RESUMEN
Total tumor clearance through immunotherapy is associated with a fully coordinated innate and adaptive immune response, but knowledge on the exact contribution of each immune cell subset is limited. We show that therapy-induced intratumoral CD8+ T cells recruited and skewed late-stage activated M1-like macrophages, which were critical for effective tumor control in two different murine models of cancer immunotherapy. The activated CD8+ T cells summon these macrophages into the tumor and their close vicinity via CCR5 signaling. Exposure of non-polarized macrophages to activated T cell supernatant and tumor lysate recapitulates the late-stage activated and tumoricidal phenotype in vitro. The transcriptomic signature of these macrophages is also detected in a similar macrophage population present in human tumors and coincides with clinical response to immune checkpoint inhibitors. The requirement of a functional co-operation between CD8+ T cells and effector macrophages for effective immunotherapy gives warning to combinations with broad macrophage-targeting strategies.
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Linfocitos T CD8-positivos , Inmunoterapia , Macrófagos , Animales , Linfocitos T CD8-positivos/inmunología , Inmunoterapia/métodos , Ratones , Humanos , Macrófagos/inmunología , Receptores CCR5/metabolismo , Receptores CCR5/genética , Ratones Endogámicos C57BL , Activación de Macrófagos/inmunología , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Activación de Linfocitos/inmunología , Femenino , Microambiente Tumoral/inmunologíaRESUMEN
CD3 bispecific antibody (CD3 bsAb) therapy is clinically approved for refractory hematological malignancies, but responses in solid tumors have been limited so far. One of the main hurdles in solid tumors is the lack of sufficient T-cell infiltrate. Here, we show that pre-treatment vaccination, even when composed of tumor-unrelated antigens, induces CXCR3-mediated T-cell influx in immunologically 'cold' tumor models in male mice. In the absence of CD3 bsAb, the infiltrate is confined to the tumor invasive margin, whereas subsequent CD3 bsAb administration induces infiltration of activated effector CD8 T cells into the tumor cell nests. This combination therapy installs a broadly inflamed Th1-type tumor microenvironment, resulting in effective tumor eradication. Multiple vaccination formulations, including synthetic long peptides and viruses, empower CD3 bsAb therapy. Our results imply that eliciting tumor infiltration with vaccine-induced tumor-(un)related T cells can greatly improve the efficacy of CD3 bsAbs in solid tumors.
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Anticuerpos Biespecíficos , Neoplasias , Vacunas , Masculino , Animales , Ratones , Linfocitos T , Complejo CD3 , Neoplasias/tratamiento farmacológico , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Antígenos de Neoplasias , Microambiente TumoralRESUMEN
The immune checkpoint NKG2A/CD94 is a promising target for cancer immunotherapy, and its ligand major histocompatibility complex E (MHC-E) is frequently upregulated in cancer. NKG2A/CD94-mediated inhibition of lymphocytes depends on the presence of specific leader peptides in MHC-E, but when and where they are presented in situ is unknown. We apply a nanobody specific for the Qdm/Qa-1b complex, the NKG2A/CD94 ligand in mouse, and find that presentation of Qdm peptide depends on every member of the endoplasmic reticulum-resident peptide loading complex. With a turnover rate of 30 min, the Qdm peptide reflects antigen processing capacity in real time. Remarkably, Qdm/Qa-1b complexes require inflammatory signals for surface expression in situ, despite the broad presence of Qa-1b molecules in homeostasis. Furthermore, we identify LILRB1 as a functional inhibition receptor for MHC-E in steady state. These data provide a molecular understanding of NKG2A blockade in immunotherapy and assign MHC-E as a convergent ligand for multiple immune checkpoints.
