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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.

International audience; Interleukin (IL)-2 is a pleiotropic cytokine that is necessary to prevent chronic inflammation in the gastrointestinal tract 1-4. The protective effects of IL-2 involve the generation, maintenance and function of regulatory T (T reg) cells 4-8 , and the use of low doses of IL-2 has emerged as a potential therapeutic strategy for patients with inflammatory bowel disease 9. However, the cellular and molecular pathways that control the production of IL-2 in the context of intestinal health are undefined. Here we show, in a mouse model, that IL-2 is acutely required to maintain T reg cells and immunological homeostasis throughout the gastrointestinal tract. Notably, lineage-specific deletion of IL-2 in T cells did not reduce T reg cells in the small intestine. Unbiased analyses revealed that, in the small intestine, group-3 innate lymphoid cells (ILC3s) are the dominant cellular source of IL-2, which is induced selectively by IL-1β. Macrophages in the small intestine produce IL-1β, and activation of this pathway involves MYD88-and NOD2-dependent sensing of the microbiota. Our loss-of-function studies show that ILC3-derived IL-2 is essential for maintaining T reg cells, immunological homeostasis and oral tolerance to dietary antigens in the small intestine. Furthermore, production of IL-2 by ILC3s was significantly reduced in the small intestine of patients with Crohn's disease, and this correlated with lower frequencies of T reg cells. Our results reveal a previously unappreciated pathway in which a microbiota-and IL-1β-dependent axis promotes the production of IL-2 by ILC3s to orchestrate immune regulation in the intestine. To determine whether IL-2 is constitutively required for the maintenance of T reg cells and immunological homeostasis in the intestine, we administered isotype-control or anti-IL-2 neutralizing antibodies every other day to adult mice for two weeks. Within this short time period, the neutralization of IL-2 promoted an enlargement of the spleen and mesenteric lymph nodes, and caused significant reductions of T reg cells and significant increases in the proliferation of CD4 + T cells throughout the gastrointestinal tract and associated lymphoid tissues, including the mesenteric lymph nodes, large intestine and small intestine (Extended Data Fig. 1a-g). Blockade of IL-2 resulted in significantly enhanced IFNγ production by CD4 + T cells in both the small and large intestine, as well as increased IL-17A production in the large intestine (Extended Data Fig. 1h-k). Previous studies have suggested that CD4 + T cells are the dominant cellular source of IL-2 1,2. Therefore, we generated mice with a lineage-specific deletion of IL-2 in T cells by crossing IL-2-floxed mice 10 with Lck cre mice. Lck cre Il2 f/f mice exhibited a complete loss of IL-2 protein staining in T cells, and we observed a significant reduction in the number of T reg cells, and an increase in CD4 + T cell proliferation and effector function in the mesenteric lymph nodes and large intestine (Extended Data Fig. 2a-g). By contrast, deletion of

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.

This Research Topic was conceived to provide a venue for investigators to document their critical contributions to our understanding of the cellular and molecular mechanisms that provide our remarkable immune system the capacity to protect us from environmental insults while simultaneously remaining unreactive to our internal molecular milieu. Although the scientific literature gives one a history of what happened, when it happened, and who were responsible, it fails to capture precisely how things came together, and why some investigators were successful, while others working contemporaneously failed. Thus, seminal contributors were asked to recount the various aspects of their experiments and people who were instrumental in making the progress that moved our understanding forward. Looking back over the brief history of immunology, a discipline that arose after the pioneering approaches of Edward Jenner introduced the smallpox vaccine in 1798 (1) and Louis Pasteur catapulted immunology into universal awareness 100 years later (2–4), the science has only become mature within the past 50 years. For almost 100 years after Pasteur, experimentalists focused on observations of the reactions of whole experimental animals or humans to the administration of putative antigenic substances. For the first time, around 1960, it was appreciated that lymphocytes are the cells that mediate the immune reaction (5–7), and experimentation moved for the first time from in vivo to in vitro, which allowed one to manipulate and investigate an immune reaction of cell populations “outside of the black box.” During the 1960s, various techniques were improved so that it was possible to discern that several different types of cells cooperated to ultimately generate a measurable immune response, usually monitored by the appearance of antibody-forming cells (AFCs) (8, 9). As detailed in this Research Topic, by the 1970s, experiments culminated in the demonstration that two distinct types of lymphocytes existed, termed thymic-derived cells (T cells) and bone marrow-derived cells (B cells), which generate AFCs. Furthermore, a third type of cell derived from myeloid cells, termed an antigen-presenting cell (APC), also played a role. Evidence was also presented that there are at least two distinct subsets of T cells. Moreover, investigators detected activities in culture supernatants of activated lymphocyte populations that seemed to enhance or suppress the generation of AFCs as well as the proliferation of various lymphocytes. However, the molecular basis of these activities remained enigmatic and essentially unapproachable, given the experimental biochemical methods then available. Four new and novel experimental methods were introduced in the 1970s that revolutionized all of biological sciences, which enabled a further reduction from cells to molecules, and led to the discipline that now can be recognized as molecular immunology. In 1972, the flow cytometer introduced a new method to identify and isolate individual cells present in cell populations (10). Also, genetic engineering approaches enabled investigators to identify and isolate complimentary DNA molecules encoding gene products, which then allowed the ready determination of their primary structures, and provided a means to generate essentially unlimited quantities of critical proteins (11). Third, the advent in 1975 of the capacity to isolate and clone individual AFCs, which could produce unlimited quantities of monoclonal antibody molecules (12), could then be used to identify and isolate both individual cells using the flow cytometer, but also new reagents that could be used to isolate and purify individual protein molecules from complex mixtures. The fourth critical technical advance, which like monoclonal antibodies was also special to immunology, was the creation in 1979 of the methods to select, clone, and grow the functional progeny of individual T cells (13). This advance, for the first time, allowed one to circumvent the tremendous heterogeneity of individual cells within T cell populations so that the molecules responsible for antigen recognition, histocompatibility restriction, and the molecular mechanisms underlying T cell function, including T cell help of antibody production and T cell mediated cytolysis, could be uncovered. The contributions comprising this compilation of the stories about how the transition from experiments on whole living organisms to cell populations to individual cells and finally to homogeneous molecules represent a unique aspect of scientific communication, in that they tell the behind the scenes dramas that are usually left out of the scientific literature. This Research Topic tells how science gets done, and what the people are like who actually do it. I hope that you enjoy the stories.

