Publications

2024
Georgiev P, Benamar M, Han SJ, Haigis MC, Sharpe AH, Chatila TA. Regulatory T cells in dominant immunologic tolerance. J Allergy Clin Immunol. 2024;153 (1) :28-41.Abstract
Regulatory T cells expressing the transcription factor forkhead box protein 3 mediate peripheral immune tolerance both to self-antigens and to the commensal flora. Their defective function due to inborn errors of immunity or acquired insults is associated with a broad range of autoimmune and immune dysregulatory diseases. Although their function in suppressing autoimmunity and enforcing commensalism is established, a broader role for regulatory T cells in tissue repair and metabolic regulation has emerged, enabled by unique programs of tissue adaptability and specialization. In this review, we focus on the myriad roles played by regulatory T cells in immunologic tolerance and host homeostasis and the potential to harness these cells in novel therapeutic approaches to human diseases.
Cohen JN, Gouirand V, Macon CE, Lowe MM, Boothby IC, Moreau JM, Gratz IK, Stoecklinger A, Weaver CT, Sharpe AH, et al. Regulatory T cells in skin mediate immune privilege of the hair follicle stem cell niche. Sci Immunol. 2024;9 (91) :eadh0152.Abstract
Immune tolerance is maintained in lymphoid organs (LOs). Despite the presence of complex immune cell networks in non-LOs, it is unknown whether self-tolerance is maintained in these tissues. We developed a technique to restrict genetic recombination to regulatory T cells (Tregs) only in skin. Selective depletion of skin Tregs resulted in T cell-mediated inflammation of hair follicles (HFs). Suppression did not rely on CTLA-4, but instead on high-affinity interleukin-2 (IL-2) receptor expression by skin Tregs, functioning exclusively in a cell-extrinsic manner. In a novel model of HF stem cell (HFSC)-driven autoimmunity, we reveal that skin Tregs immunologically protect the HFSC niche. Finally, we used spatial transcriptomics to identify aberrant IL-2 signaling at stromal-HF interfaces in a rare form of human alopecia characterized by HFSC destruction and alopecia areata. Collectively, these results reveal the fundamental biology of Tregs in skin uncoupled from the systemic pool and elucidate a mechanism of self-tolerance.
LaFleur MW, Lemmen AM, Streeter ISL, Nguyen TH, Milling LE, Derosia NM, Hoffman ZM, Gillis JE, Tjokrosurjo Q, Markson SC, et al. X-CHIME enables combinatorial, inducible, lineage-specific and sequential knockout of genes in the immune system. Nat Immunol. 2024;25 (1) :178-188.Abstract
Annotation of immunologic gene function in vivo typically requires the generation of knockout mice, which is time consuming and low throughput. We previously developed CHimeric IMmune Editing (CHIME), a CRISPR-Cas9 bone marrow delivery system for constitutive, ubiquitous deletion of single genes. Here we describe X-CHIME, four new CHIME-based systems for modular and rapid interrogation of gene function combinatorially (C-CHIME), inducibly (I-CHIME), lineage-specifically (L-CHIME) or sequentially (S-CHIME). We use C-CHIME and S-CHIME to assess the consequences of combined deletion of Ptpn1 and Ptpn2, an embryonic lethal gene pair, in adult mice. We find that constitutive deletion of both PTPN1 and PTPN2 leads to bone marrow hypoplasia and lethality, while inducible deletion after immune development leads to enteritis and lethality. These findings demonstrate that X-CHIME can be used for rapid mechanistic evaluation of genes in distinct in vivo contexts and that PTPN1 and PTPN2 have some functional redundancy important for viability in adult mice.