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Antígenos de Histocompatibilidad Clase I , Neoplasias , Ratones , Animales , Antígenos de Histocompatibilidad Clase I/metabolismo , Receptor Leucocitario Tipo Inmunoglobulina B1/metabolismo , Células Asesinas Naturales , Ligandos , Péptidos/metabolismo , Neoplasias/metabolismo , Subfamília C de Receptores Similares a Lectina de Células NK/metabolismoRESUMEN
The absence of T cells in the tumor microenvironment of solid tumors is a major barrier to cancer immunotherapy efficacy. Oncolytic viruses, including reovirus type 3 Dearing (Reo), can recruit CD8+ T cells to the tumor and thereby enhance the efficacy of immunotherapeutic strategies that depend on high T-cell density, such as CD3-bispecific antibody (bsAb) therapy. TGF-ß signaling might represent another barrier to effective Reo&CD3-bsAb therapy due to its immunoinhibitory characteristics. Here, we investigated the effect of TGF-ß blockade on the antitumor efficacy of Reo&CD3-bsAb therapy in the preclinical pancreatic KPC3 and colon MC38 tumor models, where TGF-ß signaling is active. TGF-ß blockade impaired tumor growth in both KPC3 and MC38 tumors. Furthermore, TGF-ß blockade did not affect reovirus replication in both models and significantly enhanced the Reo-induced T-cell influx in MC38 colon tumors. Reo administration decreased TGF-ß signaling in MC38 tumors but instead increased TGF-ß activity in KPC3 tumors, resulting in the accumulation of α-smooth muscle actin (αSMA+) fibroblasts. In KPC3 tumors, TGF-ß blockade antagonized the antitumor effect of Reo&CD3-bsAb therapy, even though T-cell influx and activity were not impaired. Moreover, genetic loss of TGF-ß signaling in CD8+ T cells had no effect on therapeutic responses. In contrast, TGF-ß blockade significantly improved therapeutic efficacy of Reo&CD3-bsAb in mice bearing MC38 colon tumors, resulting in a 100% complete response. Further understanding of the factors that determine this intertumor dichotomy is required before TGF-ß inhibition can be exploited as part of viroimmunotherapeutic combination strategies to improve their clinical benefit. Significance: Blockade of the pleiotropic molecule TGF-ß can both improve and impair the efficacy of viro-immunotherapy, depending on the tumor model. While TGF-ß blockade antagonized Reo&CD3-bsAb combination therapy in the KPC3 model for pancreatic cancer, it resulted in 100% complete responses in the MC38 colon model. Understanding factors underlying this contrast is required to guide therapeutic application.
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Neoplasias del Colon , Neoplasias Pancreáticas , Ratones , Animales , Linfocitos T CD8-positivos , Factor de Crecimiento Transformador beta , Neoplasias Pancreáticas/tratamiento farmacológico , Inmunoterapia , Microambiente TumoralRESUMEN
Introduction: Mycosis fungoides (MF), the most common type of Cutaneous T cell Lymphoma (CTCL), is characterized by an inflamed skin intermixed with proliferating malignant mature skin-homing CD4+ T cells. Detailed genomic analyses of MF skin biopsies revealed several candidate genes possibly involved in genesis of these tumors and/or potential targets for therapy. These studies showed, in addition to common loss of cell cycle regulator CDKN2A, activation of several oncogenic pathways, most prominently and consistently involving JAK/STAT signaling. SOCS1, an endogenous inhibitor of the JAK/STAT signaling pathway, was identified as a recurrently deleted gene in MF, already occurring in the earliest stages of the disease. Methods: To explore the mechanisms of MF, we create in vivo mouse models of autochthonous CTCLs and these genetically engineered mouse models (GEMMS) can also serve as valid experimental models for targeted therapy. We describe the impact of allelic deletion of Socs1 in CD4 T cells of the skin. To achieve this, we crossed inducible Cre-transgenic mice in the CD4 lineage with transgenic mice carrying floxed genes of Socs1. We first determined optimal conditions for Socs1 ablation with limited effects on circulating CD4 T-cells in blood. Next, we started time-course experiments mimicking sustained inflammation, typical in CTCL. FACS analysis of the blood was done every week. Skin biopsies were analyzed by immunocytochemical staining at the end of the experiment. Results: We found that the Socs1 knockout transgenic group had thicker epidermis of treated skin compared with the control group and had more CD3 and CD4 in the skin of the transgenic group compared to the control group. We also noted more activation of Stat3 by staining for P-Stat3 in Socs1 knockout compared to wt CD4+T cells in the skin. The results also indicated that single copy loss of Socs1 in combination with sustained inflammation is insufficient to start a phenotype resembling early stage mycosis fungoides within eight weeks in these mice. Conclusion: In sum, we developed and optimized an autochthonous murine model permitting selective knockout of Socs1 in skin infiltrating CD4 T-cells. This paves the way for more elaborate experiments to gain insight in the oncogenesis of CTCL.