Although we had successfully created antigen-specific cytolytic T lymphocyte lines (CTLLs) using conditioned media as a source of growth-promoting factors, we had no effective method to determine the relative activities of different batches of conditioned media. Therefore, it was crucial to create a quantitative assay for the activity we termed “T cell growth factor” (TCGF). Fortunately, Torgny Fredrickson and I had already created a bioassay for the red blood cell growth factor, erythropoietin (EPO), using murine fetal liver cells that are enriched for EPO-responsive precursors (1, 2). Thus, patterned on the EPO bioassay, it was straightforward to construct a similar assay for TCGF using as target cells our long-term CTLL. The critical elements of the assay were (1) a low density of CTLL target cells and (2) serial twofold dilutions of conditioned media samples, thereby establishing a dose–response curve that allowed comparison of separate conditioned media (3). Of note was the observation that the curve was symmetrically sigmoid when the linear responses of tritiated thymidine incorporation were plotted vs. the logarithm of the conditioned media dilutions. We arbitrarily assigned 1.0 U/mL that yielded 50% of maximal growth promotion at a dilution of 1:10. This assay represented the first ever quantitative bioassay for a lymphokine. Thus, armed with a rapid, quantitative bioassay, we next sought to generate T cell clones derived from our antigen-specific CTLL so that we could assess the potential problem of target cell heterogeneity. We tried two established cloning methods: (1) dilute cell suspensions seeded into soft agar containing TCGF-conditioned media and (2) limiting dilution (0.03–0.01 cells/well) in microtiter plates containing TCGF-conditioned media. The limiting dilution technique in suspension culture worked very well, yielding 67–100% plating efficiency. This was the first description of monoclonal antigen-specific cytolytic T cells (4). Accordingly, T cell clones permitted an unambiguous interpretation that TCGF was acting directly on cloned T cells and not indirectly through an intermediate cell type, e.g., an APC. We submitted our manuscript to Nature, which again rejected it without review [see Ref. (5)], so that we immediately reformatted it and sent it to the J. Exp. Med., which accepted it without changes, so much for non-scientist journalists (Nature) vs. peer scientists (JEM) making informed editorial decisions (6). Other investigators rapidly adopted these cloning methods, in that they not only allowed for separation of the cell clones, but also could be used to grow large numbers of progeny, which could be used for both biological and molecular characterizations. The ability to create monoclonal functional T cells was as transformative for T cells as monoclonal antibodies were for B cells. The foregoing results of these studies on the various mitogenic activities in conditioned media pointed to the overwhelming need to identify the molecules responsible for the bioactivities. In addition, the molecular mechanisms whereby the mitogenic activities interacted with their target cells loomed as a huge overriding question. Thus, armed with the quantitative TCGF bioassay, analytical biochemical experimental approaches yielded results consistent with a single, small protein (~15.5 kDa; pI = 8.2) as solely promoting the biological response (7). This was an important finding, in that it meant that further purification of the molecule responsible for the activity would be straightforward, whereas if several molecules cooperated to produce the activity, purification of each component would be difficult. These biochemical approaches permitted the separation and purification of enough biosynthetically radiolabeled TCGF to permit classic hormone binding assays, which revealed that radiolabeled TCGF-binding sites expressed all of the characteristics of true hormone receptors, i.e., the binding was restricted to TCGF-responsive cells, there was a lack of competition by other growth factors and hormones, the binding was of very high affinity, and there was a close correlation between the TCGF concentrations that bound to cells and those that mediated the proliferative response (8). These data all supported the conclusion that the binding site detected was on the receptor through which the biological effects of TCGF are initiated. This report was the first to demonstrate and characterize a cytokine receptor, and consequentially became the prototype for the identification and characterization of all subsequent cytokine receptors involved in regulating immune and inflammatory responses. Moreover, the radiolabeled TCGF binding assay was absolutely instrumental in the identification of the first monoclonal antibody reactive with a cytokine receptor molecule (9). These experimental approaches allowed us to calculate the Specific Activity of IL-2 for the first time, so that 1.0 U/mL = 150 ng/mL, which indicated that we would need to start with several liters of conditioned media to concentrate and purify adequate IL-2 protein to immunize mice with microgram amounts of TCGF protein and ultimately to develop the first monoclonal antibodies reactive with a lymphokine molecule (10). Prior to the development of quantitative bioassays and radioreceptor assays, investigators had tested only one dilution of a sample, e.g., a 1:2 or a 1:4, so that they could not quantify the amount of activity and relate it to a measured protein concentration. Thus, attempts were made to purify cytokine molecules with only 10–100 mL of starting conditioned media. In this article, we detailed the critical experimental approaches and advances that led to our success in generating monoclonal antibodies reactive with human TCGF, so that others might follow. Through rigorous biochemical analytic methods, it was possible to prove that the monoclonal antibodies were useful to immunoaffinity purify IL-2 molecules to homogeneity in milligram quantities from multiple liters of conditioned media that then could be used for unambiguous molecular and biological characteristics of the first interleukin molecule to be identified. Thus, with these new and novel cellular and molecular reagents, we could proceed to experiments that had never been done before.

