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Combination therapy with PD-1 blockade and IL-2 is highly effective during chronic lymphocytic choriomeningitis virus infection(1). Here we examine the underlying basis for this synergy. We show that PD-1 + IL-2 combination therapy, unlike PD-1 monotherapy, dramatically changes the differentiation program of the PD-1(+)TCF-1(+) stem-like CD8(+) T cells and results in the generation of transcriptionally and epigenetically distinct effector CD8(+) T cells that resemble highly functional effector CD8(+) T cells seen after an acute viral infection. The generation of these qualitatively superior CD8(+) T cells that mediate viral control underlies the synergy between PD-1 and IL-2. Our results show that the PD-1(+)TCF-1(+) stem-like CD8(+) T cells, also referred to as precursors of exhausted CD8(+) T cells, are not fate-locked into the exhaustion program and their differentiation trajectory can be changed by IL-2 signals. These virus-specific effector CD8(+) T cells emerging from the stem-like CD8(+) T cells after combination therapy expressed increased levels of the high affinity IL-2 trimeric (CD25, CD122, CD132) receptor. This was not seen after PD-1 blockade alone. Finally, we show that CD25 engagement with IL-2 plays an important role in the observed synergy between IL-2 cytokine and PD-1 blockade. Either blocking CD25 with an antibody or using a mutated version of IL-2 that does not bind CD25 but still binds CD122/CD132 almost completely abrogated the synergistic effects seen after PD-1 + IL-2 combination therapy. There is currently considerable interest in PD-1 + IL-2 combination therapy for patients with cancer(2,3) and our fundamental studies defining the underlying mechanisms of how IL-2 synergizes with PD-1 blockade should inform these human translational studies.

Differential interleukin-2 (IL-2) signaling and production are associated with disparate effector and memory fates. Whether the IL-2 signals perceived by CD8 T cells come from autocrine or paracrine sources, the timing of IL-2 signaling and their differential impact on CD8 T cell responses remain unclear. Using distinct models of germline and conditional IL-2 ablation in post-thymic CD8 T cells, this study shows that paracrine IL-2 is sufficient to drive optimal primary expansion, effector and memory differentiation, and metabolic function. In contrast, autocrine IL-2 is uniquely required during primary expansion to program robust secondary expansion potential in memory-fated cells. This study further shows that IL-2 production by antigen-specific CD8 T cells is largely independent of CD4 licensing of dendritic cells (DCs) in inflammatory infections with robust DC activation. These findings bear implications for immunizations and adoptive T cell immunotherapies, where effector and memory functions may be commandeered through IL-2 programming.

The cytokine IL-2 is critical for promoting the development, homeostasis, and function of regulatory T (Treg) cells. The cellular sources of IL-2 that promote these processes remain unclear. T cells, B cells, and dendritic cells (DCs) are known to make IL-2 in peripheral tissues. We found that T cells and DCs in the thymus also make IL-2. To identify cellular sources of IL-2 in Treg cell development and homeostasis, we used Il2FL/FL mice to selectively delete Il2 in T cells, B cells, and DCs. Because IL-15 can partially substitute for IL-2 in Treg cell development, we carried out the majority of these studies on an Il15−/− background. Deletion of Il2 in B cells, DCs, or both these subsets had no effect on Treg cell development, either in wild-type (WT) or Il15−/− mice. Deletion of Il2 in T cells had minimal effects in WT mice but virtually eliminated developing Treg cells in Il15−/− mice. In the spleen and most peripheral lymphoid organs, deletion of Il2 in B cells, DCs, or both subsets had no effect on Treg cell homeostasis. In contrast, deletion of Il2 in T cells led to a significant decrease in Treg cells in either WT or Il15−/− mice. The one exception was the mesenteric lymph nodes where significantly fewer Treg cells were observed when Il2 was deleted in both T cells and DCs. Thus, T cells are the sole source of IL-2 needed for Treg cell development, but DCs can contribute to Treg cell homeostasis in select organs.

Interleukin (IL)-2 is a pleiotropic cytokine that is necessary to prevent chronic inflammation in the gastrointestinal tract

The inhibitory receptor programmed cell death 1 (PD-1) plays a major role in functional exhaustion of T cells during chronic infections and cancer, and recent clinical data suggest that blockade of the PD-1 pathway is an effective immunotherapy in treating certain cancers. Thus, it is important to define combinatorial approaches that increase the efficacy of PD-1 blockade. To address this issue, we examined the effect of IL-2 and PD-1 ligand 1 (PD-L1) blockade in the mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection. We found that low-dose IL-2 administration alone enhanced CD8+ T cell responses in chronically infected mice. IL-2 treatment also decreased inhibitory receptor levels on virus-specific CD8+ T cells and increased expression of CD127 and CD44, resulting in a phenotype resembling that of memory T cells. Surprisingly, IL-2 therapy had only a minimal effect on reducing viral load. However, combining IL-2 treatment with blockade of the PD-1 inhibitory pathway had striking synergistic effects in enhancing virus-specific CD8+ T cell responses and decreasing viral load. Interestingly, this reduction in viral load occurred despite increased numbers of Tregs. These results suggest that combined IL-2 therapy and PD-L1 blockade merits consideration as a regimen for treating human chronic infections and cancer.

The molecular mechanisms responsible for the exquisite discrimination between self and nonself molecules have remained enigmatic despite intense investigation. However, with the availability of adequate amounts of anergic lymphocytes produced by double transgenic mice, large numbers of immature B cells from sublethaly irradiated, hematopoietically-synchronized mice, as well as critical gene-deleted mice, it has been possible for the first time to uncover plausible molecular mechanisms that lead to tolerance versus immunity. The Rel family of transcription factors is expressed at different stages of lymphocyte maturation and differentiation. C-Rel is not activated by immature lymphocytes, which undergo either anergy or apoptosis when triggered by antigen receptors, but c-Rel is activated in mature lymphocytes. Antigen receptor triggering induces c-Rel-dependent survival and proliferative genetic programs. In T cells, a critical c-Rel-dependent gene encodes the T-cell growth factor interleukin-2 (IL-2). Thus, T cells from c-Rel gene-deleted mice produce inadequate quantities of IL-2, which renders them immunocompromised and unable to mount normal T-cell proliferative and differentiative responses. In the face of absolute IL-2 deficiency from birth, severe, multiorgan autoimmunity gradually ensues. Also, with more subtle IL-2 deficiency, organ/tissue-specific autoimmune disease becomes evident. Accordingly, both c-Rel and IL-2 appear to be key molecules for tolerance versus immunity, and doubtless will become foci for continued investigation, as well as future therapeutic targets in autoimmune diseases.

The regulation of the tempo, magnitude, and duration of the immune response has been thought to reside solely with antigen for the past 50 years. However, with the discovery of the interleukins (ILs) 30 years ago, it became evident that these endogenous 'lymphocytotrophic hormones' provide the molecular mechanisms via classic hormone-receptor interactions. However, lacking in the hormonal regulatory capacity of the ILs were negative feedback mechanisms that functioned to switch off the positive driving force of the immune response, whether after antigen was cleared or when antigen persists, as with auto-antigens, tumor antigens, persistent infections, or allografts. Our recent experimental data, reviewed herein, exploring the T-cell antigen receptor (TCR) induction of the negative transcriptional regulator, forkhead box protein 3 (FOXP3), indicate that its expression is signaled by the T-cell growth factor IL-2. Once expressed, FOXP3 functions to restrict IL-2 expression in reaction to continued TCR stimulation. Thus, IL-2 regulates it own levels via a FOXP3-mediated negative feedback loop. In contrast, we found no evidence that FOXP3(+) cells actively suppress IL-2 expression, thereby failing to support the notion that such cells regulate potential effector cells.

