ABSTRACT
For many solid tumors, immune checkpoint blockade therapy has become first line treatment, yet a large proportion of patients with immunologically cold tumors do not benefit due to the paucity of tumor infiltrating lymphocytes. Here we show that the orphan G Protein-Coupled Receptor 182 (GPR182) contributes to immunotherapy resistance in cancer via scavenging chemokines that are important for lymphocyte recruitment to tumors. GPR182 is primarily upregulated in melanoma-associated lymphatic endothelial cells (LECs) during tumorigenesis, and this atypical chemokine receptor endocytoses chemokines promiscuously. In GPR182-deficient mice, T cell infiltration into transplanted melanomas increases, leading to enhanced effector T cell function and improved antitumor immunity. Ablation of GPR182 leads to increased intratumoral concentrations of multiple chemokines and thereby sensitizes poorly immunogenic tumors to immune checkpoint blockade and adoptive cellular therapies. CXCR3 blockade reverses the improved antitumor immunity and T cell infiltration characteristic of GPR182-deficient mice. Our study thus identifies GPR182 as an upstream regulator of the CXCL9/CXCL10/CXCR3 axis that limits antitumor immunity and as a potential therapeutic target in immunologically cold tumors.
Subject(s)
Chemokine CXCL10/genetics , Chemokine CXCL9/genetics , Melanoma, Experimental/genetics , Melanoma/genetics , Receptors, CXCR3/genetics , Receptors, G-Protein-Coupled/genetics , Skin Neoplasms/genetics , Animals , Cell Movement , Chemokine CXCL10/immunology , Chemokine CXCL9/immunology , Gene Expression Regulation, Neoplastic , Humans , Immunotherapy/methods , Lymphocytes, Tumor-Infiltrating/cytology , Lymphocytes, Tumor-Infiltrating/immunology , Melanoma/immunology , Melanoma/mortality , Melanoma/therapy , Melanoma, Experimental/immunology , Melanoma, Experimental/mortality , Melanoma, Experimental/therapy , Mice , Mice, Knockout , Protein Binding , Receptors, CXCR3/immunology , Receptors, G-Protein-Coupled/immunology , Signal Transduction , Skin Neoplasms/immunology , Skin Neoplasms/mortality , Skin Neoplasms/therapy , Survival Analysis , T-Lymphocytes, Cytotoxic/cytology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/transplantation , Tumor Burden , Tumor Microenvironment/genetics , Tumor Microenvironment/immunologyABSTRACT
Anti-tumor therapies that seek to exploit and redirect the cytotoxic killing and effector potential of autologous or syngeneic T cells have shown extraordinary promise and efficacy in certain clinical settings. Such cells, when engineered to express synthetic chimeric antigen receptors (CARs) acquire novel targeting and activation properties which are governed and orchestrated by, typically, antibody fragments specific for a tumor antigen of interest. However, it is becoming increasingly apparent that not all antibodies are equal in this regard, with a growing appreciation that 'optimal' CAR performance requires a consideration of multiple structural and contextual parameters. Thus, antibodies raised by classical approaches and intended for other applications often perform poorly or not at all when repurposed as CARs. With this in mind, we have explored the potential of an in vitro phenotypic CAR library discovery approach that tightly associates antibody-driven bridging of tumor and effector T cells with an informative and functionally relevant CAR activation reporter signal. Critically, we demonstrate the utility of this enrichment methodology for 'real world' de novo discovery by isolating several novel anti-mesothelin CAR-active scFv candidates.