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, 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

The TCR signals are essential for T cell activation and proliferation, primarily through the induction of cytokine and cytokine receptors. Several transcription factor families, including NF-kappaB/Rel, have been implicated in the regulation of cytokine gene expression in T cells in response to antigen, cytokine and mitogenic stimulation. In this study, we show that the mice with a null mutation in the lymphoid-specific c-Rel gene have normal development of lymphoid tissues and T cell compartment. However, T cells derived from the c-Rel knockout mice have several functional abnormalities. The c-Rel-deficient T lymphocytes fail to respond to activation and proliferation signals mediated by the TCR and mitogens in vitro. This is attributed to an impaired production of cytokines IL-2, IL-3 and granulocyte macrophage colony stimulating factor. In addition, the induction of IL-2R alpha chain is impaired in the c-Rel(-/-) T cells. The poor expression of cytokines and IL-2R alpha chain correlates with a reduced nuclear translocation of NF-kappaB components in c-Rel(-/-) T cells. Since activation is prerequisite for differentiation into effector cells, c-Rel(-/-) T cells failed to differentiate into cytotoxic T cells or Th cells without rescuing cytokines. However, upon supplement with exogenous IL-2, the c-Rel(-/-) cytotoxic T lymphocytes are able to execute cytotoxicity and the c-Rel(-/-) Th cells are capable of providing help to normal B cells. These data suggest that c-Rel is important for inducible cytokine and cytokine receptor expression, and a key regulator of early activation and proliferation in T cells.

By 1980 it was obvious that to more fully understand adaptive immunity, one needed to somehow reduce the tremendous complexity of antigen recognition by T cell populations. Thus, there were two developments that resulted in a paradigm shift in immunology, one being the generation of monoclonal antibodies (MoAbs), and the other the development of monoclonal functional antigen-specific T cell lines. For the first time, the cellular reagents became available to ask new questions as to how individual cells comprising the complex cell populations recognize and respond to changes in their molecular environments. The first successful generation of monoclonal T cells depended upon the understanding that antigen renders cells responsive to the antigen non-specific T cell growth factor that came to be termed interleukin-2 (IL-2), which could then be used in propagating large numbers of the progeny of single cells, which in turn could then be used for molecular analyses. Monoclonal functional human T cells were used to immunize mice to generate clone-specific (clonotypic) MoAbs, which then permitted the first biochemical characterizations of the antigen recognition elements of the T cell antigen receptor (TCR) complex. Moreover, the use of monoclonal cytolytic and helper/inducer human T cell clones essentially proved that the T cell-specific molecules T4 (CD4) and T8 (CD8) functioned as accessory molecules in antigen recognition by defining MHC class II or class I restriction respectively. As well, the expression of the T3 (CD3) molecules, found to be common to all T cells, were shown further to be obligatory for functional antigen-specific T cell signaling. The monoclonal IL-2-dependent T cells were also instrumental in the isolation and purification of the IL-2 molecule to homogeneity, the first interleukin molecule to be identified and characterized. These advances then led to the generation of pure radiolabeled IL-2 molecules that were used to identify the first interleukin cellular receptors, and as well the generation of the first MoAbs reactive with both IL-2 and IL-2 receptors. All of these advances led subsequently to the isolation of the first cDNA clones recognizing one of the two chains comprising the T cell antigen recognition elements (β-chain), as well cDNA clones encoding IL-2. Accordingly, armed with all of these unique cellular and molecular reagents, it was possible to determine that antigen triggering of the TCR complex initiates IL-2 production and IL-2 receptor expression, which in turn initiate the T cell clonal proliferative expansion, envisioned by Burnet in his formulation of the clonal selection theory. Thus, adaptive immunity receives antigen-specific activation signals from the environment and turns them into antigen non-specific endogenous action signals.

