Adiposity, immunity, and inflammation: interrelationships in health and disease: a report from 24th Annual Harvard Nutrition Obesity Symposium, June 2023.

The rapidly evolving field of immunometabolism explores how changes in local immune environments may affect key metabolic and cellular processes, including that of adipose tissue. Importantly, these changes may contribute to low-grade systemic inflammation. In turn, chronic low-grade inflammation affecting adipose tissue may exacerbate the outcome of metabolic diseases. Novel advances in our understanding of immunometabolic processes may critically lead to interventions to reduce disease severity and progression. An important example in this regard relates to obesity, which has a multifaceted effect on immunity, activating the proinflammatory pathways such as the inflammasome and disrupting cellular homeostasis. This multifaceted effect of obesity can be investigated through study of downstream conditions using cellular and systemic investigative techniques. To further explore this field, the National Institutes of Health P30 Nutrition Obesity Research Center at Harvard, in partnership with Harvard Medical School, assembled experts to present at its 24th Annual Symposium entitled "Adiposity, Immunity, and Inflammation: Interrelationships in Health and Disease" on 7 June, 2023. This manuscript seeks to synthesize and present key findings from the symposium, highlighting new research and novel disease-specific advances in the field. Better understanding the interaction between metabolism and immunity offers promising preventative and treatment therapies for obesity-related immunometabolic diseases.

Metformin and feeding increase levels of the appetite-suppressing metabolite Lac-Phe in humans.

Metformin, a widely used first-line treatment for type 2 diabetes (T2D), is known to reduce blood glucose levels and suppress appetite. Here we report a significant elevation of the appetite-suppressing metabolite N-lactoyl phenylalanine (Lac-Phe) in the blood of individuals treated with metformin across seven observational and interventional studies. Furthermore, Lac-Phe levels were found to rise in response to acute metformin administration and post-prandially in patients with T2D or in metabolically healthy volunteers.

Immune activation state modulates infant engram expression across development.

Infantile amnesia is possibly the most ubiquitous form of memory loss in mammals. We investigated how memories are stored in the brain throughout development by integrating engram labeling technology with mouse models of infantile amnesia. Here, we found a phenomenon in which male offspring in maternal immune activation models of autism spectrum disorder do not experience infantile amnesia. Maternal immune activation altered engram ensemble size and dendritic spine plasticity. We rescued the same apparently forgotten infantile memories in neurotypical mice by optogenetically reactivating dentate gyrus engram cells labeled during complex experiences in infancy. Furthermore, we permanently reinstated lost infantile memories by artificially updating the memory engram, demonstrating that infantile amnesia is a reversible process. Our findings suggest not only that infantile amnesia is due to a reversible retrieval deficit in engram expression but also that immune activation during development modulates innate, and reversible, forgetting switches that determine whether infantile amnesia will occur.

Diet, nutrient supply, and tumor immune responses.

Nutrients are essential for cell function. Immune cells operating in the complex tumor microenvironment (TME), which has a unique nutrient composition, face challenges of adapting their metabolism to support effector functions. We discuss the impact of nutrient availability on immune function in the tumor, competition between immune cells and tumor cells for nutrients, and how this is altered by diet. Understanding which diets can promote antitumor immune responses could open a new era of treatment, where dietary modifications can be used as an adjunct to boost the success of existing cancer therapies.

Interleukin-17 as a key player in neuroimmunometabolism.

In mammals, interleukin (IL)-17 cytokines are produced by innate and adaptive lymphocytes. However, the IL-17 family has widespread expression throughout evolution, dating as far back as cnidaria, molluscs and worms, which predate lymphocytes. The evolutionary conservation of IL-17 suggests that it is involved in innate defence strategies, but also that this cytokine family has a fundamental role beyond typical host defence. Throughout evolution, IL-17 seems to have a major function in homeostatic maintenance at barrier sites. Most recently, a pivotal role has been identified for IL-17 in regulating cellular metabolism, neuroimmunology and tissue physiology, particularly in adipose tissue. Here we review the emerging role of IL-17 signalling in regulating metabolic processes, which may shine a light on the evolutionary role of IL-17 beyond typical immune responses. We propose that IL-17 helps to coordinate the cross-talk among the nervous, endocrine and immune systems for whole-body energy homeostasis as a key player in neuroimmunometabolism.

Human Granzyme K Is a Feature of Innate T Cells in Blood, Tissues, and Tumors, Responding to Cytokines Rather than TCR Stimulation.