Having created unique and novel cellular and molecular reagents, we could approach questions that had perplexed investigators interested in cell growth for over 50 years, i.e., the basis for variable cell cycle transit times of individual cells among genetically homogeneous cell populations (1). Studies of all cell populations, both prokaryote and eukaryote, revealed that within a cell population, the cell cycle times of individual cells follow a normal distribution when examined as a function of the division rate (the rate-normal distribution). Thus, some cells proliferate slowly while others proliferate faster, with most cells distributed about the mean on a log-linear plot. Prior to the discovery of the IL-2 molecule (2) and IL-2 receptors (IL-2R) (3), T cell populations were known to follow this rate-normal distribution, common to all living cells. Mathematical analysis of proliferating cell populations indicated that individual cell variability in cell cycle transit times was stochastic, depending upon a “hidden variable.” However, once the “hidden” molecular variables of T cell cycle proliferation had been identified (the IL-2 concentration, IL-2R density, affinity of the IL-2/IL-2R interaction, and the duration of the IL-2/IL-2R interaction), experiments could be preformed for the first time revealing that as long as these crucial characteristics of T cell cycle progression were known, then the variability of cell cycle transit times were entirely predictable and deterministic, not probabilistic or left to chance. These experiments and approaches were possible because of painstaking attention to the creation of critical cell clones and homogeneous purified IL-2 molecules, as well as the development of the radiolabeled IL-2 binding assay, and monoclonal antibodies reactive with both IL-2 and its receptor. Moreover, the employment of the flow cytometer allowed us to proceed beyond studies of cell populations to quantify the intermolecular interactions of individual cells for the first time. Doreen Cantrell, a postdoctoral fellow skilled in flow cytometry, was critical to our experimental approach. Because the growth characteristics of all known cell populations are identical to those of T cell populations, it followed that individual cells of all other cell populations would demonstrate the same type of molecular determinants. Thus, the title of this article: “The interleukin-2 T cell system: A new cell growth model,” a new universal paradigm in cell and molecular biology (1). The significance of these findings was obvious. As cells of all tissues, especially of metazoans, have identical growth characteristics of T cells, it followed that the cells of all metazoans are regulated in the same molecular fashion, i.e., cells are directed to undergo all-or-none (quantal) cell fate decisions by critical molecular concentrations. Furthermore, as all malignant cells arise from a single cell, so that all malignancies are clonal in origin, a gain of function mutation of one of the genes encoding the molecular determinants of cell cycle progression could obviate the strict cytokine/receptor/signaling pathways normally controlling the decision to divide, thereby resulting in a neoplastic cell growth (4). In other words, a critical “driver mutation” can put the cell on “autopilot.” From the viewpoint of the immune system, Burnet’s “Clonal Selection Theory,” derives from the observation that antigen-reactive clones of immune cells are selected by antigen, but subsequent to selection, each clone undergoes a proliferative clonal expansion (5). It is this clonal proliferative expansion that is determined by the molecular parameters discovered in this series of experiments focused on IL-2 and T cells. Accordingly, the regulatory cells and other cytokine molecules that influence the concentrations of the IL-2 molecules, their receptors, and the molecules of their signaling pathways ultimately determine the tempo, magnitude, and duration of immune responses. That is, internally derived hormones and their receptors regulate the immune system, just as every other bodily system is regulated, and immunity is not regulated solely by environmental antigens from without, a previous central dogma of immunology. For immunologists, it is especially noteworthy that the lymphocyte antigen receptors function in an identical fashion to convert TCR signals to a digital control of cytokine gene expression (6–10). These findings and considerations, led me to propose “The Quantal Theory of Immunity” (11–14). Thus, the immune system is regulated at the systemic (population) level by the number of clones responding and the extent of the proliferative expansion of each clone as originally stated by Burnet. However, at the level of individual cells, participation in an immune response is regulated in a quantal (all-or-none) fashion, and this quantal decision is determined by the absolute number of intermolecular interactions, which the cells somehow “count.” Once the crucial number has been surpassed, the cell responds in a quantal fashion. It goes without mentioning that all of the molecules and cells that participate in an immune response obey identical principles.

IL-2 is a cytokine that functions as a growth factor and central regulator in the immune system and mediates its effects through ligand-induced hetero-trimerization of the receptor subunits IL-2Rα, IL-2Rβ, and γc. Here, we describe the crystal structure of the trimeric assembly of the human IL-2 receptor ectodomains in complex with IL-2 at 3.0 Å resolution. The quaternary structure is consistent with a stepwise assembly from IL-2/IL-2Rα to IL-2/IL-2Rα/IL-2Rβ to IL-2/IL-2Rα/IL-2Rβ/γc. The IL-2Rα subunit forms the largest of the three IL-2/IL-2R interfaces, which, together with the high abundance of charge–charge interactions, correlates well with the rapid association rate and high-affinity interaction of IL-2Rα with IL-2 at the cell surface. Surprisingly, IL-2Rα makes no contacts with IL-2Rβ or γc, and only minor changes are observed in the IL-2 structure in response to receptor binding. These findings support the principal role of IL-2Rα to deliver IL-2 to the signaling complex and act as regulator of signal transduction. Cooperativity in assembly of the final quaternary complex is easily explained by the extraordinarily extensive set of interfaces found within the fully assembled IL-2 signaling complex, which nearly span the entire length of the IL-2Rβ and γcsubunits. Helix A of IL-2 wedges tightly between IL-2Rβ and γcto form a three-way junction that coalesces into a composite binding site for the final γcrecruitment. The IL-2/γcinterface itself exhibits the smallest buried surface and the fewest hydrogen bonds in the complex, which is consistent with its promiscuous use in other cytokine receptor complexes.

Exactly how the immune system discriminates between all environmental antigens to which it reacts vs. all self-antigens to which it does not, is a principal unanswered question in immunology. As set forth in this review, because of the advances in our understanding of the immune system that have occurred in the last 50 years, for the first time it is possible to formulate a new theory, termed the "Quantal Theory of Immunity", which reduces the problem from the immune system as a whole, to the individual cells comprising the system, and finally to a molecular explanation as to how the system behaves as it does.

IL-2, the first cytokine discovered with T cell growth factor activity, is now known to have pleiotropic effects on T cells. For example, it can promote growth, survival, and differentiation of Ag-selected cells, or facilitate Ag-induced cell death of T cells when Ag persists, and in vivo, it is thought to contribute to the regulation of the size of adaptive T cell response. IL-2 is deficient in HIV-1 infection and has been used in the management of HIV-1-infected individuals undergoing antiretroviral therapy. In this study, we investigated how continuous low-dose IL-2 affected the CD4+ and CD8+ T cell response induced by two inoculations of a canarypox recombinant SIV-based vaccine candidate in healthy macaques chronically infected with SIVmac251. These macaques had normal levels of CD4+ T cells at the beginning of antiretroviral therapy treatment. Vaccination in the presence of IL-2 significantly augmented Gag-specific CD8+ T cell responses, but actually reduced Gag-specific CD4+ T cell responses. Although IL-2 at low doses did not change the overall concentration of circulating CD4+ or CD8+ T cells, it expanded the frequency of CD4+CD25+ T cells. Depletion of the CD4+CD25+ T cells in vitro, however, did not result in a reconstitution of Gag-specific CD4+ responses or augmentation of SIV-specific CD8+ T cell responses. Thus, we conclude that the decrease in virus-specific CD4+ T cell response may be due to IL-2-promoted redistribution of cells from the circulation, or due to Ag-induced cell death, rather than suppression by a T regulatory population.

