RESUMEN
Stem cells are maintained by transcriptional programs that promote self-renewal and repress differentiation. Here, we found that the transcription factor c-Myb was essential for generating and maintaining stem cells in the CD8+ T cell memory compartment. Following viral infection, CD8+ T cells lacking Myb underwent terminal differentiation and generated fewer stem cell-like central memory cells than did Myb-sufficient T cells. c-Myb acted both as a transcriptional activator of Tcf7 (which encodes the transcription factor Tcf1) to enhance memory development and as a repressor of Zeb2 (which encodes the transcription factor Zeb2) to hinder effector differentiation. Domain-mutagenesis experiments revealed that the transactivation domain of c-Myb was necessary for restraining differentiation, whereas its negative regulatory domain was critical for cell survival. Myb overexpression enhanced CD8+ T cell memory formation, polyfunctionality and recall responses that promoted curative antitumor immunity after adoptive transfer. These findings identify c-Myb as a pivotal regulator of CD8+ T cell stemness and highlight its therapeutic potential.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica/inmunología , Neoplasias Experimentales/inmunología , Proteínas Proto-Oncogénicas c-myb/inmunología , Células Madre/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Diferenciación Celular/inmunología , Línea Celular Tumoral , Células HEK293 , Humanos , Memoria Inmunológica/genética , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/metabolismo , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/fisiología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/virología , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Proto-Oncogénicas c-myb/metabolismo , Células Madre/metabolismo , Células Madre/virología , Factor 1 de Transcripción de Linfocitos T/genética , Factor 1 de Transcripción de Linfocitos T/inmunología , Factor 1 de Transcripción de Linfocitos T/metabolismoRESUMEN
Cancer cells must evade immune responses at distant sites to establish metastases. The lung is a frequent site for metastasis. We hypothesized that lung-specific immunoregulatory mechanisms create an immunologically permissive environment for tumor colonization. We found that T-cell-intrinsic expression of the oxygen-sensing prolyl-hydroxylase (PHD) proteins is required to maintain local tolerance against innocuous antigens in the lung but powerfully licenses colonization by circulating tumor cells. PHD proteins limit pulmonary type helper (Th)-1 responses, promote CD4(+)-regulatory T (Treg) cell induction, and restrain CD8(+) T cell effector function. Tumor colonization is accompanied by PHD-protein-dependent induction of pulmonary Treg cells and suppression of IFN-γ-dependent tumor clearance. T-cell-intrinsic deletion or pharmacological inhibition of PHD proteins limits tumor colonization of the lung and improves the efficacy of adoptive cell transfer immunotherapy. Collectively, PHD proteins function in T cells to coordinate distinct immunoregulatory programs within the lung that are permissive to cancer metastasis. PAPERCLIP.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/secundario , Pulmón/inmunología , Oxígeno/metabolismo , Prolil Hidroxilasas/metabolismo , Linfocitos T Reguladores/inmunología , Traslado Adoptivo , Animales , Linfocitos T CD8-positivos/enzimología , Glucólisis/inmunología , Interferón gamma/inmunología , Pulmón/patología , Neoplasias Pulmonares/terapia , Activación de Linfocitos , Ratones , Ratones Noqueados , Metástasis de la Neoplasia , Neuropilina-1/metabolismo , Prolil Hidroxilasas/genética , Linfocitos T Reguladores/enzimología , Células TH1/enzimología , Células TH1/inmunologíaRESUMEN
T cell antigen receptor (TCR) signaling drives distinct responses depending on the differentiation state and context of CD8(+) T cells. We hypothesized that access of signal-dependent transcription factors (TFs) to enhancers is dynamically regulated to shape transcriptional responses to TCR signaling. We found that the TF BACH2 restrains terminal differentiation to enable generation of long-lived memory cells and protective immunity after viral infection. BACH2 was recruited to enhancers, where it limited expression of TCR-driven genes by attenuating the availability of activator protein-1 (AP-1) sites to Jun family signal-dependent TFs. In naive cells, this prevented TCR-driven induction of genes associated with terminal differentiation. Upon effector differentiation, reduced expression of BACH2 and its phosphorylation enabled unrestrained induction of TCR-driven effector programs.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Linfocitos T CD8-positivos/fisiología , Factor de Transcripción AP-1/metabolismo , Virus Vaccinia/inmunología , Vaccinia/inmunología , Inmunidad Adaptativa , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Linfocitos T CD8-positivos/virología , Diferenciación Celular/genética , Células Cultivadas , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica , Memoria Inmunológica/genética , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Oncogénica p65(gag-jun) , Transducción de Señal/genética , Factor de Transcripción AP-1/genéticaRESUMEN
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.