Subject(s)
Neoplasms/therapy , Receptors, Chimeric Antigen/isolation & purification , T-Lymphocytes, Cytotoxic/immunology , Cell Line, Tumor , Gene Library , HEK293 Cells , Healthy Volunteers , Humans , Immunotherapy, Adoptive/methods , Neoplasms/immunology , Neoplasms/pathology , Primary Cell Culture , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/immunology , Single-Chain Antibodies/immunology , Single-Chain Antibodies/metabolism , T-Lymphocytes, Cytotoxic/metabolism , T-Lymphocytes, Cytotoxic/transplantationABSTRACT
BACKGROUND/AIM: Heat shock protein 105 (HSP105) is overexpressed in various cancers, but not in normal tissues. We investigated the expression levels of HSP105 in cervical cancer and the efficacy of immunotherapy targeting HSP105. MATERIALS AND METHODS: Previously, we established human leukocyte antigen-A*02:01 (HLA-A2) restricted HSP105 peptide-specific cytotoxic T lymphocyte (CTL) clones from a colorectal cancer patient vaccinated with an HSP105 peptide. Herein, we evaluated the expression of HSP105 in cervical cancer and cervical intraepithelial neoplasia. Moreover, we tested the effectiveness of an HLA-A2-restricted HSP105 peptide-specific CTL clone against cervical cancer cell lines. RESULTS: HSP105 was expressed in 95% (19/20) of examined cervical cancer tissues. Moreover, the HSP105 peptide-specific CTL clone recognized HSP105- and HLA-A*02:01-positive cervical cancer cell lines and also showed that cytotoxicity against the cervical cancer cell lines depends on HSP105 peptide and HLA class I restricted manners. CONCLUSION: HSP105 could be an effective target for immunotherapy in patients with cervical cancer.
Subject(s)
HSP110 Heat-Shock Proteins/immunology , Immunotherapy/methods , Uterine Cervical Neoplasms/therapy , Animals , Cell Line, Tumor , Female , HLA-A2 Antigen/immunology , HLA-A2 Antigen/metabolism , HSP110 Heat-Shock Proteins/metabolism , Humans , Mice , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/transplantation , Uterine Cervical Neoplasms/immunology , Uterine Cervical Neoplasms/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Efficacy of cytotoxic T lymphocyte (CTL)-based immunotherapy is still unsatisfactory against solid tumors, which are frequently characterized by condensed extracellular matrix. Here, using a unique 3D killing assay, we identify that the killing efficiency of primary human CTLs is substantially impaired in dense collagen matrices. Although the expression of cytotoxic proteins in CTLs remained intact in dense collagen, CTL motility was largely compromised. Using light-sheet microscopy, we found that persistence and velocity of CTL migration was influenced by the stiffness and porosity of the 3D matrix. Notably, 3D CTL velocity was strongly correlated with their nuclear deformability, which was enhanced by disruption of the microtubule network especially in dense matrices. Concomitantly, CTL migration, search efficiency, and killing efficiency in dense collagen were significantly increased in microtubule-perturbed CTLs. In addition, the chemotherapeutically used microtubule inhibitor vinblastine drastically enhanced CTL killing efficiency in dense collagen. Together, our findings suggest targeting the microtubule network as a promising strategy to enhance efficacy of CTL-based immunotherapy against solid tumors, especially stiff solid tumors.
