Epithelial organoid supports resident memory CD8 T cell differentiation.

Resident memory T cells (TRMs) play a vital role in regional immune defense. Although laboratory rodents have been extensively used to study fundamental TRM biology, poor isolation efficiency and low cell survival rates have limited the implementation of TRM-focused high-throughput assays. Here, we engineer a murine vaginal epithelial organoid (VEO)-CD8 T cell co-culture system that supports CD8 TRM differentiation. These in-vitro-generated TRMs are phenotypically and transcriptionally similar to in vivo TRMs. Pharmacological and genetic approaches showed that transforming growth factor ß (TGF-ß) signaling plays a crucial role in their differentiation. The VEOs in our model are susceptible to viral infections and the CD8 T cells are amenable to genetic manipulation, both of which will allow a detailed interrogation of antiviral CD8 T cell biology. Altogether we have established a robust in vitro TRM differentiation system that is scalable and can be subjected to high-throughput assays that will rapidly add to our understanding of TRMs.

Lessons for the Next Generation of Scientists from the Second Annual Arthur and Sandra Irving Cancer Immunology Symposium.

The Arthur and Sandra Irving Cancer Immunology Symposium has been created as a platform for established cancer immunologists to mentor trainees and young investigators as they launch their research career in the field. By sharing their different paths to success, the senior faculty mentors provide an invaluable resource to support the development of the next generation of leaders in the cancer immunology community. This Commentary describes some of the key topics that were discussed during the 2022 symposium: scientific and career trajectory, leadership, mentoring, collaborations, and publishing. For each of these topics, established investigators discussed the elements that facilitate success in these areas as well as mistakes that can hinder progress. Herein, we outline the critical points raised in these discussions for establishing a successful independent research career. These points are highly relevant for the broader scientific community.

Metabolic programs of T cell tissue residency empower tumour immunity.

Tissue resident memory CD8+ T (TRM) cells offer rapid and long-term protection at sites of reinfection1. Tumour-infiltrating lymphocytes with characteristics of TRM cells maintain enhanced effector functions, predict responses to immunotherapy and accompany better prognoses2,3. Thus, an improved understanding of the metabolic strategies that enable tissue residency by T cells could inform new approaches to empower immune responses in tissues and solid tumours. Here, to systematically define the basis for the metabolic reprogramming supporting TRM cell differentiation, survival and function, we leveraged in vivo functional genomics, untargeted metabolomics and transcriptomics of virus-specific memory CD8+ T cell populations. We found that memory CD8+ T cells deployed a range of adaptations to tissue residency, including reliance on non-steroidal products of the mevalonate-cholesterol pathway, such as coenzyme Q, driven by increased activity of the transcription factor SREBP2. This metabolic adaptation was most pronounced in the small intestine, where TRM cells interface with dietary cholesterol and maintain a heightened state of activation4, and was shared by functional tumour-infiltrating lymphocytes in diverse tumour types in mice and humans. Enforcing synthesis of coenzyme Q through deletion of Fdft1 or overexpression of PDSS2 promoted mitochondrial respiration, memory T cell formation following viral infection and enhanced antitumour immunity. In sum, through a systematic exploration of TRM cell metabolism, we reveal how these programs can be leveraged to fuel memory CD8+ T cell formation in the context of acute infections and enhance antitumour immunity.

Cancer immunosurveillance by gut T cells.

T cells primed in the gut influence immune responses to extraintestinal tumors.

Speed and Location Both Matter: Antigen Stimulus Dynamics Controls CAR-T Cell Response.