Asunto(s)
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
Mutations in KRAS are some of the most common across multiple cancer types and are thus attractive targets for therapy. Recent studies demonstrated that mutant KRAS generates immunogenic neoantigens that are targetable by adoptive T-cell therapy in metastatic diseases. To expand mutant KRAS-specific immunotherapies, it is critical to identify additional HLA-I allotypes that can present KRAS neoantigens and their cognate T-cell receptors (TCR). Here, we identified a murine TCR specific to a KRAS-G12V neoantigen (7VVVGAVGVGK16) using a vaccination approach with transgenic mice expressing HLA-A*03:01 (HLA-A3). This TCR demonstrated exquisite specificity for mutant G12V and not WT KRAS peptides. To investigate the molecular basis for neoantigen recognition by this TCR, we determined its structure in complex with HLA-A3(G12V). G12V-TCR CDR3ß and CDR1ß formed a hydrophobic pocket to interact with p6 Val of the G12V but not the WT KRAS peptide. To improve the tumor sensitivity of this TCR, we designed rational substitutions to improve TCR:HLA-A3 contacts. Two substitutions exhibited modest improvements in TCR binding avidity to HLA-A3 (G12V) but did not sufficiently improve T-cell sensitivity for further clinical development. Our study provides mechanistic insight into how TCRs detect neoantigens and reveals the challenges in targeting KRAS-G12V mutations.
RESUMEN
The transcriptional repressor Blimp-1 promotes the differentiation of CD8(+) T cells into short-lived effector cells (SLECs) that express the lectin-like receptor KLRG-1, but how it operates remains poorly defined. Here we show that Blimp-1 bound to and repressed the promoter of the gene encoding the DNA-binding inhibitor Id3 in SLECs. Repression of Id3 by Blimp-1 was dispensable for SLEC development but limited the ability of SLECs to persist as memory cells. Enforced expression of Id3 was sufficient to restore SLEC survival and enhanced recall responses. Id3 function was mediated in part through inhibition of the transcriptional activity of E2A and induction of genes regulating genome stability. Our findings identify the Blimp-1-Id3-E2A axis as a key molecular switch that determines whether effector CD8(+) T cells are programmed to die or enter the memory pool.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica/inmunología , Proteínas Inhibidoras de la Diferenciación/metabolismo , Factores de Transcripción/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular/genética , Línea Celular Tumoral , Reparación del ADN , Replicación del ADN , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Proteínas Inhibidoras de la Diferenciación/genética , Lectinas Tipo C , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Regiones Promotoras Genéticas , Receptores Inmunológicos/metabolismoRESUMEN
Tumours progress despite being infiltrated by tumour-specific effector T cells. Tumours contain areas of cellular necrosis, which are associated with poor survival in a variety of cancers. Here, we show that necrosis releases intracellular potassium ions into the extracellular fluid of mouse and human tumours, causing profound suppression of T cell effector function. Elevation of the extracellular potassium concentration ([K+]e) impairs T cell receptor (TCR)-driven Akt-mTOR phosphorylation and effector programmes. Potassium-mediated suppression of Akt-mTOR signalling and T cell function is dependent upon the activity of the serine/threonine phosphatase PP2A. Although the suppressive effect mediated by elevated [K+]e is independent of changes in plasma membrane potential (Vm), it requires an increase in intracellular potassium ([K+]i). Accordingly, augmenting potassium efflux in tumour-specific T cells by overexpressing the potassium channel Kv1.3 lowers [K+]i and improves effector functions in vitro and in vivo and enhances tumour clearance and survival in melanoma-bearing mice. These results uncover an ionic checkpoint that blocks T cell function in tumours and identify potential new strategies for cancer immunotherapy.
