RESUMO
CRISPR-enabled screening is a powerful tool for the discovery of genes that control T cell function and has nominated candidate targets for immunotherapies1-6. However, new approaches are required to probe specific nucleotide sequences within key genes. Systematic mutagenesis in primary human T cells could reveal alleles that tune specific phenotypes. DNA base editors are powerful tools for introducing targeted mutations with high efficiency7,8. Here we develop a large-scale base-editing mutagenesis platform with the goal of pinpointing nucleotides that encode amino acid residues that tune primary human T cell activation responses. We generated a library of around 117,000 single guide RNA molecules targeting base editors to protein-coding sites across 385 genes implicated in T cell function and systematically identified protein domains and specific amino acid residues that regulate T cell activation and cytokine production. We found a broad spectrum of alleles with variants encoding critical residues in proteins including PIK3CD, VAV1, LCP2, PLCG1 and DGKZ, including both gain-of-function and loss-of-function mutations. We validated the functional effects of many alleles and further demonstrated that base-editing hits could positively and negatively tune T cell cytotoxic function. Finally, higher-resolution screening using a base editor with relaxed protospacer-adjacent motif requirements9 (NG versus NGG) revealed specific structural domains and protein-protein interaction sites that can be targeted to tune T cell functions. Base-editing screens in primary immune cells thus provide biochemical insights with the potential to accelerate immunotherapy design.
Assuntos
Alelos , Edição de Genes , Mutagênese , Linfócitos T , Humanos , Aminoácidos/genética , Sistemas CRISPR-Cas/genética , Mutagênese/genética , RNA Guia de Sistemas CRISPR-Cas/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Ativação Linfocitária , Citocinas/biossíntese , Citocinas/metabolismo , Mutação com Ganho de Função , Mutação com Perda de FunçãoRESUMO
BACKGROUND: Development of interleukin (IL)-2-dependent antitumor responses focus on targeting the intermediate affinity IL-2R to stimulate memory-phenotypic CD8+ T and natural killer (NK) cells while minimizing regulatory T cell (Treg) expansion. However, this approach may not effectively engage tumor-specific T effector cells. Since tumor-antigen specific T cells upregulate the high-affinity IL-2R, we tested an IL-2 biologic, mouse IL-2/CD25, with selectivity toward the high-affinity IL-2R to support antitumor responses to tumors that vary in their immunogenicity. METHODS: Mice were first implanted with either CT26, MC38, B16.F10, or 4T1 and after a tumor mass developed, they were treated with high-dose (HD) mouse (m)IL-2/CD25 alone or in combination with anti-programmed cell death protein-1 (PD-1) checkpoint blockade. Tumor growth was monitored and in parallel the immune signature in the tumor microenvironment (TME) was determined by a combination of multiparameter flow cytometry, functional assays, and enumeration of tumor-reactive T cells. RESULTS: We show that HD mIL-2/CD25, which preferentially stimulates the high-affinity IL-2R, but not IL-2/anti-IL-2 complexes with preferential activity toward the intermediate-affinity IL-2R, supports vigorous antitumor responses to immunogenic tumors as a monotherapy that were enhanced when combined with anti-PD-1. Treatment of CT26-bearing mice with HD mIL-2/CD25 led to a high CD8+:Treg ratio in the TME, increased frequency and function of tumor-specific CD8+ T effector cells with a less exhausted phenotype, and antitumor memory responses. CONCLUSIONS: Targeting the high-affinity IL-2R on tumor-specific T cells with HD mIL-2/CD25 alone or with PD-1 blockade supports antitumor responses, where the resulting memory response may afford long-term protection against tumor re-emergence.
Assuntos
Linfócitos T CD8-Positivos , Neoplasias , Camundongos , Animais , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Linfócitos T Reguladores , Células Matadoras Naturais , Imunoterapia , Microambiente TumoralRESUMO
Cancer vaccines offer the potential to enhance T cell-mediated antitumor immunity by expanding and increasing the function of tumor-specific T cells and shaping the recall response against recurring tumors. While the use of cancer vaccines is not a new immunotherapeutic approach, the cancer vaccine field continues to evolve as new antigen types emerge and vaccine formulations and delivery strategies are developed. As monotherapies, cancer vaccines have not been very efficacious in part due to pre-existing peripheral- and tumor-mediated tolerance mechanisms that limit T cell function. Over the years, various agents including Toll-like receptor agonists, cytokines, and checkpoint inhibitors have been employed as vaccine adjuvants and immune modulators to increase antigen-mediated activation, expansion, memory formation, and T effector cell function. A renewed interest in this approach has emerged as better neoepitope discovery tools are being developed and our understanding of what constitutes an effective cancer vaccine is improved. In the coming years, cancer vaccines will likely be vital to enhance the response to current immunotherapies. In this review, we discuss the various types of therapeutic cancer vaccines, including types of antigens and approaches used to enhance cancer vaccine responses such as TLR agonists, recombinant interleukin-2 and interleukin-2 derivatives, and checkpoint inhibitors.
RESUMO
Preclinical studies of the T cell growth factor activity of IL-2 resulted in this cytokine becoming the first immunotherapy to be approved nearly 30 years ago by the US Food and Drug Administration for the treatment of cancer. Since then, we have learnt the important role of IL-2 in regulating tolerance through regulatory T cells (Treg cells) besides promoting immunity through its action on effector T cells and memory T cells. Another pivotal event in the history of IL-2 research was solving the crystal structure of IL-2 bound to its tripartite receptor, which spurred the development of cell type-selective engineered IL-2 products. These new IL-2 analogues target Treg cells to counteract the dysregulated immune system in the context of autoimmunity and inflammatory disorders or target effector T cells, memory T cells and natural killer cells to enhance their antitumour responses. IL-2 biologics have proven to be effective in preclinical studies and clinical assessment of some is now underway. These studies will soon reveal whether engineered IL-2 biologics are truly capable of harnessing the IL-2-IL-2 receptor pathway as effective monotherapies or combination therapies for autoimmunity and cancer.