Subject(s)
Cell Movement/drug effects , Collagen Type I/chemistry , Cytotoxicity, Immunologic , Immunotherapy, Adoptive , Microtubules/drug effects , Neoplasms/therapy , T-Lymphocytes, Cytotoxic/transplantation , Tubulin Modulators/pharmacology , Vinblastine/pharmacology , Cell Line, Tumor , Coculture Techniques , Elasticity , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Extracellular Matrix/pathology , Humans , Hydrogels , Microtubules/immunology , Microtubules/metabolism , Neoplasms/immunology , Neoplasms/metabolism , Neoplasms/pathology , Porosity , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolismABSTRACT
Adoptive T cell therapy (ACT) requires lymphodepletion preconditioning to eliminate immune-suppressive elements and enable efficient engraftment of adoptively transferred tumor-reactive T cells. As anti-CD4 monoclonal antibody depletes CD4+ immune-suppressive cells, the combination of anti-CD4 treatment and ACT has synergistic potential in cancer therapy. Here, we demonstrate a post-ACT conditioning regimen that involves transient anti-CD4 treatment (CD4post). Using murine melanoma, the combined effect of cyclophosphamide preconditioning (CTXpre), CD4post, and ex vivo primed tumor-reactive CD8+ T-cell infusion is presented. CTXpre/CD4post increases tumor suppression and host survival by accelerating the proliferation and differentiation of ex vivo primed CD8+ T cells and endogenous CD8+ T cells. Endogenous CD8+ T cells enhance effector profile and tumor-reactivity, indicating skewing of the TCR repertoire. Notably, enrichment of polyfunctional IL-18Rαhi CD8+ T cell subset is the key event in CTXpre/CD4post-induced tumor suppression. Mechanistically, the anti-tumor effect of IL-18Rαhi subset is mediated by IL-18 signaling and TCR-MHC I interaction. This study highlights the clinical relevance of CD4post in ACT and provides insights regarding the immunological nature of anti-CD4 treatment, which enhances anti-tumor response of CD8+ T cells.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents, Alkylating/pharmacology , CD4-Positive T-Lymphocytes/drug effects , Cyclophosphamide/pharmacology , Interleukin-18 Receptor alpha Subunit/genetics , Melanoma, Experimental/therapy , Skin Neoplasms/therapy , T-Lymphocytes, Cytotoxic/immunology , Adoptive Transfer , Animals , Antigens, CD/genetics , Antigens, CD/immunology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/pathology , Female , Gene Expression Regulation, Neoplastic , Immunotherapy, Adoptive/methods , Interleukin-18/genetics , Interleukin-18/immunology , Interleukin-18 Receptor alpha Subunit/agonists , Interleukin-18 Receptor alpha Subunit/immunology , Lymphocyte Activation , Melanoma, Experimental/genetics , Melanoma, Experimental/immunology , Melanoma, Experimental/mortality , Mice , Mice, Inbred C57BL , Receptors, CCR4/genetics , Receptors, CCR4/immunology , Receptors, CCR8/genetics , Receptors, CCR8/immunology , Receptors, Histamine H4/genetics , Receptors, Histamine H4/immunology , Signal Transduction , Skin Neoplasms/genetics , Skin Neoplasms/immunology , Skin Neoplasms/mortality , Survival Analysis , T-Lymphocytes, Cytotoxic/cytology , T-Lymphocytes, Cytotoxic/transplantation , Tumor Burden/drug effectsABSTRACT
In recent years, adoptive cell therapy of immune effector cells, such as chimeric antigen receptor-T (CAR-T) cells, natural killer (NK) cells, and epitope-specific cytotoxic T lymphocyte (CTL) cells have been employed in clinical trials. In addition, CD19 CAR-T cells have been approved by the FDA for treatment of non-Hodgkin lymphoma and diffuse large B-cell lymphoma. In this context, it is vital to detect cellular cytotoxicity and monitor the quality of ex vivo expanded immune cells before product release and patient infusion. Target cells could proliferate in parallel with effector cells during the cytotoxicity assay, making it difficult to estimate the death ratio using conventional approaches. Meanwhile, non-specific dyes or non-homogeneous biomarkers for target cells may interfere with the final readout post addition of effector cells. Here, we modified a component of the coincubation medium to suppress the spontaneous release of bis(acetoxymethyl)2,2':6',2â³-terpyridine-6,6â³-dicarboxylate and sustained the window at a stable range (~70%). Further, the optimized Eu-TDA method presented reliable outcomes compared with lactate dehydrogenase detection and was compatible with cytotoxicity tests for NK cells and specific CTLs. Finally, the reported assay can accurately detect death of target cells depending on the amount of hydrophilic complex and can be reliably applied in quality control and cell activity evaluation tests on co-suspended effector and target cells. SUMMARY: A medium component for cellular coincubations (and associated protocols) have been optimized and validated for cytotoxicity assays, which can reliably evaluate the potency of engineered CD19 CAR-T cells, NK cells, and specific CTLs. In particular, the reported method can be applied widely in routine assays for bi-suspended effector and target cells with a stable window.