Despite the success in B-cell malignancies, chimeric antigen receptor (CAR)-T cell therapies have not yet demonstrated consistent efficacy across all patients and tumor types, particularly against solid tumors. Higher rates of T cell exhaustion are associated with inferior clinical outcomes following CAR-T cell therapy, which is prevalent in solid tumors. T cell exhaustion may originate from persistent and chronic antigen stimulation by tumor cells that resist and/or evade T cell-mediated killing. We exploited CAR-T exhaustion with a classic negative feedback model (incoherent feedforward loop, IFFL) to investigate the balance between CAR-T cell activation and exhaustion under different antigen presentation dynamics. Built upon the experimental and clinical data, we hypothesize that the speed and anatomical location of antigenic stimulation are both crucial to CAR-T cell response. Chronic antigenic stimulation as well as the harsh tumor microenvironment present multiple barriers to CAR-T cell efficacy in solid tumors. Many therapeutic strategies are individually insufficient to improve of CAR-T responses against solid tumors, as they clear but one of the many barriers CAR-T cells face in solid tumors. A combination strategy targeting multiple barriers holds promise to improve CAR-T therapy in solid tumors.

Bromodomain protein BRD4 directs and sustains CD8 T cell differentiation during infection.

In response to infection, pathogen-specific CD8 T cells differentiate into functionally diverse effector and memory T cell populations critical for resolving disease and providing durable immunity. Through small-molecule inhibition, RNAi studies, and induced genetic deletion, we reveal an essential role for the chromatin modifier and BET family member BRD4 in supporting the differentiation and maintenance of terminally fated effector CD8 T cells during infection. BRD4 bound diverse regulatory regions critical to effector T cell differentiation and controlled transcriptional activity of terminal effector-specific super-enhancers in vivo. Consequentially, induced deletion of Brd4 or small molecule-mediated BET inhibition impaired maintenance of a terminal effector T cell phenotype. BRD4 was also required for terminal differentiation of CD8 T cells in the tumor microenvironment in murine models, which we show has implications for immunotherapies. Taken together, these data reveal an unappreciated requirement for BRD4 in coordinating activity of cis regulatory elements to control CD8 T cell fate and lineage stability.

Heterogenous Populations of Tissue-Resident CD8+ T Cells Are Generated in Response to Infection and Malignancy.

Tissue-resident memory CD8+ T cells (Trm) provide host protection through continuous surveillance of non-lymphoid tissues. Using single-cell RNA-sequencing (scRNA-seq) and genetic reporter mice, we identified discrete lineages of intestinal antigen-specific CD8+ T cells, including a Blimp1hiId3lo tissue-resident effector cell population most prominent in the early phase of acute viral and bacterial infections and a molecularly distinct Blimp1loId3hi tissue-resident memory population that subsequently accumulated at later infection time points. These Trm populations exhibited distinct cytokine production, secondary memory potential, and transcriptional programs including differential roles for transcriptional regulators Blimp1, T-bet, Id2, and Id3 in supporting and maintaining intestinal Trm. Extending our analysis to malignant tissue, we also identified discrete populations of effector-like and memory-like CD8+ T cell populations with tissue-resident gene-expression signatures that shared features of terminally exhausted and progenitor-exhausted T cells, respectively. Our findings provide insight into the development and functional heterogeneity of Trm cells, which has implications for enhancing vaccination and immunotherapy approaches.

Early precursors and molecular determinants of tissue-resident memory CD8+ T lymphocytes revealed by single-cell RNA sequencing.

During an immune response to microbial infection, CD8+ T cells give rise to distinct classes of cellular progeny that coordinately mediate clearance of the pathogen and provide long-lasting protection against reinfection, including a subset of noncirculating tissue-resident memory (TRM) cells that mediate potent protection within nonlymphoid tissues. Here, we used single-cell RNA sequencing to examine the gene expression patterns of individual CD8+ T cells in the spleen and small intestine intraepithelial lymphocyte (siIEL) compartment throughout the course of their differentiation in response to viral infection. These analyses revealed previously unknown transcriptional heterogeneity within the siIEL CD8+ T cell population at several stages of differentiation, representing functionally distinct TRM cell subsets and a subset of TRM cell precursors within the tissue early in infection. Together, these findings may inform strategies to optimize CD8+ T cell responses to protect against microbial infection and cancer.

Myeloid Slc2a1-Deficient Murine Model Revealed Macrophage Activation and Metabolic Phenotype Are Fueled by GLUT1.

