RESUMEN
High frequencies of stem-like memory T cells in infusion products correlate with superior patient outcomes across multiple T cell therapy trials. Herein, we analyzed a published CRISPR activation screening to identify transcriptional regulators that could be harnessed to augment stem-like behavior in CD8+ T cells. Using IFN-γ production as a proxy for CD8+ T cell terminal differentiation, LMO4 emerged among the top hits inhibiting the development of effectors cells. Consistently, we found that Lmo4 was downregulated upon CD8+ T cell activation but maintained under culture conditions facilitating the formation of stem-like T cells. By employing a synthetic biology approach to ectopically express LMO4 in antitumor CD8+ T cells, we enabled selective expansion and enhanced persistence of transduced cells, while limiting their terminal differentiation and senescence. LMO4 overexpression promoted transcriptional programs regulating stemness, increasing the numbers of stem-like CD8+ memory T cells and enhancing their polyfunctionality and recall capacity. When tested in syngeneic and xenograft tumor models, LMO4 overexpression boosted CD8+ T cell antitumor immunity, resulting in enhanced tumor regression. Rather than directly modulating gene transcription, LMO4 bound to JAK1 and potentiated STAT3 signaling in response to IL-21, inducing the expression of target genes (Tcf7, Socs3, Junb, and Zfp36) crucial for memory responses. CRISPR/Cas9-deletion of Stat3 nullified the enhanced memory signature conferred by LMO4, thereby abrogating the therapeutic benefit of LMO4 overexpression. These results establish LMO4 overexpression as an effective strategy to boost CD8+ T cell stemness, providing a new synthetic biology tool to bolster the efficacy of T cell-based immunotherapies.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Linfocitos T CD8-positivos , Proteínas con Dominio LIM , Factor de Transcripción STAT3 , Transducción de Señal , Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/inmunología , Linfocitos T CD8-positivos/inmunología , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/inmunología , Factor de Transcripción STAT3/metabolismo , Ratones , Animales , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Humanos , Transducción de Señal/inmunología , Transducción de Señal/genética , Interleucinas/genética , Interleucinas/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunologíaRESUMEN
Progress in cytokine engineering is driving therapeutic translation by overcoming these proteins' limitations as drugs. The interleukin-2 (IL-2) cytokine is a promising immune stimulant for cancer treatment but is limited by its concurrent activation of both pro-inflammatory immune effector cells and anti-inflammatory regulatory T cells, toxicity at high doses, and short serum half-life. One approach to improve the selectivity, safety, and longevity of IL-2 is complexation with anti-IL-2 antibodies that bias the cytokine towards immune effector cell activation. Although this strategy shows potential in preclinical models, clinical translation of a cytokine/antibody complex is complicated by challenges in formulating a multi-protein drug and concerns regarding complex stability. Here, we introduced a versatile approach to designing intramolecularly assembled single-agent fusion proteins (immunocytokines, ICs) comprising IL-2 and a biasing anti-IL-2 antibody that directs the cytokine towards immune effector cells. We optimized IC construction and engineered the cytokine/antibody affinity to improve immune bias. We demonstrated that our IC preferentially activates and expands immune effector cells, leading to superior antitumor activity compared to natural IL-2, both alone and combined with immune checkpoint inhibitors. Moreover, therapeutic efficacy was observed without inducing toxicity. This work presents a roadmap for the design and translation of cytokine/antibody fusion proteins.
RESUMEN
The IL-2 receptor α chain (IL-2Rα/CD25) is constitutively expressed on double-negative (DN2/DN3 thymocytes and regulatory T cells (Tregs) but induced by IL-2 on T and natural killer (NK) cells, with Il2ra expression regulated by a STAT5-dependent super-enhancer. We investigated CD25 regulation and function using a series of mice with deletions spanning STAT5-binding elements. Deleting the upstream super-enhancer region mainly affected constitutive CD25 expression on DN2/DN3 thymocytes and Tregs, with these mice developing autoimmune alopecia, whereas deleting an intronic region decreased IL-2-induced CD25 on peripheral T and NK cells. Thus, distinct super-enhancer elements preferentially control constitutive versus inducible expression in a cell type-specific manner. The mediator-1 coactivator colocalized with specific STAT5-binding sites. Moreover, both upstream and intronic regions had extensive chromatin interactions, and deletion of either region altered the super-enhancer structure in mature T cells. These results demonstrate differential functions for distinct super-enhancer elements, thereby indicating previously unknown ways to manipulate CD25 expression in a cell type-specific fashion.