Subject(s)
Cytotoxicity, Immunologic , Immunohistochemistry , Immunotherapy, Adoptive , Killer Cells, Natural/transplantation , Leukemia, Erythroblastic, Acute/therapy , T-Lymphocytes, Cytotoxic/transplantation , Antigens, CD19/genetics , Antigens, CD19/immunology , Cell Survival , Coculture Techniques , Epitopes , Flow Cytometry , Humans , K562 Cells , Killer Cells, Natural/immunology , Leukemia, Erythroblastic, Acute/immunology , Leukemia, Erythroblastic, Acute/pathology , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/immunology , T-Lymphocytes, Cytotoxic/immunologyABSTRACT
Vγ9Vδ2 T cells are attractive effector cells for immunotherapy with potent cytotoxic activity against a variety of malignant cells. However, the effect of Vγ9Vδ2 T cells on chemotherapy-resistant acute myeloid leukemia (AML) blasts, especially highly refractory leukemia stem cells (LSCs) is still unknown. In this study, we investigated the effect of cytotoxicity of allogeneic Vγ9Vδ2 T cells on chemotherapy-resistant AML cell lines, as well as on primary AML blasts and LSCs obtained from refractory AML patients. The results indicated that Vγ9Vδ2 T cells can efficiently kill drug-resistant AML cell lines in vitro and in vivo, and the sensitivity of AML cells to Vγ9Vδ2 T cell-mediated cytotoxicity is not influenced by the sensitivity of AML cells to chemotherapy. We further found that Vγ9Vδ2 T cells exhibited a comparable effect of cytotoxicity against LSCs to primary AML blasts. More importantly, we revealed that the CD226-extracellular signal-regulatory kinase1/2 (ERK1/2)-lysosome-associated membrane protein 1 (LAMP1) pathway is an important mechanism for Vγ9Vδ2 T cell-induced cytotoxicity against AML cells. First, Vγ9Vδ2 T cells recognized AML cells by receptor-ligand interaction of CD226-Nectin-2, which then induced ERK1/2 phosphorylation in Vγ9Vδ2 T cells. Finally, triggering the movement of lytic granules toward AML cells induced cytolysis of AML cells. The expression level of Nectin-2 may be used as a novel marker to predict the susceptibility/resistance of AML cells to Vγ9Vδ2 T cell treatment.
Subject(s)
Antigens, Differentiation, T-Lymphocyte/metabolism , Drug Resistance, Neoplasm , Leukemia, Myeloid, Acute/therapy , Lysosomal Membrane Proteins/metabolism , T-Lymphocytes, Cytotoxic/transplantation , Animals , Cytotoxicity, Immunologic , Female , HL-60 Cells , Humans , Immunotherapy, Adoptive , K562 Cells , Leukemia, Myeloid, Acute/immunology , MAP Kinase Signaling System , Mice , T-Lymphocytes, Cytotoxic/immunology , Treatment Outcome , Xenograft Model Antitumor AssaysSubject(s)
Epstein-Barr Virus Infections/therapy , Hematopoietic Stem Cell Transplantation , Immunotherapy, Adoptive , Lymphoproliferative Disorders/therapy , Paraproteinemias/therapy , Postoperative Complications/therapy , T-Lymphocytes, Cytotoxic/transplantation , Viremia/therapy , Blood Viscosity , Clone Cells/chemistry , Clone Cells/virology , Combined Modality Therapy , Female , Hematopoietic Stem Cell Transplantation/adverse effects , Herpesvirus 4, Human/immunology , Herpesvirus 4, Human/isolation & purification , Humans , Immunocompromised Host , Immunoglobulin M/blood , Immunosuppressive Agents/adverse effects , Lymphoproliferative Disorders/etiology , Lymphoproliferative Disorders/virology , Neoplastic Stem Cells/chemistry , Neoplastic Stem Cells/virology , Paraproteinemias/etiology , Paraproteinemias/virology , Paraproteins/analysis , Plasma Cells/chemistry , Plasma Cells/virology , Plasmapheresis , Remission Induction , T-Cell Antigen Receptor Specificity , T-Lymphocytes, Cytotoxic/immunology , Transplantation Conditioning/adverse effects , Viremia/etiology , Virus Activation , Young AdultABSTRACT
