RÉSUMÉ
New strategies that augment T cell responses are required to broaden the therapeutic arsenal against cancer. CD96, TIGIT, and CD226 are receptors that bind to a communal ligand, CD155, and transduce either inhibitory or activating signals. The function of TIGIT and CD226 is established, whereas the role of CD96 remains ambiguous. Using a panel of engineered antibodies, we discovered that the T cell stimulatory activity of anti-CD96 antibodies requires antibody cross-linking and is potentiated by Fcγ receptors. Thus, soluble "Fc silent" anti-CD96 antibodies failed to stimulate human T cells, whereas the same antibodies were stimulatory after coating onto plastic surfaces. Remarkably, the activity of soluble anti-CD96 antibodies was reinstated by engineering the Fc domain to a human IgG1 isotype, and it was dependent on antibody trans-cross-linking by FcγRI. In contrast, neither human IgG2 nor variants with increased Fcγ receptor IIB binding possessed stimulatory activity. Anti-CD96 antibodies acted directly on T cells and augmented gene expression networks associated with T cell activation, leading to proliferation, cytokine secretion, and resistance to Treg suppression. Furthermore, CD96 expression correlated with survival in HPV+ head and neck squamous cell carcinoma, and its cross-linking activated tumor-infiltrating T cells, thus highlighting the potential of anti-CD96 antibodies in cancer immunotherapy.
Sujet(s)
Cellules tueuses naturelles , Tumeurs , Antigènes CD/métabolisme , Cytokines/métabolisme , Humains , Immunoglobuline G/métabolisme , Ligands , Matières plastiques/métabolisme , Récepteurs du fragment Fc des IgG/métabolisme , Récepteurs immunologiques/métabolismeRÉSUMÉ
Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for distant metastasis and is characterised by the accumulation of cancer-associated fibroblasts in the stroma. We investigated whether the epithelial to mesenchymal transition status of CRC cells influences fibroblast phenotype, with a focus on the transfer of extracellular vesicles (EVs), as a controlled means of cell-cell communication. Epithelial CRC EVs suppressed TGF-ß-driven myofibroblast differentiation, whereas mesenchymal CRC EVs did not. This was driven by miR-200 (miR-200a/b/c, -141), which was enriched in epithelial CRC EVs and transferred to recipient fibroblasts. Ectopic miR-200 expression or ZEB1 knockdown, in fibroblasts, similarly suppressed myofibroblast differentiation. Supporting these findings, there was a strong negative correlation between miR-200 and myofibroblastic markers in a cohort of CRC patients in the TCGA dataset. This was replicated in mice, by co-injecting epithelial or mesenchymal CRC cells with fibroblasts and analysing stromal markers of myofibroblastic phenotype. Fibroblasts from epithelial tumours contained more miR-200 and expressed less ACTA2 and FN1 than those from mesenchymal tumours. As such, these data provide a new mechanism for the development of fibroblast heterogeneity in CRC, through EV-mediated transfer of miRNAs, and provide an explanation as to why CRC tumours with greater metastatic potential are CAF rich.
Sujet(s)
Tumeurs colorectales , Transition épithélio-mésenchymateuse , Vésicules extracellulaires , microARN , Animaux , Tumeurs colorectales/génétique , Transition épithélio-mésenchymateuse/génétique , Vésicules extracellulaires/génétique , Vésicules extracellulaires/métabolisme , Fibroblastes/métabolisme , Humains , Souris , microARN/génétique , microARN/métabolisme , PhénotypeRÉSUMÉ
BACKGROUND: Hypoxia is a hallmark of the tumor microenvironment (TME) and in addition to altering metabolism in cancer cells, it transforms tumor-associated stromal cells. Within the tumor stromal cell compartment, tumor-associated macrophages (TAMs) provide potent pro-tumoral support. However, TAMs can also be harnessed to destroy tumor cells by monoclonal antibody (mAb) immunotherapy, through antibody dependent cellular phagocytosis (ADCP). This is mediated via antibody-binding activating Fc gamma receptors (FcγR) and impaired by the single inhibitory FcγR, FcγRIIb. METHODS: We applied a multi-OMIC approach coupled with in vitro functional assays and murine tumor models to assess the effects of hypoxia inducible factor (HIF) activation on mAb mediated depletion of human and murine cancer cells. For mechanistic assessments, siRNA-mediated gene silencing, Western blotting and chromatin immune precipitation were utilized to assess the impact of identified regulators on FCGR2B gene transcription. RESULTS: We report that TAMs are FcγRIIbbright relative to healthy tissue counterparts and under hypoxic conditions, mononuclear phagocytes markedly upregulate FcγRIIb. This enhanced FcγRIIb expression is transcriptionally driven through HIFs and Activator protein 1 (AP-1). Importantly, this phenotype reduces the ability of macrophages to eliminate anti-CD20 monoclonal antibody (mAb) opsonized human chronic lymphocytic leukemia cells in vitro and EL4 lymphoma cells in vivo in human FcγRIIb+/+ transgenic mice. Furthermore, post-HIF activation, mAb mediated blockade of FcγRIIb can partially restore phagocytic function in human monocytes. CONCLUSION: Our findings provide a detailed molecular and cellular basis for hypoxia driven resistance to antitumor mAb immunotherapy, unveiling a hitherto unexplored aspect of the TME. These findings provide a mechanistic rationale for the modulation of FcγRIIb expression or its blockade as a promising strategy to enhance approved and novel mAb immunotherapies.