A cohort of rhesus macaques with long-standing SIVmac251 infection (≥5 mo) was treated with continuous antiretroviral therapy (ART). A group of eight macaques was vaccinated with or without simultaneous administration of low dose IL-2 with the highly attenuated poxvirus vector (NYVAC) vaccine candidate expressing the SIVmac structural gag-pol-env (gpe) genes and a novel chimeric fusion protein derived from the rev-tat-nef (rtn) regulatory genes. Control groups consisted of mock-vaccinated macaques or animals treated only with IL-2. Vaccination significantly expanded both virus-specific CD4+ and CD8+ T cell responses, and IL-2 further increased the vaccine-induced response to an immunodominant Gag epitope. Following antiretroviral treatment interruption, the viral set point was significantly lower in vaccinated than in control macaques for at least 4 consecutive mo, and viral containment was inversely correlated with vaccine-induced, virus-specific CD4+ and CD8+ T cell responses. These data provide the proof of concept that therapeutic vaccination before cessation of ART may be a feasible approach in the clinical management of HIV-1 infection.

Purpose: Previous studies with intermittent interleukin-2 (IL-2) therapy using intermediate and high levels of IL-2 have demonstrated significant increases in the CD4+ T cell count in HIV-infected patients. Intermittent regimens are amenable to outpatient use, but severe adverse events are frequently experienced with intermediate- and highdose levels of IL-2. Therefore in this study, the effect of daily, subcutaneous low-dose IL2 therapy on safety and immunological endpoints was investigated to determine whether immunological benefit could be achieved without toxicity in HIV-infected patients also receiving highly active antiretroviral therapy (HAART). Method: A total of 115 patients were enrolled in the trial. Fifty-six asymptomatic HIV-infected patients who had CD4+ T cell counts less than 300 cells/µL at screening and a stable HIV viral load received lowdose IL-2 (1.2 million IU [MIU]/m 2 beginning dose) once daily in conjunction with HAART (IL-2 group). Fifty-nine patients received HAART alone (control group). Results: A dramatic effect of IL-2 on the natural killer (NK) cell population was observed with mean increases of 156 cells/µL in the IL-2 group compared to 19.93 cells/µL in the control group (p < .001). Additionally, IL-2‐treated patients experienced a statistically significant increase in the mean percentage of CD4+ T cells (3.52% increase) when compared to control patients (1.33% increase) (p < .001). The expanded CD4+ T cell population was primarily of the naive phenotype, with mean increases of 4.53% for the IL-2 group and 0.31% for the control group (p < .001 for between-group difference). In addition, a higher proportion of IL-2‐treated patients (67%) compared to control patients (33%) achieved increases of greater than 50% in the CD4+ T cell count (p = .08). Adverse events of grade 3 or grade 4 toxicity were infrequent in the current study and were substantially lower by comparison to those in studies of intermittent dose IL-2 therapy. Also, negligible changes in the HIV viral load from baseline to final measurement were observed in both groups. A trend toward a reduced number of modifications of antiretroviral therapy was apparent in the IL-2 group when compared to control patients. Conclusion: Daily, lowdose subcutaneous IL-2 therapy in conjunction with HAART is safe and well tolerated and is effective in expanding lymphocyte cell types including NK cells and naive T cells in individuals who have

Interleukin-2-deficient mice (IL-2(-/-)) manifest severe immune system abnormalities characterized by an uncontrolled activation and proliferation of lymphocytes. A systemic autoimmune syndrome results, and hemolytic anemia leads to early death especially in mice derived from a BALB/c genotype. Remarkably, IL-2 treatment prevents both the activation of the immune system and the development of autoimmune disease. Moreover, adoptive transfer of lymphocytes from IL-2-treated IL-2(-/-) animals confers protection to IL-2(-/-) mice, suggesting that IL-2 induces a postnatal differentiation/maturation of regulatory cells necessary for self- and non-self-discrimination.

When administered in high doses to HIV positive (HIV+) individuals, interleukin 2 (IL-2) causes extreme toxicity and markedly increases plasma HIV levels. Integration of the information from the structure-activity relationships of the IL-2 receptor interaction, the cellular distribution of the different classes of IL-2 receptors, and the pharmacokinetics of IL-2 provides for the rationale that low IL-2 doses should circumvent toxicity. Therefore, to identify a nontoxic, but effective and safe IL-2 treatment regimen that does not stimulate viral replication, doses of IL-2 from 62,500 to 250,000 IU/m2/day were administered subcutaneously for 6 months to 16 HIV+ individuals with 200-500 CD4+ T cells/mm3. IL-2 was already detectable in the plasma of most HIV+ individuals even before therapy. Peak plasma IL-2 levels were near saturating for high affinity IL-2 receptors in 10 individuals who received the maximum nontoxic dose, which ranged from 187,500 to 250,000 IU/m2/day. During the 6 months of treatment at this dose range, plasma levels of proinflammatory cytokines remained undetectable, and plasma HIV RNA levels did not change significantly. However, delayed type hypersensitivity responses to common recall antigens were markedly augmented, and there were IL-2 dose-dependent increases in circulating Natural Killer cells, eosinophils, monocytes, and CD4+ T cells. Expanded clinical trials of low dose IL-2 are now warranted, especially in combination with effective antivirals to test for the prevention of immunodeficiency and the emergence of drug-resistant mutants and for the eradication of residual virions.

To explore the role of interleukin-2 (IL-2) in T cell proliferation, and to circumvent the IL-2 deficiency autoimmune syndrome of conventional il2 gene deletion, mice were created to allow conditional il2 gene deletion when treated with the estrogen analog, tamoxifen (TAM) as adults. Splenocytes from four different mouse strains, C57Bl/6 wild type (WT), conventional IL-2(-/-), TAM-treated Cre recombinase-negative (Cre-)/IL2fl/fl, and Cre recombinase-positive (Cre+)/IL2fl/fl, were activated with anti-CD3 and anti-CD28, and monitored for CD4+ and CD8+ T cell lymphocyte blastogenesis, aerobic glycolysis, BrdU incorporation into newly synthesized DNA, and CFSE dye dilution to monitor cell division. IL-2 production was monitored by quantitative ELISA and multiple additional cytokines were monitored by quantitative protein-bead arrays. Splenocytes from conventional IL-2(-/-) and TAM-treated Cre+ mice resulted in undetectable IL-2 production by ELISA, so that both strains were IL-2-deficient. As monitored by flow cytometry, activated CD4+ and CD8+ T cells from WT, Cre+, and Cre- mice all underwent blastogenesis, whereas far fewer cells from conventional IL-2(-/-) mice did so. By comparison, only cells from IL-2 sufficient WT and Cre- mice switched to aerobic glycolysis as evidenced by a drop in media pH. Blastogenesis was mirrored by BrdU incorporation and CFSE dye dilution by CD4+ and CD8+ T cells from WT, Cre+, and Cre- mice, which were all equivalent, while proliferation of cells from conventional IL-2(-/-) mice was compromised. Splenocytes from IL-2 deficient conventional IL-2(-/-) mice produced low or undetectable other γ(c)-chain cytokines (IL-4, IL-7, IL-9, IL-13, IL-15, and IL-21), whereas production of these γ(c)-chain cytokines from IL-2-deficient conditional IL-2(-/-) Cre+ mice were comparable with WT and Cre- mice. These results indicate that CD4+ and CD8+ T cell blastogenesis cannot be attributable to IL-2 alone, but a switch to aerobic glycolysis was attributable to IL-2, and proliferation after CD3/CD28 activation is dependent on γ(c)-chain cytokines, and not CD3/28 triggering per se.