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.

Acute Plasmodium yoelii murine malaria is associated with a marked depression of splenic T cell responses. The present study was undertaken to address the question if a defect in T cell proliferation results from a relative increase of a non-T cell population in the spleen or real biological changes occurring in T cells of the spleen after infection. When animals were acutely infected, the splenic cells responded poorly to cross-linked anti-CD3 mAb, Con A, and PWM stimulation. At this stage, a very limited array of cytokine was expressed. We failed to detect the transcripts for IL-2R p55, IL-2, IL-6, IL-10, and IFN-γ in mice with acute P. yoelii malaria irrespective of the number of splenocytes subjected to RT-PCR. In contrast, late in the infection when mice cleared the parasites and became resistant to reinfection, mRNAs for the above cytokines as well as for IL-4, IL-5, GM-CSF, and TNF-α were detectable. During this late phase of infection, lymphocytes proliferated vigorously in response to TCR- and T cell mitogen-mediated stimulation. Surprisingly, during an early phase (as early as 3 days postinfection) with low parasitemia, before the establishment of T cell unresponsiveness, a broad array of cytokine expression including IL-2 and IFN-γ expression as well as marked lymphoproliferative response upon T cell mitogen- and TCR-mediated stimulation was observed. When the expression of cytokine gene in freshly isolated ( ex vivo ) splenocytes from P. yoelii -infected animals was investigated, a similar pattern of cytokine profile was detected. We devised a methodology in which RNA from an increasing number of splenocytes (ranging from 1 to 16 million) was used to compensate for any difference in the frequency of splenic T cells between immune and acutely infected mice and to augment target molecules which could be measured simultaneously by PCR. The data presented in this study led us to speculate that “anergy” or relative increase of a non-T cell population cannot account solely for the T cell unresponsiveness in the acute phase of infection. We suggest that inactivation or/and ablation of reactive T cells may explain T cell hyporesponsiveness during acute malaria.

Fifty years ago Jacques Miller devised a technique to thymectomize neonatal mice to explore the hypothesis that the thymus played a role in the development of immunity. He found that if thymectomized by day 3 postpartum, the mice would develop normally for the first month, but thereafter they underwent a runting syndrome similar to that observed during Graft vs. Host Disease (GvHD) (Miller, 1962). During the second month of life the mice would lose weight and suffer from a dermatitis and generalized lymphadenopathy and splenomegaly followed by premature death. A more detailed examination of the immune system revealed that early after thymectomy the mice were lymphopenic and immunocompromized, unable to reject allogeneic or even xenogeneic skin grafts, and incapable of generating antibodies to routine antigens. Miller correctly interpreted his findings as evidence that the thymus appeared to be critical in the first few weeks of life for the development of a mature functional immune system, but he did not speculate on the cause of the later enigmatic development of lymphoproliferation and apparent autoimmunity.

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.

We have examined the mechanism of thalidomide inhibition of lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-alpha) production and found that the drug enhances the degradation of TNF-alpha mRNA. Thus, the half-life of the molecule was reduced from approximately 30 to approximately 17 min in the presence of 50 micrograms/ml of thalidomide. Inhibition of TNF-alpha production was selective, as other LPS-induced monocyte cytokines were unaffected. Pentoxifylline and dexamethasone, two other inhibitors of TNF-alpha production, are known to exert their effects by means of different mechanisms, suggesting that the three agents inhibit TNF-alpha synthesis at distinct points of the cytokine biosynthetic pathway. These observations provide an explanation for the synergistic effects of these drugs. The selective inhibition of TNF-alpha production makes thalidomide an ideal candidate for the treatment of inflammatory conditions where TNF-alpha-induced toxicities are observed and where immunity must remain intact.

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.