Sujet(s)
Leucémie chronique lymphocytaire à cellules B , Récepteurs du fragment Fc des IgG , Animaux , Anticorps monoclonaux/pharmacologie , Humains , Hypoxie/métabolisme , Immunothérapie , Leucémie chronique lymphocytaire à cellules B/métabolisme , Macrophages/métabolisme , Souris , Récepteurs du fragment Fc des IgG/génétique , Récepteurs du fragment Fc des IgG/métabolisme , Microenvironnement tumoralRÉSUMÉ
Despite advances in identifying the key immunoregulatory roles of many of the human leukocyte immunoglobulin-like receptor (LILR) family members, the function of the inhibitory molecule LILRB3 (ILT5, CD85a, LIR3) remains unclear. Studies indicate a predominant myeloid expression; however, high homology within the LILR family and a relative paucity of reagents have hindered progress toward identifying the function of this receptor. To investigate its function and potential immunomodulatory capacity, a panel of LILRB3-specific monoclonal antibodies (mAbs) was generated. LILRB3-specific mAbs bound to discrete epitopes in Ig-like domain 2 or 4. LILRB3 ligation on primary human monocytes by an agonistic mAb resulted in phenotypic and functional changes, leading to potent inhibition of immune responses in vitro, including significant reduction in T cell proliferation. Importantly, agonizing LILRB3 in humanized mice induced tolerance and permitted efficient engraftment of allogeneic cells. Our findings reveal powerful immunosuppressive functions of LILRB3 and identify it as an important myeloid checkpoint receptor.
Sujet(s)
Antigènes CD/génétique , Épitopes/immunologie , Protéines de points de contrôle immunitaires/génétique , Tolérance immunitaire , Lymphomes/génétique , Monocytes/immunologie , Récepteurs immunologiques/génétique , Animaux , Anticorps monoclonaux/biosynthèse , Anticorps monoclonaux/métabolisme , Antigènes CD/immunologie , Lignée cellulaire tumorale , Prolifération cellulaire , Cartographie épitopique , Épitopes/composition chimique , Analyse de profil d'expression de gènes , Régulation de l'expression des gènes , Hétérogreffes , Humains , Protéines de points de contrôle immunitaires/immunologie , Lymphomes/immunologie , Lymphomes/mortalité , Lymphomes/anatomopathologie , Souris , Monocytes/cytologie , Banque de peptides , Culture de cellules primaires , Récepteurs immunologiques/agonistes , Récepteurs immunologiques/immunologie , Analyse de survie , Lymphocytes T/cytologie , Lymphocytes T/immunologie , Transplantation homologueRÉSUMÉ
Therapy-resistant microenvironments represent a major barrier toward effective elimination of disseminated cancer. Many hematologic and solid tumors are resistant to therapeutic antibodies in the bone marrow (BM), but not in the periphery (e.g., spleen). We previously showed that cyclophosphamide (CTX) sensitizes the BM niche to antibody therapeutics. Here, we show that (i) BM resistance was induced not only by the tumor but also by the intrinsic BM microenvironment; (ii) CTX treatment overcame both intrinsic and extrinsic resistance mechanisms by augmenting macrophage activation and phagocytosis, including significant upregulation of activating Fcγ receptors (FcγRIII and FcγRIV) and downregulation of the inhibitory receptor, FcγRIIB; and (iii) CTX synergized with cetuximab (anti-EGFR) and trastuzumab (anti-Her2) in eliminating metastatic breast cancer in the BM of humanized mice. These findings provide insights into the mechanisms by which CTX synergizes with antibody therapeutics in resistant niche-specific organs and its applicability in treating BM-resident tumors.