Asunto(s)
Interleucina-2 , Factor de Transcripción STAT5 , Animales , Ratones , Elementos de Facilitación Genéticos/genética , Interleucina-2/genética , Interleucina-2/farmacología , Subunidad alfa del Receptor de Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Receptores de Interleucina-2 , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismoRESUMEN
Cytokines are secreted or membrane-presented molecules that mediate broad cellular functions, including development, differentiation, growth and survival. Accordingly, the regulation of cytokine activity is extraordinarily important both physiologically and pathologically. Cytokine and/or cytokine receptor engineering is being widely investigated to safely and effectively modulate cytokine activity for therapeutic benefit. IL-2 in particular has been extensively engineered, to create IL-2 variants that differentially exhibit activities on regulatory T cells to potentially treat autoimmune disease versus effector T cells to augment antitumour effects. Additionally, engineering approaches are being applied to many other cytokines such as IL-10, interferons and IL-1 family cytokines, given their immunosuppressive and/or antiviral and anticancer effects. In modulating the actions of cytokines, the strategies used have been broad, including altering affinities of cytokines for their receptors, prolonging cytokine half-lives in vivo and fine-tuning cytokine actions. The field is rapidly expanding, with extensive efforts to create improved therapeutics for a range of diseases.
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Enfermedades Autoinmunes , Interleucina-2 , Humanos , Citocinas , Enfermedades Autoinmunes/tratamiento farmacológico , Inmunosupresores , Linfocitos T ReguladoresRESUMEN
Progress in cytokine engineering is driving therapeutic translation by overcoming the inherent limitations of these proteins as drugs. The interleukin-2 (IL-2) cytokine harbors great promise as an immune stimulant for cancer treatment. However, the cytokine's concurrent activation of both pro-inflammatory immune effector cells and anti-inflammatory regulatory T cells, its toxicity at high doses, and its short serum half-life have limited clinical application. One promising approach to improve the selectivity, safety, and longevity of IL-2 is complexation with anti-IL-2 antibodies that bias the cytokine towards the activation of immune effector cells (i.e., effector T cells and natural killer cells). Although this strategy shows therapeutic potential in preclinical cancer models, clinical translation of a cytokine/antibody complex is complicated by challenges in formulating a multi-protein drug and concerns about complex stability. Here, we introduce a versatile approach to designing intramolecularly assembled single-agent fusion proteins (immunocytokines, ICs) comprising IL-2 and a biasing anti-IL-2 antibody that directs the cytokine's activities towards immune effector cells. We establish the optimal IC construction and further engineer the cytokine/antibody affinity to improve immune biasing function. We demonstrate that our IC preferentially activates and expands immune effector cells, leading to superior antitumor activity compared to natural IL-2 without inducing toxicities associated with IL-2 administration. Collectively, this work presents a roadmap for the design and translation of immunomodulatory cytokine/antibody fusion proteins.
RESUMEN
In response to external stimuli during immune responses, monocytes can have multifaceted roles such as pathogen clearance and tissue repair. However, aberrant control of monocyte activation can result in chronic inflammation and subsequent tissue damage. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces monocyte differentiation into a heterogenous population of monocyte-derived dendritic cells (moDCs) and macrophages. However, the downstream molecular signals that dictate the differentiation of monocytes under pathological conditions is incompletely understood. We report here that the GM-CSF-induced STAT5 tetramerization is a critical determinate of monocyte fate and function. Monocytes require STAT5 tetramers to differentiate into moDCs. Conversely, the absence of STAT5 tetramers results in a switch to a functionally distinct monocyte-derived macrophage population. In the dextran sulfate sodium (DSS) model of colitis, STAT5 tetramer-deficient monocytes exacerbate disease severity. Mechanistically, GM-CSF signaling in STAT5 tetramer-deficient monocytes results in the overexpression of arginase I and a reduction in nitric oxide synthesis following stimulation with lipopolysaccharide. Correspondingly, the inhibition of arginase I activity and sustained supplementation of nitric oxide ameliorates the worsened colitis in STAT5 tetramer-deficient mice. This study suggests that STAT5 tetramers protect against severe intestinal inflammation through the regulation of arginine metabolism.