Interleukin-2 (IL-2) is a 133 amino acid alpha-helical protein secreted by activated T-cells. Combinatorial cassette mutagenesis was used to investigate the functional role of a continuous five amino acid region of IL-2 suspected to interact with the intermediate-affinity IL-2 receptor. A limited random library of IL-2 mutants was constructed in which residues 17-21 (Leu-Leu-Leu-Asp-Leu) were simultaneously mutated. The proteins were produced in an Escherichia coli expression system and screened in a biological assay for their ability to mediate the proliferation of a murine IL-2-dependent cell line. From the over 2600 clones examined, only 42 exhibited significant activity, confirming the functional importance of this region. Selected clones were purified and further characterized by biological and receptor binding assays. Viewed in the context of the recently revised 2.5-A crystal structure for IL-2, these results suggest the following conclusions: both Asp20 and Leu21, as shown by their sensitivity to mutation, are the functionally more important residues in this region, but for different reasons. Asp20 is solvent-accessible and likely plays a direct receptor contact role as previous studies have indicated. Leu21, in contrast, is completely buried in the hydrophobic core of the protein. Substitutions at this position, even a conservative Leu-->Val substitution, were found to perturb the precise hydrophobic packing arrangements that are critical for activity, resulting in a significant loss of function. In addition, one of the analogs identified in the screen was found to be 2-3 times more potent than the wild-type protein.

Clonal expansion of antigen-reactive T lymphocytes is driven by the lymphokine interleukin 2 (IL-2). To further elucidate the mechanisms of IL-2 action, we have utilized a differential hybridization procedure to clone IL-2-induced immediate-early genes from an IL-2-stimulated human T-cell cDNA library. To increase the frequency of IL-2-induced transcripts represented in the library, the protein synthesis inhibitor cycloheximide was included during the 2-hr IL-2 stimulation to superinduce gene expression, and the uridine analogue 4-thiouridine was utilized to enable selective purification of newly synthesized transcripts. From the enriched library, we have isolated eight IL-2-induced genes, six of which represent previously unrecognized human sequences. Northern blot analysis revealed that the induction of seven of the genes is specific to the IL-2-mediated G1 "progression" phase of the cell cycle, in that only one gene is also induced during the T-cell receptor-triggered G0-G1 "competence" phase. These results indicate that the effects of IL-2 are mediated by the specific induction of a number of immediate-early genes and provide a means with which to further delineate the mechanisms whereby IL-2 stimulates T-lymphocyte proliferation and differentiation. The methods described in this report should also be of general utility in the dissection of the signaling pathways activated by diverse cytokine receptors.

13 patients with human immunodeficiency virus type 1 infection class II-IV, but without opportunistic infection or neoplasm, received 6 micrograms (3.6 x 10(4) IU) of polyethylene glycol recombinant human interleukin 2 (PEG IL-2) intradermally twice a week for 4 mo were then followed for an additional 6 mo. Clinical, immunological, and viral parameters were monitored in the patients, all of whom were taking zidovudine. The cutaneous administration of PEG IL-2 resulted in an indurated zone resembling a delayed-type hypersensitivity response of 26 +/- 1 mm diameter (676 mm2) at 72-96 h after injection throughout the 4 mo of administration. This dose, which was appreciably lower than in most previous trials, was not associated with local or systemic toxicity. No increase in the viral burden of circulating leukocytes or plasma occurred. A number of immunological functions were stimulated by this course of therapy. All patients demonstrated high levels of lymphokine-activated killer cell activity by cells freshly removed from the circulation and in the absence of in vitro exposure to IL-2. Natural killer cell activity was also enhanced. Limiting dilution analysis revealed an increase in the frequency of IL-2-responsive cells from abnormally low to levels above normal during the course of injections. In a subgroup of four patients with > or = 400 CD4+ T cells/microliter at entry, there was a trend to sustained increases in CD4+ T cell numbers. However, this increase did not reach statistical significance. This subset of patients also exhibited higher proliferative responses to phytohemagglutinin as mitogen. Several of these effects persisted for 3-6 mo after cessation of therapy. In conclusion, low-dose IL-2 regimens lead to sustained immune enhancement in the absence of toxicity. We suggest pursuit of this approach for further clinical trials both as prophylaxis and therapy.

Interleukin-2 (IL-2) is a key cytokine in cellular immunity. Human immunodeficiency virus type 1 (HIV-1)-infected individuals lack IL-2 because of low CD4+ T lymphocyte numbers. In an attempt to enhance cellular immunity, low-dose recombinant human (rh) IL-2 at 10 micrograms or 180,000 units or its polyethylene glycol (PEG) derivative at 9 micrograms or 36,000 units was given by intracutaneous injection to 8 HIV-1-infected men for 30 days. Participants had no evidence of opportunistic infection and received concurrent zidovudine. IL-2 treatment was nontoxic and elicited a local cellular response resembling classic delayed-type hypersensitivity (DTH) with local interferon-gamma production, even in anergic patients. Systemic responses included enhanced DTH responses to recall antigens, improved in vitro proliferative responses to mitogen, and enhanced NK cell activity. Peripheral leukocyte phenotype and virus titers were unchanged. Long-term studies of low-dose IL-2 are warranted to determine whether immunoenhancing effects can be sustained and if they are associated with improved clinical course.