Sujet(s)
Antinéoplasiques immunologiques/pharmacologie , Moelle osseuse/immunologie , Cyclophosphamide/pharmacologie , Résistance aux médicaments antinéoplasiques/effets des médicaments et des substances chimiques , Macrophages/effets des médicaments et des substances chimiques , Récepteurs du fragment Fc des IgG/génétique , Animaux , Antinéoplasiques immunologiques/immunologie , Antinéoplasiques immunologiques/usage thérapeutique , Moelle osseuse/effets des médicaments et des substances chimiques , Moelle osseuse/anatomopathologie , Cyclophosphamide/immunologie , Cyclophosphamide/usage thérapeutique , Résistance aux médicaments antinéoplasiques/immunologie , Régulation de l'expression des gènes/effets des médicaments et des substances chimiques , Humains , Immunothérapie , Activation des macrophages/effets des médicaments et des substances chimiques , Macrophages/immunologie , Macrophages/métabolisme , Souris , Tumeurs expérimentales/immunologie , Tumeurs expérimentales/thérapie , Récepteurs du fragment Fc des IgG/métabolisme , Microenvironnement tumoral/effets des médicaments et des substances chimiques , Microenvironnement tumoral/immunologieRÉSUMÉ
Purpose: PD-1 checkpoint blockade has revolutionized the field of cancer immunotherapy, yet the frequency of responding patients is limited by inadequate T-cell priming secondary to a paucity of activatory dendritic cells (DC). DC signals can be bypassed by CD27 agonists, and we therefore investigated if the effectiveness of anti-PD-1/L1 could be improved by combining with agonist anti-CD27 monoclonal antibodies (mAb).Experimental Design: The efficacy of PD-1/L1 blockade or agonist anti-CD27 mAb was compared with a dual-therapy approach in multiple tumor models. Global transcriptional profiling and flow cytometry analysis were used to delineate mechanisms underpinning the observed synergy.Results: PD-1/PD-L1 blockade and agonist anti-CD27 mAb synergize for increased CD8+ T-cell expansion and effector function, exemplified by enhanced IFNγ, TNFα, granzyme B, and T-bet. Transcriptome analysis of CD8+ T cells revealed that combination therapy triggered a convergent program largely driven by IL2 and Myc. However, division of labor was also apparent such that anti-PD-1/L1 activates a cytotoxicity-gene expression program whereas anti-CD27 preferentially augments proliferation. In tumor models, either dependent on endogenous CD8+ T cells or adoptive transfer of transgenic T cells, anti-CD27 mAb synergized with PD-1/L1 blockade for antitumor immunity. Finally, we show that a clinically relevant anti-human CD27 mAb, varlilumab, similarly synergizes with PD-L1 blockade for protection against lymphoma in human-CD27 transgenic mice.Conclusions: Our findings suggest that suboptimal T-cell invigoration in cancer patients undergoing treatment with PD-1 checkpoint blockers will be improved by dual PD-1 blockade and CD27 agonism and provide mechanistic insight into how these approaches cooperate for CD8+ T-cell activation. Clin Cancer Res; 24(10); 2383-94. ©2018 AACR.
Sujet(s)
Lymphocytes T CD8+/immunologie , Lymphocytes T CD8+/métabolisme , Tumeurs/génétique , Tumeurs/immunologie , Récepteur-1 de mort cellulaire programmée/antagonistes et inhibiteurs , Transcriptome , Antigènes CD27/agonistes , Transfert adoptif , Animaux , Antinéoplasiques immunologiques/pharmacologie , Antinéoplasiques immunologiques/usage thérapeutique , Cytotoxicité immunologique , Modèles animaux de maladie humaine , Analyse de profil d'expression de gènes , Humains , Immunomodulation/effets des médicaments et des substances chimiques , Immunomodulation/génétique , Immunomodulation/immunologie , Activation des lymphocytes/immunologie , Mélanome expérimental , Souris , Souris transgéniques , Tumeurs/traitement médicamenteux , Tumeurs/métabolismeRÉSUMÉ
Colorectal cancer is a global disease with increasing incidence. Mortality is largely attributed to metastatic spread and therefore, a mechanistic dissection of the signals which influence tumor progression is needed. Cancer stroma plays a critical role in tumor proliferation, invasion and chemoresistance. Here, we sought to identify and characterize exosomal microRNAs as mediators of stromal-tumor signaling. In vitro, we demonstrated that fibroblast exosomes are transferred to colorectal cancer cells, with a resultant increase in cellular microRNA levels, impacting proliferation and chemoresistance. To probe this further, exosomal microRNAs were profiled from paired patient-derived normal and cancer-associated fibroblasts, from an ongoing prospective biomarker study. An exosomal cancer-associated fibroblast signature consisting of microRNAs 329, 181a, 199b, 382, 215 and 21 was identified. Of these, miR-21 had highest abundance and was enriched in exosomes. Orthotopic xenografts established with miR-21-overexpressing fibroblasts and CRC cells led to increased liver metastases compared to those established with control fibroblasts. Our data provide a novel stromal exosome signature in colorectal cancer, which has potential for biomarker validation. Furthermore, we confirmed the importance of stromal miR-21 in colorectal cancer progression using an orthotopic model, and propose that exosomes are a vehicle for miR-21 transfer between stromal fibroblasts and cancer cells.
Sujet(s)
Fibroblastes associés au cancer/métabolisme , Tumeurs colorectales/anatomopathologie , Exosomes/métabolisme , microARN/génétique , Sujet âgé , Animaux , Tumeurs colorectales/génétique , Tumeurs colorectales/métabolisme , Évolution de la maladie , Exosomes/génétique , Femelle , Hétérogreffes , Humains , Mâle , Souris , microARN/métabolismeRÉSUMÉ
Monoclonal antibodies (mAbs) can destroy tumors by recruiting effectors such as myeloid cells, or targeting immunomodulatory receptors to promote cytotoxic T cell responses. Here, we examined the therapeutic potential of combining a direct tumor-targeting mAb, anti-CD20, with an extended panel of immunomodulatory mAbs. Only the anti-CD27/CD20 combination provided cures. This was apparent in multiple lymphoma models, including huCD27 transgenic mice using the anti-huCD27, varlilumab. Detailed mechanistic analysis using single-cell RNA sequencing demonstrated that anti-CD27 stimulated CD8+ T and natural killer cells to release myeloid chemo-attractants and interferon gamma, to elicit myeloid infiltration and macrophage activation. This study demonstrates the therapeutic advantage of using an immunomodulatory mAb to regulate lymphoid cells, which then recruit and activate myeloid cells for enhanced killing of mAb-opsonized tumors.