Introduction: The Evolution of Our Understanding of the Immune System Molecular Immunology The Problem: Understanding How Molecules Direct the Behavior of Cells Compromising the Immune System The Quantal Theory of Immunity The VariabILity of Cell Cycle Progression and the Competence and Progression Phases of the Cell Cycle The Quantal Nature of IL-2-Promoted T Cell Cycle Progression The Molecular Basis for Quantal IL-2/IL-2R Signaling of Cell Cycle Progression: The IL-2/Receptor Interaction The Molecular Basis for Quantal IL-2/IL-2R Signaling of Cell Cycle Progression: IL-2 and IL-2 Receptor Metabolism The Molecular Basis for Quantal IL-2/IL-2R Signaling of Cell Cycle Progression: IL-2 Receptor Signaling via the JAK/STAT Pathway The Molecular Basis for Quantal IL-2/IL-2R Signaling of Cell Cycle Progression: IL-2 Receptor Signaling via Phosphorylation of IL-2R  Chain Y338 The T Cell Antigen Receptor Complex and the Quantal Regulation of the IL-2 and IL-2R Genes Signaling via the T Cell Antigen Receptor Complex Negative Feedback Regulation of T Cell Antigen Receptor Complex Signaling: Attenuation of IL-2 Gene Expression.

A simple in vitro experimental system was devised to reflect the in vivo generation of a T cell anamnestic response so that T cell differentiation could be examined at the level of lymphokine gene expression. Comparison of neonatal and adult T cells revealed that both populations expressed the genes for interleukin 2 (IL-2) and its receptor, but only adult T cells were capable of transcribing mRNAs for IL-3, IL-4, IL-5, IL-6, interferon gamma, and granulocyte/macrophage colony-stimulating factor. However, neonatal T cells could be induced to undergo functional differentiation in vitro, thereby acquiring the capacity to express the lymphokine gene repertoire characteristic for adult T cells. These data suggest that the T cells generated from neonatal blood by a primary stimulation in vitro are functionally indistinguishable from the T cells in adult blood that presumably have undergone primary stimulation in vivo. Therefore, we propose that the term "memory cell" be applied to those T cells that can be identified by their differentiated state of inducible effector-lymphokine gene expression.

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.

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.

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)

To further the understanding of the role of T cells in immunity to the parasite Toxoplasma gondii, antigen-specific T cell clones were generated using peripheral blood mononuclear cells from seropositive individuals. Whole parasites were used to stimulate a proliferative expansion of antigen-reactive cells, followed by limiting dilution cloning in the presence of irradiated, autologous PBMC and rIL-2. Three parasite antigen-specific T cell clones expressing the CD3+ phenotype were selected for further characterization. Phenotypic analysis with monoclonal antibodies revealed two clones reactive with CD8 (RTg1 and RTg3) while the other (RTg2) phenotyped as CD4+, CD8-. When tested in a proliferation assay using a panel of different T. gondii proteins, clone RTg1 reacted with a single large protein (Mr greater than 180,000) as well as smaller components (less than 12,000), clone RTg2 reacted with a protein of Mr = 28,000 and clone RTg3 reacted with a protein of 116,000 plus smaller components (less than 12,000). Only the 28,000 = Mr antigen recognized by RTg2 was reactive on Western blot with autologous donor antisera. All three clones produced IFN-gamma and IL-2 in varying amounts upon antigenic stimulation in the presence of irradiated APC. Moreover, one clone RTg1, exhibited direct parasite cytotoxicity, inhibiting extracellular T. gondii by greater than 70% when incubated at an effector/target ratio of 40:1. This clone was alpha, beta TCR heterodimer positive and exerted its cytotoxic parasiticidal activity in the apparent absence of MHC restriction. The results provide evidence for the existence of circulating antigen-specific cytotoxic T cells in normal humans who are toxoplasma antibody seropositive.