RESUMEN
BACKGROUND: Many solid tumors do not respond to immunotherapy due to their immunologically cold tumor microenvironment (TME). We and others found that oncolytic viruses (OVs), including reovirus type 3 Dearing, can enhance the efficacy of immunotherapy by recruiting CD8+ T cells to the TME. A significant part of the incoming CD8+ T cells is directed toward reovirus itself, which may be detrimental to the efficacy of OVs. However, here we aim to exploit these incoming virus-specific T cells as anticancer effector cells. METHODS: We performed an in-depth characterization of the reovirus-induced T-cell response in immune-competent mice bearing pancreatic KPC3 tumors. The immunodominant CD8+ T-cell epitope of reovirus was identified using epitope prediction algorithms and peptide arrays, and the quantity and quality of reovirus-specific T cells after reovirus administration were assessed using high-dimensional flow cytometry. A synthetic long peptide (SLP)-based vaccination strategy was designed to enhance the intratumoral frequency of reovirus-specific CD8+ T cells. RESULTS: Reovirus administration did not induce tumor-specific T cells but rather induced high frequencies of reovirus-specific CD8+ T cells directed to the immunodominant epitope. Priming of reovirus-specific T cells required a low-frequent population of cross-presenting dendritic cells which was absent in Batf3-/- mice. While intratumoral and intravenous reovirus administration induced equal systemic frequencies of reovirus-specific T cells, reovirus-specific T cells were highly enriched in the TME exclusively after intratumoral administration. Here, they displayed characteristics of potent effector cells with high expression of KLRG1, suggesting they may be responsive against local reovirus-infected cells. To exploit these reovirus-specific T cells as anticancer effector cells, we designed an SLP-based vaccination strategy to induce a strong T-cell response before virotherapy. These high frequencies of circulating reovirus-specific T cells were reactivated on intratumoral reovirus administration and significantly delayed tumor growth. CONCLUSIONS: These findings provide proof of concept that OV-specific T cells, despite not being tumor-specific, can be exploited as potent effector cells for anticancer treatment when primed before virotherapy. This is an attractive strategy for low-immunogenic tumors lacking tumor-specific T cells.
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Neoplasias , Viroterapia Oncolítica , Virus Oncolíticos , Animales , Linfocitos T CD8-positivos , Inmunoterapia , Ratones , Viroterapia Oncolítica/métodos , Microambiente TumoralRESUMEN
The NKG2A/HLA-E axis is an immune checkpoint that suppresses immune effector activity in the tumor microenvironment. In mice, the ligand for the NKG2A/CD94 inhibitory receptor is the nonclassical MHC molecule Qa-1b, the HLA-E ortholog, which presents the peptide AMAPRTLLL, referred to as Qdm (for Qa-1 determinant modifier). This dominant peptide is derived from the leader sequences of murine classical MHC class I encoded by the H-2D and -L loci. To broaden our understanding of Qa-1b/Qdm peptide complex biology and its tumor protective role, we identified a TCR-like Ab from a single domain VHH library using yeast surface display. The TCR-like Ab (EXX-1) binds only to the Qa-1b/Qdm peptide complex and not to Qa-1b alone or Qa-1b loaded with control peptides. Conversely, currently available Abs to Qa-1b bind independent of peptide loaded. Flow cytometric results revealed that EXX-1 selectively bound to Qa-1b/Qdm-positive B16F10, RMA, and TC-1 mouse tumor cells but only after pretreatment with IFN-γ; no binding was observed following genetic knockdown of Qa-1b or Qdm peptide. Furthermore, EXX-1 Ab blockade promoted NK cell-mediated tumor cell lysis in vitro. Our findings show that EXX-1 has exquisite binding specificity for the Qa-1b/Qdm peptide complex, making it a valuable research tool for further investigation of the Qa-1b/Qdm peptide complex expression and regulation in healthy and diseased cells and for evaluation as an immune checkpoint blocking Ab in syngeneic mouse tumor models.