Sujet(s)
Anticorps monoclonaux/pharmacologie , Antinéoplasiques/pharmacologie , Lymphomes/immunologie , Antigènes CD27/agonistes , Animaux , Anticorps monoclonaux humanisés , Humains , Immunothérapie/méthodes , Activation des lymphocytes/effets des médicaments et des substances chimiques , Activation des lymphocytes/immunologie , Activation des macrophages/effets des médicaments et des substances chimiques , Activation des macrophages/immunologie , Souris , Souris transgéniquesRÉSUMÉ
Memory CD8+ T cells confer long-term immunity against tumors, and anticancer vaccines therefore should maximize their generation. Multiple memory CD8+ T-cell subsets with distinct functional and homing characteristics exist, but the signaling pathways that regulate their development are ill defined. Here we examined the role of the serine/threonine kinase Akt in the generation of protective immunity by CD8+ T cells. Akt is known to be activated by the T-cell antigen receptor and the cytokine IL-2, but its role in T-cell immunity in vivo has not been explored. Using CD8+ T cells from pdk1K465E/K465E knockin mice, we found that decreased Akt activity inhibited the survival of T cells during the effector-to-memory cell transition and abolished their differentiation into C-X-C chemokine receptor 3 (CXCR3)loCD43lo effector-like memory cells. Consequently, antitumor immunity by CD8+ T cells that display defective Akt signaling was substantially diminished during the memory phase. Reduced memory T-cell survival and altered memory cell differentiation were associated with up-regulation of the proapoptotic protein Bim and the T-box transcription factor eomesodermin, respectively. These findings suggest an important role for effector-like memory CD8+ T cells in tumor immune surveillance and identify Akt as a key signaling node in the development of protective memory CD8+ T-cell responses.
Sujet(s)
Lymphocytes T CD8+/immunologie , Mémoire immunologique/immunologie , Surveillance immunologique/immunologie , Tumeurs expérimentales/immunologie , Protéines proto-oncogènes c-akt/immunologie , Animaux , Sites de fixation/génétique , Lymphocytes T CD8+/métabolisme , Récepteur-1 de la chimiokine CX3C/immunologie , Récepteur-1 de la chimiokine CX3C/métabolisme , Lignée cellulaire tumorale , Surveillance immunologique/génétique , Souris de lignée C57BL , Souris transgéniques , Mutation , Tumeurs expérimentales/génétique , Protein-Serine-Threonine Kinases/génétique , Protein-Serine-Threonine Kinases/immunologie , Protein-Serine-Threonine Kinases/métabolisme , Protéines proto-oncogènes c-akt/métabolisme , Pyruvate dehydrogenase acetyl-transferring kinase , Récepteurs aux antigènes des cellules T/immunologie , Récepteurs aux antigènes des cellules T/métabolisme , Récepteurs CXCR3/immunologie , Récepteurs CXCR3/métabolisme , Transduction du signal/immunologieRÉSUMÉ
Purpose In squamous cell carcinomas of the head and neck (HNSCC), the increasing incidence of oropharyngeal squamous cell carcinomas (OPSCCs) is attributable to human papillomavirus (HPV) infection. Despite commonly presenting at late stage, HPV-driven OPSCCs are associated with improved prognosis compared with HPV-negative disease. HPV DNA is also detectable in nonoropharyngeal (non-OPSCC), but its pathogenic role and clinical significance are unclear. The objectives of this study were to determine whether HPV plays a causal role in non-OPSCC and to investigate whether HPV confers a survival benefit in these tumors. Methods Meta-analysis was used to build a cross-tissue gene-expression signature for HPV-driven cancer. Classifiers trained by machine-learning approaches were used to predict the HPV status of 520 HNSCCs profiled by The Cancer Genome Atlas project. DNA methylation data were similarly used to classify 464 HNSCCs and these analyses were integrated with genomic, histopathology, and survival data to permit a comprehensive comparison of HPV transcript-positive OPSCC and non-OPSCC. Results HPV-driven tumors accounted for 4.1% of non-OPSCCs. Regardless of anatomic site, HPV+ HNSCCs shared highly similar gene expression and DNA methylation profiles; nonkeratinizing, basaloid histopathological features; and lack of TP53 or CDKN2A alterations. Improved overall survival, however, was largely restricted to HPV-driven OPSCCs, which were associated with increased levels of tumor-infiltrating lymphocytes compared with HPV-driven non-OPSCCs. Conclusion Our analysis identified a causal role for HPV in transcript-positive non-OPSCCs throughout the head and neck. Notably, however, HPV-driven non-OPSCCs display a distinct immune microenvironment and clinical behavior compared with HPV-driven OPSCCs.