In the past 50 years, immunologists have accumulated an amazing amount of information as to how the immune system functions. However, one of the most fundamental aspects of immunity, how the immune system discriminates between self vs. non-self, still remains an enigma. Any attempt to explain this most intriguing and fundamental characteristic must account for this decision at the level of the whole immune system, but as well, at the level of the individual cells making up the immune system. Moreover, it must provide for a molecular explanation as to how and why the cells behave as they do. The "Quantal Theory", proposed herein, is based upon the "Clonal Selection Theory", first proposed by Sir McFarland Burnet in 1955, in which he explained the remarkable specificity as well as diversity of recognition of everything foreign in the environment. The "Quantal Theory" is built upon Burnet's premise that after antigen selection of cell clones, a proliferative expansion of the selected cells ensues. Furthermore, it is derived from experiments which indicate that the proliferation of antigen-selected cell clones is determined by a quantal, "all-or-none", decision promulgated by a critical number of cellular receptors triggered by the T Cell Growth Factor (TCGF), interleukin 2 (IL2). An extraordinary number of experiments reported especially in the past 20 years, and detailed herein, indicate that the T cell Antigen Receptor (TCR) behaves similarly, and also that there are several critical numbers of triggered TCRs that determine different fates of the T cells. Moreover, the fates of the cells appear ultimately to be determined by the TCR triggering of the IL2 and IL2 receptor (IL2R) genes, which are also expressed in a very quantal fashion. The "Quantal Theory" states that the fundamental decisions of the T cell immune system are dependent upon the cells receiving a critical number of triggered TCRs and IL2Rs and that the cells respond in an all-or-none fashion. The "Quantal Theory" accounts fully for the development of T cells in the thymus, and such fundamental cellular fates as both "positive" and "negative" selection, as well as the decision to differentiate into a "Regulatory T cell" (T-Reg). In the periphery, the "Quantal Theory" accounts for the decision to proliferate or not in response to the presence of an antigen, either non-self or self, or to differentiate into a T-Reg. Since the immune system discriminates between self and non-self antigens by the accumulated number of triggered TCRs and IL2Rs, therapeutic manipulation of the determinants of these quantal decisions should permit new approaches to either enhance or dampen antigen-specific immune responses.

Lymphocyte activation culminates in blastogenesis, cell cycle progression, DNA replication and mitosis. These complex cellular changes are programmed almost simultaneously by multiple ligands and receptors that trigger specific signal transduction pathways and transcription factors. Until now, the discovery of the genes regulated by each ligand/receptor pair has been hampered by the technologies available. To identify interleukin-2 (IL-2)-responsive genes, human peripheral blood mononuclear cells (PBMC) were pre-activated with anti-CD3, rested, and restimulated with IL-2 for 4 hr. Gene expression was analyzed using Affymetrix U95Av2 oligonucleotide arrays. To determine the most stringent parameters to score a gene as a bona fide IL-2 target, the expression of 19 known IL-2-regulated genes was examined first. All were induced at least 2-fold, with a difference in fluorescent intensity of ≥ 100 at p < 0.05. An additional 53 unique genes met these criteria. To determine which of these were immediate/early IL-2 targets in T cells, purified T cells were stimulated with IL-2 for 4 hr in the presence of cycloheximide to prevent secondary gene expression. Of the 72 genes identified in PBMCs, 20 were detected as immediate/early IL-2-regulated genes in purified T cells. In addition, 27 unique genes were IL-2-regulated in T cells but not in PBMCs. For a successful reductionist approach to the analysis of gene expression in lymphocyte activation, it is necessary to examine purified cell populations and immediate/early gene expression regulated by each ligand/receptor pair involved. This approach should allow the discovery of genes regulated by all of the ligand/receptor pairs involved in lymphocyte activation.

Measurement of glucocorticoid receptors appears to be useful for selecting which patients with leukemia and lymphoma should receive glucocorticoid therapy. To determine the effect of recent or concurrent glucocorticoid therapy on the number of measured tumor glucocorticoid receptor sites, 18 patients with leukemia and lymphoma were studied. Baseline determinations of numbers of glucocorticoid receptors were performed on the malignant cells circulating in the patients' peripheral blood. Glucocorticoid therapy was then instituted consisting of dexamethasone 4 mg p.o. every 6 hr. Repeat determinations of the number of glucocorticoid receptor sites were performed within 24 hr and at various subsequent times from the start of therapy. When compared to baseline receptor numbers, 16 of 18 patients demonstrated a decrease in receptor number (median decrease 1651 sites/cell) after the start of glucocorticoid therapy. The magnitude of the change in receptor number was independent on the initial number of receptors. Our results suggest that in order accurately interpret glucocorticoid receptor numbers in patients with leukemia and lymphoma, glucocorticoid should not be administered for 3 wk prior to determinations of receptor levels.

Glucocortoid receptor (GR) levels have not been extensively studied in untreated patients with chronic lymphocytic leukemia (CLL). We have measured tumor GR levels using a whole cell assay in 18 untreated patients with CLL and correlated these levels with various in-vitro and in-vivo data. In 12 of these patients, tumor cells from more than one tissue were simultaneously studied. Measurable GR levels were found in all 36 specimens studied with median total GR levels in blood, bone marrow, and lymph nodes being 3018 (n = 17), 3524 (n = 12) and 3102 (n = 7) sites/cell respectively. GR levels did not correlate with in-vitro sensitivity, as measured by inhibition by dexamethasone of radiolabelled leucine, uridine, and thymidine uptake, nor with the clinical or laboratory features examined. Nine patients were treated with dexamethasone as a single agent; blood GR levels were not predictive of antitumor response. We conclude that in patients with CLL, malignant cells from all involved tissues have measurable numbers of GRs, but these are not correlated with in-vitro sensitivity to glucocorticoid. Blood GR levels do not correlate with antitumor response; further studies are required to see if bone marrow or lymph node receptor levels correlate with antitumor response to steroids.

