RESUMEN
Regulatory T (Treg) cells require (interleukin-2) IL-2 for their homeostasis by affecting their proliferation, survival and activation. Here we investigated transcriptional and epigenetic changes after acute, periodic and persistent IL-2 receptor (IL-2R) signaling in mouse peripheral Treg cells in vivo using IL-2 or the long-acting IL-2-based biologic mouse IL-2-CD25. We show that initially IL-2R-dependent STAT5 transcription factor-dependent pathways enhanced gene activation, chromatin accessibility and metabolic reprogramming to support Treg cell proliferation. Unexpectedly, at peak proliferation, less accessible chromatin prevailed and was associated with Treg cell contraction. Restimulation of IL-2R signaling after contraction activated signature IL-2-dependent genes and others associated with effector Treg cells, whereas genes associated with signal transduction were downregulated to somewhat temper expansion. Thus, IL-2R-dependent Treg cell homeostasis depends in part on a shift from more accessible chromatin and expansion to less accessible chromatin and contraction. Mouse IL-2-CD25 supported greater expansion and a more extensive transcriptional state than IL-2 in Treg cells, consistent with greater efficacy to control autoimmunity.
Asunto(s)
Ensamble y Desensamble de Cromatina , Interleucina-2 , Linfocitos T Reguladores , Animales , Cromatina/metabolismo , Interleucina-2/metabolismo , Ratones , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/metabolismo , Transducción de SeñalRESUMEN
Chronic antigen exposure during viral infection or cancer promotes an exhausted T cell (Tex) state with reduced effector function. However, whether all antigen-specific T cell clones follow the same Tex differentiation trajectory remains unclear. Here, we generate a single-cell multiomic atlas of T cell exhaustion in murine chronic viral infection that redefines Tex phenotypic diversity, including two late-stage Tex subsets with either a terminal exhaustion (Texterm) or a killer cell lectin-like receptor-expressing cytotoxic (TexKLR) phenotype. We use paired single-cell RNA and T cell receptor sequencing to uncover clonal differentiation trajectories of Texterm-biased, TexKLR-biased or divergent clones that acquire both phenotypes. We show that high T cell receptor signaling avidity correlates with Texterm, whereas low avidity correlates with effector-like TexKLR fate. Finally, we identify similar clonal differentiation trajectories in human tumor-infiltrating lymphocytes. These findings reveal clonal heterogeneity in the T cell response to chronic antigen that influences Tex fates and persistence.
Asunto(s)
Linfocitos T CD8-positivos , Virosis , Humanos , Ratones , Animales , Receptores de Antígenos de Linfocitos T/genética , Diferenciación Celular , Linfocitos Infiltrantes de TumorRESUMEN
Soon after activation, CD4+ T cells are segregated into BCL6+ follicular helper (Tfh) and BCL6- effector (Teff) T cells. Here, we explored how these subsets are maintained during chronic antigen stimulation using the mouse chronic LCMV infection model. Using single cell-transcriptomic and epigenomic analyses, we identified a population of PD-1+ TCF-1+ CD4+ T cells with memory-like features. TCR clonal tracing and adoptive transfer experiments demonstrated that these cells have self-renewal capacity and continue to give rise to both Teff and Tfh cells, thus functioning as progenitor cells. Conditional deletion experiments showed Bcl6-dependent development of these progenitors, which were essential for sustaining antigen-specific CD4+ T cell responses to chronic infection. An analogous CD4+ T cell population developed in draining lymph nodes in response to tumors. Our study reveals the heterogeneity and plasticity of CD4+ T cells during persistent antigen exposure and highlights their population dynamics through a stable, bipotent intermediate state.
