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.

Differential regulation of nucleus accumbens glutamate and GABA in obesity-prone and obesity-resistant rats.

Obesity is one of the leading health concerns in the United States. Studies from human and rodent models suggest that inherent differences in the function of brain motivation centers, including the nucleus accumbens (NAc), contribute to overeating and thus obesity. For example, there are basal enhancements in the excitability of NAc GABAergic medium spiny neurons (MSN) and reductions in basal expression of AMPA-type glutamate receptors in obesity-prone vs obesity-resistant rats. However, very little is known about the regulation of extracellular glutamate and GABA within the NAc of these models. Here we gave obesity-prone and obesity-resistant rats stable isotope-labeled glucose (13 C6 -glucose) and used liquid chromatography mass spectrometry (LC-MS) analysis of NAc dialysate to examine the real-time incorporation of 13 C6 -glucose into glutamate, glutamine, and GABA. This novel approach allowed us to identify differences in glucose utilization for neurotransmitter production between these selectively bred lines. We found that voluntarily ingested or gastrically infused 13 C6 -glucose rapidly enters the NAc and is incorporated into 13 C2 -glutamine, 13 C2 -glutamate, and 13 C2 -GABA in both groups within minutes. However, the magnitude of increases in NAc 13 C2 -glutamine and 13 C2 -GABA were lower in obesity-prone than in obesity-resistant rats, while basal levels of glutamate were elevated. This suggested that there may be differences in the astrocytic regulation of these analytes. Thus, we next examined NAc glutamine synthetase, GAD67, and GLT-1 protein expression. Consistent with reduced 13 C2 -glutamine and 13 C2 -GABA, NAc glutamine synthetase and GLT-1 protein expression were reduced in obesity-prone vs obesity-resistant groups. Taken together, these data show that NAc glucose utilization differs dramatically between obesity-prone and obesity-resistant rats, favoring glutamate over GABA production in obesity-prone rats and that reductions in NAc astrocytic recycling of glutamate contribute to these differences. These data are discussed in light of established differences in NAc function between these models and the role of the NAc in feeding behavior.

Sudden death after being in a bounce house: a late complication of congenital diaphragmatic hernia.

A 3-year-old white female with no significant past medical history was found unresponsive and apneic after several hours of vomiting. The patient had reportedly felt unwell since jumping in a bounce house at a festival earlier in the day. After one hour of attempted resuscitation by EMS and hospital staff, the patient was pronounced deceased. On autopsy there was a 3 cm opening in the left posterolateral hemidiaphragm with the spleen, the stomach, and portions of the small and large bowel displaced into the left chest cavity, resulting in compression of both lungs and the heart to the right side of the chest. The right lung weighed 295 g while the left lung weighed 73 g. Histologic examination revealed atelectasis in the left lung and congestion in the right lung, the spleen, the stomach, and the bowel. The cause of death was ultimately ruled to be respiratory compromise and associated gastrointestinal complications due to diaphragmatic herniation of abdominal contents into the chest cavity. It is hypothesized that herniation occurred in this case secondary to increased intra-abdominal pressure from jumping in a bounce house, an etiology that we were unable to find elsewhere despite a thorough literature search.

Identification of Cellular Sources of IL-2 Needed for Regulatory T Cell Development and Homeostasis.

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.

Prolonged interleukin-2Ralpha expression on virus-specific CD8+ T cells favors terminal-effector differentiation in vivo.

CD25, the high-affinity interleukin-2 (IL-2) receptor alpha chain, is rapidly upregulated by antigen-specific CD8(+) T cells after T cell receptor stimulation. Here, we demonstrate that during an acute viral infection, CD25 expression is quite dynamic-after initial upregulation, a subset of virus-specific T cells sustains CD25 expression longer than the rest. At this time when there is distinct heterogeneity in CD25 expression, examination of the in vivo fate of effector cells revealed that CD25(lo) cells, which are relatively less sensitive to IL-2, preferentially upregulate CD127 and CD62L and give rise to functional long-lived memory cells. In contrast, CD25(hi) cells perceiving prolonged IL-2 signals proliferate more rapidly, are prone to apoptosis, exhibit a more pronounced effector phenotype, and appear to be terminally differentiated. Consistent with this, sustained IL-2 receptor signaling during expansion drove terminal-effector differentiation. These data support the hypothesis that prolonged IL-2 signals during priming promote terminal-effector differentiation.