Prognostic factors for acute lymphocytic leukemia (ALL), including initial white blood count (WBC), age at diagnosis, sex, cytology, lymphocyte surface markers, and glucocorticoid receptors, have been evaluated in 12 patients presenting with ALL and the Philadelphia chromosome (Ph 1 + ALL). Ph 1 + ALL patients had a median initial WBC of 89 × 10 9 liter, a median age of 23 years and a male: female ratio of 1:1. The cytology of the blasts was L1 in 50% and L2 in 50%. The blasts in all cases studied were characteristic of non-T, non-B, common ALL, had high terminal deoxynucleotidyl transference levels, and low numbers of glucocorticoid receptors. The prognostic factors in these Ph 1 + ALL patients were compared with those of a concurrent group of adults with Ph 1 − ALL. Patients with Ph 1 + ALL more commonly had unfavorable prognostic features (high WBC, older age, male sex, L2 cytology, and low glucocorticoid receptors). They also had fewer complete remissions (58 vs. 100%, p=0.07), shorter remission durations (median 10 vs. 44+ months, p=0.001) and shorter survival times (median 11 vs. 48.5+ months, p=0.00008). However, even when patients with similar prognostic features were compared, those with the Ph 1 had fewer and shorter remissions and shorter survival times than ALL patients without the Ph 1 . These data suggest that the Ph 1 is an independent unfavorable prognostic factor in adults with ALL.

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.

Virus infected, fixed, replicate monolayer cell cultures can be pre-tested, stored, then withdrawn for use in the radioimmunoassay (RIA) as solid phase viral antigens. Ready availability of uniform, potent viral antigens permits quantitative determination of viral antibody concentrations simply, rapidly and precisely. Where cell destruction by cytolytic viruses might prevent in situ fixation of cell monolayers, a rapid and simple RIA procedure is described which utilizes imitation pearls as antigen adsorbents.

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.

Cycochemical, morphological, immunologic, and cytogenetic studies were carried out on hand-mirror cells (HMC) from a mentally retarded patient with a constitutional chromosome abnormality, 46,XX,r(21), and acute lymphoblastic leukemia. Scanning electron and differential interference contrast microscopy showed microspikes on the uropodia, but little evidence of cellular motility, despite formation and disappearance of individual uropodia in cell suspensions. The cells rosetted with sheep erythrocytes, suggesting T-cell origin. Cells derived from the bone marrow (80% HMC) showed a high degree of polyploidy (60%) and a bimodal chromosome number of 49 (49,XX,+10,-21, +3 rings) and 94 (6 no. 10, 3 no. 18, 2 no. 21 chromosomes, 3 ring chromosomes, plus 4 copies of each other chromosome).

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.

Levels of elastin production in explant culture of fetal sheep nuchal ligament and corresponding levels of translatable elastin mRNA were determined in parallel studies during a period of rapid growth of the embryo. The identity of the explant culture and cell-free products was confirmed by peptide mapping, immunoprecipitation, and the characteristic lack of histidine and methionine. Elastin production was quantitated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and radioimmune precipitation. The translation products could be labeled with methionine only when NH2-terminally donated as f-Met-tRNAfMet. Explant cultures showed a large rise in elastin production from 70 days after conception to 150 days after conception. Cell-free translation of RNA demonstrated a parallel in elastin mRNA levels and in elastin mRNA per cell. It appears, therefore, that the marked emphasis the differentiating nuchal ligament places on elastin production is modulated, at least in part, by the quantities of available elastin mRNA.

A rat X mouse T cell hybrid (PC60) proliferates in the absence of T cell growth factor (TCGF) and its cytolytic activity can be induced by culture in mixed leukocyte culture supernatants or concanavalin A-activated rat spleen cell supernatant (CS) to lyse 51Cr-labeled tumor target cells. To characterize the factor(s) responsible for this reversible induction, serum-free CS was fractionated by reverse phase high performance liquid chromatography and by phenyl-Sepharose chromatography. A cytotoxicity-inducing activity (CIA) was separated from TCGF and macrophage-activating factor/interferon-gamma. CIA was found to be a macromolecule with an apparent molecular weight of 12,000-18,000 and a pI of 5.0 and 6.2. Its activity on PC60 cells depended on the addition of TCGF. Thus TCGF may have other effects on T cells than the induction of entry into cell cycle. The number of TCGF surface receptors on PC60 cells was measured using purified 3H-TCGF. TCGF receptors were undetectable on noninduced cells but appeared during induction. The expression of TCGF receptors was not induced either by TCGF or by CIA-containing supernatants or fractions alone, only by a combination of both. These results show that TCGF plays a role in the regulation of the expression of its own receptors.