Advances in immunotherapy with T cells armed with chimeric antigen receptors (CAR-Ts), opened up new horizons for the treatment of B-cell lymphoid malignancies. However, the lack of appropriate targetable antigens on the malignant myeloid cell deprives patients with refractory acute myeloid leukaemia of effective CAR-T therapies. Although non-engineered T cells targeting multiple leukaemia-associated antigens [i.e. leukaemia-specific T cells (Leuk-STs)] represent an alternative approach, the prerequisite challenge to obtain high numbers of dendritic cells (DCs) for large-scale Leuk-ST generation, limits their clinical implementation. We explored the feasibility of generating bivalent-Leuk-STs directed against Wilms tumour 1 (WT1) and preferentially expressed antigen in melanoma (PRAME) from umbilical cord blood units (UCBUs) disqualified for allogeneic haematopoietic stem cell transplantation. By repurposing non-transplantable UCBUs and optimising culture conditions, we consistently produced at clinical scale, both cluster of differentiation (CD)34+ cell-derived myeloid DCs and subsequently polyclonal bivalent-Leuk-STs. Those bivalent-Leuk-STs contained CD8+ and CD4+ T cell subsets predominantly of effector memory phenotype and presented high specificity and cytotoxicity against both WT1 and PRAME. In the present study, we provide a paradigm of circular economy by repurposing unusable UCBUs and a platform for future banking of Leuk-STs, as a 'third-party', 'off-the-shelf' T-cell product for the treatment of acute leukaemias.
Subject(s)
Antigens, Neoplasm/immunology , Dendritic Cells/immunology , Fetal Blood/cytology , Immunotherapy, Adoptive/methods , Leukemia/therapy , T-Cell Antigen Receptor Specificity , T-Lymphocyte Subsets/immunology , WT1 Proteins/immunology , Antigens, CD/analysis , Blood Banks/economics , Cell Differentiation , Cells, Cultured , Cord Blood Stem Cell Transplantation/standards , Cytotoxicity, Immunologic , Dendritic Cells/cytology , Dendritic Cells/transplantation , Humans , Immunomagnetic Separation , Immunophenotyping , Immunotherapy, Adoptive/economics , Leukemia/economics , Memory T Cells/immunology , Memory T Cells/transplantation , T-Lymphocyte Subsets/transplantation , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/transplantationABSTRACT
Post-transplantation lymphoproliferative disorder (PTLD) is a severe complication of haematopoietic stem cell transplantation (HSCT), occurring in a setting of immune suppression and dysregulation. The disease is in most cases driven by the reactivation of the Epstein-Barr virus (EBV), which induces B cell proliferation through different pathomechanisms. Beyond EBV, many factors, variably dependent on HSCT-related immunosuppression, contribute to the disease development. PTLDs share several features with primary lymphomas, though clinical manifestations may be different, frequently depending on extranodal involvement. According to the WHO classification, histologic examination is required for diagnosis, allowing also to distinguish among PTLD subtypes. However, in cases of severe and abrupt presentation, a diagnosis based on a combination of imaging studies and EBV-load determination is accepted. Therapies include prophylactic and pre-emptive interventions, aimed at eradicating EBV proliferation before symptoms onset, and targeted treatments. Among them, rituximab has emerged as first-line option, possibly combined with a reduction of immunosuppression, while EBV-specific cytotoxic T lymphocytes are effective and safe alternatives. Though prognosis remains poor, survival has markedly improved following the adoption of the aforementioned treatments. The validation of innovative, combined approaches is the future challenge.