HIV alters neuronal mitochondrial fission/fusion in the brain during HIV-associated neurocognitive disorders.

HIV-associated neurocognitive disorders (HAND) still occur in approximately 50% of HIV patients, and therapies to combat HAND progression are urgently needed. HIV proteins are released from infected cells and cause neuronal damage, possibly through mitochondrial abnormalities. Altered mitochondrial fission and fusion is implicated in several neurodegenerative disorders. Here, we hypothesized that mitochondrial fission/fusion may be dysregulated in neurons during HAND. We have identified decreased mitochondrial fission protein (dynamin 1-like; DNM1L) in frontal cortex tissues of HAND donors, along with enlarged and elongated mitochondria localized to the soma of damaged neurons. Similar pathology was observed in the brains of GFAP-gp120 tg mice. In vitro, recombinant gp120 decreased total and active DNM1L levels, reduced the level of Mitotracker staining, and increased extracellular acidification rate (ECAR) in primary neurons. DNM1L knockdown enhanced the effects of gp120 as measured by reduced Mitotracker signal in the treated cells. Interestingly, overexpression of DNM1L increased the level of Mitotracker staining in primary rat neurons and reduced neuroinflammation and neurodegeneration in the GFAP-gp120-tg mice. These data suggest that mitochondrial biogenesis dynamics are shifted towards mitochondrial fusion in brains of HAND patients and this may be due to gp120-induced reduction in DNM1L activity. Promoting mitochondrial fission during HIV infection of the CNS may restore mitochondrial biogenesis and prevent neurodegeneration.

Conjugated block copolymer photovoltaics with near 3% efficiency through microphase separation.

Organic electronic materials have the potential to impact almost every aspect of modern life including how we access information, light our homes, and power personal electronics. Nevertheless, weak intermolecular interactions and disorder at junctions of different organic materials limit the performance and stability of organic interfaces and hence the applicability of organic semiconductors to electronic devices. Here, we demonstrate control of donor-acceptor heterojunctions through microphase-separated conjugated block copolymers. When utilized as the active layer of photovoltaic cells, block copolymer-based devices demonstrate efficient photoconversion well beyond devices composed of homopolymer blends. The 3% block copolymer device efficiencies are achieved without the use of a fullerene acceptor. X-ray scattering results reveal that the remarkable performance of block copolymer solar cells is due to self-assembly into mesoscale lamellar morphologies with primarily face-on crystallite orientations. Conjugated block copolymers thus provide a pathway to enhance performance in excitonic solar cells through control of donor-acceptor interfaces.

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.

Smallpox: can we still learn from the journey to eradication?

One of the most celebrated achievements of immunology and modern medicine is the eradication of the dreaded plague smallpox. From the introduction of smallpox vaccination by Edward Jenner, to its popularization by Louis Pasteur, to the eradication effort led by Donald Henderson, this story has many lessons for us today, including the characteristics of the disease and vaccine that permitted its eradication, and the obviousness of the vaccine as a vector for other intractable Infectious diseases. The disease itself, interpreted in the light of modern molecular immunology, is an obvious immunopathological disease, which occurs after a latent interval of 1-2 weeks, and manifests as a systemic cell-mediated delayed type hypersensitivity (DTH) syndrome. The vaccine that slayed this dragon was given the name vaccinia, and was thought to have evolved from cowpox virus, but is now known to be most closely related to a poxvirus isolated from a horse. Of interest is the fact that of the various isolates of orthopox viruses, only variola, vaccinia and monkeypox viruses can infect humans. In contrast to the systemic disease of variola, vaccinia only replicates locally at the site of inoculation, and causes a localized DTH response that usually peaks after 7-10 days. This difference in the pathogenicity of variola vs. vaccinia is thought to be due to the capacity of variola to circumvent innate immunity, which allows it to disseminate widely before the adaptive immune response occurs. Thus, the fact that vaccinia virus is attenuated compared to variola, but is still replication competent, makes for its remarkable efficacy as a vaccine, as the localized infection activates all of the cells and molecules of both innate and adaptive immunity. Accordingly vaccinia itself, and not modified replication incompetent vaccina, is the hope for use as a vector in the eradication of additional pathogenic microbes from the globe.