Assuntos
Produtos Biológicos , Neoplasias , Autoimunidade , Produtos Biológicos/farmacologia , Humanos , Fatores Imunológicos , Imunoterapia/métodos , Interleucina-2/uso terapêutico , Receptores de Interleucina-2 , Linfócitos T ReguladoresRESUMO
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.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/imunologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Neoplasias/genética , Receptores de Interleucina-2/metabolismo , Linfócitos T/metabolismo , Animais , Feminino , Humanos , Masculino , Camundongos , Neoplasias/patologiaRESUMO
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.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Subunidade alfa de Receptor de Interleucina-2/administração & dosagem , Interleucina-2/administração & dosagem , Células Matadoras Naturais/imunologia , Melanoma Experimental/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Linfócitos T Reguladores/imunologia , Animais , Antígenos de Neoplasias , Feminino , Humanos , Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Recombinantes de Fusão/imunologia , Transdução de Sinais , Microambiente TumoralRESUMO
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.
Assuntos
Subunidade alfa de Receptor de Interleucina-2/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/imunologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Animais , Citocinas/metabolismo , Feminino , Inflamação/metabolismo , Subunidade alfa de Receptor de Interleucina-2/genética , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Transcrição STAT5/genética , Transdução de Sinais/genética , Linfócitos T Reguladores/metabolismoRESUMO
Placental immune responses are highly regulated to strike a balance between protection and tolerance. For relatively mild infections, protection encompasses both the mother and fetus; however, during worsening conditions, protection becomes exclusively reserved for the mother. Previously, we and others have shown that the host factor perforin-2 plays a central role in protecting mice and cells against infection. In this study, we analyzed perforin-2 activity in the mouse placenta to determine whether perforin-2 plays a similarly protective role. We show that perforin-2 is critical for inhibiting Listeria monocytogenes colonization of the placenta and fetus and that this protection is due to both maternal and fetal-encoded perforin-2. Perforin-2 mRNA is readily detectable in individual immune cells of the decidua, and these levels are further enhanced specifically in decidual macrophages during high-dose infections that result in fetal expulsion. Unexpectedly, inductive perforin-2 expression in decidual macrophages did not occur during milder infections in which fetal viability remained intact. This pattern of expression significantly differed from that observed in splenic macrophages in which inductive perforin-2 expression was observed in both high and mild infection conditions. In the placenta, inductive perforin-2 expression in decidual macrophages was coincident with their polarization from a CD206+ MHC class IIlo to CD206- MHC class IIhi phenotype that normally occurs in the placenta during high-burden infections. Our results suggest that perforin-2 is part of a host response that is protective either for both the mother and fetus in milder infections or exclusively for the mother during high-dose infections.
Assuntos
Feto/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Placenta/imunologia , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Complicações Infecciosas na Gravidez/imunologia , Animais , Patógenos Transmitidos pelo Sangue , Células Cultivadas , Feminino , Humanos , Imunidade Materno-Adquirida , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Placenta/microbiologia , Proteínas Citotóxicas Formadoras de Poros/genética , Gravidez , Análise de Célula ÚnicaRESUMO
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.
Assuntos
Diabetes Mellitus/metabolismo , Subunidade alfa de Receptor de Interleucina-2 , Interleucina-2/farmacologia , Receptores de Interleucina-2/metabolismo , Transdução de Sinais/fisiologia , Animais , Autoanticorpos , Feminino , Humanos , Interleucina-2/química , Camundongos , Camundongos Endogâmicos , Proteínas Recombinantes de Fusão/farmacologia , Subpopulações de Linfócitos T/efeitos dos fármacosRESUMO
Yersinia pestis, the causative agent of plague, possesses a number of virulence mechanisms that allows it to survive and proliferate during its interaction with the host. To discover additional infection-specific Y. pestis factors, a transposon site hybridization (TraSH)-based genome-wide screen was employed to identify genomic regions required for its survival during cellular infection. In addition to several well-characterized infection-specific genes, this screen identified three chromosomal genes (y3397, y3399, and y3400), located in an apparent operon, that promoted successful infection. Each of these genes is predicted to encode a leucine-rich repeat family protein with or without an associated ubiquitin E3 ligase domain. These genes were designated Yersinia leucine-rich repeat gene A (ylrA), B (ylrB), and C (ylrC). Engineered strains with deletions of y3397 (ylrC), y3399 (ylrB), or y3400 (ylrA), exhibited infection defects both in cultured cells and in the mouse. C-terminal FLAG-tagged YlrA, YlrB, and YlrC were secreted by Y. pestis in the absence but not the presence of extracellular calcium and deletions of the DNA sequences encoding the predicted N-terminal type III secretion signals of YlrA, YlrB, and YlrC prevented their secretion, indicating that these proteins are substrates of the type III secretion system (T3SS). Further strengthening the connection with the T3SS, YlrB was readily translocated into HeLa cells and expression of the YlrA and YlrC proteins in yeast inhibited yeast growth, indicating that these proteins may function as anti-host T3S effector proteins.