2023
Han SJ, Georgiev P, Ringel AE, Sharpe AH, Haigis MC. Age-associated remodeling of T cell immunity and metabolism. Cell Metab. 2023;35 (1) :36-55.Abstract
Aging results in remodeling of T cell immunity and is associated with poor clinical outcomes in age-related diseases such as cancer. Among the hallmarks of aging, changes in host and cellular metabolism critically affect the development, maintenance, and function of T cells. Although metabolic perturbations impact anti-tumor T cell responses, the link between age-associated metabolic dysfunction and anti-tumor immunity remains unclear. In this review, we summarize recent advances in our understanding of aged T cell metabolism, with a focus on the bioenergetic and immunologic features of T cell subsets unique to the aging process. We also survey insights into mechanisms of metabolic T cell dysfunction in aging and discuss the impacts of aging on the efficacy of cancer immunotherapy. As the average life expectancy continues to increase, understanding the interplay between age-related metabolic reprogramming and maladaptive T cell immunity will be instrumental for the development of therapeutic strategies for older patients.
Iorgulescu BJ, Ruthen N, Ahn R, Panagioti E, Gokhale PC, Neagu M, Speranza MC, Eschle BK, Soroko KM, Piranlioglu R, et al. Antigen presentation deficiency, mesenchymal differentiation, and resistance to immunotherapy in the murine syngeneic CT2A tumor model. Front Immunol. 2023;14 :1297932.Abstract
BACKGROUND: The GL261 and CT2A syngeneic tumor lines are frequently used as immunocompetent orthotopic mouse models of human glioblastoma (huGBM) but demonstrate distinct differences in their responses to immunotherapy. METHODS: To decipher the cell-intrinsic mechanisms that drive immunotherapy resistance in CT2A-luc and to define the aspects of human cancer biology that these lines can best model, we systematically compared their characteristics using whole exome and transcriptome sequencing, and protein analysis through immunohistochemistry, Western blot, flow cytometry, immunopeptidomics, and phosphopeptidomics. RESULTS: The transcriptional profiles of GL261-luc2 and CT2A-luc tumors resembled those of some huGBMs, despite neither line sharing the essential genetic or histologic features of huGBM. Both models exhibited striking hypermutation, with clonal hotspot mutations in RAS genes (Kras p.G12C in GL261-luc2 and Nras p.Q61L in CT2A-luc). CT2A-luc distinctly displayed mesenchymal differentiation, upregulated angiogenesis, and multiple defects in antigen presentation machinery (e.g. Tap1 p.Y488C and Psmb8 p.A275P mutations) and interferon response pathways (e.g. copy number losses of loci including IFN genes and reduced phosphorylation of JAK/STAT pathway members). The defect in MHC class I expression could be overcome in CT2A-luc by interferon-γ treatment, which may underlie the modest efficacy of some immunotherapy combinations. Additionally, CT2A-luc demonstrated substantial baseline secretion of the CCL-2, CCL-5, and CCL-22 chemokines, which play important roles as myeloid chemoattractants. CONCLUSION: Although the clinical contexts that can be modeled by GL261 and CT2A for huGBM are limited, CT2A may be an informative model of immunotherapy resistance due to its deficits in antigen presentation machinery and interferon response pathways.
Bod L, Kye Y-C, Shi J, Triglia ET, Schnell A, Fessler J, Ostrowski SM, Von-Franque MY, Kuchroo JR, Barilla RM, et al. B-cell-specific checkpoint molecules that regulate anti-tumour immunity. Nature. 2023;619 (7969) :348-356.Abstract
The role of B cells in anti-tumour immunity is still debated and, accordingly, immunotherapies have focused on targeting T and natural killer cells to inhibit tumour growth1,2. Here, using high-throughput flow cytometry as well as bulk and single-cell RNA-sequencing and B-cell-receptor-sequencing analysis of B cells temporally during B16F10 melanoma growth, we identified a subset of B cells that expands specifically in the draining lymph node over time in tumour-bearing mice. The expanding B cell subset expresses the cell surface molecule T cell immunoglobulin and mucin domain 1 (TIM-1, encoded by Havcr1) and a unique transcriptional signature, including multiple co-inhibitory molecules such as PD-1, TIM-3, TIGIT and LAG-3. Although conditional deletion of these co-inhibitory molecules on B cells had little or no effect on tumour burden, selective deletion of Havcr1 in B cells both substantially inhibited tumour growth and enhanced effector T cell responses. Loss of TIM-1 enhanced the type 1 interferon response in B cells, which augmented B cell activation and increased antigen presentation and co-stimulation, resulting in increased expansion of tumour-specific effector T cells. Our results demonstrate that manipulation of TIM-1-expressing B cells enables engagement of the second arm of adaptive immunity to promote anti-tumour immunity and inhibit tumour growth.