Subject(s)
Hematopoietic Stem Cell Transplantation/adverse effects , Lymphoproliferative Disorders/etiology , Adult , Aged , B-Lymphocytes/pathology , B-Lymphocytes/virology , Child , Diagnosis, Differential , Epstein-Barr Virus Infections/complications , Epstein-Barr Virus Infections/immunology , Graft vs Host Disease/drug therapy , Graft vs Host Disease/etiology , Graft vs Host Disease/prevention & control , Herpesvirus 4, Human/pathogenicity , Herpesvirus 4, Human/physiology , Humans , Immunocompromised Host , Immunosuppressive Agents/adverse effects , Immunosuppressive Agents/therapeutic use , Lymphoproliferative Disorders/epidemiology , Lymphoproliferative Disorders/therapy , Lymphoproliferative Disorders/virology , MicroRNAs/genetics , Middle Aged , Mutation , Prognosis , Risk Factors , Rituximab/therapeutic use , T-Lymphocytes, Cytotoxic/transplantation , Transplantation Conditioning/adverse effects , Transplantation, Homologous/adverse effects , Virus ActivationABSTRACT
NY-ESO-1-specific T cells have shown promising activity in the treatment of soft tissue sarcoma (STS). However, standardized protocols for their generation are limited. Particularly, cost-effectiveness considerations of cell production protocols are of importance for conducting clinical studies. In this study, two different NY-ESO-1-specific T cell production protocols were compared. Major differences between protocols 1 and 2 include culture medium, interleukin-2 and retronectin concentrations, T cell activation strategy, and the transduction process. NY-ESO-1-specific T cells generated according to the two protocols were investigated for differences in cell viability, transduction efficiency, T cell expansion, immunophenotype as well as functionality. NY-ESO-1-specific T cells showed similar viability and transduction efficiency between both protocols. Protocol 1 generated higher absolute numbers of NY-ESO-1-specific T cells. However, there was no difference in absolute numbers of NY-ESO-1-specific T cell subsets with less-differentiated phenotypes accounting for efficient in vivo expansion and engraftment. Furthermore, cells generated according to protocol 1 displayed higher capacity of TNF-α generation, but lower cytotoxic capacities. Overall, both protocols provided functional NY-ESO-1-specific T cells. However, compared to protocol 1, protocol 2 is advantageous in terms of cost-effectiveness. Cell production protocols should be designed diligently to achieve a cost-effective cellular product for further clinical evaluation.
Subject(s)
Antigens, Neoplasm/immunology , Membrane Proteins/immunology , Sarcoma/immunology , T-Lymphocytes, Cytotoxic/immunology , Adoptive Transfer , Cell Culture Techniques , Cell Line , Humans , Sarcoma/therapy , T-Lymphocytes, Cytotoxic/transplantationABSTRACT
Clinical successes demonstrated by chimeric antigen receptor T-cell immunotherapy have facilitated further development of T-cell immunotherapy against wide variety of diseases. One approach is the development of "off-the-shelf" T-cell sources. Technologies to generate T-cells from pluripotent stem cells (PSCs) may offer platforms to produce "off-the-shelf" and synthetic allogeneic T-cells. However, low differentiation efficiency and poor scalability of current methods may compromise their utilities. Here we show improved differentiation efficiency of T-cells from induced PSCs (iPSCs) derived from an antigen-specific cytotoxic T-cell clone, or from T-cell receptor (TCR)-transduced iPSCs, as starting materials. We additionally describe feeder-free differentiation culture systems that span from iPSC maintenance to T-cell proliferation phases, enabling large-scale regenerated T-cell production. Moreover, simultaneous addition of SDF1α and a p38 inhibitor during T-cell differentiation enhances T-cell commitment. The regenerated T-cells show TCR-dependent functions in vitro and are capable of in vivo anti-tumor activity. This system provides a platform to generate a large number of regenerated T-cells for clinical application and investigate human T-cell differentiation and biology.
