 days in culture. None of them gave rise to phenotypically and functionally competent lymphocytes during this period. TNC alone and the supernatant of TNC had no effect on activation. The presence of both TNC and rIL2 was necessary for the growth of fetal thymocytes in our system. The proliferation of fetal thymocytes was inhibited by a monoclonal antibody against mouse IL2 receptors (IL2R). The fetal thymocytes could be maintained further in this co-culture condition. The prolonged cultivation of fetal thymocytes resulted in the establishment of the fetal thymocyte line and its several clones. CD4 single-positive cells of activated fetal thymocytes first appeared 14 days after the onset of culture and their number increased, whereas CD8+ cells or CD4CD8 double-positive cells were not observed. These results indicate that fetal CD4-CD8- thymocytes underwent phenotypic change after long periods of culture. All established clones of fetal thymocytes are CD4 single positive showing lymphocyte-TNC interactions but do not express CD3 complex. Northern blot analysis detected mRNA for the gamma T cell receptor, but no messages for the delta, alpha or beta T cell receptor. Chemical cross-linking of 125I-labelled IL2 revealed that the 90-kDa band (presumably considered to be the IL2R beta chain) was clearly present in IL2-responsive fetal clones, whereas freshly isolated day 14-15 fetal thymocytes lacked the band. Taken together, TNC might be involved in the differentiation and/or expansion of murine fetal thymocytes by inducing IL2R beta chain, which forms the functional IL2R together with IL2R alpha chain and CD4, one of the T cell accessory molecules, on the cell surface through direct cell-cell interaction.

Different Nature of the Proliferation Defects of GLD, LPR and ME V C57BL/6 Mouse Lymphoid Cells

The three non-allelic gld, lpr and mev mutations in the mouse all lead to profound immunodeficiency besides a splenomegaly and a generalized autoimmunity. Spleen cells from young B6 gld, B6 lpr and B6 mev mice all display a decreased proliferative response to the T-cell mitogen concanavalin A (ConA), but the nature of the deficiency seems very different. No restoration of proliferation could be obtained by adding exogenous recombinant rIL2 to ConA-treated mev spleen cells, this lack of IL2-responsiveness suggesting a lack of (functional) IL2-receptors. In young mice of both gld and lpr strains, a B6 wild-type level of proliferation could be reached by rIl2 addition to ConA-treated spleen cells, this normal responsiveness to exogenous IL2 suggesting a normal expression of IL2-receptors. The endogenous IL2 production by ConA-treated spleen cells decreased very much with ageing in both B6 gld and B6 lpr mice. Yet, IL2 production in young mice revealed an earlier deficiency of the B6 lpr mice: the young B6 gld IL2 levels reached about 60% of age-matched B6 wild cell levels, but the B6 lpr levels reached 14% only. Finally the addition of exogenous rIL2 to ConA-pretreated cells from old B6 gld and B6 lpr mice, while enhancing the proliferative responses, could not restore the B6 wild-type levels. This suggests that, with ageing, the expression of functional IL2-receptors may become as abnormal in these gld and lpr mutants as it is from birth in the mev mutant mice.

A murine thymocyte clone expressing γδ T cell receptor mediates natural killer-like cytolytic function and TH1-like lymphokine production

The murine CD4 − CD8 − (double negative, DN) thymocyte cell line and clones expressing T cell receptor γδ chains in association with CD3 complex have been established and characterized. This line and a representative clone (DN7.12.11) which appear to derive from the minor population of CD3 + DN thymocytes can be stimulated to proliferate and to produce lymphokines by antiCD3 or anti-Thy-1 antibodies or calcium ionophore plus phorbol ester. Autocrine proliferation is dependent on binding of interleukin (IL)2 to functional IL2 receptor. Upon stimulation, these cells produce IL2 and IFN-γ but not IL4, resembling conventional CD4 + TH1 cells in this regard. The cloned line also mediates spontaneous cytolysis against a variety of tumor targets without regard for the presence of conventional major histocompatibility complex molecules on the target cell surface. Blocking and modulation experiments suggest that target recognition by the γδ/CD3 complex is not involved in the spontaneous lysis, resembling natural killer (NK) cells. The results suggest that γδ +DN T cells are able to have mature functions such as NK-like cytotoxicity and lymphokine secretion as peripheral γδ + T cells. They also provide a possible role of γδ + DN thymocytes in establishing a intrathymic environment for differentiation and selection of αβ-expressing T cells.