Rowe JH, Elia I, Shahid O, Gaudiano EF, Sifnugel NE, Johnson S, Reynolds AG, Fung ME, Joshi S, LaFleur MW, et al. Formate Supplementation Enhances Antitumor CD8+ T-cell Fitness and Efficacy of PD-1 Blockade. Cancer Discov. 2023;13 (12) :2566-2583.Abstract
UNLABELLED: The tumor microenvironment (TME) restricts antitumor CD8+ T-cell function and immunotherapy responses. Cancer cells compromise the metabolic fitness of CD8+ T cells within the TME, but the mechanisms are largely unknown. Here we demonstrate that one-carbon (1C) metabolism is enhanced in T cells in an antigen-specific manner. Therapeutic supplementation of 1C metabolism using formate enhances CD8+ T-cell fitness and antitumor efficacy of PD-1 blockade in B16-OVA tumors. Formate supplementation drives transcriptional alterations in CD8+ T-cell metabolism and increases gene signatures for cellular proliferation and activation. Combined formate and anti-PD-1 therapy increases tumor-infiltrating CD8+ T cells, which are essential for enhanced tumor control. Our data demonstrate that formate provides metabolic support to CD8+ T cells reinvigorated by anti-PD-1 to overcome a metabolic vulnerability in 1C metabolism in the TME to further improve T-cell function. SIGNIFICANCE: This study identifies that deficiencies in 1C metabolism limit the efficacy of PD-1 blockade in B16-OVA tumors. Supplementing 1C metabolism with formate during anti-PD-1 therapy enhances CD8+ T-cell fitness in the TME and CD8+ T-cell-mediated tumor clearance. These findings demonstrate that formate supplementation can enhance exhausted CD8+ T-cell function. See related commentary by Lin et al., p. 2507. This article is featured in Selected Articles from This Issue, p. 2489.
Helou DG, Quach C, Fung M, Painter JD, Hurrell BP, Eddie Loh Y-H, Howard E, Shafiei-Jahani P, Soroosh P, Sharpe AH, et al. Human PD-1 agonist treatment alleviates neutrophilic asthma by reprogramming T cells. J Allergy Clin Immunol. 2023;151 (2) :526-538.e8.Abstract
BACKGROUND: Neutrophilic asthma is associated with disease severity and corticosteroid insensitivity. Novel therapies are required to manage this life-threatening asthma phenotype. Programmed cell death protein-1 (PD-1) is a key homeostatic modulator of the immune response for T-cell effector functions. OBJECTIVE: We sought to investigate the role of PD-1 in the regulation of acute neutrophilic inflammation in a murine model of airway hyperreactivity (AHR). METHODS: House dust mite was used to induce and compare neutrophilic AHR in wild-type and PD-1 knockout mice. Then, the therapeutic potential of a human PD-1 agonist was tested in a humanized mouse model in which the PD-1 extracellular domain is entirely humanized. Single-cell RNA sequencing and flow cytometry were mainly used to investigate molecular and cellular mechanisms. RESULTS: PD-1 was highly induced on pulmonary T cells in our inflammatory model. PD-1 deficiency was associated with an increased neutrophilic AHR and high recruitment of inflammatory cells to the lungs. Consistently, PD-1 agonist treatment dampened AHR, decreased neutrophil recruitment, and modulated cytokine production in a humanized PD-1 mouse model. Mechanistically, we demonstrated at the transcriptional and protein levels that the inhibitory effect of PD-1 agonist is associated with the reprogramming of pulmonary effector T cells that showed decreased number and activation. CONCLUSIONS: PD-1 agonist treatment is efficient in dampening neutrophilic AHR and lung inflammation in a preclinical humanized mouse model.