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Colitis , Monocitos , Factor de Transcripción STAT5 , Animales , Ratones , Arginasa/metabolismo , Diferenciación Celular , Sulfato de Dextran/efectos adversos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Inflamación , Óxido Nítrico/metabolismo , Factor de Transcripción STAT5/metabolismoRESUMEN
The interleukin-4 (IL-4) cytokine plays a critical role in modulating immune homeostasis. Although there is great interest in harnessing this cytokine as a therapeutic in natural or engineered formats, the clinical potential of native IL-4 is limited by its instability and pleiotropic actions. Here, we design IL-4 cytokine mimetics (denoted Neo-4) based on a de novo engineered IL-2 mimetic scaffold and demonstrate that these cytokines can recapitulate physiological functions of IL-4 in cellular and animal models. In contrast with natural IL-4, Neo-4 is hyperstable and signals exclusively through the type I IL-4 receptor complex, providing previously inaccessible insights into differential IL-4 signaling through type I versus type II receptors. Because of their hyperstability, our computationally designed mimetics can directly incorporate into sophisticated biomaterials that require heat processing, such as three-dimensional-printed scaffolds. Neo-4 should be broadly useful for interrogating IL-4 biology, and the design workflow will inform targeted cytokine therapeutic development.
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Citocinas , Interleucina-4 , Animales , Transducción de SeñalRESUMEN
Interleukin-15 is a pleiotropic cytokine type I four alpha-helical bundle cytokine that along with IL-2, IL-4, IL-7, IL-9, and IL-21 shares the common cytokine receptor γ chain, γc. IL-15 is vital for the development, survival, and expansion of natural killer cells and for the development of CD8+ memory T cells. Whereas other family γc cytokines signal by directly binding to their target cells, IL-15 is distinctive in that it binds to IL-15Rα, a sushi domain containing binding protein that is expressed on a number of cell types, including monocytes and dendritic cells as well as T cells, and then is trans-presented to responding cells that express IL-2Rß and γc. This distinctive mechanism for IL-15 relates to its role in signaling in the context of cell-cell interactions and signaling synapses. The actions of IL-15 and ways of manipulating its actions to potential therapeutic benefit are discussed.
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Subunidad gamma Común de Receptores de Interleucina , Interleucina-15 , Humanos , Animales , Interleucina-15/química , Interleucina-15/metabolismo , Subunidad gamma Común de Receptores de Interleucina/metabolismo , Unión Proteica , Transducción de Señal , InmunidadRESUMEN
Combination therapy with PD-1 blockade and IL-2 is highly effective during chronic lymphocytic choriomeningitis virus infection1. Here we examine the underlying basis for this synergy. We show that PD-1 + IL-2 combination therapy, in contrast to PD-1 monotherapy, substantially changes the differentiation program of the PD-1+TCF1+ stem-like CD8+ T cells and results in the generation of transcriptionally and epigenetically distinct effector CD8+ T cells that resemble highly functional effector CD8+ T cells seen after an acute viral infection. The generation of these qualitatively superior CD8+ T cells that mediate viral control underlies the synergy between PD-1 and IL-2. Our results show that the PD-1+TCF1+ stem-like CD8+ T cells, also referred to as precursors of exhausted CD8+ T cells, are not fate-locked into the exhaustion program and their differentiation trajectory can be changed by IL-2 signals. These virus-specific effector CD8+ T cells emerging from the stem-like CD8+ T cells after combination therapy expressed increased levels of the high-affinity IL-2 trimeric (CD25-CD122-CD132) receptor. This was not seen after PD-1 blockade alone. Finally, we show that CD25 engagement with IL-2 has an important role in the observed synergy between IL-2 cytokine and PD-1 blockade. Either blocking CD25 with an antibody or using a mutated version of IL-2 that does not bind to CD25 but still binds to CD122 and CD132 almost completely abrogated the synergistic effects observed after PD-1 + IL-2 combination therapy. There is considerable interest in PD-1 + IL-2 combination therapy for patients with cancer2,3, and our fundamental studies defining the underlying mechanisms of how IL-2 synergizes with PD-1 blockade should inform these human translational studies.