The sensitivity of T cells to interleukin-2 (IL-2) can vary by three orders of magnitude and is determined by the surface densities of the IL-2 receptor α subunits. Regulatory T cells inflict a double hit on effector T cells by lowering the bulk IL-2 concentration as well as the sensitivity of effector T cells to this crucial cytokine. This double hit deprives weakly activated effector T cells of pSTAT5 survival signals while having only minimal effects on strongly activated effector cells that express increased levels of the IL-2 receptor. Short-term signaling differences lead to a differential functional in terms of proliferation and cell division: regulatory T cell specifically suppress weakly activated effector T cells even at large numbers; small numbers of strongly activated effector T cells overcome the suppression., Self-/non-self-discrimination in the adaptive immune system relies, to a large extent, on distinctions between self-antigens and foreign antigens as made by individual T cells. As such, single-cell decisions are prone to errors a reliable immune response can be expected to incorporate further proofreading schemes. One such scheme involves long time scale, population-level interactions between effector (Teff) and regulatory (Treg) T cells. Treg cells are often described as immune suppressors; their role as immune regulators can be understood by mapping out the scenarios in which Treg suppression is either significant or insignificant. In this study, we have focused on one mechanism that allows Treg cells to suppress Teff survival, namely, interleukin-2 (IL-2) deprivation. Following antigen activation, Teff cells secrete IL-2 and express the α subunit of the IL-2 receptor (IL-2r). The binding of extracellular IL-2 to the IL-2r is crucial for Teff survival and proliferation and consequently for a full-blown immune response. Treg cells deplete this IL-2 from the environment and deprive the Teff cells of this important survival signal. In this tug-of-war for IL-2, we sought to quantitatively describe those scenarios in which IL-2 uptake by Treg cells suffices to suppress Teff cell activation and those where it does not. The core of this competition for IL-2 lies in the fact that IL-2rα is expressed on both Teff and Treg cells. To understand how IL-2 binds to its receptor, we measured IL-2r subunit levels on single cells, together with STAT5 phosphorylation as evoked by varied IL-2. Contrary to previous descriptions that set the EC50 of IL-2/IL-2r interaction at 10 pM, we found that the sensitivity of T cells to IL-2 varies over three orders of magnitude concentrations (Figure 1E, experiment). Teff cells with higher levels of IL-2rα receptor subunit are more sensitive to IL-2, Treg cells with higher levels of IL-2rα are more efficient in the scavenging of IL-2. IL-2rβ levels, on the other hand, determine response amplitudes. We describe a short time scale, two-step model to quantitatively describe IL-2 binding onto individual cells (Figure 1E, theory). IL-2r expression levels are therefore a crucial parameter for determining the outcome of the competition for IL-2. We measured the regulation of IL-2r subunits on longer time scales in cultures of either Teff or Treg cells. For both cell types, IL-2r levels depend on the exposure to IL-2. For Teff cells, there is a further dependence on the concentration of antigen by which they were activated. We then measured IL-2r expression in cocultures of Treg and Teff cells. We show how IL-2 secreted by activated Teff cells suffices in inducing IL-2rα upregulation in the Treg population. We further show that the presence of Treg cells decreased IL-2r upregulation in cocultured weakly activated Teff cells. Treg cells thus inflict a double hit on Teff cells by reducing not only extracellular IL-2 concentrations but also the Teff cells' ability to sense IL-2. Teff cells activated by high-antigen concentrations exhibit sustained IL-2rα expression that is less prone to this effect. We compared IL-2r levels on Treg cells and Teff activated by varied antigen concentrations and found a critical crossover: at low-antigen concentrations Treg cells have higher IL-2rα than Teff cells, but this is reversed at high-antigen concentrations. We constructed a long time scale computational model to quantify the significance of this crossover. The model describes IL-2/IL-2r binding and the regulation of IL-2 and IL-2r expression in populations of Treg and Teff cells. For a pure Teff population, our model predicted a ‘quorum-sensing' threshold implying that sustained pSTAT5 signaling requires a minimal concentration of cells that increases with decreasing activation strength. The model further predicts that the addition of Treg cells will greatly increase the quorum concentration for weakly activated Teff cells but have no effect on strongly activated Teff cells. We validated the model's predictions in vitro. We show a quorum-sensing threshold for activated Teff cells. We also show that the presence of a Treg population suppressed pSTAT5 signaling in a large number of weakly but has little effect on even a few strongly activated Teff cells (Figure 6C and D). On longer time scales, this translates to the suppression of cell division (Figure 6G and H) and proliferation (Figure 6I) in a manner that discriminates between strongly and weakly activated cells. We then went to demonstrate that IL-2 deprivation by Treg cells takes place in vivo. We used IL-2 injections to upregulate IL-2rα levels in Treg cells. As predicted by our in vitro results, such treatment leads to a suppressive environment in which Teff cells activated by subsequent antigen/LPS immunization proliferate to a lesser extent. We were able to reverse this suppressive effect by continuing IL-2 treatment post-immunization. This highlights IL-2 as a limiting factor for Teff proliferation and renders its scavenging by Treg cells an important mechanism of suppression in vivo. In conclusion, we formulated a quantitative description of IL-2/IL-2r regulation in mixed population of Treg and Teff cells. Population feedback loops that depend on cell numbers, molecular cell surface densities, free molecular densities and timing critically affect the outcome of the competition for IL-2. Such a description allows us to precisely identify the scenarios in which IL-2 deprivation by Treg cells has a major suppressive role in vitro and better understand the role of this mechanism in vivo., Understanding how the immune system decides between tolerance and activation by antigens requires addressing cytokine regulation as a highly dynamic process. We quantified the dynamics of interleukin-2 (IL-2) signaling in a population of T cells during an immune response by combining in silico modeling and single-cell measurements in vitro. We demonstrate that IL-2 receptor expression levels vary widely among T cells creating a large variability in the ability of the individual cells to consume, produce and participate in IL-2 signaling within the population. Our model reveals that at the population level, these heterogeneous cells are engaged in a tug-of-war for IL-2 between regulatory (Treg) and effector (Teff) T cells, whereby access to IL-2 can either increase the survival of Teff cells or the suppressive capacity of Treg cells. This tug-of-war is the mechanism enforcing, at the systems level, a core function of Treg cells, namely the specific suppression of survival signals for weakly activated Teff cells but not for strongly activated cells. Our integrated model yields quantitative, experimentally validated predictions for the manipulation of Treg suppression.

Interleukin 2 (IL-2), a T lymphocyte product released upon antigen stimulation, has been used for cancer therapy in high doses for more than five years. More recently, its potential as a stimulant of cell-mediated immunity in infectious diseases, particularly those caused by intracellular microbes, has become appreciated. Drawing on the extensive information available as to the structure, cellular and molecular effects of IL-2, this review focuses on its use in patients with lepromatous leprosy and AIDS in low, physiologic doses. The data indicate that IL-2 is effective in stimulating cell-mediated immunity without systemic toxicity.

As interleukin-2 (IL2) is central to the clonal expansion of antigen-selected T cells, we investigated the relationship between IL2 and the negative regulatory transcription factor FOXP3. We found IL2 to be responsible for T cell antigen receptor (TCR)-activated FOXP3 expression by both CD4+ and CD8+ human T cells, and as anticipated, FOXP3 expression restricted TCR-stimulated IL2 expression. However, no evidence could be found that FOXP3+ cells actively suppress IL2 expression by FOXP3- cells. These data are consistent with an IL2/FOXP3-dependent negative feedback loop that normally regulates the T cell immune response. It follows that a defect in this negative feedback loop as a result of a deficiency of either IL2 or FOXP3 will lead to a hyperproliferative autoimmune syndrome, without the necessity of invoking an active suppressive function for FOXP3+ T cells.

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.

Interleukin (IL)-2 is currently used to enhance T-cell immunity but can have both positive and negative effects on T cells. To determine whether these opposing results are due to IL-2 acting differently on T cells depending on their stage of differentiation, we examined the effects of IL-2 therapy during the expansion, contraction and memory phases of the T-cell response in lymphocytic choriomeningitis virus (LCMV)-infected mice. IL-2 treatment during the expansion phase was detrimental to the survival of rapidly dividing effector T cells. In contrast, IL-2 therapy was highly beneficial during the death phase, resulting in increased proliferation and survival of virus-specific T cells. IL-2 treatment also increased proliferation of resting memory T cells in mice that controlled the infection. Virus-specific T cells in chronically infected mice also responded to IL-2 resulting in decreased viral burden. Thus, timing of IL-2 administration and differentiation status of the T cell are critical parameters in designing IL-2 therapies.

