AMPK Drives both Glycolytic and Oxidative Metabolism in Murine and Human T Cells During Graft-versus-host Disease.

Allogeneic T cells reprogram their metabolism during acute graft-versus-host disease (GVHD) in a process involving the cellular energy sensor AMP-activated protein kinase (AMPK). Deletion of AMPK in donor T cells limits GVHD but still preserves homeostatic reconstitution and graft-versus-leukemia (GVL) effects. In the current studies, murine AMPK KO T cells decreased oxidative metabolism at early timepoints post-transplant and lacked a compensatory increase in glycolysis following inhibition of the electron transport chain. Immunoprecipitation using an antibody specific to phosphorylated targets of AMPK determined that AMPK modified interactions of several glycolytic enzymes including aldolase, enolase, pyruvate kinase M (PKM), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and enzyme assays indicated impaired aldolase and GAPDH activity in AMPK KO T cells. Importantly, these changes in glycolysis correlated with both an impaired ability of AMPK KO T cells to produce significant amounts of interferon gamma (IFNγ) upon antigenic re-stimulation and a decrease in the total number of donor CD4 T cells recovered at later time points post-transplant. Human T cells lacking AMPK gave similar results, with glycolytic compensation impaired both in vitro and following expansion in vivo. GVHD results also mirrored those of the murine model, with reduced CD4/CD8 ratios and a significant improvement in disease severity. Together these data highlight a significant role for AMPK in controlling oxidative and glycolytic metabolism in both murine and human T cells and endorse further study of AMPK inhibition as a potential clinical target for future GVHD therapies.

Monocyte Production of C1q Potentiates CD8+ T Cell Function Following Respiratory Viral Infection.

Respiratory viral infections remain a leading cause of morbidity and mortality. Using a murine model of human metapneumovirus (HMPV), we identified recruitment of a C1q-expressing inflammatory monocyte population concomitant with viral clearance by adaptive immune cells. Genetic ablation of C1q led to reduced CD8+ T cell function. Production of C1q by a myeloid lineage was necessary to enhance CD8+ T cell function. Activated and dividing CD8+ T cells expressed a C1q receptor, gC1qR. Perturbation of gC1qR signaling led to altered CD8+ T cell IFN-γ production, metabolic capacity, and cell proliferation. Autopsy specimens from fatal respiratory viral infections in children demonstrated diffuse production of C1q by an interstitial population. Humans with severe COVID-19 infection also demonstrated upregulation of gC1qR on activated and rapidly dividing CD8+ T cells. Collectively, these studies implicate C1q production from monocytes as a critical regulator of CD8+ T cell function following respiratory viral infection. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Asparaginase-specific basophil recognition and activation predict Asparaginase hypersensitivity in mice.

Background: Asparaginase (ASNase) is a crucial part of acute leukemia treatment, but immune responses to the agent can reduce its effectiveness and increase the risk of relapse. Currently, no reliable and validated biomarker predicts ASNase-induced hypersensitivity reactions during therapy. We aimed to identify predictive biomarkers and determine immune cells responsible for anaphylaxis using a murine model of ASNase hypersensitivity. Methods: Our preclinical study uses a murine model to investigate predictive biomarkers of ASNase anaphylaxis, including anti-ASNase antibody responses, immune complex (IC) levels, ASNase-specific binding to leukocytes or basophils, and basophil activation. Results: Our results indicate that mice immunized to ASNase exhibited dynamic IgM, IgG, and IgE antibody responses. The severity of ASNase-induced anaphylaxis was found to be correlated with levels of IgG and IgE, but not IgM. Basophils from immunized mice were able to recognize and activate in response to ASNase ex vivo, and the extent of recognition and activation also correlated with the severity of anaphylaxis observed. Using a multivariable model that included all biomarkers significantly associated with anaphylaxis, independent predictors of ASNase-induced hypersensitivity reactions were found to be ASNase IC levels and ASNase-specific binding to leukocytes or basophils. Consistent with our multivariable analysis, we found that basophil depletion significantly protected mice from ASNase-induced hypersensitivity reactions, supporting that basophils are essential and can be used as a predictive marker of ASNase-induced anaphylaxis. Conclusions: Our study demonstrates the need for using tools that can detect both IC- and IgE-mediated hypersensitivity reactions to mitigate the risk of ASNase-induced hypersensitivity reactions during treatment.

Overexpression of AMPKγ2 increases AMPK signaling to augment human T cell metabolism and function.