Functional consequences of interleukin 2 receptor expression on resting human lymphocytes. Identification of a novel natural killer cell subset with high affinity receptors.

In this study, we have used radiolabeled IL-2 binding assays, Northern blot analysis, immunofluorescent flow cytometry and cell sorting, as well as proliferation and cytotoxicity assays to perform an extensive phenotypic and functional characterization of the IL-2 receptor in normal resting human peripheral blood lymphocytes. Our results indicate that almost all T cells (greater than 98%) express neither the high affinity IL-2 receptor nor the functional intermediate affinity p75 chain of the IL-2 receptor without prior activation. In contrast, most NK cells constitutively express the isolated intermediate affinity p75 IL-2 receptor. In addition, a subpopulation of NK cells, distinguished by high density expression of the NKH1 antigen, constitutively express the high affinity IL-2 receptor, in addition to an excess of the isolated intermediate affinity p75 IL-2 receptor. These NKH1bright+ cells exhibit a brisk proliferative response to IL-2, similar to that seen with antigen-activated T cells, yet do so in the absence of any known antigenic stimuli. No other resting peripheral blood lymphocyte population, including CD4+, CD8+, and CD20 cells, exhibits this property. The intermediate affinity p75 IL-2 receptor, as it exists in its isolated form on resting NK cells, does not transduce a growth signal equivalent to that seen in NK cells expressing the high affinity IL-2 receptor, despite doses of IL-2 that are known to fully saturate the isolated p75 chain. This strongly suggests that additional structural or functional components are involved in generating the proliferative response following the binding of IL-2 to the high affinity heterodimeric form of the IL-2 receptor. The constitutive expression of this functional high affinity IL-2 receptor on a small population of resting NK cells provides further evidence in support of a role for these cells in the host's early defense against viral infection or malignant transformation, before the more delayed but specific T cell response.

Molecular signals in B cell activation: II. IL-2-mediated signals are required in late G 1 for transition to S phase after lonomycin and PMA treatment

We report that sustained increase of intracellular calcium ion concentration and protein kinase C (PKC) activation maintained throughout the G 1 phase of cell cycle do not provide sufficient signals to cause S-phase entry in rabbit B cells, and that additional signals transduced by IL-2 and IL-2 receptor interaction are essential for G 1 to S transition. We have shown earlier that rabbit B cells can be activated to produce IL-2 and express functional IL-2 receptors after treatment with ionomycin and PMA. Herein we have compared the response of rabbit PBLs, which contain about 50% T cells, with those of purified B cells. After activation with ionomycin or PMA, comparable numbers of PBLs and B cells entered the cell cycle; but DNA synthesis by the PBL cultures was three to four times higher than that of cultures of purified B cells. Interestingly, IL-2 production by the PBL cultures was also three to four times higher than in B cell cultures, suggesting an involvement of IL-2 in inducing DNA synthesis in these cells. The hypothesis that IL-2, which is produced in early G 1 acts in late G 1 and is required for G 1 to S transition in B cells was supported by the following observations: (i) IL-2 production by B cells was detected as early as 6 hr after activation and preceded DNA synthesis by at least 24 hr. (ii) B cell blasts in G 1 (produced by treatment of resting B cells with ionomycin and PMA) showed DNA synthesis in response to IL-2, but showed very little DNA synthesis in response to restimulation with ionomycin and PMA. (iii) A polyclonal rabbit anti-human IL-2 antibody caused nearly complete inhibition of DNA synthesis by B cells activated by ionomycin and PMA. (iv) A PKC inhibitor, K252b, inhibited DNA synthesis in ionomycin and PMA-stimulated cells if added at the beginning of culture but was not inhibitory if added 16 hr later. We conclude that increased [Ca 2+] i and PKC activation are not sufficient signals for G 1 to S transition in B cells; entry into S is signaled by IL-2, and IL-2-mediated signal transduction probably does not involve increased [Ca 2+] i or PKC activation.