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Antígenos de Histocompatibilidad Clase I , Células Asesinas Naturales , Animales , Anticuerpos/metabolismo , Ratones , Subfamília C de Receptores Similares a Lectina de Células NK/metabolismo , Péptidos , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) has the characteristics of high-density desmoplastic stroma, a distinctive immunosuppressive microenvironment and is profoundly resistant to all forms of chemotherapy and immunotherapy, leading to a 5-year survival rate of 9%. Our study aims to add novel small molecule therapeutics for the treatment of PDAC. DESIGN: We have studied whether TAK-981, a novel highly selective and potent small molecule inhibitor of the small ubiquitin like modifier (SUMO) activating enzyme E1 could be used to treat a preclinical syngeneic PDAC mouse model and we have studied the mode of action of TAK-981. RESULTS: We found that SUMOylation, a reversible post-translational modification required for cell cycle progression, is increased in PDAC patient samples compared with normal pancreatic tissue. TAK-981 decreased SUMOylation in PDAC cells at the nanomolar range, thereby causing a G2/M cell cycle arrest, mitotic failure and chromosomal segregation defects. TAK-981 efficiently limited tumour burden in the KPC3 syngeneic mouse model without evidence of systemic toxicity. In vivo treatment with TAK-981 enhanced the proportions of activated CD8 T cells and natural killer (NK) cells but transiently decreased B cell numbers in tumour, peripheral blood, spleen and lymph nodes. Single cell RNA sequencing revealed activation of the interferon response on TAK-981 treatment in lymphocytes including T, B and NK cells. TAK-981 treatment of CD8 T cells ex vivo induced activation of STAT1 and interferon target genes. CONCLUSION: Our findings indicate that pharmacological inhibition of the SUMO pathway represents a potential strategy to target PDAC via a dual mechanism: inhibiting cancer cell cycle progression and activating anti-tumour immunity by inducing interferon signalling.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Carcinoma Ductal Pancreático/patología , Ciclo Celular , Proliferación Celular , Interferones , Células Asesinas Naturales , Ratones , Neoplasias Pancreáticas/patología , Sumoilación , Microambiente Tumoral , Enzimas Activadoras de Ubiquitina , Ubiquitinas/metabolismo , Neoplasias PancreáticasRESUMEN
The surface inhibitory receptor NKG2A forms heterodimers with the invariant CD94 chain and is expressed on a subset of activated CD8 T cells. As antibodies to block NKG2A are currently tested in several efficacy trials for different tumor indications, it is important to characterize the NKG2A+ CD8 T cell population in the context of other inhibitory receptors. Here we used a well-controlled culture system to study the kinetics of inhibitory receptor expression. Naïve mouse CD8 T cells were synchronously and repeatedly activated by artificial antigen presenting cells in the presence of the homeostatic cytokine IL-7. The results revealed NKG2A as a late inhibitory receptor, expressed after repeated cognate antigen stimulations. In contrast, the expression of PD-1, TIGIT and LAG-3 was rapidly induced, hours after first contact and subsequently down regulated during each resting phase. This late, but stable expression kinetics of NKG2A was most similar to that of TIM-3 and CD39. Importantly, single-cell transcriptomics of human tumor-infiltrating lymphocytes (TILs) showed indeed that these receptors were often coexpressed by the same CD8 T cell cluster. Furthermore, NKG2A expression was associated with cell division and was promoted by TGF-ß in vitro, although TGF-ß signaling was not necessary in a mouse tumor model in vivo. In summary, our data show that PD-1 reflects recent TCR triggering, but that NKG2A is induced after repeated antigen stimulations and represents a late inhibitory receptor. Together with TIM-3 and CD39, NKG2A might thus mark actively dividing tumor-specific TILs.
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Proteínas de Punto de Control Inmunitario/fisiología , Subfamília C de Receptores Similares a Lectina de Células NK/fisiología , Animales , Antígenos CD/fisiología , Linfocitos T CD8-positivos/inmunología , División Celular , Receptor 2 Celular del Virus de la Hepatitis A/fisiología , Humanos , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T/fisiología , Receptores Inmunológicos/fisiología , Factor de Crecimiento Transformador beta/farmacología , Microambiente Tumoral , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
BACKGROUND: T-cell mediated immunotherapy brought clinical success for many cancer patients. Nonetheless, downregulation of MHC class I antigen presentation, frequently occurring in solid cancers, limits the efficacy of these therapies. Unraveling the mechanisms underlying this type of immune escape is therefore of great importance. We here investigated the immunological effects of metabolic stress in cancer cells as a result of nutrient deprivation. METHODS: TC1 and B16F10 tumor cell lines were cultured under oxygen- and glucose-deprivation conditions that mimicked the tumor microenvironment of solid tumors. Presentation of peptide antigens by MHC class I molecules was measured by flow cytometry and via activation of tumor-specific CD8 T cell clones. The proficiency of the IFNy-STAT1 pathway was investigated by Western blots on phosphorylated proteins, transfection of constitutive active STAT1 constructs and qPCR of downstream targets. Kinase inhibitors for PI3K were used to examine its role in IFNy receptor signal transduction. RESULTS: Combination of oxygen- and glucose-deprivation resulted in decreased presentation of MHC class I antigens on cancer cells, even in the presence of the stimulatory cytokine IFNy. This unresponsiveness to IFNy was the result of failure to phosphorylate the signal transducer STAT1. Forced expression of constitutive active STAT1 fully rescued the MHC class I presentation. Furthermore, oxygen- and glucose-deprivation increased PI3K activity in tumor cells. Pharmacological inhibition of this pathway not only restored signal transduction through IFNy-STAT1 but also improved MHC class I presentation. Importantly, PI3K inhibitors also rendered tumor cells sensitive for recognition by CD8 T cells in culture conditions of metabolic stress. CONCLUSIONS: These data revealed a strong impact of metabolic stress on the presentation of tumor antigens by MHC class I and suggest that this type of tumor escape takes place at hypoxic areas even during times of active T cell immunity and IFNy release.