Cellular therapies are currently employed to treat a variety of disease processes. For T cell-based therapies, success often relies on the metabolic fitness of the T cell product, where cells with enhanced metabolic capacity demonstrate improved in vivo efficacy. AMP-activated protein kinase (AMPK) is a cellular energy sensor which combines environmental signals with cellular energy status to enforce efficient and flexible metabolic programming. We hypothesized that increasing AMPK activity in human T cells would augment their oxidative capacity, creating an ideal product for adoptive cellular therapies. Lentiviral transduction of the regulatory AMPKγ2 subunit stably enhanced intrinsic AMPK signaling and promoted mitochondrial respiration with increased basal oxygen consumption rates, higher maximal oxygen consumption rate, and augmented spare respiratory capacity. These changes were accompanied by increased proliferation and inflammatory cytokine production, particularly within restricted glucose environments. Introduction of AMPKγ2 into bulk CD4 T cells decreased RNA expression of canonical Th2 genes, including the cytokines interleukin (IL)-4 and IL-5, while introduction of AMPKγ2 into individual Th subsets universally favored proinflammatory cytokine production and a downregulation of IL-4 production in Th2 cells. When AMPKγ2 was overexpressed in regulatory T cells, both in vitro proliferation and suppressive capacity increased. Together, these data suggest that augmenting intrinsic AMPK signaling via overexpression of AMPKγ2 can improve the expansion and functional potential of human T cells for use in a variety of adoptive cellular therapies.

Terminally exhausted CD8+ T cells contribute to age-dependent severity of respiratory virus infection.

BACKGROUND: Lower respiratory infections are a leading cause of severe morbidity and mortality among older adults. Despite ubiquitous exposure to common respiratory pathogens throughout life and near universal seropositivity, antibodies fail to effectively protect the elderly. Therefore, we hypothesized that severe respiratory illness in the elderly is due to deficient CD8+ T cell responses. RESULTS: Here, we establish an aged mouse model of human metapneumovirus infection (HMPV) wherein aged C57BL/6 mice exhibit worsened weight loss, clinical disease, lung pathology and delayed viral clearance compared to young adult mice. Aged mice generate fewer lung-infiltrating HMPV epitope-specific CD8+ T cells. Those that do expand demonstrate higher expression of PD-1 and other inhibitory receptors and are functionally impaired. Transplant of aged T cells into young mice and vice versa, as well as adoptive transfer of young versus aged CD8+ T cells into Rag1-/- recipients, recapitulates the HMPV aged phenotype, suggesting a cell-intrinsic age-associated defect. HMPV-specific aged CD8+ T cells exhibit a terminally exhausted TCF1/7- TOX+ EOMES+ phenotype. We confirmed similar terminal exhaustion of aged CD8+ T cells during influenza viral infection. CONCLUSIONS: This study identifies terminal CD8+ T cell exhaustion as a mechanism of severe disease from respiratory viral infections in the elderly.

AMPK Drives Both Glycolytic and Oxidative Metabolism in T Cells During Graft-versus-host Disease.

Allogeneic T cells reprogram their metabolism during acute graft-versus-host disease (GVHD) in a process reliant on the cellular energy sensor AMP-activated protein kinase (AMPK). Deletion of AMPK in donor T cells limits GVHD but still preserves homeostatic reconstitution and graft-versus-leukemia (GVL) effects. In the current studies, murine T cells lacking AMPK decreased oxidative metabolism at early timepoints post-transplant and were also unable to mediate a compensatory increase in glycolysis following inhibition of the electron transport chain. Human T cells lacking AMPK gave similar results, with glycolytic compensation impaired both in vitro and following expansion in vivo in a modified model of GVHD. Immunoprecipitation of proteins from day 7 allogeneic T cells, using an antibody specific to phosphorylated AMPK targets, recovered lower levels of multiple glycolysis-related proteins including the glycolytic enzymes aldolase, enolase, pyruvate kinase M (PKM), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Functionally, murine T cells lacking AMPK exhibited impaired aldolase activity following anti-CD3/CD28 stimulation and a decrease in GAPDH activity on day 7 post-transplant. Importantly, these changes in glycolysis correlated with an impaired ability of AMPK KO T cells to produce significant amounts of interferon gamma (IFNγ) upon antigenic re-stimulation. Together these data highlight a significant role for AMPK in controlling oxidative and glycolytic metabolism in both murine and human T cells during GVHD and endorse further study of AMPK inhibition as a potential target for future clinical therapies. KEY POINTS: AMPK plays a key role in driving both and oxidative and glycolytic metabolism in T cells during graft-versus-host disease (GVHD)Absence of AMPK simultaneously impairs both glycolytic enzyme activity, most notably by aldolase, and interferon gamma (IFNγ) production.

Monocyte production of C1q potentiates CD8 + T cell effector function following respiratory viral infection.