Analysis of structure and function of IL-2 receptor .BETA. chain.

Analysis of structure and function of IL-2 receptor .BETA. chain.

Phorbol myristate acetate induces both high affinity and low affinity interleukin 2-receptors on a pre-B leukemic cell line

The human pre-B leukemic cell line Reh6 does not constitutively express the Tac molecule (p55), which is one of the two polypeptide chains of the interleukin 2-receptor (IL2-R). Cell incubation with Phorbol Myristate Acetate (PMA) induces expression of the Tac molecule in a dose and time-dependent manner. Binding experiments with radiolabelled recombinant IL2 (rIL2) revealed both high and low affinity IL2-R (225 ± 105 sites/cell with a K d of 130 ± 51 pM and 24060 sites/cell with a K d of 17.3 nM respectively), whereas unstimulated Reh6 cells only expressed intermediate affinity IL2-R (58 sites per cell with a K d of 460 pM). Cross-linking of radiolabelled rIL2 to PMA-incubated Reh6 cells revealed the presence of two polypeptide chains of mol. wts 55,000 (Tac molecule) and 70,000, as in normal activated T cells, while the 70,000 mol. wt chain alone was observed in unstimulated Reh6 cells. IL2-R-bearing Reh6 cells could absorb rIL2 in a dose-dependent manner and this absorption was inhibited by a monoclonal antibody against the Tac molecule (anti-Tac). Moreover, partial internalization of IL2 bound under high affinity conditions occurred at 37°C. IL2-R expressed on PMA-induced cells were functional since rIL2 specifically enhanced the proliferation in vitro of PMA-treated cells in semi-solid but not liquid cultures. These findings thus demonstrate an IL2-dependent mechanism of proliferation in vitro of pre-B leukemic cells induced by PMA, which can express high affinity, functional IL2-R.

High affinity IL-2 receptors on a Hodgkin's derived cell line

Hodgkin and Sternberg Reed (H and SR) cells, the putative malignant cells of Hodgkin's disease carry regularly T-cell activation antigens, like CD30 and CD25 (low affinity IL-2 receptor). We have investigated the Hodgkin cell line L540, bearing characteristic markers of H and SR cells for its expression of the low affinity IL-2 receptor (IL-2R) and for IL-2. Expression of the low affinity IL-2R was found on mRNA level, by detection of specific 3.5 kb and 1.4 kb mRNA and on the protein level by immunoprecipitation of a 55,000 mol. wt molecule from detergent extracts of surface iodinated cells, however IL-2 specific mRNA was not detected. Scatchard plot analysis revealed the presence of 2 × 10 3 high affinity IL-2Rs. Crosslinking experiments directly demonstrated the high affinity IL-2R to consist of the 55,000 mol.wt light chain (L), and the 70/75,000 (H1/H2) heavy chains. IL-2 was rapidly internalized by these receptors, suggesting that they can be functional. The expression of functional IL-2Rs might be involved in induction or differentiation of Hodgkin's disease.

Reconstitution of a functional IL-2 receptor by the beta-chain cDNA. A newly acquired receptor transduces negative signal.