Burke KP, Patterson DG, Liang D, Sharpe AH. Immune checkpoint receptors in autoimmunity. Curr Opin Immunol. 2023;80 :102283.Abstract
Immune checkpoint receptors such as programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte associated protein 4 (CTLA-4), lymphocyte-activation gene 3 (LAG-3), and T cell immunoglobulin and ITIM domain (TIGIT) have distinct and overlapping inhibitory functions that regulate Tcell activation, differentiation, and function. These inhibitory receptors also mediate tolerance, and dysregulation of these receptors can result in a breach of tolerance and the development of autoimmune syndromes. Similarly, antibody blockade of immune checkpoint receptors or their ligands for cancer immunotherapy may trigger a spectrum of organ inflammation that resembles autoimmunity, termed immune-related adverse events (irAE). In this review, we discuss recent advances in the regulation of autoimmunity by immune checkpoint receptors. We highlight coordinated gene expression programs linking checkpoint receptors, heterogeneity within autoreactive T-cell populations, parallels between irAE and autoimmunity, and bidirectional functional interactions between immune checkpoint receptors and their ligands.
Datta M, Chatterjee S, Perez EM, Gritsch S, Roberge S, Duquette M, Chen IX, Naxerova K, Kumar AS, Ghosh M, et al. Losartan controls immune checkpoint blocker-induced edema and improves survival in glioblastoma mouse models. Proc Natl Acad Sci U S A. 2023;120 (6) :e2219199120.Abstract
Immune checkpoint blockers (ICBs) have failed in all phase III glioblastoma trials. Here, we found that ICBs induce cerebral edema in some patients and mice with glioblastoma. Through single-cell RNA sequencing, intravital imaging, and CD8+ T cell blocking studies in mice, we demonstrated that this edema results from an inflammatory response following antiprogrammed death 1 (PD1) antibody treatment that disrupts the blood-tumor barrier. Used in lieu of immunosuppressive corticosteroids, the angiotensin receptor blocker losartan prevented this ICB-induced edema and reprogrammed the tumor microenvironment, curing 20% of mice which increased to 40% in combination with standard of care treatment. Using a bihemispheric tumor model, we identified a "hot" tumor immune signature prior to losartan+anti-PD1 therapy that predicted long-term survival. Our findings provide the rationale and associated biomarkers to test losartan with ICBs in glioblastoma patients.
Kurmi K, Liang D, van de Ven R, Georgiev P, Gassaway BM, Han SJ, Notarangelo G, Harris IS, Yao C-H, Park JS, et al. Metabolic modulation of mitochondrial mass during CD4 T cell activation. Cell Chem Biol. 2023;30 (9) :1064-1075.e8.Abstract
Mitochondrial biogenesis initiates within hours of T cell receptor (TCR) engagement and is critical for T cell activation, function, and survival; yet, how metabolic programs support mitochondrial biogenesis during TCR signaling is not fully understood. Here, we performed a multiplexed metabolic chemical screen in CD4+ T lymphocytes to identify modulators of metabolism that impact mitochondrial mass during early T cell activation. Treatment of T cells with pyrvinium pamoate early during their activation blocks an increase in mitochondrial mass and results in reduced proliferation, skewed CD4+ T cell differentiation, and reduced cytokine production. Furthermore, administration of pyrvinium pamoate at the time of induction of experimental autoimmune encephalomyelitis, an experimental model of multiple sclerosis in mice, prevented the onset of clinical disease. Thus, modulation of mitochondrial biogenesis may provide a therapeutic strategy for modulating T cell immune responses.