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Linfocitos T CD8-positivos , Interleucina-2 , Receptor de Muerte Celular Programada 1 , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/efectos de los fármacos , Quimioterapia Combinada , Humanos , Subunidad gamma Común de Receptores de Interleucina , Interleucina-2/inmunología , Interleucina-2/farmacología , Interleucina-2/uso terapéutico , Subunidad alfa del Receptor de Interleucina-2 , Subunidad beta del Receptor de Interleucina-2 , Coriomeningitis Linfocítica/tratamiento farmacológico , Coriomeningitis Linfocítica/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Factor 1 de Transcripción de Linfocitos TRESUMEN
Newborn screening for severe combined immunodeficiency (SCID) has not only accelerated diagnosis and improved treatment for affected infants, but also led to identification of novel genes required for human T cell development. A male proband had SCID newborn screening showing very low T cell receptor excision circles (TRECs), a biomarker for thymic output of nascent T cells. He had persistent profound T lymphopenia, but normal numbers of B and natural killer (NK) cells. Despite an allogeneic hematopoietic stem cell transplant from his brother, he failed to develop normal T cells. Targeted resequencing excluded known SCID genes; however, whole exome sequencing (WES) of the proband and parents revealed a maternally inherited X-linked missense mutation in MED14 (MED14V763A), a component of the mediator complex. Morpholino (MO)-mediated loss of MED14 function attenuated T cell development in zebrafish. Moreover, this arrest was rescued by ectopic expression of cDNA encoding the wild type human MED14 ortholog, but not by MED14V763A , suggesting that the variant impaired MED14 function. Modeling of the equivalent mutation in mouse (Med14V769A) did not disrupt T cell development at baseline. However, repopulation of peripheral T cells upon competitive bone marrow transplantation was compromised, consistent with the incomplete T cell reconstitution experienced by the proband upon transplantation with bone marrow from his healthy male sibling, who was found to have the same MED14V763A variant. Suspecting that the variable phenotypic expression between the siblings was influenced by further mutation(s), we sought to identify genetic variants present only in the affected proband. Indeed, WES revealed a mutation in the L1 cell adhesion molecule (L1CAMQ498H); however, introducing that mutation in vivo in mice did not disrupt T cell development. Consequently, immunodeficiency in the proband may depend upon additional, unidentified gene variants.
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Linfopenia , Inmunodeficiencia Combinada Grave , Animales , Humanos , Lactante , Recién Nacido , Linfopenia/genética , Masculino , Ratones , Tamizaje Neonatal , Inmunodeficiencia Combinada Grave/diagnóstico , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/terapia , Linfocitos T , Pez CebraRESUMEN
BACKGROUND: Enhanced recovery after surgery advocates that consuming carbohydrates two hours before anesthesia is beneficial to the patient's recovery. Patients with diabetes are prone to delayed gastric emptying. Different guidelines for preoperative carbohydrate consumption in patients with diabetes remain controversial due to concerns about the risk of regurgitation, aspiration and hyperglycemia. Ultrasonic gastric volume (GV) assessment and blood glucose monitoring can comprehensively evaluate the safety and feasibility of preoperative carbohydrate intake in type 2 diabetes (T2D) patients. AIM: To evaluate the impact of preoperative carbohydrate loading on GV before anesthesia induction in T2D patients. METHODS: Patients with T2D receiving surgery under general anesthesia from December 2019 to December 2020 were included. A total of 78 patients were randomly allocated to 4 groups receiving 0, 100, 200, or 300 mL of carbohydrate loading 2 h before anesthesia induction. Gastric volume per unit weight (GV/W), Perlas grade, changes in blood glucose level, and risk of reflux and aspiration were evaluated before anesthesia induction. RESULTS: No significant difference was found in GV/W among the groups before anesthesia induction (P > 0.05). The number of patients with Perlas grade II and GV/W > 1.5 mL/kg did not differ among the groups (P > 0.05). Blood glucose level increased by > 2 mmol/L in patients receiving 300 mL carbohydrate drink, which was significantly higher than that in groups 1 and 2 (P < 0.05). CONCLUSION: Preoperative carbohydrate loading < 300 mL 2 h before induction of anesthesia in patients with T2D did not affect GV or increase the risk of reflux and aspiration. Blood glucose levels did not change significantly with preoperative carbohydrate loading of < 200 mL. However, 300 mL carbohydrate loading may increase blood glucose levels in patients with T2D before induction of anesthesia.