Respiratory viral infections remain a leading cause of morbidity and mortality. Using a murine model of human metapneumovirus (HMPV), we identified recruitment of a C1q-producing inflammatory monocyte population concomitant with viral clearance by adaptive immune cells. Genetic ablation of C1q led to reduced CD8 + T cell function. Production of C1q by a myeloid lineage was sufficient to enhance CD8 + T cell function. Activated and dividing CD8 + T cells expressed a putative C1q receptor, gC1qR. Perturbation of gC1qR signaling led to altered CD8 + T cell IFN-γ production and metabolic capacity. Autopsy specimens from fatal respiratory viral infections in children demonstrated diffuse production of C1q by an interstitial population. Humans with severe COVID-19 infection also demonstrated upregulation of gC1qR on activated and rapidly dividing CD8 + T cells. Collectively, these studies implicate C1q production from monocytes as a critical regulator of CD8 + T cell function following respiratory viral infection.

Mechanistic studies of PEG-asparaginase-induced liver injury and hepatic steatosis in mice.

PEGylated-l-asparaginase (PEG-ASNase) is a chemotherapeutic agent used to treat pediatric acute lymphoblastic leukemia (ALL). Its use is avoided in adults due to its high risk of liver injury including hepatic steatosis, with obesity and older age considered risk factors of the injury. Our study aims to elucidate the mechanism of PEG-ASNase-induced liver injury. Mice received 1500 U/kg of PEG-ASNase and were sacrificed 1, 3, 5, and 7 days after drug administration. Liver triglycerides were quantified, and plasma bilirubin, ALT, AST, and non-esterified fatty acids (NEFA) were measured. The mRNA and protein levels of genes involved in hepatic fatty acid synthesis, ß-oxidation, very low-density lipoprotein (VLDL) secretion, and white adipose tissue (WAT) lipolysis were determined. Mice developed hepatic steatosis after PEG-ASNase, which associated with increases in bilirubin, ALT, and AST. The hepatic genes Ppara, Lcad/Mcad, Hadhb, Apob100, and Mttp were upregulated, and Srebp-1c and Fas were downregulated after PEG-ASNase. Increased plasma NEFA, WAT loss, and adipose tissue lipolysis were also observed after PEG-ASNase. Furthermore, we found that PEG-ASNase-induced liver injury was exacerbated in obese and aged mice, consistent with clinical studies of ASNase-induced liver injury. Our data suggest that PEG-ASNase-induced liver injury is due to drug-induced lipolysis and lipid redistribution to the liver.

Adalimumab Immunogenicity Is Negatively Correlated with Anti-Hinge Antibody Levels in Patients with Rheumatoid Arthritis.

Patients with rheumatoid arthritis (RA) are frequently treated with anti-tumor necrosis factor-α immunoglobulin therapy but develop neutralizing antibodies against these drugs, necessitating therapeutic monitoring of drug concentrations and anti-drug antibodies. Patients with RA have multiple factors related to their autoimmune disposition that interfere with conventionally used methods to detect anti-drug antibodies. Currently deployed analytical methods have significant limitations that hinder clinical interpretation and/or routine use, and no method can detect immunogenicity and drug levels simultaneously to provide clinically meaningful recommendations. Given these limitations, the objective of this study was to identify sources of and associations with assay interference in patients with RA. We designed a modular immunogenicity and drug concentration detection technology to identify the factors that interfere with the detection of adalimumab and anti-adalimumab antibodies in a cohort of 206 patients with RA. Patients were included from the University of Pittsburgh Rheumatoid Arthritis Comparative Effectiveness Research registry. In this cohort, we analyzed clinical and plasma factors associated with anti-adalimumab and anti-hinge antibodies. A novel flow cytometry-based assay was developed and validated that simultaneously measures adalimumab and anti-adalimumab antibody concentrations, overcoming many of the interference factors that are limitations of conventional assays, including anti-fragment crystallizable (Fc) and anti-hinge antibodies. C-reactive protein (P = 0.035), Disease Activity Score-28 (DAS28) score (P = 0.002), and disease activity category (P = 0.009) were significantly associated with anti-adalimumab antibodies but not with anti-hinge antibodies (P > 0.05). Anti-hinge antibodies were inversely associated with drug-neutralizing antibodies (P = 0.002). In patients with RA, anti-hinge antibodies may have a potential protective effect against the development of anti-adalimumab antibodies. SIGNIFICANCE STATEMENT: Using a novel cytometric assay that simultaneously measures drug and anti-drug antibodies, we overcame many interferences that hinder the clinical interpretation of adalimumab immunogenicity testing. Our investigation in patients with RA demonstrated that immunogenicity impaired the pharmacological action of adalimumab via analysis of RA disease severity markers. We also demonstrate that patients with anti-hinge antibodies had lower anti-adalimumab antibody levels and decreased drug neutralization. Our results suggest that anti-hinge antibodies can predict adalimumab immunogenicity before the start of therapy.

Genetic inhibition of NFATC2 attenuates asparaginase hypersensitivity in mice.