Gender, Generations, and Guilt: Defendant Gender and Age Affect Jurors' Decisions and Perceptions in an Intimate Partner Homicide Trial.

Using the context of an intimate partner homicide trial, the study explored the effects of defendant gender and age on mock-jurors' verdicts, sentences, and culpability ratings-and whether defendant credibility and juror anger mediate these effects. The study used a 2 (Defendant Gender: male vs. female) × 3 (Defendant Age: 25, 45, or 65 years) between-subjects design. Participants (N = 513 community members) completed the experiment online. Participants were randomly assigned to one of the six Defendant Gender × Age Conditions. Participants read the trial transcripts that included the age and gender manipulations, provided verdicts and sentences, and completed the following measures: culpability, anger, credibility, and manipulation checks. Consistent with our hypotheses mock-jurors were more likely to find the male defendant guilty and give him longer sentences than the female defendant. Additionally, when the defendant was male (vs. female) mock-jurors provided higher anger ratings and rated the defendant as more culpable in the victim's death. Also consistent with our hypotheses, mock-jurors were more likely to find the youngest defendant guilty and view him as more culpable and less credible than the oldest defendant. The mechanisms responsible for jurors' biased decisions varied as a function of the extra-legal variable (defendant gender vs. age). The defendant age effect was mediated by defendant credibility and the gender effect by juror anger. A defendant's right to a fair trial is dependent on a court's ability to limit extra-legal variables from influencing jurors' decisions. Understanding the mechanism responsible for such bias is required before the courts can effectively remedy bias.

American Delirium Society 2022 Year in Review: Highlighting the Year's Most Impactful Delirium Research.

Background: Since 2015, the American Delirium Society (ADS) Research Committee has conducted an annual survey of the delirium literature for presentation in its year-in-review session. Our objectives were to describe the review process used for the 2021-2022 and to summarise the selected publications. Methods: Each member of the ADS Research Committee nominated up to 6 publications considered to be the most impactful primary delirium research published from September 1, 2021, to July 31, 2022. The 24 nominated studies were divided into three categories balanced by number of articles: medical intervention trials, non-medical intervention trials, and delirium detection/basic science studies. Each ADS Research Committee member ranked all studies in their assigned category for methodological rigor and for impact, each being scored as 0-10, for a total score of 0-20. It was decided a priori to select the top three highest-scoring articles in each category for presentation, with ties adjudicated by Committee consensus. Results: Nineteen Research Committee members served as reviewers. Scores for each category were similar: medical interventions mean (standard deviation) 12.8 (1.1), non-medical interventions 13.1 (1.1), and detection/basic science 12.6 (1.0). We summarise the results of the papers presented in the 2022 ADS year-in-review session. Conclusion: The diversity of studies presented for the 2022 ADS year-in-review session illustrates the breadth of the delirium field and the growing number of clinical trials. The dissemination of publications across a broad, diverse array of journals provides further justification of the need for delirium-specific journals.

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.

Autocrine and paracrine IL-2 signals collaborate to regulate distinct phases of CD8 T cell memory.

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

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 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.

Single-cell quantification of IL-2 response by effector and regulatory T cells reveals critical plasticity in immune response.

Understanding how the immune system decides between tolerance and activation by antigens requires addressing cytokine regulation as a highly dynamic process. We quantified the dynamics of interleukin-2 (IL-2) signaling in a population of T cells during an immune response by combining in silico modeling and single-cell measurements in vitro. We demonstrate that IL-2 receptor expression levels vary widely among T cells creating a large variability in the ability of the individual cells to consume, produce and participate in IL-2 signaling within the population. Our model reveals that at the population level, these heterogeneous cells are engaged in a tug-of-war for IL-2 between regulatory (T(reg)) and effector (T(eff)) T cells, whereby access to IL-2 can either increase the survival of T(eff) cells or the suppressive capacity of T(reg) cells. This tug-of-war is the mechanism enforcing, at the systems level, a core function of T(reg) cells, namely the specific suppression of survival signals for weakly activated T(eff) cells but not for strongly activated cells. Our integrated model yields quantitative, experimentally validated predictions for the manipulation of T(reg) suppression.

Therapeutic use of IL-2 to enhance antiviral T-cell responses in vivo.

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.