Sujet(s)
Marqueurs biologiques tumoraux/génétique , Carcinome épidermoïde/virologie , Tumeurs de la tête et du cou/virologie , Papillomaviridae/génétique , Infections à papillomavirus/virologie , Méthylation de l'ADN , Tests de détection de l'ADN du virus du papillome humain , Humains , Tumeurs de l'oropharynx/virologie , Pronostic , Analyse de survieRÉSUMÉ
Antibodies that block T-cell-regulatory checkpoints have recently emerged as a transformative approach to cancer treatment. However, the clinical efficacy of checkpoint blockade depends upon inherent tumor immunogenicity, with variation in infiltrating T cells contributing to differences in objective response rates. Here, we sought to understand the molecular correlates of tumor-infiltrating T lymphocytes (TIL) in squamous cell carcinoma (SCC), using a systems biologic approach to integrate publicly available omics datasets with histopathologic features. We provide evidence that links TIL abundance and therapeutic outcome to the regulation of tumor glycolysis by EGFR and HIF, both of which are attractive molecular targets for use in combination with immunotherapeutics. Cancer Res; 76(14); 4136-48. ©2016 AACR.
Sujet(s)
Lymphocytes T CD8+/immunologie , Carcinome épidermoïde/immunologie , Glucose/métabolisme , Tumeurs de la tête et du cou/immunologie , Lymphocytes TIL/immunologie , Animaux , Carcinome épidermoïde/métabolisme , Carcinome épidermoïde/mortalité , Récepteurs ErbB/génétique , Récepteurs ErbB/physiologie , Femelle , Transporteur de glucose de type 1/analyse , Tumeurs de la tête et du cou/métabolisme , Tumeurs de la tête et du cou/mortalité , Sous-unité alpha du facteur-1 induit par l'hypoxie/physiologie , Souris , Souris de lignée C57BL , Carcinome épidermoïde de la tête et du cou , Transcriptome , Régulation positiveRÉSUMÉ
Antigenic stimulation via the B-cell receptor (BCR) is a major driver of the proliferation and survival of chronic lymphocytic leukemia (CLL) cells. However, the precise mechanisms by which BCR stimulation leads to accumulation of malignant cells remain incompletely understood. Here, we investigated the ability of BCR stimulation to increase messenger RNA (mRNA) translation, which can promote carcinogenesis by effects on both global mRNA translation and upregulated expression of specific oncoproteins. Re-analysis of gene expression profiles revealed striking upregulation of pathways linked to mRNA translation both in CLL cells derived from lymph nodes, the major site of antigen stimulation in vivo, and after BCR stimulation in vitro. Anti-IgM significantly increased mRNA translation in primary CLL cells, measured using bulk metabolic labeling and a novel flow cytometry assay to quantify responses at a single-cell level. These translational responses were suppressed by inhibitors of BTK (ibrutinib) and SYK (tamatinib). Anti-IgM-induced mRNA translation was associated with increased expression of translation initiation factors eIF4A and eIF4GI, and reduced expression of the eIF4A inhibitor, PDCD4. Anti-IgM also increased mRNA translation in normal blood B cells, but without clear modulatory effects on these factors. In addition, anti-IgM increased translation of mRNA-encoding MYC, a major driver of disease progression. mRNA translation is likely to be an important mediator of the growth-promoting effects of antigen stimulation acting, at least in part, via translational induction of MYC. Differences in mechanisms of translational regulation in CLL and normal B cells may provide opportunities for selective therapeutic attack.
Sujet(s)
Régulation de l'expression des gènes dans la leucémie , Leucémie chronique lymphocytaire à cellules B/génétique , Protéines proto-oncogènes c-myc/génétique , ARN messager/génétique , Récepteurs pour l'antigène des lymphocytes B/immunologie , Adénine/analogues et dérivés , Agammaglobulinaemia tyrosine kinase , Anticorps anti-idiotypiques/immunologie , Lymphocytes B/métabolisme , Lymphocytes B/anatomopathologie , Lignée cellulaire tumorale , Humains , Protéines et peptides de signalisation intracellulaire/antagonistes et inhibiteurs , Leucémie chronique lymphocytaire à cellules B/traitement médicamenteux , Leucémie chronique lymphocytaire à cellules B/anatomopathologie , Pipéridines , Biosynthèse des protéines/effets des médicaments et des substances chimiques , Inhibiteurs de protéines kinases/pharmacologie , Protein-tyrosine kinases/antagonistes et inhibiteurs , Pyrazoles/pharmacologie , Pyrimidines/pharmacologie , Récepteurs pour l'antigène des lymphocytes B/génétique , Syk kinase , Cellules cancéreuses en cultureRÉSUMÉ
Langerhans cells (LCs) are professional antigen-presenting cells (APCs) residing in the epidermis. Despite their high potential to activate T lymphocytes, current understanding of human LC biology is limited. Genome-wide comparison of the transcriptional profiles of human skin migratory CD1a+ LCs and CD11c+ dermal dendritic cells (DDCs) demonstrated significant differences between these "dendritic cell (DC)" types, including preferential expression of 625 genes (P<0.05) in LC and 914 genes (P<0.05) in DDC. Analysis of the temporal regulation of molecular networks activated after stimulation with tumor necrosis factor-α (TNF-α) confirmed the unique molecular signature of LCs. Although LCs conformed to the phenotype of professional APC, inflammatory signaling activated primarily genes associated with cellular metabolism and mitochondrial activation (e.g., CYB561 and MRPS35), cell membrane re-organization, and antigen acquisition and degradation (CAV1 and PSMD14; P<0.05-P<0.0001). Conversely, TNF-α induced classical activation in DDCs with early downregulation of surface receptors (mannose receptor-1 (MRC1) and C-type lectins), and subsequent upregulation of cytokines, chemokines (IL1a, IL1b, and CCL18), and matrix metalloproteinases (MMP1, MMP3, and MMP9; P<0.05-P<0.0001). Functional interference of caveolin abrogated LCs superior ability to cross-present antigens to CD8+ T lymphocytes, highlighting the importance of these networks to biological function. Taken together, these observations support the idea of distinct biological roles of cutaneous DC types.