Asunto(s)
Cationes Monovalentes/metabolismo , Melanoma/inmunología , Potasio/metabolismo , Linfocitos T/inmunología , Escape del Tumor/inmunología , Microambiente Tumoral/inmunología , Animales , Humanos , Tolerancia Inmunológica/inmunología , Inmunoterapia/métodos , Canal de Potasio Kv1.3/metabolismo , Masculino , Melanoma/metabolismo , Melanoma/patología , Melanoma/terapia , Potenciales de la Membrana , Ratones , Necrosis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Análisis de Supervivencia , Linfocitos T/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Th17 cells have been described as short lived, but this view is at odds with their capacity to trigger protracted damage to normal and transformed tissues. We report that Th17 cells, despite displaying low expression of CD27 and other phenotypic markers of terminal differentiation, efficiently eradicated tumors and caused autoimmunity, were long lived, and maintained a core molecular signature resembling early memory CD8(+) cells with stem cell-like properties. In addition, we found that Th17 cells had high expression of Tcf7, a direct target of the Wnt and ß-catenin signaling axis, and accumulated ß-catenin, a feature observed in stem cells. In vivo, Th17 cells gave rise to Th1-like effector cell progeny and also self-renewed and persisted as IL-17A-secreting cells. Multipotency was required for Th17 cell-mediated tumor eradication because effector cells deficient in IFN-γ or IL-17A had impaired activity. Thus, Th17 cells are not always short lived and are a less-differentiated subset capable of superior persistence and functionality.
Asunto(s)
Células Madre/metabolismo , Células Th17/inmunología , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Supervivencia Celular/genética , Perfilación de la Expresión Génica , Interleucina-17/biosíntesis , Ratones , Ratones Transgénicos , Neoplasias/inmunología , Células Madre/citología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Células TH1/citología , Células TH1/inmunología , Células Th17/citología , Células Th17/metabolismo , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
Antibodies targeting the T-cell immune checkpoint cytotoxic T-lymphocyte antigen-4 (CTLA4) enhance the effectiveness of radiotherapy for melanoma patients, but many remain resistant. To further improve response rates, we explored combining anti-CTLA4 blockade with antisense suppression of CD47, an inhibitory receptor on T cells that limit T-cell receptor signaling and killing of irradiated target cells. Human melanoma data from The Cancer Genome Atlas revealed positive correlations between CD47 mRNA expression and expression of T-cell regulators including CTLA4 and its counter receptors CD80 and CD86. Antisense suppression of CD47 on human T cells in vitro using a translational blocking morpholino (CD47 m) alone or combined with anti-CTLA4 enhanced antigen-dependent killing of irradiated melanoma cells. Correspondingly, the treatment of locally irradiated B16F10 melanomas in C57BL/6 mice using combined blockade of CD47 and CTLA4 significantly increased the survival of mice relative to either treatment alone. CD47 m alone or in combination with anti-CTLA4 increased CD3+ T-cell infiltration in irradiated tumors. Anti-CTLA4 also increased CD3+ and CD8+ T-cell infiltration as well as markers of NK cells in non-irradiated tumors. Anti-CTLA4 combined with CD47 m resulted in the greatest increase in intratumoral granzyme B, interferon-γ, and NK-cell marker mRNA expression. These data suggest that combining CTLA4 and CD47 blockade could provide a survival benefit by enhancing adaptive T- and NK-cell immunity in irradiated tumors.