Chronic infection with HIV renders individuals incapable of mounting an effective host antiviral response, as defined by in vitro assays. Therefore, to determine whether antiviral reactivity could be detected in vivo, we interrupted effective antiviral treatment prospectively in nine chronically infected aviremic individuals. Low-dose interleukin-2 (IL-2) was administered before and after treatment interruption to compensate for any potential IL-2 production deficiency. In vivo antiviral reactivity was monitored subsequent to the interruption of antiviral therapy via viral and lymphocyte dynamics. The study was terminated when the plasma HIV RNA concentration reached a plateau, defined as four successive determinations that were25% from the mean.Plasma viral relapse occurred in all participants; reaching a peak concentration within 2.5 weeks. However, over the subsequent 2 weeks viremia was reduced an order of magnitude coincident with a 2-fold lymphocytosis of the CD8 + T cell subset. A second treatment interruption resulted in attenuation of the peak and trough virus concentrations by10-fold in 3 of 4 participants, while the CD8 + T cell concentrations remained elevated.These findings indicate that chronic HIV infection prior to successful antiviral therapy does not preclude host antiviral reactivity. In addition, in vivo antiviral reactivity as revealed by viral and lymphocyte dynamics after antiviral treatment interruption can be useful to monitor the efficacy of different therapies.

There is now a great deal of interest in therapies focused on improving the function of the immune system in the treatment of individuals infected with the HIV. Although the antiviral drugs effectively suppress replication of the virus, they cannot cure the infection. Therefore, it now appears that both antivirals and immune system stimulants will be necessary to maximally suppress residual latent virus, thereby allowing the discontinuation of the antivirals without relapse of detectable plasma virus. Interleukin-2 (IL-2) the first cytokine to be discovered at the molecular level has been used as a therapeutic in HIV infection, because it is critical for a normal functioning immune response. IL-2 is essential for the survival and proliferative expansion of antigen-activated T cells and natural killer (NK) cells, and also for promoting their differentiated functions of cytokine secretion and cytolysis. However, as IL-2 stimulates both the innate and acquired immune responses, when used as a therapeutic it can lead to severe toxicity when given in high doses. This review focuses on low dose, daily IL-2 therapy, used to accelerate the recovery of the immune system when viral replication is suppressed maximally with antivirals. In addition, the principles of the use of IL-2 to activate HIV-specific immune reactivity are discussed. At least two signals are required to promote the proliferative expansion and function of antiviral effector lymphocytes, HIV antigens and IL-2.

Cellular proliferation and survival are modulated by the expression of specific genes. Cytokine response gene 8 (CR8), which was originally cloned as an IL-2-induced gene in human T lymphocytes, encodes a basic helix--loop--helix (bHLH) transcription factor. The CR8 gene product is highly conserved among human, mouse and rat, and contains sequence motifs that distinguish it from other bHLH families. The CR8 gene is ubiquitously expressed, and CR8 gene expression is induced by both growth-promoting as well as growth-inhibitory stimuli. As bHLH proteins have been found to regulate both the G1-S phase cell cycle transition, as well as cellular survival, the effects of CR8 on these processes were investigated. Ectopic CR8 expression in asynchronous U2OS cell cultures reduces the percentage of cells in the cell cycle S phase, and also slows the entry of G1-synchronized cells into S phase. The prolonged G1 interval correlates with impaired elevation of cyclin E protein and prolonged p21 protein expression in G1. CR8 expression also protects U2OS cells from serum-withdrawal induced apoptosis. These results indicate that CR8 is an important modulator of both the G1-S phase cell cycle transition, and cellular survival.

The baculovirus-mediated eukaryotic insect cell expression system was used to prepare large quantities of the beta-subunit ectodomain of the high-affinity interleukin-2 receptor (IL-2R beta x). We describe the expression, purification, and biophysical characterization of this ligand binding domain. The human cDNA encoding IL-2R beta x was inserted into baculovirus transfer vectors. High titer recombinant baculovirus was produced in Spodoptera frugiperda (Sf9) insect cells, and the viral supernatants were subsequently used to infect monolayers of Trichoplusia ni (High Five) insect cells in serum-free culture. Maximal expression of the recombinant protein excreted into the cell culture supernatants was determined by SDS/PAGE analysis, where a band migrating with an apparent molecular mass of 31 kDa was identified by immunostaining. One-step purification was achieved by affinity chromatography on either a monoclonal antibody (TIC-1) column or an IL-2 column, with a final yield of approximately 5 mg/L of culture supernatant. Interestingly, partial purification was also demonstrated using metal chelate affinity chromatography. Amino-terminal sequence analysis of the protein matched the published sequence. Both equilibrium sedimentation analysis and gel filtration chromatography indicated that IL-2R beta x remains monomeric. Deconvolution of far-UV circular dichroism (CD) spectra indicated the predominant secondary structural element to be beta-sheet, consistent with structural analysis and predictions for other members of the hematopoietic receptor family. A dissociation constant (Kd) for IL-2R beta x in solution of 5.3 x 10(-7) M was calculated from competitive receptor binding assays.(ABSTRACT TRUNCATED AT 250 WORDS)

The development of efficient cloning methods for the isolation of cytokine-response genes is vital to our understanding of how cytokines elicit distinct cellular responses. Here, Carol Beadling and Kendall Smith describe a straightforward and rapid method that has been used to clone interleukin 2 (IL-2)-induced genes.

Interleukin-2 (IL-2), a 15 kDa protein secreted by antigen-activated T lymphocytes, plays a central role in the generation of an immune response. Initially characterized as a T cell growth factor, IL-2 has subsequently been found to act on all lymphoid cell types, inducing both their proliferation and functional differentiation. The effects of IL-2 are mediated by the activation of a specific cell-surface receptor (IL- 2R), which is comprised of at least three distinct proteins. Early biochemical signaling events triggered upon IL-2R activation have recently begun to be elucidated, and a number of genes have been identified which are specifically induced by IL-2. The task now at hand is to link the biochemical signaling pathways activated by IL-2 with the regulation of gene expression, and to delineate the roles of these gene products in IL-2-mediated proliferation and differentiation.

Selected parameters of cellular immunity relating to cytokine gene activation and responsiveness to interleukin-2 (IL-2) were analyzed in 27 patients with active pulmonary tuberculosis and no human immunodeficiency virus type 1 infection. Cytokine mRNAs were not expressed by peripheral blood mononuclear cells (PBMC) of normal controls. In PBMC of tuberculosis patients, messages for IL-1, IL-8, and tumor necrosis factor-alpha were uniformly expressed, whereas PBMC of only 5 of 18 patients expressed IL-6. PBMC of 7 patients (all of those with systemic symptoms) expressed interferon-gamma mRNA and none expressed IL-2 mRNA. Most patients' cells demonstrated IL-4 mRNA. Limiting dilution analysis of IL-2-responsive cells in PBMC revealed that tuberculosis patients had 10-fold fewer IL-2-responsive cells than did controls.