Sujet(s)
Cellules présentatrices d'antigène/immunologie , Cellules dendritiques/immunologie , Régulation de l'expression des gènes/immunologie , Cellules de Langerhans/immunologie , Peau/immunologie , Lymphocytes T/immunologie , Antigènes CD1/immunologie , Antigènes CD1/métabolisme , Antigènes CD11c/métabolisme , Cavéoline-1/génétique , Cavéoline-1/immunologie , Cavéoline-1/métabolisme , Mouvement cellulaire/immunologie , Cellules dendritiques/cytologie , Cellules dendritiques/métabolisme , Derme/cytologie , Derme/immunologie , Cellules épidermiques , Épiderme/immunologie , Humains , Cellules de Langerhans/cytologie , Cellules de Langerhans/métabolisme , Peau/cytologie , TranscriptomeRÉSUMÉ
A major contributing factor to the final magnitude and breadth of CD8(+) T-cell responses to complex antigens is immunodomination, where CD8(+) T cells recognizing their cognate ligand inhibit the proliferation of other CD8(+) T cells engaged with the same APC. In this study, we examined how the half-life of cell surface peptide-MHC class I complexes influences this phenomenon. We found that primary CD8(+) T-cell responses to DNA vaccines in mice are shaped by competition among responding CD8(+) T cells for nonspecific stimuli early after activation and prior to cell division. The susceptibility of CD8(+) T cells to 'domination' was a direct correlate of higher kinetic stability of the competing CD8(+) T-cell cognate ligand. When high affinity competitive CD8(+) T cells were deleted by self-antigen expression, competition was abrogated. These findings show, for the first time to our knowledge, the existence of regulatory mechanisms that direct the responding CD8(+) T-cell repertoire toward epitopes with high-stability interactions with MHC class I molecules. They also provide an insight into factors that facilitate CD8(+) T-cell coexistence, with important implications for vaccine design and delivery.
Sujet(s)
Cellules présentatrices d'antigène/immunologie , Lymphocytes T CD8+/immunologie , Antigènes d'histocompatibilité de classe I/immunologie , Vaccins à ADN/immunologie , Animaux , Cellules présentatrices d'antigène/cytologie , Lymphocytes T CD8+/cytologie , Déterminants antigéniques des lymphocytes T/immunologie , Période , Immunisation , Épitopes immunodominants/immunologie , Cinétique , Activation des lymphocytes/immunologie , Souris , Souris de lignée C57BL , Souris transgéniquesRÉSUMÉ
Antigen-specific CD4(+) T cells are central to natural and vaccine-induced immunity. An ongoing antigen-specific T-cell response can, however, influence surrounding T cells with unrelated antigen specificities. We previously observed this bystander effect in healthy human subjects following recall vaccination with tetanus toxoid (TT). Since this interplay could be important for maintenance of memory, we have moved to a mouse model for further analysis. We investigated whether boosting memory CD4(+) T cells against TT in vivo would influence injected CD4(+) TCR transgenic T cells (OT-II) specific for an unrelated OVA peptide. If OT-II cells were pre-activated with OVA peptide in vitro, these cells showed a bystander proliferative response during the ongoing parallel TT-specific response. Bystander proliferation was dependent on boosting of the TT-specific memory response in the recipients, with no effect in naive mice. Bystander stimulation was also proportional to the strength of the TT-specific memory T-cell response. T cells activated in vitro displayed functional receptors for IL-2 and IL-7, suggesting these as potential mediators. This crosstalk between a stimulated CD4(+) memory T-cell response and CD4(+) T cells activated by an unrelated antigen could be important in human subjects continually buffeted by environmental antigens.