Asunto(s)
Antígenos , Linfocitos T Colaboradores-Inductores , Traslado Adoptivo , Animales , Diferenciación Celular , Ratones , Proteínas Proto-Oncogénicas c-bcl-6/genética , Células MadreRESUMEN
T cells are activated by antigen and co-stimulatory receptor signaling and undergo robust proliferation and differentiation into effector cells with protective function. Such quantitatively and qualitatively amplified T cell responses are effective in controlling acute infection and are followed by contraction of the effector population and the formation of resting memory T cells for enhanced protection against previously experienced antigens. However, in the face of persistent antigen during chronic viral infection, in autoimmunity, or in the tumor microenvironment, T cells exhibit distinct responses relative to those in acute insult in several aspects, including reduced clonal expansion and impaired effector function associated with inhibitory receptor expression, a state known as exhaustion. Nevertheless, their responses to chronic infection and tumors are sustained through the establishment of hierarchical heterogeneity, which preserves the duration of the response by generating newly differentiated effector cells. In this review, we highlight recent findings on distinct dynamics of T cell responses under "exhausting" conditions and the roles of the transcription factors that support attenuated yet long-lasting T cell responses as well as the establishment of dysfunctional states.
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Linfocitos T CD8-positivos , Virosis , Humanos , Regulación de la Expresión Génica , Diferenciación Celular , Dinámica PoblacionalRESUMEN
High-dose IL-2 induces cancer regression but its therapeutic use is limited due to high toxicities resulting from its broad cell targeting. In one strategy to overcome this limitation, IL-2 has been modified to selectively target the intermediate affinity IL-2R that broadly activates memory-phenotypic CD8+ T and NK cells, while minimizing Treg-associated tolerance. In this study, we modeled an alternative strategy to amplify tumor antigen-specific TCR transgenic CD8+ T cells through limited application of a long-acting IL-2 fusion protein, mIL-2/mCD25, which selectively targets the high-affinity IL-2R. Here, mice were vaccinated with a tumor antigen and high-dose mIL-2/mCD25 was applied to coincide with the induction of the high affinity IL-2R on tumor-specific T cells. A single high dose of mIL-2/mCD25, but not an equivalent amount of IL-2, amplified the frequency and function of tumor-reactive CD8+ T effector (Teff) and memory cells. These mIL-2/mCD25-dependent effects relied on distinctive requirements for TLR signals during priming of CD8+ tumor-specific T cells. The mIL-2/mCD25-amplified tumor-reactive effector and memory T cells supported long-lasting antitumor responses to B16-F10 melanoma. This regimen only transiently increased Tregs, yielding a favorable Teff-Treg ratio within the tumor microenvironment. Notably, mIL-2/mCD25 did not increase non-tumor-specific Teff or NK cells within tumors, further substantiating the specificity of mIL-2/mCD25 for tumor antigen-activated T cells. Thus, the selectivity and persistence of mIL-2/mCD25 in conjunction with a tumor vaccine supports antitumor immunity through a mechanism that is distinct from recombinant IL-2 or IL-2-based biologics that target the intermediate affinity IL-2R.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Subunidad alfa del Receptor de Interleucina-2/administración & dosificación , Interleucina-2/administración & dosificación , Células Asesinas Naturales/inmunología , Melanoma Experimental/inmunología , Proteínas Recombinantes de Fusión/administración & dosificación , Linfocitos T Reguladores/inmunología , Animales , Antígenos de Neoplasias , Femenino , Humanos , Interleucina-2/inmunología , Subunidad alfa del Receptor de Interleucina-2/inmunología , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Recombinantes de Fusión/inmunología , Transducción de Señal , Microambiente TumoralRESUMEN
Low-dose IL-2 represents an immunotherapy to selectively expand regulatory T cells (Tregs) to promote tolerance in patients with autoimmunity. In this article, we show that a fusion protein (FP) of mouse IL-2 and mouse IL-2Rα (CD25), joined by a noncleavable linker, has greater in vivo efficacy than rIL-2 at Treg expansion and control of autoimmunity. Biochemical and functional studies support a model in which IL-2 interacts with CD25 in the context of this FP in trans to form inactive head-to-tail dimers that slowly dissociate into an active monomer. In vitro, IL-2/CD25 has low sp. act. However, in vivo IL-2/CD25 is long lived to persistently and selectively stimulate Tregs. In female NOD mice, IL-2/CD25 administration increased Tregs within the pancreas and reduced the instance of spontaneous diabetes. Thus, IL-2/CD25 represents a distinct class of IL-2 FPs with the potential for clinical development for use in autoimmunity or other disorders of an overactive immune response.