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Synthetic receptor signalling has the potential to endow adoptively transferred T cells with new functions that overcome major barriers in the treatment of solid tumours, including the need for conditioning chemotherapy1,2. Here we designed chimeric receptors that have an orthogonal IL-2 receptor extracellular domain (ECD) fused with the intracellular domain (ICD) of receptors for common γ-chain (γc) cytokines IL-4, IL-7, IL-9 and IL-21 such that the orthogonal IL-2 cytokine elicits the corresponding γc cytokine signal. Of these, T cells that signal through the chimeric orthogonal IL-2Rß-ECD-IL-9R-ICD (o9R) are distinguished by the concomitant activation of STAT1, STAT3 and STAT5 and assume characteristics of stem cell memory and effector T cells. Compared to o2R T cells, o9R T cells have superior anti-tumour efficacy in two recalcitrant syngeneic mouse solid tumour models of melanoma and pancreatic cancer and are effective even in the absence of conditioning lymphodepletion. Therefore, by repurposing IL-9R signalling using a chimeric orthogonal cytokine receptor, T cells gain new functions, and this results in improved anti-tumour activity for hard-to-treat solid tumours.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , Inmunoterapia Adoptiva , Subunidad gamma Común de Receptores de Interleucina , Neoplasias , Receptores de Interleucina-9 , Proteínas Recombinantes de Fusión , Linfocitos T , Animales , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Inmunoterapia Adoptiva/métodos , Subunidad gamma Común de Receptores de Interleucina/genética , Subunidad gamma Común de Receptores de Interleucina/inmunología , Interleucinas/genética , Interleucinas/inmunología , Melanoma/inmunología , Ratones , Neoplasias/genética , Neoplasias/inmunología , Neoplasias Pancreáticas/inmunología , Receptores de Interleucina-9/genética , Receptores de Interleucina-9/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Factores de Transcripción STAT/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Signal tranducer and activator of transcription 5 (STAT5) plays a critical role in mediating cellular responses following cytokine stimulation. STAT proteins critically signal via the formation of dimers, but additionally, STAT tetramers serve key biological roles, and we previously reported their importance in T and natural killer (NK) cell biology. However, the role of STAT5 tetramerization in autoimmune-mediated neuroinflammation has not been investigated. Using the STAT5 tetramer-deficient Stat5a-Stat5b N-domain double knockin (DKI) mouse strain, we report here that STAT5 tetramers promote the pathogenesis of experimental autoimmune encephalomyelitis (EAE). The mild EAE phenotype observed in DKI mice correlates with the impaired extravasation of pathogenic T-helper 17 (Th17) cells and interactions between Th17 cells and monocyte-derived cells (MDCs) in the meninges. We further demonstrate that granulocyte-macrophage colony-stimulating factor (GM-CSF)-mediated STAT5 tetramerization regulates the production of CCL17 by MDCs. Importantly, CCL17 can partially restore the pathogenicity of DKI Th17 cells, and this is dependent on the activity of the integrin VLA-4. Thus, our study reveals a GM-CSF-STAT5 tetramer-CCL17 pathway in MDCs that promotes autoimmune neuroinflammation.
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Enfermedades Autoinmunes/metabolismo , Enfermedades Neuroinflamatorias/metabolismo , Factor de Transcripción STAT5 , Animales , Quimiocina CCL17/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Macrófagos/metabolismo , Ratones , Multimerización de Proteína , Factor de Transcripción STAT5/química , Factor de Transcripción STAT5/metabolismo , Células Th17/metabolismoRESUMEN
Adoptive transfer of antigen-specific T cells represents a major advance in cancer immunotherapy, with robust clinical outcomes in some patients1. Both the number of transferred T cells and their differentiation state are critical determinants of effective responses2,3. T cells can be expanded with T cell receptor (TCR)-mediated stimulation and interleukin-2, but this can lead to differentiation into effector T cells4,5 and lower therapeutic efficacy6, whereas maintenance of a more stem-cell-like state before adoptive transfer is beneficial7. Here we show that H9T, an engineered interleukin-2 partial agonist, promotes the expansion of CD8+ T cells without driving terminal differentiation. H9T led to altered STAT5 signalling and mediated distinctive downstream transcriptional, epigenetic and metabolic programs. In addition, H9T treatment sustained the expression of T cell transcription factor 1 (TCF-1) and promoted mitochondrial fitness, thereby facilitating the maintenance of a stem-cell-like state. Moreover, TCR-transgenic and chimeric antigen receptor-modified CD8+ T cells that were expanded with H9T showed robust anti-tumour activity in vivo in mouse models of melanoma and acute lymphoblastic leukaemia. Thus, engineering cytokine variants with distinctive properties is a promising strategy for creating new molecules with translational potential.