NK cells and CD8 T cells use cytotoxic molecules to kill virally infected and tumor cell targets. While perforin and granzyme B (GzmB) are the most commonly studied lytic molecules, less is known about granzyme K (GzmK). However, this granzyme has been recently associated with improved prognosis in solid tumors. In this study, we show that, in humans, GzmK is predominantly expressed by innate-like lymphocytes, as well as a newly identified population of GzmK+CD8+ non- mucosal-associated invariant T cells with innate-like characteristics. We found that GzmK+ T cells are KLRG1+EOMES+IL-7R+CD62L-Tcf7int, suggesting that they are central memory T and effector memory T cells. Furthermore, GzmK+ cells are absent/low in cord blood, suggesting that GzmK is upregulated with immune experience. Surprisingly, GzmK+ cells respond to cytokine stimuli alone, whereas TCR stimulation downregulates GzmK expression, coinciding with GzmB upregulation. GzmK+ cells have reduced IFN-γ production compared with GzmB+ cells in each T cell lineage. Collectively, this suggests that GzmK+ cells are not naive, and they may be an intermediate memory-like or preterminally differentiated population. GzmK+ cells are enriched in nonlymphoid tissues such as the liver and adipose. In colorectal cancer, GzmK+ cells are enriched in the tumor and can produce IFN-γ, but GzmK+ expression is mutually exclusive with IL-17a production. Thus, in humans, GzmK+ cells are innate memory-like cells that respond to cytokine stimulation alone and may be important effector cells in the tumor.

γδ T cell dichotomy with opposing cytotoxic and wound healing functions in human solid tumors.

γδ T cells are important tissue-resident, innate T cells that are critical for tissue homeostasis. γδ cells are associated with positive prognosis in most tumors; however, little is known about their heterogeneity in human cancers. Here, we phenotyped innate and adaptive cells in human colorectal (CRC) and endometrial cancer. We found striking differences in γδ subsets and function in tumors compared to normal tissue, and in the γδ subsets present in tumor types. In CRC, an amphiregulin (AREG)-producing subset emerges, while endometrial cancer is infiltrated by cytotoxic cells. In humanized CRC models, tumors induced this AREG phenotype in Vδ1 cells after adoptive transfer. To exploit the beneficial roles of γδ cells for cell therapy, we developed an expansion method that enhanced cytotoxic function and boosted metabolic flexibility, while eliminating AREG production, achieving greater tumor infiltration and tumor clearance. This method has broad applications in cellular therapy as an 'off-the-shelf' treatment option.

Adipocytes regulate fibroblast function, and their loss contributes to fibroblast dysfunction in inflammatory diseases.

Fibroblasts play critical roles in tissue homeostasis, but in pathologic states can drive fibrosis, inflammation, and tissue destruction. In the joint synovium, fibroblasts provide homeostatic maintenance and lubrication. Little is known about what regulates the homeostatic functions of fibroblasts in healthy conditions. We performed RNA sequencing of healthy human synovial tissue and identified a fibroblast gene expression program characterized by enhanced fatty acid metabolism and lipid transport. We found that fat-conditioned media reproduces key aspects of the lipid-related gene signature in cultured fibroblasts. Fractionation and mass spectrometry identified cortisol in driving the healthy fibroblast phenotype, confirmed using glucocorticoid receptor gene ( NR3C1 ) deleted cells. Depletion of synovial adipocytes in mice resulted in loss of the healthy fibroblast phenotype and revealed adipocytes as a major contributor to active cortisol generation via Hsd11 ß 1 expression. Cortisol signaling in fibroblasts mitigated matrix remodeling induced by TNFα- and TGFß, while stimulation with these cytokines repressed cortisol signaling and adipogenesis. Together, these findings demonstrate the importance of adipocytes and cortisol signaling in driving the healthy synovial fibroblast state that is lost in disease.

Immunometabolism at the crossroads of obesity and cancer-a Keystone Symposia report.

Immunometabolism considers the relationship between metabolism and immunity. Typically, researchers focus on either the metabolic pathways within immune cells that affect their function or the impact of immune cells on systemic metabolism. A more holistic approach that considers both these viewpoints is needed. On September 5-8, 2022, experts in the field of immunometabolism met for the Keystone symposium "Immunometabolism at the Crossroads of Obesity and Cancer" to present recent research across the field of immunometabolism, with the setting of obesity and cancer as an ideal example of the complex interplay between metabolism, immunity, and cancer. Speakers highlighted new insights on the metabolic links between tumor cells and immune cells, with a focus on leveraging unique metabolic vulnerabilities of different cell types in the tumor microenvironment as therapeutic targets and demonstrated the effects of diet, the microbiome, and obesity on immune system function and cancer pathogenesis and therapy. Finally, speakers presented new technologies to interrogate the immune system and uncover novel metabolic pathways important for immunity.

Unveiling the epigenomic mechanisms of acquired platinum-resistance in high-grade serous ovarian cancer.