Subject(s)
Cell Culture Techniques/methods , Immunotherapy, Adoptive/methods , Induced Pluripotent Stem Cells/physiology , Neoplasms/therapy , T-Lymphocytes, Cytotoxic/transplantation , Animals , Cell Differentiation/drug effects , Cell Line, Tumor , Chemokine CXCL12/metabolism , Culture Media/metabolism , Culture Media/pharmacology , Female , Humans , Imidazoles/pharmacology , Mice , Neoplasms/immunology , Pyridines/pharmacology , Receptors, Chimeric Antigen/immunology , T-Lymphocytes, Cytotoxic/immunology , Xenograft Model Antitumor AssaysABSTRACT
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
Adoptively transferred T cell receptor (TCR)-transgenic T cells (TCR-T cells) are not restricted by cell surface expression of their targets and are therefore poised to become a main pillar of cellular cancer immunotherapies. Addressing clinical and laboratory data, we discuss emerging features for the efficient deployment of novel TCR-T therapies, such as selection of ideal TCRs targeting validated epitopes with well-characterized cancer cell expression and processing, enhancing TCR-T effector function, trafficking, expansion, persistence, and memory formation by strategic selection of substrate cells, and gene-engineering with synthetic co-stimulatory circuits. Overall, a better understanding of the relevant mechanisms of action and resistance will help prioritize the vast array of potential TCR-T optimizations for future clinical products.
Subject(s)
Immunotherapy, Adoptive/methods , Neoplasms/therapy , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes, Cytotoxic/transplantation , T-Lymphocytes, Helper-Inducer/transplantation , Animals , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , Antigens, Neoplasm/metabolism , Autoantigens/genetics , Autoantigens/immunology , Autoantigens/metabolism , Clinical Trials as Topic , Disease Models, Animal , Humans , Mice , Mutation , Neoplasms/genetics , Neoplasms/immunology , Protein Engineering , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Helper-Inducer/metabolism , Treatment OutcomeABSTRACT
Hsp60sp, a signal peptide derived from the leader sequence of heat shock protein 60 kDa (Hsp60), is a Qa-1/HLA-E-binding peptide. We previously showed that Hsp60sp-specific CD8+ T cells are involved in the immunoregulation of autoimmune diseases by controlling the response of self-reactive lymphocytes. Here, we report that Hsp60sp-specific CD8+ T cells killed malignant lymphocytes in vitro independently of transporter associated with antigen processing (TAP) and classical MHC-I expression. Induction of this cytotoxic T lymphocyte (CTL) response in vivo, either by adoptive transfer of in vitro-amplified CTLs or peptide-loaded dendritic cell immunization, resulted in effective control of lymphoid tumors, including TAP- or classical MHC-I-deficient cells. Hsp60sp-specific immune activation combined with programmed cell death protein 1 (PD-1) blocking synergistically restrained mouse lymphoma development. Importantly, Hsp60sp-specific CD8+ T cells did not negatively affect normal tissues and cells. Our data suggest that Hsp60sp-based immunotherapy is an inviting strategy to control lymphoid malignancies.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Chaperonin 60/chemistry , Dendritic Cells/immunology , Immune Checkpoint Inhibitors/administration & dosage , Lymphoma/therapy , Mitochondrial Proteins/chemistry , Protein Sorting Signals/physiology , T-Lymphocytes, Cytotoxic/immunology , Animals , Antigen Presentation , CD8-Positive T-Lymphocytes/transplantation , Cell Line , Chaperonin 60/immunology , Combined Modality Therapy , Dendritic Cells/transplantation , Female , Histocompatibility Antigens Class I/metabolism , Immune Checkpoint Inhibitors/pharmacology , Immunization , Lymphoma/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mitochondrial Proteins/immunology , T-Lymphocytes, Cytotoxic/transplantationABSTRACT