Park JS, Gazzaniga FS, Kasper DL, Sharpe AH. Microbiota-dependent regulation of costimulatory and coinhibitory pathways via innate immune sensors and implications for immunotherapy. Exp Mol Med. 2023;55 (9) :1913-1921.Abstract
Our bodies are inhabited by trillions of microorganisms. The host immune system constantly interacts with the microbiota in barrier organs, including the intestines. Over decades, numerous studies have shown that our mucosal immune system is dynamically shaped by a variety of microbiota-derived signals. Elucidating the mediators of these interactions is an important step for understanding how the microbiota is linked to mucosal immune homeostasis and gut-associated diseases. Interestingly, the efficacy of cancer immunotherapies that manipulate costimulatory and coinhibitory pathways has been correlated with the gut microbiota. Moreover, adverse effects of these therapies in the gut are linked to dysregulation of the intestinal immune system. These findings suggest that costimulatory pathways in the immune system might serve as a bridge between the host immune system and the gut microbiota. Here, we review mechanisms by which commensal microorganisms signal immune cells and their potential impact on costimulation. We highlight how costimulatory pathways modulate the mucosal immune system through not only classical antigen-presenting cells but also innate lymphocytes, which are highly enriched in barrier organs. Finally, we discuss the adverse effects of immune checkpoint inhibitors in the gut and the possible relationship with the gut microbiota.
Denize T, Jegede OA, Matar S, El Ahmar N, West DJ, Walton E, Bagheri AS, Savla V, Nabil Laimon Y, Gupta S, et al. PD-1 Expression on Intratumoral Regulatory T Cells is Associated with Lack of Benefit from Anti-PD-1 Therapy in Metastatic Clear Cell Renal Cell Carcinoma Patients. Clin Cancer Res. 2023.Abstract
PURPOSE: PD-1 expression on CD8+TIM-3-LAG3- tumor-infiltrating cells predicts positive response to PD-1 blockade in metastatic clear cell renal cell carcinoma (mccRCC). Since PD-1 signaling inhibition in regulatory T cells (Tregs) augments their immunosuppressive function, we hypothesized that PD-1 expression on tumor-infiltrating Tregs would predict resistance to PD-1 inhibitors. EXPERIMENTAL DESIGN: PD-1+ Tregs were phenotyped using multiparametric immunofluorescence in ccRCC tissues from the CheckMate-025 trial (nivolumab: n=91, everolimus: n=90). Expression of CD8, PD-1, TIM-3, and LAG-3 was previously determined (Ficial et al, 2021). Clinical endpoints included progression-free survival (PFS), overall survival (OS), and objective response rate (ORR). RESULTS: In the nivolumab (but not everolimus) arm, high percentage of PD-1+ Tregs was associated with shorter PFS (3.19 versus 5.78 months; p=0.021), shorter OS (18.1 versus 27.7 months, p=0.013) and marginally lower ORR (12.5% versus 31.3%, p=0.059). An integrated biomarker (PD-1 Treg/CD8 ratio) was developed by calculating the ratio between percentage of PD-1+Tregs (marker of resistance) and percentage of CD8+PD-1+TIM-3-LAG-3- cells (marker of response). In the nivolumab (but not everolimus) arm, patients with high-PD-1 ratio experienced shorter PFS (3.48 versus 9.23 months, p<0.001), shorter OS (18.14 versus 38.21 months, p<0.001) and lower ORR (15.69% versus 40.00%; p=0.009). Compared to the individual biomarkers, the PD-1 ratio showed improved ability to predict outcomes to nivolumab versus everolimus. CONCLUSIONS: PD-1 expression on Tregs is associated with resistance to PD-1 blockade in mccRCC, suggesting that targeting Tregs may synergize with PD-1 inhibition. A model that integrates PD-1 expression on Tregs and CD8+TIM-3-LAG3- cells has higher predictive value.