Macrophages (MΦs) are heterogeneous and metabolically flexible, with metabolism strongly affecting immune activation. A classic response to proinflammatory activation is increased flux through glycolysis with a downregulation of oxidative metabolism, whereas alternative activation is primarily oxidative, which begs the question of whether targeting glucose metabolism is a viable approach to control MΦ activation. We created a murine model of myeloid-specific glucose transporter GLUT1 (Slc2a1) deletion. Bone marrow-derived MΦs (BMDM) from Slc2a1M-/- mice failed to uptake glucose and demonstrated reduced glycolysis and pentose phosphate pathway activity. Activated BMDMs displayed elevated metabolism of oleate and glutamine, yet maximal respiratory capacity was blunted in MΦ lacking GLUT1, demonstrating an incomplete metabolic reprogramming. Slc2a1M-/- BMDMs displayed a mixed inflammatory phenotype with reductions of the classically activated pro- and anti-inflammatory markers, yet less oxidative stress. Slc2a1M-/- BMDMs had reduced proinflammatory metabolites, whereas metabolites indicative of alternative activation-such as ornithine and polyamines-were greatly elevated in the absence of GLUT1. Adipose tissue MΦs of lean Slc2a1M-/- mice had increased alternative M2-like activation marker mannose receptor CD206, yet lack of GLUT1 was not a critical mediator in the development of obesity-associated metabolic dysregulation. However, Ldlr-/- mice lacking myeloid GLUT1 developed unstable atherosclerotic lesions. Defective phagocytic capacity in Slc2a1M-/- BMDMs may have contributed to unstable atheroma formation. Together, our findings suggest that although lack of GLUT1 blunted glycolysis and the pentose phosphate pathway, MΦ were metabolically flexible enough that inflammatory cytokine release was not dramatically regulated, yet phagocytic defects hindered MΦ function in chronic diseases.

Transcriptional programming of tissue-resident memory CD8+ T cells.

Tissue-resident memory CD8+ T cells (TRM) are localized in non-lymphoid tissues throughout the body where they mediate long-lived protective immunity at common sites of pathogen exposure. As the signals controlling TRM differentiation are uncovered, it is becoming apparent that the dynamic activities of numerous transcription factors are intricately involved in TRM formation. Here, we highlight known transcriptional regulators of TRM differentiation and discuss how understanding the transcriptional programming of CD8+ T cell residency in non-lymphoid tissues can be leveraged to prevent or treat disease.

Erratum: Runx3 programs CD8+ T cell residency in non-lymphoid tissues and tumours.

This corrects the article DOI: 10.1038/nature24993.

Runx3 programs CD8+ T cell residency in non-lymphoid tissues and tumours.

Tissue-resident memory CD8+ T (TRM) cells are found at common sites of pathogen exposure, where they elicit rapid and robust protective immune responses. However, the molecular signals that control TRM cell differentiation and homeostasis are not fully understood. Here we show that mouse TRM precursor cells represent a unique CD8+ T cell subset that is distinct from the precursors of circulating memory cell populations at the levels of gene expression and chromatin accessibility. Using computational and pooled in vivo RNA interference screens, we identify the transcription factor Runx3 as a key regulator of TRM cell differentiation and homeostasis. Runx3 was required to establish TRM cell populations in diverse tissue environments, and supported the expression of crucial tissue-residency genes while suppressing genes associated with tissue egress and recirculation. Furthermore, we show that human and mouse tumour-infiltrating lymphocytes share a core tissue-residency gene-expression signature with TRM cells that is associated with Runx3 activity. In a mouse model of adoptive T cell therapy for melanoma, Runx3-deficient CD8+ tumour-infiltrating lymphocytes failed to accumulate in tumours, resulting in greater rates of tumour growth and mortality. Conversely, overexpression of Runx3 enhanced tumour-specific CD8+ T cell abundance, delayed tumour growth, and prolonged survival. In addition to establishing Runx3 as a central regulator of TRM cell differentiation, these results provide insight into the signals that promote T cell residency in non-lymphoid sites, which could be used to enhance vaccine efficacy or adoptive cell therapy treatments that target cancer.

B Cell Activity Is Impaired in Human and Mouse Obesity and Is Responsive to an Essential Fatty Acid upon Murine Influenza Infection.