The high-affinity IL-2R results from the noncovalent association between at least two subunits; alpha (p55) and beta (p70), both of which are capable of binding IL-2 with a low and intermediate affinity, respectively. Although the alpha-chain itself has been shown to be nonfunctional, suggestions have been made that the beta-chain mediates an IL-2 signal. To directly study the role of the beta-chain in the signal transduction, we transfected with the cDNA encoding the IL-2R beta-chain a human T lymphotropic virus-I-transformed T cell line, MT-1 originally expressing low-affinity alpha-chain alone, and established a stable transformant (designated MT-beta 7) which expressed both alpha- and beta-chains simultaneously. We showed 1) MT-beta 7 manifested the high-affinity IL-2 binding, which was completely disrupted by the anti-beta chain mAb (Mik-beta 1), 2) the 125I-IL-2 crosslinking patterns of MT-beta 7 were indistinguishable from those of cells expressing the native high-affinity IL-2R, 3) MT-beta 7, but not parental MT-1, internalized the bound IL-2 and responded to IL-2 with a negative signal, i.e., inhibition of the de novo DNA synthesis. These results clearly demonstrate that the beta-chain not only participates in forming the high-affinity IL-2R with the alpha-chain but also is directly involved in the IL-2 signal transduction.

T-Cell Ontogeny After Autologous Bone Marrow Transplantation: Failure to Synthesize Interleukin-2 (IL-2) and Lack of CD2- and CD3-Mediated Proliferation by Both CD4+ and CD8+ Cells Even in the Presence of Exogeneous IL-2

T cells generated during a second round of ontogeny after autologous bone marrow transplantation (ABMT) represent a unique model of early T-cell ontogeny in an autologous situation. Since grafted bone marrows were pretreated in vitro with the cyclophosphamide derivative ASTA Z 7557, circulating T cells had to be regenerated from reinfused hematopoietic progenitor cells. The T-cell population derived from 25 patients post-ABMT was phenotypically characterized: an increase in CD8+ cells, a low percentage of CD4+ cells, and a median of 12% CD56+ (NKH1+) cells were found. When the T cells were stimulated with phytohemagglutinin (PHA) and phorbol myristate acetate (PMA), defective interleukin-2 (IL-2) secretion was observed. In addition, proliferative responses of the T cells after activation through the antigen-receptor-dependent CD3 pathway, through the CD2 dependent alternative T-cell pathway, and by the lectin PHA were investigated. Despite the presence of CD2, CD3, alpha/beta chains of the T-cell receptor, and CD25+ IL-2 surface receptors, abnormal proliferative responses were obtained even in the presence of exogeneous IL-2. In experiments where the T-cell population was separated into CD4+ cells and CD8+ cells, both the CD4- and CD8+ subsets were unable to respond to activating and proliferating signals. Thus, T cells at early stages of ontogeny not only possess an intrinsic defect in IL-2 synthesis but, in addition, were unable to express functional IL-2 receptors in response to mitogenic stimuli.

T-cell ontogeny after autologous bone marrow transplantation: failure to synthesize interleukin-2 (IL-2) and lack of CD2- and CD3-mediated proliferation by both CD4- and CD8+ cells even in the presence of exogeneous IL-2

T cells generated during a second round of ontogeny after autologous bone marrow transplantation (ABMT) represent a unique model of early T- cell ontogeny in an autologous situation. Since grafted bone marrows were pretreated in vitro with the cyclophosphamide derivative ASTA Z 7557, circulating T cells had to be regenerated from reinfused hematopoietic progenitor cells. The T-cell population derived from 25 patients post-ABMT was phenotypically characterized: an increase in CD8+ cells, a low percentage of CD4+ cells, and a median of 12% CD56+ (NKH1+) cells were found. When the T cells were stimulated with phytohemagglutinin (PHA) and phorbol myristate acetate (PMA), defective interleukin-2 (IL-2) secretion was observed. In addition, proliferative responses of the T cells after activation through the antigen-receptor- dependent CD3 pathway, through the CD2 dependent alternative T-cell pathway, and by the lectin PHA were investigated. Despite the presence of CD2, CD3, alpha/beta chains of the T-cell receptor, and CD25+ IL-2 surface receptors, abnormal proliferative responses were obtained even in the presence of exogeneous IL-2. In experiments where the T-cell population was separated into CD4+ cells and CD8+ cells, both the CD4- and CD8+ subsets were unable to respond to activating and proliferating signals. Thus, T cells at early stages of ontogeny not only possess an intrinsic defect in IL-2 synthesis but, in addition, were unable to express functional IL-2 receptors in response to mitogenic stimuli.