Collier JL, Pauken KE, Lee CAA, Patterson DG, Markson SC, Conway TS, Fung ME, France JA, Mucciarone KN, Lian CG, et al. Single-cell profiling reveals unique features of diabetogenic T cells in anti-PD-1-induced type 1 diabetes mice. J Exp Med. 2023;220 (10).Abstract
Immune-related adverse events (irAEs) are a notable complication of PD-1 cancer immunotherapy. A better understanding of how these iatrogenic diseases compare with naturally arising autoimmune diseases is needed for treatment and monitoring of irAEs. We identified differences in anti-PD-1-induced type 1 diabetes (T1D) and spontaneous T1D in non-obese diabetic (NOD) mice by performing single-cell RNA-seq and TCR-seq on T cells from the pancreas, pancreas-draining lymph node (pLN), and blood of mice with PD-1-induced T1D or spontaneous T1D. In the pancreas, anti-PD-1 resulted in expansion of terminally exhausted/effector-like CD8+ T cells, an increase in T-bethi CD4+FoxP3- T cells, and a decrease in memory CD4+FoxP3- and CD8+ T cells in contrast to spontaneous T1D. Notably, anti-PD-1 caused increased TCR sharing between the pancreas and the periphery. Moreover, T cells in the blood of anti-PD-1-treated mice expressed markers that differed from spontaneous T1D, suggesting that the blood may provide a window to monitor irAEs rather than relying exclusively on the autoimmune target organ.
Park JS, Gazzaniga FS, Wu M, Luthens AK, Gillis J, Zheng W, LaFleur MW, Johnson SB, Morad G, Park EM, et al. Targeting PD-L2-RGMb overcomes microbiome-related immunotherapy resistance. Nature. 2023;617 (7960) :377-385.Abstract
The gut microbiota is a crucial regulator of anti-tumour immunity during immune checkpoint inhibitor therapy. Several bacteria that promote an anti-tumour response to immune checkpoint inhibitors have been identified in mice1-6. Moreover, transplantation of faecal specimens from responders can improve the efficacy of anti-PD-1 therapy in patients with melanoma7,8. However, the increased efficacy from faecal transplants is variable and how gut bacteria promote anti-tumour immunity remains unclear. Here we show that the gut microbiome downregulates PD-L2 expression and its binding partner repulsive guidance molecule b (RGMb) to promote anti-tumour immunity and identify bacterial species that mediate this effect. PD-L1 and PD-L2 share PD-1 as a binding partner, but PD-L2 can also bind RGMb. We demonstrate that blockade of PD-L2-RGMb interactions can overcome microbiome-dependent resistance to PD-1 pathway inhibitors. Antibody-mediated blockade of the PD-L2-RGMb pathway or conditional deletion of RGMb in T cells combined with an anti-PD-1 or anti-PD-L1 antibody promotes anti-tumour responses in multiple mouse tumour models that do not respond to anti-PD-1 or anti-PD-L1 alone (germ-free mice, antibiotic-treated mice and even mice colonized with stool samples from a patient who did not respond to treatment). These studies identify downregulation of the PD-L2-RGMb pathway as a specific mechanism by which the gut microbiota can promote responses to PD-1 checkpoint blockade. The results also define a potentially effective immunological strategy for treating patients who do not respond to PD-1 cancer immunotherapy.
Burke KP, Markson SC, Sharpe AH. Tracking tumor-specific CD8 T cell responses. Trends Immunol. 2023;44 (5) :326-328.Abstract
In a recent article, Puig-Saus et al. computationally predict and experimentally validate neoantigen-specific T cell responses in patients with melanoma. They identify a restricted set of neoantigens recognized by polyclonal CD8+ T cells as a unique feature of anti-PD-1 responders and engineer autologous tumor-responsive T cells expressing neoantigen-specific TCRs, providing proof-of-concept for future cellular therapies.
Yao C-H, Park JS, Kurmi K, Hu S-H, Notarangelo G, Crowley J, Jacobson H, Hui S, Sharpe AH, Haigis MC. Uncoupled glycerol-3-phosphate shuttle in kidney cancer reveals that cytosolic GPD is essential to support lipid synthesis. Mol Cell. 2023;83 (8) :1340-1349.e7.Abstract
The glycerol-3-phosphate shuttle (G3PS) is a major NADH shuttle that regenerates reducing equivalents in the cytosol and produces energy in the mitochondria. Here, we demonstrate that G3PS is uncoupled in kidney cancer cells where the cytosolic reaction is ∼4.5 times faster than the mitochondrial reaction. The high flux through cytosolic glycerol-3-phosphate dehydrogenase (GPD) is required to maintain redox balance and support lipid synthesis. Interestingly, inhibition of G3PS by knocking down mitochondrial GPD (GPD2) has no effect on mitochondrial respiration. Instead, loss of GPD2 upregulates cytosolic GPD on a transcriptional level and promotes cancer cell proliferation by increasing glycerol-3-phosphate supply. The proliferative advantage of GPD2 knockdown tumor can be abolished by pharmacologic inhibition of lipid synthesis. Taken together, our results suggest that G3PS is not required to run as an intact NADH shuttle but is instead truncated to support complex lipid synthesis in kidney cancer.