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Diabetes Mellitus/prevención & control , Tolerancia Inmunológica/inmunología , Subunidad alfa del Receptor de Interleucina-2/genética , Interleucina-2/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Linfocitos T Reguladores/inmunología , Animales , Línea Celular , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
Runx1 and Cbfß are critical for the establishment of definitive hematopoiesis and are implicated in leukemic transformation. Despite the absolute requirements for these factors in the development of hematopoietic stem cells and lymphocytes, their roles in the development of bone marrow progenitor subsets have not been defined. Here, we demonstrate that Cbfß is essential for the development of Flt3(+) macrophage-dendritic cell (DC) progenitors in the bone marrow and all DC subsets in the periphery. Besides the loss of DC progenitors, pan-hematopoietic Cbfb-deficient mice also lack CD105(+) erythroid progenitors, leading to severe anemia at 3 to 4 months of age. Instead, Cbfb deficiency results in aberrant progenitor differentiation toward granulocyte-macrophage progenitors (GMPs), resulting in a myeloproliferative phenotype with accumulation of GMPs in the periphery and cellular infiltration of the liver. Expression of the transcription factor Irf8 is severely reduced in Cbfb-deficient progenitors, and overexpression of Irf8 restors DC differentiation. These results demonstrate that Runx proteins and Cbfß restrict granulocyte lineage commitment to facilitate multilineage hematopoietic differentiation and thus identify their novel tumor suppressor function in myeloid leukemia.
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Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Células Dendríticas/metabolismo , Trastornos Mieloproliferativos/metabolismo , Células Madre/metabolismo , Tirosina Quinasa 3 Similar a fms/metabolismo , Anemia/genética , Anemia/metabolismo , Anemia/patología , Animales , Células de la Médula Ósea/metabolismo , Diferenciación Celular/genética , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Subunidad beta del Factor de Unión al Sitio Principal/genética , Citometría de Flujo , Expresión Génica , Granulocitos/metabolismo , Hematopoyesis/genética , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/patología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Immunization with tumor neoantigens is a promising vaccine approach to promote antitumor immunity due to their high immunogenicity, lack of expression in normal tissue, and preferential induction of tumor neoantigen-specific T cells, which are central mediators of the anti-cancer response. A drawback to targeting tumor neoantigen-specific T cells is that these cells are found at a low frequency in patients with cancer, limiting their therapeutic benefit. Interleukin-2 (IL-2) promotes expansion and persistence of tumor-reactive T cells. However, its clinical use has been hampered by toxicities arising from its multiple cellular targets. Thus, new engineered IL-2 receptor (IL-2R) agonists with distinctive cell type selectivity have been designed to harness the potential of IL-2 for tumor immunotherapy. METHODS: We investigated the potential to amplify neoantigen-specific CD4+ and CD8+ T cell immune responses to promote antitumor immunity through vaccination with tumor neoantigens. Following T cell receptor (TCR)-mediated induction of the high-affinity IL-2R on these T cells, amplification of the neoantigen-specific T cell response was achieved using a high dose of the mouse IL-2/CD25 (mIL-2/CD25) fusion protein, an IL-2R agonist with more favorable pharmacokinetics and pharmacodynamics than IL-2 and selectivity toward the high-affinity IL-2R. RESULTS: Administration of a high dose of mIL-2/CD25 shortly after antigen-dependent induction of the high-affinity IL-2R amplified the numbers and function of TCR transgenic tumor-reactive tyrosinase-related protein-1 (TRP-1) CD4+ T cells, leading to antitumor immunity to B16-F10 melanoma. This approach was adapted to amplify endogenous polyclonal B16-F10 neoantigen-specific T cells. Maximal expansion of these cells required prime/boost neoantigen vaccinations, where mIL-2/CD25 was optimal when administered only after the boosting steps. The ensuing mIL-2/CD25-driven immune response supported antitumor immunity to B16-F10 and was more effective than treatment with a similar amount of IL-2. Optimal antitumor effects required amplification of CD4+ and CD8+ neoantigen-specific T cells. High-dose mIL-2/CD25 supported a tumor microenvironment with higher numbers of CD4+ and CD8+ T effectors cells with increased granzyme B expression and importantly a more robust expansion of neoantigen-specific T cells. CONCLUSION: These results indicate that neoantigen-based vaccines are optimized by potentiating IL-2R signaling in CD4+ and CD8+ neoantigen-reactive T cells by using high-dose mIL-2/CD25, leading to more effective tumor clearance.