Asunto(s)
Antígeno CD47/antagonistas & inhibidores , Antígeno CTLA-4/antagonistas & inhibidores , Ipilimumab/administración & dosificación , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma Experimental/mortalidad , Linfocitos T Citotóxicos/inmunología , Animales , Antígeno CD47/genética , Antígeno CD47/inmunología , Terapia Combinada , Humanos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/efectos de la radiación , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos C57BL , Dosis de Radiación , Tasa de Supervivencia , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/efectos de la radiación , Células Tumorales CultivadasRESUMEN
Through their functional diversification, distinct lineages of CD4(+) T cells can act to either drive or constrain immune-mediated pathology. Transcription factors are critical in the generation of cellular diversity, and negative regulators antagonistic to alternate fates often act in conjunction with positive regulators to stabilize lineage commitment. Genetic polymorphisms within a single locus encoding the transcription factor BACH2 are associated with numerous autoimmune and allergic diseases including asthma, Crohn's disease, coeliac disease, vitiligo, multiple sclerosis and type 1 diabetes. Although these associations point to a shared mechanism underlying susceptibility to diverse immune-mediated diseases, a function for BACH2 in the maintenance of immune homeostasis has not been established. Here, by studying mice in which the Bach2 gene is disrupted, we define BACH2 as a broad regulator of immune activation that stabilizes immunoregulatory capacity while repressing the differentiation programs of multiple effector lineages in CD4(+) T cells. BACH2 was required for efficient formation of regulatory (Treg) cells and consequently for suppression of lethal inflammation in a manner that was Treg-cell-dependent. Assessment of the genome-wide function of BACH2, however, revealed that it represses genes associated with effector cell differentiation. Consequently, its absence during Treg polarization resulted in inappropriate diversion to effector lineages. In addition, BACH2 constrained full effector differentiation within TH1, TH2 and TH17 cell lineages. These findings identify BACH2 as a key regulator of CD4(+) T-cell differentiation that prevents inflammatory disease by controlling the balance between tolerance and immunity.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Homeostasis/inmunología , Linfocitos T Reguladores/inmunología , Animales , Autoinmunidad/inmunología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/deficiencia , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Femenino , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Homeostasis/genética , Humanos , Tolerancia Inmunológica/genética , Tolerancia Inmunológica/inmunología , Inflamación/genética , Inflamación/inmunología , Inflamación/mortalidad , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
CD8(+) T cells develop increased sensitivity following Ag experience, and differences in sensitivity exist between T cell memory subsets. How differential TCR signaling between memory subsets contributes to sensitivity differences is unclear. We show in mouse effector memory T cells (TEM) that >50% of lymphocyte-specific protein tyrosine kinase (Lck) exists in a constitutively active conformation, compared with <20% in central memory T cells (TCM). Immediately proximal to Lck signaling, we observed enhanced Zap-70 phosphorylation in TEM following TCR ligation compared with TCM Furthermore, we observed superior cytotoxic effector function in TEM compared with TCM, and we provide evidence that this results from a lower probability of TCM reaching threshold signaling owing to the decreased magnitude of TCR-proximal signaling. We provide evidence that the differences in Lck constitutive activity between CD8(+) TCM and TEM are due to differential regulation by SH2 domain-containing phosphatase-1 (Shp-1) and C-terminal Src kinase, and we use modeling of