We recently reported that the spleen focus-forming virus [(SFFV), a replication-defective leukemogenic virus] coded for a cell surface neoantigen which could be detected by lymphocyte-mediated cytolysis. Because the SFFV genome has recently been characterized as a recombinant between both ecotropic and xenotropic viral information, we examined whether the SFFV-specific neoantigen, as defined by in vitro cytolysis, shared determinants with antigens coded for by mink cell focus (MCF)-inducing viruses (also xenotropic/ecotropic recombinants). As detected by direct cytolysis of SFFV- and MCF-infected target cells, the SFFV-specific neoantigen was found to be partially cross-reactive with determinants on the surface of MCF virus-infected cells. The cross-reactive determinants appeared to be coded for by the envelope region of both AKR and Moloney MCF viruses, in that antisera specific for MCF gp 70 were capable of blocking SFFV-specific neoantigen-directed cytolysis. These results provide further evidence in support of the recombinant nature of the SFFV genome and its close relationship to MCF viral genetic information. However, in that the SFFV-specific neoantigen was not totally cross-reactive with MCF gp70, it remains possible that additional antigenic determinants associated with SFFV-encoded transformation may be present.

T lymphocytes are stimulated to proliferate in an autocrine/paracrine manner by the lymphokine interleukin 2 (IL-2). In seeking further insight into the mechanisms by which IL-2 induces progression of T cells through the G1 phase of the cell cycle, studies were performed with agents that increase cellular adenosine 3',5'-cyclic monophosphate (cAMP), a well-known inhibitor of lymphocyte growth. The addition of dibutyryl-cAMP, cholera toxin, forskolin, or 3-isobutyl-1-methylxanthine to an IL-2-dependent murine T-cell line evoked a dose-related suppression of S-phase transition without affecting cellular viability. Moreover, elevation of cAMP levels led to an accumulation of uniformly small cells, suggesting an arrest in early G1. Consistent with these findings, dibutyryl-cAMP inhibited the incorporation of both [3H]-uridine and [3H]thymidine by IL-2-stimulated, synchronized normal human T cells. Furthermore, maximal inhibition occurred during early G1, as indicated by experiments where the addition of dibutyryl-cAMP was delayed with respect to IL-2 stimulation. Quantitative flow cytometric analysis of RNA and DNA content of IL-2-stimulated cells affirmed that increased cAMP inhibits RNA accumulation and S-phase transition. In addition, exposure of IL-2-dependent, asynchronously proliferating normal human T cells to dibutyryl-cAMP resulted in uniform growth arrest in early G1, the point at which cycling T cells accumulate when they are deprived of IL-2. These results indicate that increased cAMP inhibits G1 progression stimulated by IL-2 and provide a rationale for the use of cAMP analogues as pharmacologic probes for the dissection of molecular events occurring during IL-2 signaling and T-cell G1 transit. They also suggest the possibility of therapeutic immunosuppression by a combination of agents that act at different stages of the T-cell cycle.

Murine spleen cells activated by concanavalin A (Con A) in culture produce a class of lymphokine molecules which possess biological activity in a number of lymphocyte response assays. Lymphokines with a mol wt of 30,000, as estimated from gel filtration studies, can be resolved into two components which differ by charge, with isoelectric point (pI) values of 4.3 and 4.9, respectively. Both components stimulate (a) the growth of established T-cell lines in culture, (b) the proliferation of thymocytes in the presence of Con A under culture conditions where Con A alone is nonmitogenic, (c) the induction of antibody responses to heterologous erythrocyte antigens in athymic (nude) spleen cultures, (d) the generation of cytotoxic T lymphocytes (CTL) in thymocyte cultures, and (e) the generation of CTL in nude spleen cultures. In each of these culture systems we suggest that the assays are detecting a single class of lymphokine which acts directly on activated T cells. Nonactivated T cells must be stimulated by either antigen or mitogen before becoming responsive to lymphokine, but do not require antigen or mitogen for continued growth with lymphokine. The two molecular species, separable by isoelectric focusing are referred to as the T-cell growth factor (TCGF). A lymphokine, similar in size (30,000 daltons) to TCGF but heterogeneous in charge (pI 3.0--4.0), stimulates immune responses to erythrocyte antigens in T-cell-depleted spleen cultures but has no stimulatory activity in the other lymphocyte assay systems described. The data have been interpreted as showing the two molecular forms of murine TCGF (pI 4.3 and 4.9) are responsible for many of the lymphokine activities described elsewhere as thymocyte mitogenic factor, nonspecific T-cell-replacing factor and killer helper factor or costimulator. The other lymphokine, separable from TCGF by charge, appears to have true T-cell-replacing activity.