Resistance to platinum-based chemotherapy is the major cause of death from high-grade serous ovarian cancer (HGSOC). We hypothesise that detection of specific DNA methylation changes may predict platinum resistance in HGSOC. Using a publicly available "discovery" dataset we examined epigenomic and transcriptomic alterations between primary platinum-sensitive (n = 32) and recurrent acquired drug resistant HGSOC (n = 28) and identified several genes involved in immune and chemoresistance-related pathways. Validation via high-resolution melt analysis of these findings, in cell lines and HGSOC tumours, demonstrated the most consistent changes were observed in three of the genes: APOBEC3A, NKAPL and PDCD1. Plasma samples from an independent HGSOC cohort (n = 17) were analysed using droplet digital PCR. Hypermethylation of NKAPL was detected in 46% and hypomethylation of APOBEC3A in 69% of plasma samples taken from women with relapsed HGSOC (n = 13), with no alterations identified in disease-free patients (n = 4). Following these results, and using a CRISPR-Cas9 approach, we were also able to demonstrate that in vitro NKAPL promoter demethylation increased platinum sensitivity by 15%. Overall, this study demonstrates the importance of aberrant methylation, especially of the NKAPL gene, in acquired platinum resistance in HGSOC.

γδ T cells unveil invisible tumors.

Tumors can evade conventional T cell recognition by rendering the HLA class I antigen presentation system defective. In a recent study, de Vries et al. reveal γδ T cells as key contributors to the efficacy of immune checkpoint blockade (ICB) against HLA-I-silenced cancers, highlighting a novel layer of surveillance against immune escape by tumors.

Diverse effects of obesity on antitumor immunity and immunotherapy.

Currently, obesity is one of the biggest health burdens facing society because it causes several comorbidities, such as type 2 diabetes, atherosclerosis, and heart disease. Obesity is also linked to multiple types of cancer. Obesity is the second most common preventable cause of cancer after smoking; the rates of obesity are increasing worldwide, as are the rates of obesity-associated cancer. Multiple factors link obesity to cancer, such as increased levels of growth hormones and adipokines, gut dysbiosis, altered tumor metabolism, and chronic low-grade inflammation. More recently, obesity has been shown to also affect the immune response against cancer. In this review we discuss the interplay between obesity, the immune system, and cancer.

Longitudinal analysis of invariant natural killer T cell activation reveals a cMAF-associated transcriptional state of NKT10 cells.

Innate T cells, including CD1d-restricted invariant natural killer T (iNKT) cells, are characterized by their rapid activation in response to non-peptide antigens, such as lipids. While the transcriptional profiles of naive, effector, and memory adaptive T cells have been well studied, less is known about the transcriptional regulation of different iNKT cell activation states. Here, using single-cell RNA-sequencing, we performed longitudinal profiling of activated murine iNKT cells, generating a transcriptomic atlas of iNKT cell activation states. We found that transcriptional signatures of activation are highly conserved among heterogeneous iNKT cell populations, including NKT1, NKT2, and NKT17 subsets, and human iNKT cells. Strikingly, we found that regulatory iNKT cells, such as adipose iNKT cells, undergo blunted activation and display constitutive enrichment of memory-like cMAF+ and KLRG1+ populations. Moreover, we identify a conserved cMAF-associated transcriptional network among NKT10 cells, providing novel insights into the biology of regulatory and antigen-experienced iNKT cells.

Human Hepatic CD56bright NK Cells Display a Tissue-Resident Transcriptional Profile and Enhanced Ability to Kill Allogenic CD8+ T Cells.

Liver-resident CD56brightCD16- natural killer (NK) cells are enriched in the human liver and are phenotypically distinct from their blood counterparts. Although these cells are capable of rapid cytotoxic effector activity, their functional role remains unclear. We hypothesise that they may contribute to immune tolerance in the liver during transplantation. RNA sequencing was carried out on FACS sorted NK cell subpopulations from liver perfusates (n=5) and healthy blood controls (n=5). Liver-resident CD56brightCD16+/- NK cells upregulate genes associated with tissue residency. They also upregulate expression of CD160 and LY9, both of which encode immune receptors capable of activating NK cells. Co-expression of CD160 and Ly9 on liver-resident NK cells was validated using flow cytometry. Hepatic NK cell cytotoxicity against allogenic T cells was tested using an in vitro co-culture system of liver perfusate-derived NK cells and blood T cells (n=10-13). In co-culture experiments, hepatic NK cells but not blood NK cells induced significant allogenic T cell death (p=0.0306). Allogenic CD8+ T cells were more susceptible to hepatic NK cytotoxicity than CD4+ T cells (p<0.0001). Stimulation of hepatic CD56bright NK cells with an anti-CD160 agonist mAb enhanced this cytotoxic response (p=0.0382). Our results highlight a role for donor liver NK cells in regulating allogenic CD8+ T cell activation, which may be important in controlling recipient CD8+ T cell-mediated rejection post liver-transplant.

Invariant NKT cell-augmented GM-CSF-secreting tumor vaccine is effective in advanced prostate cancer model.