early TCR signaling to reveal the significance of these differences. We show that inhibition of Shp-1 results in increased constitutive Lck activity in TCM to levels similar to TEM, as well as increased cytotoxic effector function in TCM Collectively, this work demonstrates a role for constitutive Lck activity in controlling Ag sensitivity, and it suggests that differential activities of TCR-proximal signaling components may contribute to establishing the divergent effector properties of TCM and TEM. This work also identifies Shp-1 as a potential target to improve the cytotoxic effector functions of TCM for adoptive cell therapy applications.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica/fisiología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Western Blotting , Linfocitos T CD8-positivos/metabolismo , Citotoxicidad Inmunológica/inmunología , Citometría de Flujo , Humanos , Activación de Linfocitos/inmunología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Cadenas de Markov , Ratones , Reacción en Cadena de la Polimerasa , Subgrupos de Linfocitos T/metabolismo , Imagen de Lapso de TiempoRESUMEN
Lymphodepleting regimens are used before adoptive immunotherapy to augment the antitumor efficacy of transferred T cells by removing endogenous homeostatic "cytokine sinks." These conditioning modalities, however, are often associated with severe toxicities. We found that microRNA-155 (miR-155) enabled tumor-specific CD8(+) T cells to mediate profound antitumor responses in lymphoreplete hosts that were not potentiated by immune-ablation. miR-155 enhanced T-cell responsiveness to limited amounts of homeostatic γc cytokines, resulting in delayed cellular contraction and sustained cytokine production. miR-155 restrained the expression of the inositol 5-phosphatase Ship1, an inhibitor of the serine-threonine protein kinase Akt, and multiple negative regulators of signal transducer and activator of transcription 5 (Stat5), including suppressor of cytokine signaling 1 (Socs1) and the protein tyrosine phosphatase Ptpn2. Expression of constitutively active Stat5a recapitulated the survival advantages conferred by miR-155, whereas constitutive Akt activation promoted sustained effector functions. Our results indicate that overexpression of miR-155 in tumor-specific T cells can be used to increase the effectiveness of adoptive immunotherapies in a cell-intrinsic manner without the need for life-threatening, lymphodepleting maneuvers.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Citocinas/inmunología , MicroARNs/genética , MicroARNs/inmunología , Animales , Secuencia de Bases , Línea Celular Tumoral , Citocinas/biosíntesis , Células HEK293 , Humanos , Inmunoterapia Adoptiva , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Antígeno gp100 del Melanoma/genética , Antígeno gp100 del Melanoma/inmunologíaRESUMEN
Programmed cell death-1 (PD-1) is expressed on activated T cells and represents an attractive target for gene-editing of tumor targeted T cells prior to adoptive cell transfer (ACT). We used zinc finger nucleases (ZFNs) directed against the gene encoding human PD-1 (PDCD-1) to gene-edit melanoma tumor infiltrating lymphocytes (TIL). We show that our clinical scale TIL production process yielded efficient modification of the PD-1 gene locus, with an average modification frequency of 74.8% (n = 3, range 69.9-84.1%) of the alleles in a bulk TIL population, which resulted in a 76% reduction in PD-1 surface-expression. Forty to 48% of PD-1 gene-edited cells had biallelic PD-1 modification. Importantly, the PD-1 gene-edited TIL product showed improved in vitro effector function and a significantly increased polyfunctional cytokine profile (TNFα, GM-CSF, and IFNγ) compared to unmodified TIL in two of the three donors tested. In addition, all donor cells displayed an effector memory phenotype and expanded approximately 500-2,000-fold in vitro. Thus, further study to determine the efficiency and safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma is warranted.