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Melanoma Experimental/inmunología , Melanoma Experimental/metabolismo , Fosfatidilinositol 3-Quinasas/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores de Interferón/antagonistas & inhibidores , Animales , Presentación de Antígeno , Hipoxia de la Célula , Línea Celular Tumoral , Glucosa/deficiencia , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interferón gamma/farmacología , Ratones , Ratones Endogámicos C57BL , Fosforilación , Receptores de Interferón/inmunología , Receptores de Interferón/metabolismo , Factor de Transcripción STAT1/inmunología , Factor de Transcripción STAT1/metabolismo , Transducción de Señal , Estrés Fisiológico/inmunología , Escape del Tumor , Receptor de Interferón gammaRESUMEN
Immunotherapy of cancer with CD3-targeting bispecific antibodies (CD3 bsAb) is a fast developing field, and multiple tumor-associated antigens (TAA) are evaluated for hematologic and solid malignancies. The efficacy of these CD3 bsAb is usually examined in xenograft mouse tumor models with human T cells or in genetically engineered mouse models, where human TAA are introduced. These models often fail to fully recapitulate the natural tumor environment, especially for solid cancers, because of interspecies differences. Here, we investigated the systemic and intratumoral effects of a mouse CD3 bsAb in a fully immune-competent mouse melanoma model. Systemic administration of 0.5 mg/kg antibody induced a brief overall T-cell activation that was selectively sustained in the tumor microenvironment for several days. A fast subsequent influx of inflammatory macrophages into the tumor microenvironment was observed, followed by an increase in the number of CD4+ and CD8+ T cells. Although the capacity to directly kill melanoma cells in vitro was very modest, optimal tumor elimination was observed in vivo, even in the absence of CD8+ T cells, implying a redundancy in T-cell subsets for therapeutic efficacy. Finally, we took advantage of the full immune competence of our mouse model and tested immune memory induction. Despite a strong initial immunity against melanoma, treatment with the CD3 bsAb did not install protective memory responses. The observed mechanisms of action revealed in this immune-competent mouse model might form a rational basis for combinatorial approaches.
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Anticuerpos Biespecíficos/administración & dosificación , Complejo CD3/antagonistas & inhibidores , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Melanoma/tratamiento farmacológico , Animales , Anticuerpos Biespecíficos/farmacología , Antígenos de Neoplasias/genética , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Línea Celular Tumoral , Femenino , Humanos , Memoria Inmunológica , Activación de Linfocitos , Melanoma/inmunología , Ratones , Microambiente Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A recurrent theme in viral immune evasion is the sabotage of MHC-I antigen presentation, which brings virus the concomitant issue of 'missing-self' recognition by NK cells that use inhibitory receptors to detect surface MHC-I proteins. Here, we report that rodent herpesvirus Peru (RHVP) encodes a Qa-1 like protein (pQa-1) via RNA splicing to counteract NK activation. While pQa-1 surface expression is stabilized by the same canonical peptides presented by murine Qa-1, pQa-1 is GPI-anchored and resistant to the activity of RHVP pK3, a ubiquitin ligase that targets MHC-I for degradation. pQa-1 tetramer staining indicates that it recognizes CD94/NKG2A receptors. Consistently, pQa-1 selectively inhibits NKG2A+ NK cells and expression of pQa-1 can protect tumor cells from NK control in vivo. Collectively, these findings reveal an innovative NK evasion strategy wherein RHVP encodes a modified Qa-1 mimic refractory to MHC-I sabotage and capable of specifically engaging inhibitory receptors to circumvent NK activation.
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Citotoxicidad Inmunológica/inmunología , Herpesviridae/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Células Asesinas Naturales/inmunología , Subfamília C de Receptores Similares a Lectina de Células NK/inmunología , Subfamília D de Receptores Similares a Lectina de las Células NK/inmunología , Secuencia de Aminoácidos , Animales , Presentación de Antígeno/inmunología , Secuencia de Bases , Citotoxicidad Inmunológica/genética , Células HEK293 , Herpesviridae/genética , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Células Asesinas Naturales/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Imitación Molecular/genética , Imitación Molecular/inmunología , Subfamília C de Receptores Similares a Lectina de Células NK/genética , Subfamília C de Receptores Similares a Lectina de Células NK/metabolismo , Subfamília D de Receptores Similares a Lectina de las Células NK/genética , Subfamília D de Receptores Similares a Lectina de las Células NK/metabolismo , Unión Proteica/inmunología , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
Tumor-infiltrating CD8 T cells were found to frequently express the inhibitory receptor NKG2A, particularly in immune-reactive environments and after therapeutic cancer vaccination. High-dimensional cluster analysis demonstrated that NKG2A marks a unique immune effector subset preferentially co-expressing the tissue-resident CD103 molecule, but not immune checkpoint inhibitors. To examine whether NKG2A represented an adaptive resistance mechanism to cancer vaccination, we blocked the receptor with an antibody and knocked out its ligand Qa-1b, the conserved ortholog of HLA-E, in four mouse tumor models. The impact of therapeutic vaccines was greatly potentiated by disruption of the NKG2A/Qa-1b axis even in a PD-1 refractory mouse model. NKG2A blockade therapy operated through CD8 T cells, but not NK cells. These findings indicate that NKG2A-blocking antibodies might improve clinical responses to therapeutic cancer vaccines.