2022
LaFleur MW, Sharpe AH. CRISPR Screens to Identify Regulators of Tumor Immunity. Annu Rev Cancer Biol. 2022;6 :103-122.Abstract
Cancer immunotherapies, such as immune checkpoint blockade (ICB), have been used in a wide range of tumor types with immense clinical benefit. However, ICB does not work in all patients, and attempts to combine ICB with other immune-based therapies have not lived up to their initial promise. Thus, there is a significant unmet need to discover new targets and combination therapies to extend the benefits of immunotherapy to more patients. Systems biology approaches are well suited for addressing this problem because these approaches enable evaluation of many gene targets simultaneously and ranking their relative importance for a phenotype of interest. As such, loss-of-function CRISPR screens are an emerging set of tools being used to prioritize gene targets for modulating pathways of interest in tumor and immune cells. This review describes the first screens performed to discover cancer immunotherapy targets and the technological advances that will enable next-generation screens.
Boytz RM, Seitz S, Gaudiano E, Patten JJ, Keiser PT, Connor JH, Sharpe AH, Davey RA. Inactivation of Ebola Virus and SARS-CoV-2 in Cell Culture Supernatants and Cell Pellets by Gamma Irradiation. Viruses. 2022;15 (1).Abstract
Viral pathogens with the potential to cause widespread disruption to human health and society continue to emerge or re-emerge around the world. Research on such viruses often involves high biocontainment laboratories (BSL3 or BSL4), but the development of diagnostics, vaccines and therapeutics often uses assays that are best performed at lower biocontainment. Reliable inactivation is necessary to allow removal of materials to these spaces and to ensure personnel safety. Here, we validate the use of gamma irradiation to inactivate culture supernatants and pellets of cells infected with a representative member of the Filovirus and Coronavirus families. We show that supernatants and cell pellets containing SARS-CoV-2 are readily inactivated with 1.9 MRad, while Ebola virus requires higher doses of 2.6 MRad for supernatants and 3.8 MRad for pellets. While these doses of radiation inactivate viruses, proinflammatory cytokines that are common markers of virus infection are still detected with low losses. The doses required for virus inactivation of supernatants are in line with previously reported values, but the inactivation of cell pellets has not been previously reported and enables new approaches for analysis of protein-based host responses to infection.
Notarangelo G, Spinelli JB, Perez EM, Baker GJ, Kurmi K, Elia I, Stopka SA, Baquer G, Lin J-R, Golby AJ, et al. Oncometabolite d-2HG alters T cell metabolism to impair CD8 T cell function. Science. 2022;377 (6614) :1519-1529.Abstract
Gain-of-function mutations in isocitrate dehydrogenase (IDH) in human cancers result in the production of d-2-hydroxyglutarate (d-2HG), an oncometabolite that promotes tumorigenesis through epigenetic alterations. The cancer cell-intrinsic effects of d-2HG are well understood, but its tumor cell-nonautonomous roles remain poorly explored. We compared the oncometabolite d-2HG with its enantiomer, l-2HG, and found that tumor-derived d-2HG was taken up by CD8+ T cells and altered their metabolism and antitumor functions in an acute and reversible fashion. We identified the glycolytic enzyme lactate dehydrogenase (LDH) as a molecular target of d-2HG. d-2HG and inhibition of LDH drive a metabolic program and immune CD8+ T cell signature marked by decreased cytotoxicity and impaired interferon-γ signaling that was recapitulated in clinical samples from human patients with IDH1 mutant gliomas.

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