Asunto(s)
Endorribonucleasas/genética , Regulación Neoplásica de la Expresión Génica , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/terapia , Receptor de Muerte Celular Programada 1/genética , Dedos de Zinc , Alelos , Animales , Separación Celular , Citocinas/metabolismo , Femenino , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Memoria Inmunológica , Inmunoterapia Adoptiva , Interferón gamma/metabolismo , Activación de Linfocitos/inmunología , Ratones , Metástasis de la Neoplasia , Trasplante de Neoplasias , Fenotipo , Receptor de Muerte Celular Programada 1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
T cells expressing antigen-specific T-cell receptors (TCRs) can mediate effective tumor regression, but they often also are accompanied by autoimmune responses. To determine the TCR affinity threshold defining the optimal balance between effective antitumor activity and autoimmunity in vivo, we used a unique self-antigen system comprising seven human melanoma gp100(209-217)-specific TCRs spanning physiological affinities (1-100 µM). We found that in vitro and in vivo T-cell responses are determined by TCR affinity, except in one case that was compensated by substantial CD8 involvement. Strikingly, we found that T-cell antitumor activity and autoimmunity are closely coupled but plateau at a defined TCR affinity of 10 µM, likely due to diminished contribution of TCR affinity to avidity above the threshold. Together, these results suggest that a relatively low-affinity threshold is necessary for the immune system to avoid self-damage, given the close relationship between antitumor activity and autoimmunity. The low threshold, in turn, indicates that adoptive T-cell therapy treatment strategies using in vitro-generated high-affinity TCRs do not necessarily improve efficacy.
Asunto(s)
Autoinmunidad/inmunología , Inmunoterapia Adoptiva/métodos , Neoplasias/inmunología , Neoplasias/terapia , Receptores de Antígenos de Linfocitos T/metabolismo , Análisis de Varianza , Pruebas Inmunológicas de Citotoxicidad , Ensayo de Inmunoadsorción Enzimática , Fluorescencia , Humanos , Inmunohistoquímica , Transducción Genética , Antígeno gp100 del Melanoma/inmunologíaRESUMEN
Enthusiasm for therapeutic cancer vaccines has been rejuvenated with the recent completion of several large, randomized phase III clinical trials that in some cases have reported an improvement in progression free or overall survival. However, an honest appraisal of their efficacy reveals modest clinical benefit and a frequent requirement for patients with relatively indolent cancers and minimal or no measurable disease. Experience with adoptive cell transfer-based immunotherapies unequivocally establishes that T cells can mediate durable complete responses, even in the setting of advanced metastatic disease. Further, these findings reveal that the successful vaccines of the future must confront: (i) a corrupted tumor microenvironment containing regulatory T cells and aberrantly matured myeloid cells, (ii) a tumor-specific T-cell repertoire that is prone to immunologic exhaustion and senescence, and (iii) highly mutable tumor targets capable of antigen loss and immune evasion. Future progress may come from innovations in the development of selective preparative regimens that eliminate or neutralize suppressive cellular populations, more effective immunologic adjuvants, and further refinement of agents capable of antagonizing immune check-point blockade pathways.
Asunto(s)
Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/uso terapéutico , Neoplasias/terapia , Subgrupos de Linfocitos T/inmunología , Adyuvantes Inmunológicos , Traslado Adoptivo , Antígenos de Diferenciación de Linfocitos T/inmunología , Vacunas contra el Cáncer/inmunología , Humanos , Inmunoterapia , Neoplasias/inmunología , Ensayos Clínicos Controlados Aleatorios como Asunto , Receptores Toll-Like/agonistas , Resultado del TratamientoRESUMEN
Engineering CD8⺠T cells to deliver interleukin 12 (IL-12) to the tumor site can lead to striking improvements in the ability of adoptively transferred T cells to induce the regression of established murine cancers. We have recently shown that IL-12 triggers an acute inflammatory environment that reverses dysfunctional antigen presentation by myeloid-derived cells within tumors and leads to an increase in the infiltration of adoptively transferred antigen-specific CD8⺠T cells. Here, we find that local delivery of IL-12 increased the expression of Fas within tumor-infiltrating macrophages, dendritic cells, and myeloid-derived suppressor cells (MDSC), and that these changes were abrogated in mice deficient in IL-12-receptor signaling. Importantly, upregulation of Fas in host mice played a critical role in the proliferation and antitumor activity of adoptively transferred IL-12-modified CD8⺠T cells. We also observed higher percentages of myeloid-derived cell populations within tumors in Fas-deficient mice, indicating that tumor stromal destruction was dependent on the Fas death receptor. Taken together, these results describe the likely requirement for costimulatory reverse signaling through Fasl on T cells that successfully infiltrate tumors, a mechanism triggered by the induction of Fas expression on myeloid-derived cells by IL-12 and the subsequent collapse of the tumor stroma.