The family of nuclear factor of activated T cells (NFAT) transcription factors plays a critical role in mediating immune responses. Our previous clinical pharmacogenetic studies suggested that NFATC2 is associated with the risk of hypersensitivity reactions to the chemotherapeutic agent L-asparaginase (ASNase) that worsen outcomes during the treatment of pediatric acute lymphoblastic leukemia. We therefore hypothesized that the genetic inhibition of NFATC2 would protect against the development of anti-ASNase antibodies and ASNase hypersensitivity. Our study demonstrates that ASNase-immunized NFATC2-deficient mice are protected against ASNase hypersensitivity and develop lower antigen-specific and total immunoglobulin E (IgE) levels compared with wild-type (WT) controls. Furthermore, ASNase-immunized NFATC2-deficient mice develop more CD4+ regulatory T cells, fewer CD4+ interleukin-4-positive (IL-4+) cells, higher IL-10/TGF-ß1 levels, and lower IL-4/IL-13 levels relative to WT mice. Basophils and peritoneal mast cells from ASNase-immunized, but not naïve, NFATC2-deficient mice had lower FcεRI expression and decreased IgE-mediated mast cell activation than WT mice. Furthermore, ASNase-immunized, but not naïve, NFATC2-deficient mice developed less severe shock than WT mice after induction of passive anaphylaxis or direct histamine administration. Thus, inhibition of NFATC2 protects against ASNase hypersensitivity by impairing T helper 2 responses, which may provide a novel strategy for attenuating hypersensitivity and the development of antidrug antibodies, including to ASNase.

Asparaginase immune complexes induce Fc-γRIII-dependent hypersensitivity in naive mice.

Asparaginase (ASNase) is an important drug for the treatment of leukemias. However, hypersensitivity to ASNase can increase the risk of leukemia relapse. Two mechanisms of ASNase hypersensitivity have been identified in mice. The existence of a pathway involving anti-ASNase IgG and Fc-γ receptor III (Fc-γRIII) implies that IgG and ASNase immune complexes (ICs) could directly induce hypersensitivity. The aim of this study was to detect ASNase ICs in mice after hypersensitivity reactions and determine their role in hypersensitivity. Protein G beads were used to detect plasma ASNase ICs by flow cytometry. Anti-ASNase IgG was purified from the plasma of sensitized mice, and ASNase ICs were prepared ex vivo at various ratios of ASNase to anti-ASNase IgG. The levels of ASNase ICs detected after hypersensitivity reactions correlated with reaction severity (R2 = 0.796; P = 0.0005). ASNase ICs prepared ex vivo required high levels of anti-ASNase IgG for formation, and binding to naive and sensitized immune cells depended on soluble anti-ASNase IgG, antigen:antibody ratio, and Fc-γRIII. Similarly, basophil activation by ASNase ICs depended on the antigen:antibody ratio and Fc-γRIII. Consistent with the ex vivo results, naive mice receiving ASNase ICs developed hypersensitivity reactions. Our data demonstrate that ASNase ICs can directly contribute to the onset and severity of ASNase hypersensitivity.-Rathod, S., Ramsey, M., DiGiorgio, D., Berrios, R., Finkelman, F. D., Fernandez, C. A. Asparaginase immune complexes induce Fc-γRIII-dependent hypersensitivity in naive mice.

Hypersensitivity reactions to asparaginase in mice are mediated by anti-asparaginase IgE and IgG and the immunoglobulin receptors FcεRI and FcγRIII.

Asparaginase is an important drug for the treatment of leukemias. However, anti-asparaginase antibodies often develop, which can decrease asparaginase drug levels and increase the risk of relapse. The aim of this study is to identify the immunoglobulin isotypes and receptors responsible for asparaginase hypersensitivities. Mice immunized with asparaginase developed anti-asparaginase IgG1 and IgE antibodies, and challenging the sensitized mice with asparaginase induced severe hypersensitivity reactions. Flow cytometry analysis indicated that macrophages/monocytes, neutrophils, and basophils bind asparaginase ex vivo through FcγRIII. In contrast, asparaginase binding to basophils was dependent on FcγRIII and IgE. Consistent with the asparaginase binding data, basophil activation by asparaginase occurred via both IgG/FcγRIII and IgE/FcεRI. Depleting >95% of B cells suppressed IgG but not IgE-dependent hypersensitivity, while depleting CD4+ T cells provided complete protection. Combined treatment with either anti-IgE mAb plus a platelet-activating factor receptor antagonist or anti-FcγRIII mAb plus a H1 receptor antagonist suppressed asparaginase hypersensitivity. The observations indicate that asparaginase hypersensitivity is mediated by antigen-specific IgG and/or IgE through the immunoglobulin receptors FcγRIII and FcεRI, respectively. Provided that these results apply to humans, they emphasize the importance of monitoring both IgE- and IgG-mediated asparaginase hypersensitivities in patients receiving this agent.