The biological effects of interleukin-2 (IL-2) were examined in T lymphocytes. IL-2 binding induced the rapid activation of diacylglycerol kinase in both cytosolic and membrane subfractions. This enzyme utilized diacylglycerol from multiple endogenous and exogenous sources for the synthesis of phosphatidic acid. Phosphatidic acid was, in turn, shown to stimulate the accumulation of c-myc mRNA and augment cellular proliferation when added to IL-2-dependent cell lines. These results link previous observations of IL-2 and glycosylphosphatidylinositol-dependent diacylglycerol production to phosphatidic acid accumulation, and suggest that diacylglycerol kinase activation is part of an intricate IL-2 signaling cascade that utilizes phosphatidic acid as an effector molecule.

To gain further insight into the role of Raf-1 in normal cell growth, c-raf-1 mRNA expression, Raf-1 protein production, and Raf-1-associated kinase activity in normal human T cells were analyzed. In contrast to the constitutive expression of Raf-1 in continuously proliferating cell lines, c-raf-1 mRNA and Raf-1 protein levels were barely detectable in freshly isolated G0 T lymphocytes. Previous work with fibroblasts has suggested that Raf-1 plays a signaling role in the G0-G1 phase transition. In T cells, triggering via the T-cell antigen receptor (TCR)-CD3 complex (TCR/CD3) resulted in an approximately fourfold increase in c-raf-1 mRNA. In addition, the promotion of G1 progression by interleukin 2 (IL-2) was associated with a 5- to 10-fold immediate/early induction of c-raf-1 mRNA, resulting in up to a 12-fold increase in Raf-1 protein expression. TCR/CD3 activation did not alter the phosphorylation state of Raf-1, whereas interleukin 2 receptor stimulation resulted in a rapid increase in the phosphorylation state of a subpopulation of Raf-1 molecules progressively increasing throughout G1. These findings were complemented by assays for Raf-1-associated kinase activity which revealed a gradual accumulation of serine and threonine autokinase activity in Raf-1 immunoprecipitates during G1, which remained elevated throughout DNA replication.

14 patients with lepromatous leprosy received twice daily injections of 10 micrograms recombinant interleukin 2 (rIL-2), by the intradermal route, in the skin of the back for 8 d (total dose, 160 micrograms). Lymphokine administration was accomplished without drug toxicity, or the development of acute nerve damage. The majority of patients developed nontender axillary lymphadenopathy during the course of treatment. Local injection sites showed progressively larger zones of induration, peaking at 24 h and persisting for many days. Early 12-h reactions were of a macular, erythematous nature and exhibited an increasingly striking diurnal variation. The morning injection sites were three- to fourfold larger in diameter than those placed in the evening (9 am to 9 pm). Systemic manifestations of intradermal rIL-2 administration were noted. Peripheral blood T cells, including CD4+ and CD8+ phenotypes, increased 2-2.5-fold and NK cells increased sixfold. Elevations in [3H]TdR incorporation into peripheral blood mononuclear cells occurred to a variety of mycobacterial antigens, but not to those of Mycobacterium leprae. Within 2 wk, biopsies at sites far removed from the back showed increased infiltration of mononuclear cells in 12 of 14 patients. Immunocytochemistry revealed the presence of newly emigrated CD4+ T cells, monocytes, and dermal CD1+ Langerhans cells. Endothelial cells of small dermal vessels expressed major histocompatibility complex class II determinants on their surface. Transmission electron microscopy of these specimens revealed markedly enlarged endothelial cells with many surface projections extending into the lumen as well as extravasating lymphoid cells. The numbers of acid-fast M. leprae in the peripheral sites were examined by slit smear and in biopsies of matched leprosy lesions taken before and after IL-2 administration. Within 2 mo, slit smears showed a 0.5 log or greater reduction in 12 of 14 patients, with a mean for all patients tested of 0.5 log units. Biopsy specimens showed a 1 log unit or greater reduction in the bacterial index (B.I.) in 6 of 14 patients. Historical controls in this Nepalese population showed a 0.5 log unit reduction after multidrug therapy over a period of 12 mo. Thus, after 8 d of IL-2 injections, a fivefold reduction in B.I. was observed during the first 2 mo of the study. Antibody levels against M. leprae phenolic glycolipid 1 (PGL-1) and lipoarabinomanan B were markedly elevated after IL-2 injections, while PGL-1 antigen levels were reduced. We conclude that the administration of rIL-2 has had a significant effect in decreasing the total body burden of M. leprae.(ABSTRACT TRUNCATED AT 400 WORDS)

Interleukin-2 (IL-2) is a representative of a growing family of small proteins termed lymphokines which are responsible for mediating cell differentiation, growth and function in the immune system. Many of these proteins are being evaluated for their clinical potential. From the perspective of drug development, structure-function analysis of these molecules and their receptors require the use methodologies different than those traditionally employed for small peptides and other natural products. However, similar pharmacologic principles apply and an understanding of ligand-receptor interactions and the associated responses is required in order to efficiently pursue agonist and antagonist design. Although IL-2 is a protein of only 133 amino acid residues for which a low resolution X-ray structure does exist, the complexity of its receptor system has provided an added challenge to structure-function studies. Consequently, little is known concerning the receptor contact residues for this protein. We have attempted to utilize established principles of protein and peptide structure to manipulate the conformation of IL-2 in a manner which has provided analogs helpful for receptor interaction studies. These proteins have not only providing useful information on the nature of the IL-2 receptor but have also revealed potential strategies for the design of IL-2 agonists and antagonists.

The receptor binding requirements for entry of the NAD+ ADP-ribosyltransferase component of DAB486-IL 2 into target cells were examined. Experiments utilizing cell lines bearing either high-affinity or individual subunits of the interleukin 2 receptor (IL 2R) as well as human peripheral blood mononuclear cells with natural killer activity demonstrate that the high-affinity receptor facilitates delivery of fragment A from DAB486-IL 2 to the cytosol approximately 1000 times more efficiently than either the intermediate-(p75) or low-affinity (p55) forms of the IL 2R. We show that elongation factor 2 (EF-2) in these cells is not quantitatively or qualitatively altered indicating that the relative resistance to intoxication displayed by IL 2R variant cell lines cannot be attributed to an altered intracellular target of the hybrid toxin. We also demonstrate that an alteration in the binding of DAB486-IL 2 to the p75 subunit of the IL 2R may account for the selective cytotoxicity of DAB486-IL 2 for cells bearing the heterodimeric high-affinity IL 2R.

Although the role of interleukin 2 (IL 2) in mature T lymphocyte function is well documented, its effect on hematopoietic progenitor cells is less well characterized. Here we have used recombinant retroviruses to transduce and express a cDNA clone encoding the p55 component of the human IL 2 receptor (h-p55), in a murine IL 3-dependent cell line, BAF3. While the parental cells do not respond to IL 2, the h-p55-expressing cells proliferate upon treatment with recombinant IL 2 after an initial lag period. The responsiveness of individual cell clones is correlated with their level of h-p55 expression, and can be inhibited by Tac monoclonal antibody. Furthermore, growth at limiting IL 2 concentrations selects a subset of cells expressing higher h-p55 levels from a bulk population. Detailed 125I-labeled IL 2 binding analysis on the highest h-p55-expressing clone detects the presence of 200 high-affinity (KD = 25 pM) IL 2 receptors. We therefore propose that the level of h-p55 expression governs the formation of high-affinity receptors, and hence IL 2 responsiveness in BAF3 cells.