Asunto(s)
Interleucina-12/uso terapéutico , Melanoma Experimental/metabolismo , Receptor fas/metabolismo , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Células Dendríticas/metabolismo , Femenino , Citometría de Flujo , Interleucina-12/administración & dosificación , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptor fas/genéticaRESUMEN
BACKGROUND: Tumor-infiltrating lymphocytes (TILs) targeting neoantigens can effectively treat a selected set of metastatic solid cancers. However, harnessing TILs for cancer treatments remains challenging because neoantigen-reactive T cells are often rare and exhausted, and ex vivo expansion can further reduce their frequencies. This complicates the identification of neoantigen-reactive T-cell receptors (TCRs) and the development of TIL products with high reactivity for patient treatment. METHODS: We tested whether TILs could be in vitro stimulated against neoantigens to achieve selective expansion of neoantigen-reactive TILs. Given their prevalence, mutant p53 or RAS were studied as models of human neoantigens. An in vitro stimulation method, termed "NeoExpand", was developed to provide neoantigen-specific stimulation to TILs. 25 consecutive patient TILs from tumors harboring p53 or RAS mutations were subjected to NeoExpand. RESULTS: We show that neoantigenic stimulation achieved selective expansion of neoantigen-reactive TILs and broadened the neoantigen-reactive CD4+ and CD8+ TIL clonal repertoire. This allowed the effective isolation of novel neoantigen-reactive TCRs. Out of the 25 consecutive TIL samples, neoantigenic stimulation enabled the identification of 16 unique reactivities and 42 TCRs, while conventional TIL expansion identified 9 reactivities and 14 TCRs. Single-cell transcriptome analysis revealed that neoantigenic stimulation increased neoantigen-reactive TILs with stem-like memory phenotypes expressing IL-7R, CD62L, and KLF2. Furthermore, neoantigenic stimulation improved the in vivo antitumor efficacy of TILs relative to the conventional OKT3-induced rapid TIL expansion in p53-mutated or KRAS-mutated xenograft mouse models. CONCLUSIONS: Taken together, neoantigenic stimulation of TILs selectively expands neoantigen-reactive TILs by frequencies and by their clonal repertoire. NeoExpand led to improved phenotypes and functions of neoantigen-reactive TILs. Our data warrant its clinical evaluation. TRIAL REGISTRATION NUMBER: NCT00068003, NCT01174121, and NCT03412877.
Asunto(s)
Antígenos de Neoplasias , Linfocitos Infiltrantes de Tumor , Receptores de Antígenos de Linfocitos T , Humanos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Antígenos de Neoplasias/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Ratones , Memoria Inmunológica , Animales , Femenino , Fenotipo , Neoplasias/inmunologíaRESUMEN
The genetic modification of CD8+ T cells using anti-tumor T-cell receptors (TCR) or chimeric antigen receptors is a promising approach for the adoptive cell therapy of patients with cancer. We previously developed a simplified method for the clinical-scale generation of central memory-like (Tcm) CD8+ T cells following transduction with lentivirus encoding anti-tumor TCR and culture in the presence of IL-2. In this study, we compared different cytokines or combinations of IL-2, IL-7, IL-12, IL-15, and IL-21 to expand genetically engineered CD8+ T cells. We demonstrated that specific cytokine combinations IL-12 plus IL-7 or IL-21 for 3 days followed by withdrawal of IL-12 yielded the phenotype of CD62L(high)CD28(high) CD127(high)CD27(high)CCR7(high), which is associated with less-differentiated T cells. Genes associated with stem cells (SOX2, NANOG, OCT4, and LIN28A), were also up-regulated by this cytokine cocktail. Moreover, the use of IL-12 plus IL-7 or IL-21 yielded CD8 T cells showing enhanced persistence in the NOD/SCID/γc-/- mouse model. This defined cytokine combination could also alter highly differentiated TIL from melanoma patients into cells with a less-differentiated phenotype. The methodology that we developed for generating a less-differentiated anti-tumor CD8+ T cells ex vivo may be ideal for the adoptive immunotherapy of cancer.
