ABSTRACT
Although immunotherapy has achieved impressive durable clinical responses, many cancers respond only temporarily or not at all to immunotherapy. To find novel, targetable mechanisms of resistance to immunotherapy, patient-derived melanoma cell lines were transduced with 576 open reading frames, or exposed to arrayed libraries of 850 bioactive compounds, prior to co-culture with autologous tumor-infiltrating lymphocytes (TILs). The synergy between the targets and TILs to induce apoptosis, and the mechanisms of inhibiting resistance to TILs were interrogated. Gene expression analyses were performed on tumor samples from patients undergoing immunotherapy for metastatic melanoma. Finally, the effect of inhibiting the top targets on the efficacy of immunotherapy was investigated in multiple preclinical models. Aurora kinase was identified as a mediator of melanoma cell resistance to T-cell-mediated cytotoxicity in both complementary screens. Aurora kinase inhibitors were validated to synergize with T-cell-mediated cytotoxicity in vitro. The Aurora kinase inhibition-mediated sensitivity to T-cell cytotoxicity was shown to be partially driven by p21-mediated induction of cellular senescence. The expression levels of Aurora kinase and related proteins were inversely correlated with immune infiltration, response to immunotherapy and survival in melanoma patients. Aurora kinase inhibition showed variable responses in combination with immunotherapy in vivo, suggesting its activity is modified by other factors in the tumor microenvironment. These data suggest that Aurora kinase inhibition enhances T-cell cytotoxicity in vitro and can potentiate antitumor immunity in vivo in some but not all settings. Further studies are required to determine the mechanism of primary resistance to this therapeutic intervention.
Subject(s)
Aurora Kinase A/metabolism , Aurora Kinase B/metabolism , Drug Resistance, Neoplasm/immunology , Immunotherapy/methods , Lymphocytes, Tumor-Infiltrating/immunology , Melanoma/immunology , T-Lymphocytes, Cytotoxic/transplantation , Animals , Apoptosis , Aurora Kinase A/antagonists & inhibitors , Aurora Kinase A/genetics , Aurora Kinase B/antagonists & inhibitors , Aurora Kinase B/genetics , Cell Proliferation , Female , Humans , Melanoma/genetics , Melanoma/metabolism , Melanoma/therapy , Mice , Prognosis , Survival Rate , T-Lymphocytes, Cytotoxic/immunology , Tumor Cells, Cultured , Tumor Microenvironment/immunology , Xenograft Model Antitumor AssaysABSTRACT
Neoantigen burden is a major determinant of tumor immunogenicity, underscored by recent clinical experience with checkpoint blockade therapy. Yet the majority of patients do not express, or express too few, neoantigens, and hence are less responsive to immune therapy. Here we describe an approach whereby a common set of new antigens are induced in tumor cells in situ by transient downregulation of the transporter associated with antigen processing (TAP). Administration of TAP siRNA conjugated to a broad-range tumor-targeting nucleolin aptamer inhibited tumor growth in multiple tumor models without measurable toxicity, was comparatively effective to vaccination against prototypic mutation-generated neoantigens, potentiated the antitumor effect of PD-1 antibody or Flt3 ligand, and induced the presentation of a TAP-independent peptide in human tumor cells. Treatment with the chemically-synthesized nucleolin aptamer-TAP siRNA conjugate represents a broadly-applicable approach to increase the antigenicity of tumor lesions and thereby enhance the effectiveness of immune potentiating therapies.
Subject(s)
ATP-Binding Cassette Transporters/immunology , Antigens, Neoplasm/immunology , Immunotherapy , Neoplasms/immunology , Neoplasms/prevention & control , ATP-Binding Cassette Transporters/genetics , Animals , Antigens, Neoplasm/genetics , Aptamers, Nucleotide , CD4-Positive T-Lymphocytes , CD8-Positive T-Lymphocytes , Cancer Vaccines , Cell Line, Tumor , Down-Regulation , Epitopes/immunology , Female , Humans , Immunization , Immunogenicity, Vaccine , Male , Membrane Proteins/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular Targeted Therapy , NIH 3T3 Cells , Neoplasms, Experimental , Oligodeoxyribonucleotides , Phosphoproteins , Programmed Cell Death 1 Receptor/immunology , RNA, Small Interfering/administration & dosage , RNA-Binding Proteins , Spleen/immunology , Spleen/pathology , Vaccination , NucleolinABSTRACT
T-cell based immunotherapies through checkpoint blockade or adoptive transfer are effective treatments for a wide range of cancers like melanomas and lung carcinomas that harbor a high mutational load. The HLA class I and class II (HLA-I and HLA-II) presented neoantigens arise from genetic mutations in the cancerous cells and are ideal non-self targets for the T cell-based treatments. Although some cancer patients responded with complete regression, many others are irresponsive to checkpoint blockade treatments, or relapse after initial success. One of the mechanisms by which tumors evade T cell recognition is by acquiring deficiencies in the HLA-I antigen-processing pathway, leading to downregulation of HLA-I molecules at the cell surface and thereby creating an 'invisible' tumor phenotype. Interestingly, an alternative antigen repertoire arises on these HLA-Ilow cancer cells. We refer to this alternative antigen repertoire as TEIPP: T cell epitopes associated with impaired peptide processing. TEIPP antigens are curious non-mutated peptides from housekeeping proteins that are not presented in homeostasis. In this review, for the first time we recapitulate all our published work on TEIPP antigens, including our recent understanding of the CD8 T cell repertoire. We are convinced that TEIPP-directed T cells will be valuable resources to target immune-edited tumors that have acquired resistance to checkpoint blockade therapy.