Invariant natural killer T cells (iNKT cells) express a semi-invariant T cell receptor that recognizes certain glycolipids (including α-galactosylceramide, αGC) bound to CD1d, and can induce potent antitumor responses. Here, we assessed whether αGC could enhance the efficacy of a GM-CSF-producing tumor cell vaccine in the transgenic SV40 T antigen-driven TRAMP prostate cancer model. In healthy mice, we initially found that optimal T cell responses were obtained with αGC-pulsed TRAMP-C2 cells secreting GM-CSF and milk fat globule epidermal growth factor protein-8 (MFG-E8) with an RGD to RGE mutation (GM-CSF/RGE TRAMP-C2), combined with systemic low dose IL-12. In a therapeutic model, transgenic TRAMP mice were then castrated at ~ 20 weeks, followed by treatment with the combination vaccine. Untreated mice succumbed to tumor by ~ 40 weeks, but survival was markedly prolonged by vaccine treatment, with most mice surviving past 80 weeks. Prostates in the treated mice were heavily infiltrated with T cells and iNKT cells, which both secreted IFNγ in response to tumor cells. The vaccine was not effective if the αGC, IL-12, or GM-CSF secretion was eliminated. Finally, immunized mice were fully resistant to challenge with TRAMP-C2 cells. Together these findings support further development of therapeutic vaccines that exploit iNKT cell activation.

Suppressive effects of the obese tumor microenvironment on CD8 T cell infiltration and effector function.

Obesity is one of the leading preventable causes of cancer; however, little is known about the effects of obesity on anti-tumor immunity. Here, we investigated the effects of obesity on CD8 T cells in mouse models and patients with endometrial cancer. Our findings revealed that CD8 T cell infiltration is suppressed in obesity, which was associated with a decrease in chemokine production. Tumor-resident CD8 T cells were also functionally suppressed in obese mice, which was associated with a suppression of amino acid metabolism. Similarly, we found that a high BMI negatively correlated with CD8 infiltration in human endometrial cancer and that weight loss was associated with a complete pathological response in six of nine patients. Moreover, immunotherapy using anti-PD-1 led to tumor rejection in lean and obese mice and partially restored CD8 metabolism and anti-tumor immunity. These findings highlight the suppressive effects of obesity on CD8 T cell anti-tumor immunity, which can partially be reversed by weight loss and/or immunotherapy.

High-Fat Diet Rapidly Modifies Trafficking, Phenotype, and Function of Plasmacytoid Dendritic Cells in Adipose Tissue.

Plasmacytoid dendritic cells (pDCs) display an increased abundance in visceral adipose tissue (VAT) of humans with obesity. In the current study, we set out to decipher the molecular mechanisms of their recruitment to VAT and the functional relevance of this process. We observed increased pDC numbers in murine blood, liver, spleen, and VAT after feeding a high-fat diet (HFD) for 3 wk when compared with a standard diet. pDCs were enriched in fat-associated lymphoid clusters representing highly specific lymphoid regions within VAT. HFD led to an enlargement of fat-associated lymphoid clusters with an increased density and migratory speed of pDCs as shown by intravital multiphoton microscopy. For their recruitment into VAT, pDCs employed P-selectin with E-selectin and L-selectin being only critical in response to HFD, indicating that the molecular cues underlying pDC trafficking were dependent on the nutritional state. Subsequent recruitment steps required α4ß1 and α4ß7 integrins and engagement of CCR7. Application of fingolimod (FTY720) abrogated egress of pDCs from VAT, indicating the involvement of sphingosine-1-phosphate in this process. Furthermore, HFD altered pDC functions by promoting their activation and type 1 IFN expression. Blocking pDC infiltration into VAT prevented weight gain and improved glucose tolerance during HFD. In summary, a HFD fundamentally alters pDC biology by promoting their trafficking, retention, and activation in VAT, which in turn seems to regulate metabolism.

Diet, lipids, and antitumor immunity.

Tumour growth and dissemination is largely dependent on nutrient availability. It has recently emerged that the tumour microenvironment is rich in a diverse array of lipids that increase in abundance with tumour progression and play a role in promoting tumour growth and metastasis. Here, we describe the pro-tumorigenic roles of lipid uptake, metabolism and synthesis and detail the therapeutic potential of targeting lipid metabolism in cancer. Additionally, we highlight new insights into the distinct immunosuppressive effects of lipids in the tumour microenvironment. Lipids threaten an anti-tumour environment whereby metabolic adaptation to lipid metabolism is linked to immune dysfunction. Finally, we describe the differential effects of commondietary lipids on cancer growth which may uncover a role for specific dietary regimens in association with traditional cancer therapies. Understanding the relationship between dietary lipids, tumour, and immune cells is important in the context of obesity which may reveal a possibility to harness the diet in the treatment of cancers.