Obesity is associated with increased risk for infections and poor responses to vaccinations, which may be due to compromised B cell function. However, there is limited information about the influence of obesity on B cell function and underlying factors that modulate B cell responses. Therefore, we studied B cell cytokine secretion and/or Ab production across obesity models. In obese humans, B cell IL-6 secretion was lowered and IgM levels were elevated upon ex vivo anti-BCR/TLR9 stimulation. In murine obesity induced by a high fat diet, ex vivo IgM and IgG were elevated with unstimulated B cells. Furthermore, the high fat diet lowered bone marrow B cell frequency accompanied by diminished transcripts of early lymphoid commitment markers. Murine B cell responses were subsequently investigated upon influenza A/Puerto Rico/8/34 infection using a Western diet model in the absence or presence of docosahexaenoic acid (DHA). DHA, an essential fatty acid with immunomodulatory properties, was tested because its plasma levels are lowered in obesity. Relative to controls, mice consuming the Western diet had diminished Ab titers whereas the Western diet plus DHA improved titers. Mechanistically, DHA did not directly target B cells to elevate Ab levels. Instead, DHA increased the concentration of the downstream specialized proresolving lipid mediators (SPMs) 14-hydroxydocosahexaenoic acid, 17-hydroxydocosahexaenoic acid, and protectin DX. All three SPMs were found to be effective in elevating murine Ab levels upon influenza infection. Collectively, the results demonstrate that B cell responses are impaired across human and mouse obesity models and show that essential fatty acid status is a factor influencing humoral immunity, potentially through an SPM-mediated mechanism.

Metabolic reprogramming of macrophages: glucose transporter 1 (GLUT1)-mediated glucose metabolism drives a proinflammatory phenotype.

Glucose is a critical component in the proinflammatory response of macrophages (MΦs). However, the contribution of glucose transporters (GLUTs) and the mechanisms regulating subsequent glucose metabolism in the inflammatory response are not well understood. Because MΦs contribute to obesity-induced inflammation, it is important to understand how substrate metabolism may alter inflammatory function. We report that GLUT1 (SLC2A1) is the primary rate-limiting glucose transporter on proinflammatory-polarized MΦs. Furthermore, in high fat diet-fed rodents, MΦs in crown-like structures and inflammatory loci in adipose and liver, respectively, stain positively for GLUT1. We hypothesized that metabolic reprogramming via increased glucose availability could modulate the MΦ inflammatory response. To increase glucose uptake, we stably overexpressed the GLUT1 transporter in RAW264.7 MΦs (GLUT1-OE MΦs). Cellular bioenergetics analysis, metabolomics, and radiotracer studies demonstrated that GLUT1 overexpression resulted in elevated glucose uptake and metabolism, increased pentose phosphate pathway intermediates, with a complimentary reduction in cellular oxygen consumption rates. Gene expression and proteome profiling analysis revealed that GLUT1-OE MΦs demonstrated a hyperinflammatory state characterized by elevated secretion of inflammatory mediators and that this effect could be blunted by pharmacologic inhibition of glycolysis. Finally, reactive oxygen species production and evidence of oxidative stress were significantly enhanced in GLUT1-OE MΦs; antioxidant treatment blunted the expression of inflammatory mediators such as PAI-1 (plasminogen activator inhibitor 1), suggesting that glucose-mediated oxidative stress was driving the proinflammatory response. Our results indicate that increased utilization of glucose induced a ROS-driven proinflammatory phenotype in MΦs, which may play an integral role in the promotion of obesity-associated insulin resistance.

Role of HGF in obesity-associated tumorigenesis: C3(1)-TAg mice as a model for human basal-like breast cancer.