Asunto(s)
Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Citocinas/inmunología , Citocinas/farmacología , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Animales , Linfocitos T CD8-positivos/citología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Humanos , Interleucinas/inmunología , Interleucinas/farmacología , Linfocitos Infiltrantes de Tumor/citología , Melanoma/inmunología , Melanoma/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , FenotipoRESUMEN
Cluster of differentiation (CD)8(+) T cells exist as naive, central memory, and effector memory subsets, and any of these populations can be genetically engineered into tumor-reactive effector cells for adoptive immunotherapy. However, the optimal subset from which to derive effector CD8(+) T cells for patient treatments is controversial and understudied. We investigated human CD8(+) T cells and found that naive cells were not only the most abundant subset but also the population most capable of in vitro expansion and T-cell receptor transgene expression. Despite increased expansion, naive-derived cells displayed minimal effector differentiation, a quality associated with greater efficacy after cell infusion. Similarly, the markers of terminal differentiation, killer cell lectin-like receptor G1 and CD57, were expressed at lower levels in cells of naive origin. Finally, naive-derived effector cells expressed higher CD27 and retained longer telomeres, characteristics that suggest greater proliferative potential and that have been linked to greater efficacy in clinical trials. Thus, these data suggest that naive cells resist terminal differentiation, or "exhaustion," maintain high replicative potential, and therefore may be the superior subset for use in adoptive immunotherapy.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica/inmunología , Inmunoterapia Adoptiva/métodos , Subgrupos de Linfocitos T/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/trasplante , Diferenciación Celular/inmunología , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Citocinas/metabolismo , Citometría de Flujo , Expresión Génica , Vectores Genéticos/genética , Humanos , Retroviridae/genética , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/trasplante , Telómero/genética , Transducción Genética , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
Adoptive immunotherapy using TCR-engineered PBLs against melanocyte differentiation Ags mediates objective tumor regression but is associated with on-target toxicity. To avoid toxicity to normal tissues, we targeted cancer testis Ag (CTA) MAGE-A3, which is widely expressed in a range of epithelial malignancies but is not expressed in most normal tissues. To generate high-avidity TCRs against MAGE-A3, we employed a transgenic mouse model that expresses the human HLA-A*0201 molecule. Mice were immunized with two HLA-A*0201-restricted peptides of MAGE-A3: 112-120 (KVAELVHFL) or MAGE-A3: 271-279 (FLWGPRALV), and T cell clones were generated. MAGE-A3-specific TCR α- and ß-chains were isolated and cloned into a retroviral vector. Expression of both TCRs in human PBLs demonstrated Ag-specific reactivity against a range of melanoma and nonmelanoma tumor cells. The TCR against MAGE-A3: 112-120 was selected for further development based on superior reactivity against tumor target cells. Interestingly, peptide epitopes from MAGE-A3 and MAGE-A12 (and to a lesser extent, peptides from MAGE-A2 and MAGE-A6) were recognized by PBLs engineered to express this TCR. To further improve TCR function, single amino acid variants of the CDR3 α-chain were generated. Substitution of alanine to threonine at position 118 of the α-chain in the CDR3 region of the TCR improved its functional avidity in CD4 and CD8 cells. On the basis of these results, a clinical trial is planned in which patients bearing a variety of tumor histologies will receive autologous PBLs that have been transduced with this optimized anti-MAGE-A3 TCR.