Epithelial IL-2 is critical for NK cell-mediated cancer immunosurveillance in mammary glands.

Interleukin 2 (IL-2) is the first identified cytokine and its interaction with receptors has been known to shape the immune responses in many lymphoid or non-lymphoid tissues for more than four decades. Active T cells are the primary cellular source for IL-2 production and epithelial cells have never been considered the major cellular source of IL-2 under physiological conditions. It is, however, tempting to speculate that epithelial cells could potentially express IL-2 that regulates the intricate interactions between epithelial cells and lymphocytes. Datamining our recently published single-cell RNAseq in the mouse mammary gland identified IL-2 expression in mammary epithelial cells, which is induced by prolactin via the STAT5 signaling pathway. Furthermore, epithelial IL-2 plays a crucial role in maintaining the physiological functions of natural killer (NK) cells within the mammary glands. IL-2 deletion in the mammary epithelial cells leads to a significant reduction in the number and function of NK cells, which in turn results in defective immunosurveillance, expansion of luminal epithelial cells, and tumor development. Interestingly, T cells in the mammary glands are not changed, indicating the specific regulation of NK cells by epithelial IL-2 production. In agreement, we also found that human epithelial cells express IL-2 and NK cells express the highest level of IL2RB among all the immune cells. Here, we provide the first evidence that epithelial cells produce IL-2, which is critical for maintaining the physiological functions of NK cells in immunosurveillance.

PD-1 combination therapy with IL-2 modifies CD8+ T cell exhaustion program.

Combination therapy with PD-1 blockade and IL-2 is highly effective during chronic lymphocytic choriomeningitis virus infection1. Here we examine the underlying basis for this synergy. We show that PD-1 + IL-2 combination therapy, in contrast to PD-1 monotherapy, substantially changes the differentiation program of the PD-1+TCF1+ 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+TCF1+ 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 has 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 to CD25 but still binds to CD122 and CD132 almost completely abrogated the synergistic effects observed after PD-1 + IL-2 combination therapy. There is considerable interest in PD-1 + IL-2 combination therapy for patients with cancer2,3, and our fundamental studies defining the underlying mechanisms of how IL-2 synergizes with PD-1 blockade should inform these human translational studies.

Innate lymphoid cells support regulatory T cells in the intestine through interleukin-2.

Interleukin (IL)-2 is a pleiotropic cytokine that is necessary to prevent chronic inflammation in the gastrointestinal tract1-4. The protective effects of IL-2 involve the generation, maintenance and function of regulatory T (Treg) cells4-8, and the use of low doses of IL-2 has emerged as a potential therapeutic strategy for patients with inflammatory bowel disease9. 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 Treg cells and immunological homeostasis throughout the gastrointestinal tract. Notably, lineage-specific deletion of IL-2 in T cells did not reduce Treg 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-1ß. Macrophages in the small intestine produce IL-1ß, 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 Treg 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 Treg cells. Our results reveal a previously unappreciated pathway in which a microbiota- and IL-1ß-dependent axis promotes the production of IL-2 by ILC3s to orchestrate immune regulation in the intestine.

PD-L1 blockade synergizes with IL-2 therapy in reinvigorating exhausted T cells.

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 roles of c-rel and interleukin-2 in tolerance: a molecular explanation of self-nonself discrimination.

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.

Following the cytokine signaling pathway to leukemogenesis: a chronology.

Studies over the past 50 years revealing the molecular events that promote normal T lymphocyte cycle competence and progression led to a detailed understanding of how cytokines function to regulate normal hematopoietic cell proliferation. During that same period, the molecular and genetic changes introduced by the Philadelphia chromosome in chronic myelogenous leukemia were unraveled, and these have led to an understanding of how mutations that constitutively activate normal cytokine signaling pathways can cause unregulated cell proliferation and malignant transformation. Based on the paradigm established by these data, it is inescapable that going forward, investigators will operate under the hypothesis that transformation of additional cells and tissues will have a similar pathogenesis.

The quantal theory of immunity and the interleukin-2-dependent negative feedback regulation of the immune response.

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.

Manipulating both the inhibitory and stimulatory immune system towards the success of therapeutic vaccination against chronic viral infections.

SUMMARY: One potentially promising strategy to control chronic infections such as human immunodeficiency virus, hepatitis B virus, and hepatitis C virus is therapeutic vaccination, which aims to reduce persisting virus by stimulating a patient's own antiviral immune responses. However, this approach has fallen short of expectations, because antiviral T cells generated during chronic infections often become functionally exhausted and thus do not respond properly to therapeutic vaccination. Therefore, it is necessary to develop a therapeutic vaccine strategy to more effectively boost endogenous T-cell responses to control persistent viral infections. Studies to elucidate the cause of impaired T-cell function have pointed to sustained inhibitory receptor signaling through T-cell expression of programmed death 1 (PD-1). Recently, another inhibitory molecule, cytotoxic T lymphocyte antigen 4 (CTLA-4), and also an immunosuppressive cytokine, interleukin 10 (IL-10), have been reported to be potential factors of establishing immune suppression and viral persistence. Blocking these negative signaling pathways could restore the host immune system, enabling it to respond to further stimulation. Indeed, combining therapeutic vaccination along with the blockade of inhibitory signals could synergistically enhance functional CD8(+) T-cell responses and improve viral control in chronically infected mice, providing a promising strategy for the treatment of chronic viral infections. Furthermore, not only the ablation of negative signals but also the addition of stimulatory signals, such as interleukin 2 (IL-2), might prove to be a potentially promising strategy to augment the efficacy of therapeutic vaccination against chronic viral infections.

Determining to divide: how do cells decide?

A cell's decision to divide must be regulated with the highest fidelity. Otherwise, abnormalities occurring in the replication of genetic material and cytokinesis would be incompatible with life. It has been known for almost a century that cells comprising a population undergo cellular division at extremely variable rates, even though genetically identical cell clones have been examined. Studies with T lymphocytes at the single cell level have revealed that the rate of cellular division is determined by the accumulation of a critical number of ligand-triggered interleukin-2 (IL2) receptors at the cell surface throughout the G(1) phase of the cell cycle. Thus, the cell "counts" the number of triggered IL2 receptors, and only decides to divide when the critical number has been attained. This information is then transferred to the cellular interior via intracellular sensors comprised of D-type cyclins, which ultimately determine when the cell surpasses the "Restriction Point" in late G(1), and which commits the cell irrevocably to initiate DNA replication. Beyond the R-point, the cell assembles a definite number of macromolecular pre-replication complexes (Pre-RCs) comprised of at least 6 distinct proteins at sites of the origin of replication on DNA. Complete assembly of the Pre-RCs is a prerequisite for their subsequent disassembly, which must occur before the initiation of DNA strand replication, and which occurs asynchronously throughout the S-phase of the cell cycle and only terminates when the entire DNA has been duplicated. Thus, the fidelity of the decision to divide is exquisitely regulated by macromolecular mechanisms initiated at the cell surface and transferred to the cellular interior so that the cell can make the decision in a quantal (all-or-none) fashion. The question before us is how this quantal decision is made at the molecular level. The available data indicate that the assembly and disassembly of a definite number of large multicomponent macromolecular complexes make the quantal decisions. Here, it is postulated that all fundamental cellular decisions, i.e. survival, death, proliferation and differentiation, are regulated in this fashion. It remains to be determined how the cell counts the signals it receives, and what the molecular forces are that dictate the behavior of macromolecular complexes.