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Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer , Inmunidad Celular , Subfamília C de Receptores Similares a Lectina de Células NK , Proteínas de Neoplasias , Neoplasias Experimentales , Vacunación , Animales , Anticuerpos Antineoplásicos/inmunología , Antígenos CD/inmunología , Linfocitos T CD8-positivos/patología , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/farmacología , Línea Celular Tumoral , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Cadenas alfa de Integrinas/inmunología , Ratones , Subfamília C de Receptores Similares a Lectina de Células NK/antagonistas & inhibidores , Subfamília C de Receptores Similares a Lectina de Células NK/inmunología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/inmunología , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Neoplasias Experimentales/terapia , Antígenos HLA-ERESUMEN
Therapy with tumor-specific Abs is common in the clinic but has limited success against solid malignancies. We aimed at improving the efficacy of this therapy by combining a tumor-specific Ab with immune-activating compounds. In this study, we demonstrate in the aggressive B16F10 mouse melanoma model that concomitant application of the anti-TRP1 Ab (clone TA99) with TLR3-7/8 or -9 ligands, and IL-2 strongly enhanced tumor control in a therapeutic setting. Depletion of NK cells, macrophages, or CD8+ T cells all mitigated the therapeutic response, showing a coordinated immune rejection by innate and adaptive immune cells. FcγRs were essential for the therapeutic effect, with a dominant role for FcγRI and a minor role for FcγRIII and FcγRIV. FcγR expression on NK cells and granulocytes was dispensable, indicating that other tumoricidal functions of NK cells were involved and implicating that FcγRI, -III, and -IV exerted their activity on macrophages. Indeed, F4/80+Ly-6C+ inflammatory macrophages in the tumor microenvironment displayed high levels of these receptors. Whereas administration of the anti-TRP1 Ab alone reduced the frequency of these macrophages, the combination with a TLR agonist retained these cells in the tumor microenvironment. Thus, the addition of innate stimulatory compounds, such as TLR ligands, to tumor-specific Ab therapy could greatly enhance its efficacy in solid cancers via optimal exploitation of FcγRs.
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Anticuerpos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Inmunoterapia/métodos , Interleucina-2/uso terapéutico , Células Asesinas Naturales/inmunología , Macrófagos/inmunología , Melanoma/terapia , Receptores de IgG/metabolismo , Animales , Antígenos de Neoplasias/inmunología , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunización , Masculino , Melanoma/inmunología , Melanoma Experimental , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidorreductasas/inmunología , Receptores de IgG/genética , Receptores Toll-Like/agonistasRESUMEN
Human cancers frequently display defects in Ag processing and presentation allowing for immune evasion, and they therefore constitute a significant challenge for T cell-based immunotherapy. We have previously demonstrated that the antigenicity of tumor-associated Ags can be significantly enhanced through unconventional residue modifications as a novel tool for MHC class I (MHC-I)-based immunotherapy approaches. We have also previously identified a novel category of cancer neo-epitopes, that is, T cell epitopes associated with impaired peptide processing (TEIPP), that are selectively presented by MHC-I on cells lacking the peptide transporter TAP. In this study, we demonstrate that substitution of the nonanchoring position 3 into a proline residue of the first identified TEIPP peptide, the murine Trh4, results in significantly enhanced recognition by antitumor CTLs toward the wild-type epitope. Although higher immunogenicity has in most cases been associated with increased MHC/peptide complex stability, our results demonstrate that the overall stability of H-2Db in complex with the highly immunogenic altered peptide ligand Trh4-p3P is significantly reduced compared with wild-type H-2Db/Trh4. Comparison of the crystal structures of the H-2Db/Trh4-p3P and H-2Db/Trh4 complexes revealed that the conformation of the nonconventional methionine anchor residue p5M is altered, deleting its capacity to form adequate sulfur-π interactions with H-2Db residues, thus reducing the overall longevity of the complex. Collectively, our results indicate that vaccination with Thr4-p3P significantly enhances T cell recognition of targets presenting the wild-type TEIPP epitope and that higher immunogenicity is not necessarily directly related to MHC/peptide complex stability, opening for the possibility to design novel peptide vaccines with reduced MHC/peptide complex stability.