Subject(s)
Antigens, Neoplasm/immunology , CD8-Positive T-Lymphocytes/immunology , Epitopes, T-Lymphocyte/immunology , Histocompatibility Antigens Class I/immunology , Neoplasms/immunology , Animals , Humans , Immunotherapy/methodsABSTRACT
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.
Subject(s)
Antigens, Neoplasm/immunology , Cancer Vaccines/chemistry , Cancer Vaccines/immunology , Membrane Proteins/chemistry , Membrane Proteins/immunology , Animals , Antigens, Neoplasm/chemistry , Epitopes, T-Lymphocyte/immunology , Histocompatibility Antigen H-2D/chemistry , Histocompatibility Antigen H-2D/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Peptides , Proline , Protein StabilityABSTRACT
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.
Subject(s)
Carrier Proteins/metabolism , Histocompatibility Antigens Class I/immunology , Lipoproteins/metabolism , Peptides/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Trans-Activators/metabolism , Animals , Biological Transport , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Carrier Proteins/genetics , Cell Line , Conserved Sequence , Histocompatibility Antigens Class I/metabolism , Humans , Ligands , Lipoproteins/genetics , Lymphocyte Activation , Mice , Mice, Knockout , Mice, Transgenic , Peptides/metabolism , Receptors, Antigen, T-Cell/metabolism , Thymus Gland/immunology , Thymus Gland/metabolism , Trans-Activators/geneticsABSTRACT
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.
ABSTRACT
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.
Subject(s)
Killer Cells, Natural/immunology , Major Histocompatibility Complex/immunology , Membrane Glycoproteins/immunology , Neoplasms/immunology , Toll-Like Receptor 7/immunology , Tumor Microenvironment/immunology , Aminoquinolines/administration & dosage , Aminoquinolines/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Chemokine CXCL10/immunology , Chemokine CXCL9/immunology , Humans , Imiquimod , Lymphocyte Activation/immunology , Membrane Glycoproteins/agonists , Mice , Neoplasms/pathology , Neoplasms/therapy , Receptors, CXCR3/immunology , Toll-Like Receptor 3/immunology , Toll-Like Receptor 7/agonists , Toll-Like Receptor 9/immunologyABSTRACT
MHC class I downregulation represents a significant challenge for successful T cell-based immunotherapy. T cell epitopes associated with impaired peptide processing (TEIPP) constitute a novel category of immunogenic Ags that are selectively presented on transporter associated with Ag processing-deficient cells. The TEIPP neoepitopes are CD8 T cell targets, derived from nonmutated self-proteins that might be exploited to prevent immune escape. In this study, the crystal structure of H-2D(b) in complex with the first identified TEIPP Ag (MCLRMTAVM) derived from the Trh4 protein has been determined to 2.25 Å resolution. In contrast to prototypic H-2D(b) peptides, Trh4 takes a noncanonical peptide-binding pattern with extensive sulfur-π interactions that contribute to the overall complex stability. Importantly, the noncanonical methionine at peptide position 5 acts as a main anchor, altering only the conformation of the H-2D(b) residues Y156 and H155 and thereby forming a unique MHC/peptide conformer that is essential for recognition by TEIPP-specific T cells. Substitution of peptide residues p2C and p5M to the conservative α-aminobutyric acid and norleucine, respectively, significantly reduced complex stability, without altering peptide conformation or T cell recognition. In contrast, substitution of p5M to a conventional asparagine abolished recognition by the H-2D(b)/Trh4-specific T cell clone LnB5. We anticipate that the H-2D(b)/Trh4 complex represents the first example, to our knowledge, of a broader repertoire of alternative MHC class I binders.