Sujet(s)
Effet bystander/immunologie , Lymphocytes T CD4+/immunologie , Rappel de vaccin , Mémoire immunologique/immunologie , Activation des lymphocytes , Sous-populations de lymphocytes/immunologie , Transfert adoptif , Animaux , Lymphocytes T CD4+/effets des médicaments et des substances chimiques , Cytokines/pharmacologie , Humains , Sous-unité gamma commune aux récepteurs des interleukines/immunologie , Sous-unité alpha du récepteur à l'interleukine-2/immunologie , Sous-unité bêta du récepteur à l'interleukine-2/immunologie , Interleukines/biosynthèse , Interleukines/génétique , Interleukines/pharmacologie , Interleukines/physiologie , Sous-populations de lymphocytes/effets des médicaments et des substances chimiques , Souris , Souris de lignée C57BL , Souris transgéniques , Ovalbumine/immunologie , Fragments peptidiques/immunologie , Récepteurs aux interleukines/immunologie , Protéines recombinantes/pharmacologie , Spécificité antigénique des récepteurs des lymphocytes T , Anatoxine tétanique/immunologieRÉSUMÉ
Peptide loading of MHC class I molecules involves multiple cofactors including tapasin. We showed previously in vitro that tapasin edits the peptide repertoire by favoring the binding of peptides with slow dissociation rates. Here, using tapasin-deficient mice and a DNA vaccine that primes directly, we confirm that tapasin establishes hierarchical responses in vivo according to peptide-MHC stability. In contrast, this hierarchy is lost when the peptides are cross-presented via an alternative DNA vaccine. By regulating transgene expression, we found that the dominant response modifier was antigen persistence. Our findings reveal strategies for activating T cells against low-affinity peptides, of potential importance for patients with repertoires narrowed by deletional tolerance.
Sujet(s)
Lymphocytes T CD8+/immunologie , Lymphocytes T CD8+/métabolisme , Protéines d'oeuf/pharmacocinétique , Antigènes d'histocompatibilité de classe I/métabolisme , Épitopes immunodominants/métabolisme , Protéines de transport membranaire/physiologie , Ovalbumine/pharmacocinétique , Animaux , Cross-priming/immunologie , Protéines d'oeuf/immunologie , Antigènes d'histocompatibilité de classe I/immunologie , Humains , Épitopes immunodominants/immunologie , Protéines de transport membranaire/déficit , Protéines de transport membranaire/génétique , Protéines de transport membranaire/pharmacocinétique , Souris , Souris de lignée C57BL , Souris knockout , Ovalbumine/immunologie , Fragments peptidiques , Transduction du signal/immunologie , Vaccins à ADN/administration et posologie , Vaccins à ADN/immunologie , Vaccins à ADN/pharmacocinétiqueRÉSUMÉ
After priming, naive T cells undergo a program of expansion, contraction, and memory formation. Numerous studies have indicated that only a brief period of antigenic stimulation is required to fully commit CD8+ T cells to this program. Nonetheless, the persistence of Ag may modulate the eventual fate of CD8+ T cells. Using DNA delivery, we showed previously that direct presentation primes high levels of effector CD8+ T cells as compared with cross-presentation. One explanation now revealed is that prolonged cross-presentation limits effector cell expansion and function. To analyze this, we used a drug-responsive system to regulate Ag expression after DNA injection. Reducing expression to a single burst expanded greater numbers of peptide-specific effector CD8+ T cells than sustained Ag. Consequences for memory development were assessed after boosting and showed that, although persistent Ag maintained higher numbers of tetramer-positive CD8+ T cells, these expanded less (approximately 4-fold) than those induced by transient Ag expression (approximately 35-fold). Transient expression at priming therefore led to a net higher secondary response. In terms of vaccine design, we propose that the most effective DNA-based CD8+ T cell vaccines will be those that deliver a short burst of Ag.
Sujet(s)
Présentation d'antigène , Lymphocytes T CD8+/immunologie , Cross-priming , Mémoire immunologique , Vaccins à ADN/immunologie , Animaux , Souris , Souris de lignée C57BL , VaccinationRÉSUMÉ
The route for presentation of Ag to CD8+ or CD4+ T cells following DNA vaccination is critical for determining outcome, but the pathways involved are unclear. In this study, we compare two different DNA vaccine designs aimed to elicit CD8+ T cell responses against a specific peptide-epitope either by direct- or cross-presentation. Each carries sequences from tetanus toxin (TT) to provide essential CD4+ T cell help. In the first already proven design, the peptide-epitope is fused to the N-terminal domain of fragment C from TT. This appears to act mainly by cross-presentation. In the second design, the peptide-epitope is encoded by a minigene, with induction of Th responses mediated by coexpression of a hybrid invariant chain molecule, incorporating a single determinant from TT (p30) in exchange for class II-associated invariant chain peptide. This design appears to act mainly via direct presentation from transfected APCs. Both vaccines mediated Th-dependent priming of CD8+ T cells in mice, but the kinetics and level of the responses differed markedly, consistent with engagement of distinct pathways of Ag presentation. Importantly, the vaccines could be combined in an alternating prime-boost regime, in either order, generating substantially expanded memory CD8+ T cells, with potent effector function. Taken together, these results demonstrate that vaccination protocols involving different modes of Ag presentation at prime and boost can significantly improve the effectiveness of immunization.