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Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Agonismo Parcial de Drogas , Interleucina-2/análogos & derivados , Interleucina-2/agonistas , Proteínas Mutantes/farmacología , Células Madre/efectos de los fármacos , Animales , Linfocitos T CD8-positivos/inmunología , Interleucina-2/química , Interleucina-2/genética , Melanoma/metabolismo , Ratones , Mitocondrias/efectos de los fármacos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Factor de Transcripción STAT5/metabolismo , Células Madre/citología , Factor 1 de Transcripción de Linfocitos T/metabolismo , Investigación Biomédica TraslacionalRESUMEN
Adoptive transfer of Tregs has been shown to improve alloengraftment in animal models. However, it is technically challenging to expand Tregs ex vivo for the purpose of infusing large numbers of cells in the clinic. We demonstrate an innovative approach to engineering an orthogonal IL-2/IL-2 receptor (IL-2R) pair, the parts of which selectively interact with each other, transmitting native IL-2 signals, but do not interact with the natural IL-2 or IL-2R counterparts, thereby enabling selective stimulation of target cells in vivo. Here, we introduced this orthogonal IL-2R into Tregs. Upon adoptive transfer in a murine mixed hematopoietic chimerism model, orthogonal IL-2 injection significantly promoted orthogonal IL-2R+Foxp3GFP+CD4+ cell proliferation without increasing other T cell subsets and facilitated donor hematopoietic cell engraftment followed by acceptance of heart allografts. Our data indicate that selective target cell stimulation enabled by the engineered orthogonal cytokine receptor improves Treg potential for the induction of organ transplantation tolerance.
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Interleucina-2/inmunología , Activación de Linfocitos , Receptores de Interleucina-2/inmunología , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunología , Tolerancia al Trasplante , Animales , Interleucina-2/genética , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Receptores de Interleucina-2/genética , Transducción de Señal/genética , Linfocitos T Reguladores/citologíaRESUMEN
Interleukin (IL)-2 and IL-21 dichotomously shape CD8+ T cell differentiation. IL-2 drives terminal differentiation, generating cells that are poorly effective against tumors, whereas IL-21 promotes stem cell memory T cells (TSCM) and antitumor responses. Here we investigated the role of metabolic programming in the developmental differences induced by these cytokines. IL-2 promoted effector-like metabolism and aerobic glycolysis, robustly inducing lactate dehydrogenase (LDH) and lactate production, whereas IL-21 maintained a metabolically quiescent state dependent on oxidative phosphorylation. LDH inhibition rewired IL-2-induced effects, promoting pyruvate entry into the tricarboxylic acid cycle and inhibiting terminal effector and exhaustion programs, including mRNA expression of members of the NR4A family of nuclear receptors, as well as Prdm1 and Xbp1 While deletion of Ldha prevented development of cells with antitumor effector function, transient LDH inhibition enhanced the generation of memory cells capable of triggering robust antitumor responses after adoptive transfer. LDH inhibition did not significantly affect IL-21-induced metabolism but caused major transcriptomic changes, including the suppression of IL-21-induced exhaustion markers LAG3, PD1, 2B4, and TIM3. LDH inhibition combined with IL-21 increased the formation of TSCM cells, resulting in more profound antitumor responses and prolonged host survival. These findings indicate a pivotal role for LDH in modulating cytokine-mediated T cell differentiation and underscore the therapeutic potential of transiently inhibiting LDH during adoptive T cell-based immunotherapy, with an unanticipated cooperative antitumor effect of LDH inhibition and IL-21.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Inhibidores Enzimáticos/farmacología , Interleucinas/metabolismo , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Melanoma Experimental/terapia , Células Madre/inmunología , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/trasplante , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Línea Celular Tumoral/trasplante , Humanos , Memoria Inmunológica , Inmunoterapia Adoptiva/métodos , Interleucina-2/inmunología , Interleucina-2/metabolismo , Interleucinas/inmunología , L-Lactato Deshidrogenasa/metabolismo , Melanoma Experimental/inmunología , Ratones , Cultivo Primario de Células , Células Madre/efectos de los fármacos , Células Madre/metabolismoRESUMEN
T cell dysfunction is a characteristic feature of chronic viral infection and cancer. Recent studies in chronic lymphocytic choriomeningitis virus (LCMV) infection have defined a PD-1+ Tcf-1+ CD8+ T cell subset capable of self-renewal and differentiation into more terminally differentiated cells that downregulate Tcf-1 and express additional inhibitory molecules such as Tim3. Here, we demonstrated that expression of the glycoprotein CD101 divides this terminally differentiated population into two subsets. Stem-like Tcf-1+ CD8+ T cells initially differentiated into a transitory population of CD101-Tim3+ cells that later converted into CD101+ Tim3+ cells. Recently generated CD101-Tim3+ cells proliferated in vivo, contributed to viral control, and were marked by an effector-like transcriptional signature including expression of the chemokine receptor CX3CR1, pro-inflammatory cytokines, and granzyme B. PD-1 pathway blockade increased the numbers of CD101-Tim3+ CD8+ T cells, suggesting that these newly generated transitional cells play a critical role in PD-1-based immunotherapy.