It is well established that the majority of murine fetal thymocytes (day 15 of gestation) express receptors for interleukin 2 (IL-2), but the functional significance of these IL-2 receptors (IL-2Rs) is not clear. In situ hybridization data show a developmentally regulated expression of IL-2 and IL-2R mRNA. IL-2 binding studies were performed on fetal thymocytes and the results show the presence of both high (kD approximately equal to 20 pM) and low (kD approximately equal to 10 nM) affinity IL-2Rs. These IL-2Rs are indeed functional: intact fetal thymic lobes (but not cell suspensions) cultured in IL-2 exhibited an in vitro proliferative response at 20 pM of IL-2, corresponding with the presence of a functional high-affinity IL-2R on fetal thymocytes. The IL-2-dependent growth was primarily observed in the IL-2R+ thymic subset, which contains the CD3-/CD4-/CD8- precursor thymocytes. Furthermore, in vitro blocking of IL-2 in intact fetal thymic lobes resulted in a reduction in the cell yield, which predominantly affected the expansion of the immature CD3-/CD4-/CD8- thymocytes. Our findings strongly support the concept that the IL-2/IL-2R pathway is responsible for the proliferation of precursor cells within the fetal thymus.

The mechanism of the lymphoproliferative effect of the macrophage product lymphocyte-activating factor [LAF(IL1] appears to be mediated by the stimulation of the release of T cell growth factor [TCGF(IL2)] by T cells. The magnitude of the resultant T cell proliferative clonal expansion is thus dependent upon the quantity of both LAF(IL1) and TCGF(IL2) induced by antigen or lectin stimulation. These observations, coupled with the ability to measure the production and actions of these hormone-like lymphokines, should allow for increased insight into the mode of action of immunoenhancing and immunosuppressive agents, as well as for new therapeutic approaches to disease states involving T lymphocytes.

Interleukin-2 (IL-2), the first of a series of lymphocytotrophic hormones to be recognized and completely characterized, is pivotal for the generation and regulation of the immune response. A T lymphocyte product, IL-2 also stimulates T cells to undergo cell cycle progression via a finite number of interactions with its specific membrane receptors. Because T cell clonal proliferation after antigen challenge is obligatory for immune responsiveness and immune memory, the IL-2-T cell system has opened the way to a molecular understanding of phenomena that are fundamental to biology, immunology, and medicine.

T lymphocyte mitosis results from the interaction of interleukin 2 (IL-2) with specific receptors that appear only after appropriate immune stimulation. To assess the potential role of IL-2 receptor levels in determining the rate and magnitude of T cell proliferation, the expression of IL-2 receptors by lectin-stimulated human peripheral blood T cells was examined and correlated with T cell growth. Using biosynthetically radiolabeled IL-2 and anti-Tac, a monoclonal antibody that blocks IL-2 receptor binding, IL-2 receptors were found to accumulate slowly and asynchronously among lectin-stimulated T cells and to precede the onset of DNA synthesis. Moreover, a critical threshold of IL-2 receptor density appeared to be required before the commitment to cell cycle progression, as analyzed quantitatively by tritiated thymidine incorporation and flow cytometric analysis of cellular DNA content. Once maximal IL-2 receptor expression occurred, continued proliferation was IL-2 concentration dependent as assessed using homogenous immunoaffinity-purified IL-2. Upon removal of the activating lectin, IL-2 receptor levels progressively declined, and, in parallel, the rate of proliferation diminished. The decay of IL-2 receptors could not be attributed to IL-2-mediated down-regulation. Instead, renewed IL-2 receptor expression was dependent upon the reintroduction of the initial activating signal. Repetitive exposure to lectin resulted in a more rapid reexpression of maximal IL-2 receptor levels, which was then followed by an accelerated resumption of proliferation. Thus, the extent of T cell proliferation after immune stimulation depends upon the interplay of the IL-2 concentration available and the density of IL-2 receptors expressed, both of which are ultimately determined by antigen/lectin stimulation. The awareness of the transience and the antigen/lectin dependence of IL-2 receptor expression, together with the capacity to monitor T cell cultures for IL-2 receptor levels, should facilitate the initiation and maintenance of cloned, antigen-specific T cells in long-term culture. In addition, these findings suggest that, in vivo, the rapidity of acquisition of maximum IL-2 receptor levels by activated T cells and the duration of IL-2 receptor expression may well direct the magnitude of T cell clonal expansion and resultant immune responses.

Molecular biological techniques have revealed the interleukin-2 receptor to be a dimer composed of one α-subunit and one β-subunit which interact non-covalently in a cooperative manner. Site-directed mutagenesis, in conjunction with structural analysis, is beginning to clarify the relationship between structural components of the receptor and their function, and Thomas Ciardelli and Kendall Smith explain why this is bringing drug developers closer to the design of IL-2 agonists and antagonists .

In studies to determine the biochemical mechanisms responsible for cell proliferation, synchronized T cells were used as a model for cellular growth control. By metabolic and morphologic criteria, it was found that activation of the T-cell antigen receptor rendered the cells responsive to interleukin-2 (IL-2), but did not move them through the cell cycle. Instead, IL-2 stimulated G1 progression to S phase, or lymphocyte "blastic transformation." During IL-2-promoted G1 progression, expression of the cellular proto-oncogene c-myb was induced transiently at six to seven times basal levels, maximal levels occurring at the midpoint of G1.

To examine directly the hypothesis that T cell growth factor (TCGF) interacts with target cells in a fashion similar to polypeptide hormones, the binding of radiolabeled TCGF to various cell populations was investigated. The results indicate that TCGF interacts with activated T cells via a receptor through which it initiates the T cell proliferative response. Internally radiolabeled TCGF, prepared from a human T leukemia cell line and purified by gel filtration and isoelectric focusing, retained biological activity and was uniform with respect to size and charge. Binding of radiolabeled TCGF to TCGF-dependent cytolytic T cells occurred rapidly (within 15 rain at 37 degrees C) and was both saturable and largely reversible. In addition, at 37 degrees C, a receptor- and lysosome-dependent degradation of TCGF occurred. Radiolabeled TCGF binding was specific for activated, TCGF-responsive T cells. Whereas unstimulated lymphocytes of human or murine origin and lipopolysaccharide-activated B cell blasts expressed few if any detectable binding sites, lectin- or alloantigen-activated cells had easily detectable binding sites. Moreover, compared with lectin- or alloantigen-activated T cells, long-term TCGF-dependent cytolytic and helper T cell lines and TCGF-dependent neo-plastic T cell lines bound TCGF with a similar affinity (dissociation constant of 5-25 pM) and expressed a similar number of receptor sites per cell (5,000-15,000). In contrast, a number of TCGF-independent cell lines of T cell, B cell, or myeloid origin did not bind detectable quantities of radiolabeled TCGF. Binding of radiolabeled TCGF to TCGF-responsive cells was specific, in that among several growth factors and polypeptide hormones tested, only TCGF competed for binding. Finally, the relative magnitude of T cell proliferation induced by a given concentration of TCGF closely paralleled the fraction of occupied receptor sites. As the extent of T cell clonal expansion depends on TCGF and on the TCGF receptor, the dissection of the molecular events surrounding the interaction of TCGF and its receptor that these studies permit, should provide new insight into the hormonelike regulation of the immune response by this lymphokine.