Asunto(s)
Antígenos de Neoplasias/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Vacunas contra el Cáncer/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Neoplasias/genética , Receptores de Interleucina-2/metabolismo , Linfocitos T/metabolismo , Animales , Femenino , Humanos , Masculino , Ratones , Neoplasias/patologíaRESUMEN
IL-2R signaling is essential for the development and homeostasis of CD4+Foxp3+ regulatory T cells (Tregs). Low-dose IL-2 is being advanced as a therapy for autoimmune diseases because of its ability to expand Tregs. Although Treg stability and function is diminished by chronic inflammation, the impact of inflammation on proximal IL-2R signaling and/or responsiveness to low-dose IL-2 is poorly understood. In this study, we show that acute inflammation induced by LPS, analogous to responses to acute bacterial infection, led to decreased endogenous STAT5 signaling and proliferative potential as measured by Ki67 in mouse Tregs. This impaired Treg activity was transient, did not lead to a reduction in Treg numbers or function, and was due to TLR signaling by non-Tregs. Although acute LPS induced high levels of IL-1 and IL-6, these cytokines did not solely mediate dysregulated Treg activity. Global gene expression analyses demonstrated that acute LPS-induced inflammation substantially and rapidly altered the Treg transcriptome. In the presence of an IL-2R agonist, the mouse IL-2/CD25 fusion protein (mIL-2/CD25), this type of inflammatory response tempered the transcription of IL-2R-dependent genes in vivo. Gene enrichment and pathway analyses are consistent with LPS attenuating mIL-2/CD25-dependent genes related to the cell cycle, DNA replication, and cholesterol biosynthesis while enhancing mRNAs that mediated Treg suppression in vivo. Acute LPS-induced inflammation diminished some responses by Tregs to mIL-2/CD25 treatment in vivo. Together, these results suggest a role for persistent IL-2R signaling in mitigating some but not all of the deleterious effects of inflammation on Treg proliferation while supporting their function.
Asunto(s)
Subunidad alfa del Receptor de Interleucina-2/metabolismo , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Animales , Citocinas/metabolismo , Femenino , Inflamación/metabolismo , Subunidad alfa del Receptor de Interleucina-2/genética , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Factor de Transcripción STAT5/genética , Transducción de Señal/genética , Linfocitos T Reguladores/metabolismoRESUMEN
Low-dose interleukin-2 (IL-2) represents a new therapeutic approach to regulate immune homeostasis to promote immune tolerance in patients with autoimmune diseases, including type 1 diabetes. We have developed a new IL-2-based biologic, an IL-2/CD25 fusion protein, with greatly improved pharmacokinetics and pharmacodynamics when compared with recombinant IL-2 to enhance this type of immunotherapy. In this study, we show that low-dose mouse IL-2/CD25 (mIL-2/CD25), but not an equivalent amount of IL-2, prevents the onset of diabetes in NOD mice and controls diabetes in hyperglycemic mice. mIL-2/CD25 acts not only to expand regulatory T cells (Tregs) but also to increase their activation and migration into lymphoid tissues and the pancreas. Lower incidence of diabetes is associated with increased serum levels of IL-10, a cytokine readily produced by activated Tregs. These effects likely act in concert to lower islet inflammation while increasing Tregs in the remaining inflamed islets. mIL-2/CD25 treatment is also associated with lower anti-insulin autoantibody levels in part by inhibition of T follicular helper cells. Thus, long-acting mIL-2/CD25 represents an improved IL-2 analog that persistently elevates Tregs to maintain a favorable Treg/effector T cell ratio that limits diabetes by expansion of activated Tregs that readily migrate into lymphoid tissues and the pancreas while inhibiting autoantibodies.