Sujet(s)
Présentation d'antigène/immunologie , Lymphocytes T CD8+/immunologie , Rappel de vaccin , Fragments peptidiques/administration et posologie , Fragments peptidiques/immunologie , Transduction du signal/immunologie , Vaccins à ADN/administration et posologie , Vaccins à ADN/immunologie , Animaux , Présentation d'antigène/génétique , Antigènes de différenciation des lymphocytes B/administration et posologie , Antigènes de différenciation des lymphocytes B/génétique , Antigènes de différenciation des lymphocytes B/immunologie , Lymphocytes T CD8+/cytologie , Lymphocytes T CD8+/métabolisme , Cytotoxicité immunologique/génétique , Protéines d'oeuf/administration et posologie , Protéines d'oeuf/génétique , Protéines d'oeuf/immunologie , Antigènes H-2/immunologie , Antigènes H-2/métabolisme , Antigènes d'histocompatibilité de classe II/administration et posologie , Antigènes d'histocompatibilité de classe II/génétique , Antigènes d'histocompatibilité de classe II/immunologie , Rappel de vaccin/méthodes , Interféron gamma/biosynthèse , Numération des lymphocytes , Souris , Souris de lignée C57BL , Souris transgéniques , Ovalbumine/administration et posologie , Ovalbumine/génétique , Ovalbumine/immunologie , Fragments peptidiques/génétique , Transduction du signal/génétique , Lymphocytes T auxiliaires/immunologie , Toxine tétanique/administration et posologie , Toxine tétanique/génétique , Toxine tétanique/immunologie , Vaccins à ADN/synthèse chimique , Vaccins synthétiques/administration et posologie , Vaccins synthétiques/génétique , Vaccins synthétiques/immunologieRÉSUMÉ
Vaccination against target antigens expressed by cancer cells has now become a realistic goal. DNA vaccines provide a direct link between identification of genetic markers in tumors and vaccine formulation. Simplicity of manufacture facilitates construction of vaccines against disease subsets or even for individual patients. To engage an immune system that exists to fight pathogens, we have developed fusion gene vaccines encoding tumor antigens fused to pathogen-derived sequences. This strategy activates high levels of T-cell help, the key to induction and maintenance of effective immunity. We have dissected the immunogenic tetanus toxin to obtain specific sequences able to activate antibody, CD4+, or CD8+ T cells to attack selected fused tumor antigens. Principles established in preclinical models are now being tested in patients. So far, objective immune responses against idiotypic antigen of neoplastic B cells have been observed in patients with B-cell malignancies and in normal transplant donors. These responses provide a platform for testing physical methods to improve DNA delivery and strategies to boost responses. For cancer, demands are high, because vaccines have to activate powerful immunity against weak antigens, often in a setting of immune damage or tolerance. Vaccination strategies against cancer and against microbes are sharing knowledge and technology for mutual benefit.
Sujet(s)
Vaccins anticancéreux/génétique , Vaccins anticancéreux/immunologie , Tumeurs/thérapie , Vaccins à ADN/génétique , Vaccins à ADN/immunologie , Antigènes néoplasiques/immunologie , Fusion artificielle de gènes , Lymphocytes T CD4+/immunologie , Lymphocytes T CD8+/immunologie , Humains , Immunotoxines/génétique , Immunotoxines/immunologie , Immunotoxines/usage thérapeutique , Myélome multiple/immunologie , Myélome multiple/thérapie , Fragments peptidiques/génétique , Fragments peptidiques/immunologie , Protéines de fusion recombinantes/génétique , Protéines de fusion recombinantes/immunologie , Protéines de fusion recombinantes/usage thérapeutique , Toxine tétanique/génétique , Toxine tétanique/immunologie , Vaccination/méthodesRÉSUMÉ
The clonotypic T-cell antigen receptor (TCR) provides unique Valpha and Vbeta sequences with potential as idiotypic targets for immunoregulation. For T-cell malignancies, vaccination with the TCR could induce therapeutic anti-idiotypic responses. To facilitate this approach, we have developed DNA vaccines that include the genes encoding TCR sequences from a T-cell lymphoma (TCL). To combine requirements for stable folding with a simple minimized single-chain construction, we used a three-domain ValphaVbetaCbeta sequence. To promote anti-TCR immunity, we fused a pathogen-derived sequence from tetanus toxin to the 3'-end of the single-chain TCR. The fusion gene vaccine induced anti-idiotypic antibodies and generated protection against the TCL. The critical requirement for the conformational integrity of the delivered TCR antigen was highlighted by the observation that DNA fusion vaccines containing either ValphaVbeta or VbetaCbeta sequences failed to generate antibodies reactive with the native TCR or provide protection. This is the first report of a DNA vaccine able to induce anti-idiotypic immunity against TCL, and it presents a simple strategy for selectively eliminating T-cell clones in vivo.