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Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/química , Vacunas contra el Cáncer/inmunología , Proteínas de la Membrana/química , Proteínas de la Membrana/inmunología , Animales , Antígenos de Neoplasias/química , Epítopos de Linfocito T/inmunología , Antígeno de Histocompatibilidad H-2D/química , Antígeno de Histocompatibilidad H-2D/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Péptidos , Prolina , Estabilidad ProteicaRESUMEN
The HLA-E homolog in the mouse (Qa-1b) is a conserved MHC class Ib molecule presenting monomorphic peptides to germline-encoded natural killer receptor CD94/NKG2A. Previously, we demonstrated the replacement of this canonical peptide by a diverse peptidome upon deficiency of the TAP peptide transporter. Analysis of this Qa-1b-restricted T cell repertoire against these non-mutated neoantigens revealed characteristics of conventional hypervariable CD8+ T cells, but also of invariant T cell receptor (TCR)αß T cells. A shared TCR Vα chain was used by this subset in combination with a variety of Vß chains. The TCRs target peptide ligands that are conserved between mouse and man, like the identified peptide derived from the transcriptional cofactor Med15. The thymus selection was studied in a TCR-transgenic mouse and emerging naïve CD8+ T cells displayed a slightly activated phenotype, as witnessed by higher CD122 and Ly6C expression. Moreover, the Qa-1b protein was dispensable for thymus selection. Importantly, no self-reactivity was observed as reported for other MHC class Ib-restricted subsets. Naïve Qa-1b restricted T cells expanded, contracted, and formed memory cells in vivo upon peptide vaccination in a similar manner as conventional CD8+ T cells. Based on these data, the Qa-1b restricted T cell subset might be positioned closest to conventional CD8+ T cells of all MHC class Ib populations.
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Proteínas Portadoras/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Lipoproteínas/metabolismo , Péptidos/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Transactivadores/metabolismo , Animales , Transporte Biológico , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Proteínas Portadoras/genética , Línea Celular , Secuencia Conservada , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Ligandos , Lipoproteínas/genética , Activación de Linfocitos , Ratones , Ratones Noqueados , Ratones Transgénicos , Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Timo/inmunología , Timo/metabolismo , Transactivadores/genéticaRESUMEN
Cancers frequently evade immune-recognition by lowering peptide:MHC-I complexes on their cell surface. Limited peptide supply due to TAP-deficiency results in such MHC-Ilow immune-escape variants. Previously, we reported on a category of TAP-independent self-peptides, called TEIPP, with selective presentation by these tumors. Here we demonstrate that in contrast to T cells specific for conventional tumor antigens, TEIPP-directed T cells remain naïve in mice bearing immune-escaped tumors. This unaffected state was caused by low levels of MHC-I on the tumors and the failure to cross-present low levels of antigenic protein by host APCs. Importantly, increased levels of MHC-I, antigen or co-stimulation resulted in potent activation of TEIPP-specific T cells via direct presentation. Genetic knockdown by CRISPR/Cas9 technology of the relevant MHC-I allele in tumor cells indeed abrogated T cell activation. Vaccine-mediated priming of TEIPP-specific T cells induced efficient homing to MHC-Ilow tumors and subsequently protected mice against outgrowth of their MHC-Ilow tumor. Thus, our data open up the search of TEIPP-specific T cells in cancer patients to explore their application against MHC-Ilow tumor cells.
RESUMEN
One of the next challenges in cancer immunotherapy is the resistance of tumors to T-cell-based treatments through loss of MHC class I. Here, we show that under these circumstances, the Toll-like receptor (TLR)-7/8 ligand imiquimod, but not the TLR3 ligand poly I:C or TLR9 ligand CpG, mediated an effective antitumor response. The rejection of these immune-escaped cancers was mediated by NK cells and CD4+ T cells, whereas activated CD8+ T cells were dispensable. Application of the innate immune stimulator at a distant site activated NK cells and thereby elicited tumor-specific T-cell responses in tumor-bearing mice. Mechanistically, imiquimod activated NK cells to kill tumor cells, resulting in release of tumor antigens and induction of tumor-specific CD4+ T cells. These T helper cells provoked a strong induction of CXCL9 and CXCL10 in the tumor environment. Simultaneously, imiquimod induced the expression of the cognate chemokine receptor CXCR3 on peripheral lymphocytes. This ignited intratumoral CD4+ T-cell infiltration and accumulation, which was critical for tumor rejection; CXCR3 blocking antibodies mitigated the clinical response. In the effector phase, NK cell recruitment to tumors and their activation depended on CD4+ T cells. Together, we have uncovered a potent immune axis of tumor-specific CD4+ T cells and NK cells that eliminates escaped MHC-Ilow tumors. Cancer Immunol Res; 5(8); 642-53. ©2017 AACR.