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Antígenos CD/metabolismo , Linfocitos T CD8-positivos/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Animales , Biomarcadores/metabolismo , Receptor 1 de Quimiocinas CX3C/genética , Receptor 1 de Quimiocinas CX3C/metabolismo , Femenino , Granzimas/genética , Granzimas/metabolismo , Receptor 2 Celular del Virus de la Hepatitis A/biosíntesis , Factor Nuclear 1-alfa del Hepatocito/genética , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Coriomeningitis Linfocítica/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1/genéticaRESUMEN
We have recently defined a novel population of PD-1 (programmed cell death 1)+ TCF1 (T cell factor 1)+ virus-specific CD8 T cells that function as resource cells during chronic LCMV infection and provide the proliferative burst seen after PD-1 blockade. Such CD8 T cells have been found in other chronic infections and also in cancer in mice and humans. These CD8 T cells exhibit stem-like properties undergoing self-renewal and also differentiating into the terminally exhausted CD8 T cells. Here we compared the epigenetic signature of stem-like CD8 T cells with exhausted CD8 T cells. ATAC-seq analysis showed that stem-like CD8 T cells had a unique signature implicating activity of HMG (TCF) and RHD (NF-κB) transcription factor family members in contrast to higher accessibility to ETS and RUNX motifs in exhausted CD8 T cells. In addition, regulatory regions of the transcription factors Tcf7 and Id3 were more accessible in stem-like cells whereas Prdm1 and Id2 were more accessible in exhausted CD8 T cells. We also compared the epigenetic signatures of the 2 CD8 T cell subsets from chronically infected mice with effector and memory CD8 T cells generated after an acute LCMV infection. Both CD8 T cell subsets generated during chronic infection were strikingly different from CD8 T cell subsets from acute infection. Interestingly, the stem-like CD8 T cell subset from chronic infection, despite sharing key functional properties with memory CD8 T cells, had a very distinct epigenetic program. These results show that the chronic stem-like CD8 T cell program represents a specific adaptation of the T cell response to persistent antigenic stimulation.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Factor Nuclear 1-alfa del Hepatocito/genética , Coriomeningitis Linfocítica/genética , Receptor de Muerte Celular Programada 1/genética , Animales , Linfocitos T CD8-positivos/patología , Linaje de la Célula/inmunología , Epigénesis Genética , Regulación de la Expresión Génica/inmunología , Humanos , Inmunoterapia , Proteína 2 Inhibidora de la Diferenciación/genética , Proteínas Inhibidoras de la Diferenciación/genética , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/genética , Virus de la Coriomeningitis Linfocítica/patogenicidad , Ratones , Factor 1 de Unión al Dominio 1 de Regulación Positiva/genética , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patologíaRESUMEN
The common cytokine receptor γ chain, γc, is a component of the receptors for interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15, and IL-21. Mutation of the gene encoding γc results in X-linked severe combined immunodeficiency in humans, and γc family cytokines collectively regulate development, proliferation, survival, and differentiation of immune cells. Here, we review the basic biology of these cytokines, highlighting mechanisms of signaling and gene regulation that have provided insights for immunodeficiency, autoimmunity, allergic diseases, and cancer. Moreover, we discuss how studies of this family stimulated the development of JAK3 inhibitors and present an overview of current strategies targeting these pathways in the clinic, including novel antibodies, antagonists, and partial agonists. The diverse roles of these cytokines on a range of immune cells have important therapeutic implications.