Subject(s)
Epitopes, T-Lymphocyte/immunology , Histocompatibility Antigen H-2D/metabolism , Membrane Proteins/immunology , Membrane Proteins/metabolism , Neoplasms/immunology , Neoplasms/metabolism , Peptides/immunology , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Amino Acid Sequence , Animals , Binding Sites , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/metabolism , Histocompatibility Antigen H-2D/immunology , Hydrophobic and Hydrophilic Interactions , Mice , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Peptides/chemistry , Peptides/metabolism , Protein Binding , Protein Conformation , Protein Multimerization , Protein Stability , Receptors, Antigen, T-Cell, alpha-beta/chemistry , ThermodynamicsABSTRACT
Immune escape strategies aimed to avoid T-cell recognition, including the loss of tumor MHC class I expression, are commonly found in malignant cells. Tumor immune escape has proven to have a negative effect on the clinical outcome of cancer immunotherapy, including treatment with antibodies blocking immune checkpoint molecules. Hence, there is an urgent need to develop novel approaches to overcome tumor immune evasion. MHC class I antigen presentation is often affected in human cancers and the capacity to induce upregulation of MHC class I cell surface expression is a critical step in the induction of tumor rejection. This review focuses on characterization of rejection, escape, and dormant profiles of tumors and its microenvironment with a special emphasis on the tumor MHC class I expression. We also discuss possible approaches to recover MHC class I expression on tumor cells harboring reversible/'soft' or irreversible/'hard' genetic lesions. Such MHC class I recovery approaches might well synergize with complementary forms of immunotherapy.
Subject(s)
Histocompatibility Antigens Class I/immunology , Immunotherapy , Neoplasms/immunology , Neoplasms/therapy , Animals , HumansABSTRACT
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.
Subject(s)
ATP-Binding Cassette Transporters/immunology , Antigen Presentation , Antigens, Neoplasm/immunology , Autoantigens/immunology , Epitopes, T-Lymphocyte/immunology , Neoplasms, Experimental/immunology , Peptides/immunology , Tumor Escape , ATP Binding Cassette Transporter, Subfamily B, Member 2 , ATP-Binding Cassette Transporters/genetics , Animals , Antigens, Neoplasm/genetics , Autoantigens/genetics , Cell Line, Tumor , Epitopes, T-Lymphocyte/genetics , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Mice , Mice, Knockout , Neoplasms, Experimental/genetics , Neoplasms, Experimental/pathology , Peptides/geneticsABSTRACT
The association of B7-1/CD28 between antigen presenting cells (APCs) and T-cells provides a second signal to proliferate and activate T-cell immunity at the induction phase. Many reports indicate that tumor cells transfected with B7-1 induced augmented antitumor immunity at the induction phase by mimicking APC function; however, the function of B7-1 on antitumor immunity at the effector phase is unknown. Here, we report direct evidence of enhanced T-cell antitumor immunity at the effector phase by the B7-1 molecule. Our experiments in vivo and in vitro indicated that reactivity of antigen-specific monoclonal and polyclonal T-cell effectors against a Lass5 epitope presented by RMA-S cells is increased when the cells expressed B7-1. Use of either anti-B7-1 or anti-CD28 antibodies to block the B7-1/CD28 association reduced reactivity of the T effectors against B7-1 positive RMA-S cells. Transfection of Lass5 cDNA into or pulse of Lass5 peptide onto B7-1 positive RMA-S cells overcomes the requirement of the B7-1/CD28 signal for T effector response. To our knowledge, the data offers, for the first time, strong evidence that supports the requirement of B7-1/CD28 secondary signal at the effector phase of antitumor T-cell immunity being dependent on the density of an antigenic peptide.
Subject(s)
Antigen-Presenting Cells/immunology , B7-1 Antigen/immunology , CD28 Antigens/immunology , Immunity, Cellular/immunology , Membrane Proteins/immunology , Signal Transduction/immunology , T-Lymphocytes/immunology , Animals , B7-1 Antigen/metabolism , CD28 Antigens/metabolism , Cell Line, Tumor , Cell Proliferation/physiology , Flow Cytometry , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Polymerase Chain ReactionABSTRACT
Central nervous system (CNS) autoimmunity is regulated by the balance of pro-inflammatory cytokines and IL-10. Here we identify the transcriptional regulator Blimp1 as crucial to induce IL-10 in inflammatory T helper cells. Pre-committed Th17 cells respond to IL-27 and IL-12 by upregulating Blimp1 and adopt a Tr-1-like phenotype characterized by IL-10 and IFN-γ production. Accordingly, Blimp1-deficient effector T cells fail to produce IL-10, and deficiency in Tr-1 cell function leads to uncontrolled Th17 cell-driven CNS pathology without the need to stabilize the Th17 phenotype with IL-23. IL-23 counteracts IL-27 and IL-12-mediated effects on Tr-1-development reinforcing the pro-inflammatory phenotype of Th17 cells. Thus, the balance of IL-23 vs IL-12/IL-27 signals into CD4(+) effector T cells determines whether tissue inflammation is perpetuated or resolves.