Asunto(s)
Células Asesinas Naturales/inmunología , Complejo Mayor de Histocompatibilidad/inmunología , Glicoproteínas de Membrana/inmunología , Neoplasias/inmunología , Receptor Toll-Like 7/inmunología , Microambiente Tumoral/inmunología , Aminoquinolinas/administración & dosificación , Aminoquinolinas/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Quimiocina CXCL10/inmunología , Quimiocina CXCL9/inmunología , Humanos , Imiquimod , Activación de Linfocitos/inmunología , Glicoproteínas de Membrana/agonistas , Ratones , Neoplasias/patología , Neoplasias/terapia , Receptores CXCR3/inmunología , Receptor Toll-Like 3/inmunología , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 9/inmunologíaRESUMEN
Tumor cells frequently escape from CD8+ T cell recognition by abrogating MHC-I antigen presentation. Deficiency in processing components, like the transporter associated with antigen processing (TAP), results in strongly decreased surface display of peptide/MHC-I complexes. We previously identified a class of hidden self-antigens known as T cell epitopes associated with impaired peptide processing (TEIPP), which emerge on tumor cells with such processing defects. In the present study, we analyzed thymus selection and peripheral behavior of T cells with specificity for the prototypic TEIPP antigen, the "self" TRH4 peptide/Db complex. TEIPP T cells were efficiently selected in the thymus, egressed with a naive phenotype, and could be exploited for immunotherapy against immune-escaped, TAP-deficient tumor cells expressing low levels of MHC-I (MHC-Ilo). In contrast, overt thymus deletion and functionally impaired TEIPP T cells were observed in mice deficient for TAP1 due to TEIPP antigen presentation on all body cells in these mice. Our results strongly support the concept that TEIPPs derive from ubiquitous, nonmutated self-antigens and constitute a class of immunogenic neoantigens that are unmasked during tumor immune evasion. These data suggest that TEIPP-specific CD8+ T cells are promising candidates in the treatment of tumors that have escaped from conventional immunotherapies.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/inmunología , Presentación de Antígeno , Antígenos de Neoplasias/inmunología , Autoantígenos/inmunología , Epítopos de Linfocito T/inmunología , Neoplasias Experimentales/inmunología , Péptidos/inmunología , Escape del Tumor , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Animales , Antígenos de Neoplasias/genética , Autoantígenos/genética , Línea Celular Tumoral , Epítopos de Linfocito T/genética , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Ratones , Ratones Noqueados , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Péptidos/genéticaRESUMEN
Abundant macrophage infiltration of solid cancers commonly correlates with poor prognosis. Tumor-promoting functions of macrophages include angiogenesis, metastasis formation, and suppression of Th1-type immune responses. Here, we show that successful treatment of cervical carcinoma in mouse models with synthetic long peptide (SLP) vaccines induced influx of cytokine-producing CD8 T cells that strongly altered the numbers and phenotype of intratumoral macrophages. On the basis of the expression of CD11b, CD11c, F4/80, Ly6C, Ly6G, and MHC II, we identified four myeloid subpopulations that increased in numbers from 2.0-fold to 8.7-fold in regressing tumors. These changes of the intratumoral myeloid composition coincided with macrophage recruitment by chemokines, including CCL2 and CCL5, and were completely dependent on a vaccine-induced influx of tumor-specific CD8 T cells. CD4 T cells were dispensable. Incubation of tumor cells with T cell-derived IFNγ and TNFα recapitulated the chemokine profile observed in vivo, confirming the capacity of antitumor CD8 T cells to mediate macrophage infiltration of tumors. Strikingly, complete regressions of large established tumors depended on the tumor-infiltrating macrophages that were induced by this immunotherapy, because a small-molecule drug inhibitor targeting CSF-1R diminished the number of intratumoral macrophages and abrogated the complete remissions. Survival rates after therapeutic SLP vaccination deteriorated in the presence of CSF-1R blockers. Together, these results show that therapeutic peptide vaccination could induce cytokine-producing T cells with strong macrophage-skewing capacity necessary for tumor shrinkage, and suggest that the development of macrophage-polarizing, rather than macrophage-depleting, agents is warranted.