Obesity is associated with basal-like breast cancer (BBC), an aggressive breast cancer subtype. The objective of this study was to determine whether obesity promotes BBC onset in adulthood and to evaluate the role of stromal-epithelial interactions in obesity-associated tumorigenesis. We hypothesized that hepatocyte growth factor (HGF) plays a promoting role in BBC, which express the HGF receptor, c-Met. In C3(1)-T(Ag) mice, a murine model of BBC, we demonstrated that obesity leads to a significant increase in HGF secretion and an associated decrease in tumor latency. By immunohistochemical analysis, normal mammary gland exhibited obesity-induced HGF, c-Met and phospho-c-Met, indicating that the activation of the cascade was obesity-driven. HGF secretion was also increased from primary mammary fibroblasts isolated from normal mammary glands and tumors of obese mice compared to lean. These results demonstrate that obesity-induced elevation of HGF expression is a stable phenotype, maintained after several passages, and after removal of dietary stimulation. Conditioned media from primary tumor fibroblasts from obese mice drove tumor cell proliferation. In co-culture, neutralization of secreted HGF blunted tumor cell migration, further linking obesity-mediated HGF-dependent effects to in vitro measures of tumor aggressiveness. In sum, these results demonstrate that HGF/c-Met plays an important role in obesity-associated carcinogenesis. Understanding the effects of obesity on risk and progression is important given that epidemiologic studies imply a portion of BBC could be eliminated by reducing obesity.

The inflammation highway: metabolism accelerates inflammatory traffic in obesity.

As humans evolved, perhaps the two strongest selection determinants of survival were a robust immune response able to clear bacterial, viral, and parasitic infection and an ability to efficiently store nutrients to survive times when food sources were scarce. These traits are not mutually exclusive. It is now apparent that critical proteins necessary for regulating energy metabolism, such as peroxisome proliferator-activated receptors, Toll-like receptors, and fatty acid-binding proteins, also act as links between nutrient metabolism and inflammatory pathway activation in immune cells. Obesity in humans is a symptom of energy imbalance: the scale has been tipped such that energy intake exceeds energy output and may be a result, in part, of evolutionary selection toward a phenotype characterized by efficient energy storage. As discussed in this review, obesity is a state of low-grade, chronic inflammation that promotes the development of insulin resistance and diabetes. Ironically, the formation of systemic and/or local, tissue-specific insulin resistance upon inflammatory cell activation may actually be a protective mechanism that co-evolved to repartition energy sources within the body during times of stress during infection. However, the point has been reached where a once beneficial adaptive trait has become detrimental to the health of the individual and an immense public health and economic burden. This article reviews the complex relationship between obesity, insulin resistance/diabetes, and inflammation, and although the liver, brain, pancreas, muscle, and other tissues are relevant, we focus specifically on how the obese adipose microenvironment can promote immune cell influx and sustain damaging inflammation that can lead to the onset of insulin resistance and diabetes. Finally, we address how substrate metabolism may regulate the immune response and discuss how fuel uptake and metabolism may be a targetable approach to limit or abrogate obesity-induced inflammation.

The impact of obesity on the immune response to infection.

There is strong evidence indicating that excess adiposity negatively impacts immune function and host defence in obese individuals. This is a review of research findings concerning the impact of obesity on the immune response to infection, including a discussion of possible mechanisms. Obesity is characterised by a state of low-grade, chronic inflammation in addition to disturbed levels of circulating nutrients and metabolic hormones. The impact of these metabolic abnormalities on obesity-related comorbidities has undergone intense scrutiny over the past decade. However, relatively little is known of how the immune system and host defence are influenced by the pro-inflammatory and excess energy milieu of the obese. Epidemiological data suggest obese human subjects are at greater risk for nosocomial infections, especially following surgery. Additionally, the significance of altered immunity in obese human subjects is emphasised by recent studies reporting obesity to be an independent risk factor for increased morbidity and mortality following infection with the 2009 pandemic influenza A (H1N1) virus. Rodent models offer important insight into how metabolic abnormalities associated with excess body weight can impair immunity. However, more research is necessary to understand the specific aspects of immunity that are impaired and what factors are contributing to reduced immunocompetence in the obese. Additionally, special consideration of how infection in this at-risk population is managed is required, given that this population may not respond optimally to antimicrobial drugs and vaccination. Obesity impacts millions globally, and greater understanding of its associated physiological disturbances is a key public health concern.