Renal cell carcinoma (RCC) is not sensitive to conventional radiotherapy and chemotherapy, and the effectiveness rate of molecular targeted therapy is low. Therefore, it is urgent to identify new treatment methods. Recently, adoptive Tcell therapy has provided a new option for cancer treatment. Furthermore, lowdose chemotherapy not only has no evident side effects and inhibitory effects on the human immune system, but can also enhance the immune activity of some effector cells. Therefore, it is surmised that the combination of different mechanisms of chemotherapy and immunotherapy could be a new treatment concept. In the present study, the effects of lowdose chemotherapy combined with T cells in the treatment of renal cell carcinoma were explored using cytotoxicity assays, enzymelinked immunosorbent assay (ELISA), western blot analysis and flow cytometric analysis. The results revealed that lowdose chemotherapy and T cells had synergistic effects on tumor cell elimination in vitro. The transforming growth factor (TGF)ß signaling pathway may be involved in the inhibition of Tcell functions. The targeted inhibition of TGFß signals may be a promising therapeutic strategy for the treatment of renal cancer. The present results provided a novel strategy for the combination of lowdose chemotherapy and T cells to enhance the therapeutic efficacy of RCC treatment.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Carcinoma, Renal Cell/therapy , Immunotherapy, Adoptive/methods , Kidney Neoplasms/therapy , T-Lymphocytes, Cytotoxic/transplantation , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Renal Cell/immunology , Carcinoma, Renal Cell/pathology , Cell Line, Tumor , Chemotaxis, Leukocyte/drug effects , Chemotaxis, Leukocyte/immunology , Cisplatin/pharmacology , Cisplatin/therapeutic use , Combined Modality Therapy/methods , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Female , Humans , Kidney Neoplasms/immunology , Kidney Neoplasms/pathology , Mice , Mitomycin/pharmacology , Mitomycin/therapeutic use , Primary Cell Culture , Signal Transduction/drug effects , Signal Transduction/immunology , T-Lymphocytes, Cytotoxic/drug effects , T-Lymphocytes, Cytotoxic/immunology , Transforming Growth Factor beta/antagonists & inhibitors , Transforming Growth Factor beta/metabolismABSTRACT
The success of cancer immunotherapy is limited by resistance to immune checkpoint blockade. We therefore conducted a genetic screen to identify genes that mediated resistance against CTLs in anti-PD-L1 treatment-refractory human tumors. Using PD-L1-positive multiple myeloma cells cocultured with tumor-reactive bone marrow-infiltrating CTL as a model, we identified calcium/calmodulin-dependent protein kinase 1D (CAMK1D) as a key modulator of tumor-intrinsic immune resistance. CAMK1D was coexpressed with PD-L1 in anti-PD-L1/PD-1 treatment-refractory cancer types and correlated with poor prognosis in these tumors. CAMK1D was activated by CTL through Fas-receptor stimulation, which led to CAMK1D binding to and phosphorylating caspase-3, -6, and -7, inhibiting their activation and function. Consistently, CAMK1D mediated immune resistance of murine colorectal cancer cells in vivo The pharmacologic inhibition of CAMK1D, on the other hand, restored the sensitivity toward Fas-ligand treatment in multiple myeloma and uveal melanoma cells in vitro Thus, rapid inhibition of the terminal apoptotic cascade by CAMK1D expressed in anti-PD-L1-refractory tumors via T-cell recognition may have contributed to tumor immune resistance.
Subject(s)
B7-H1 Antigen/antagonists & inhibitors , Calcium-Calmodulin-Dependent Protein Kinase Type 1/immunology , Immunotherapy/methods , Neoplasms/immunology , Neoplasms/therapy , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/transplantation , Animals , B7-H1 Antigen/biosynthesis , B7-H1 Antigen/immunology , Calcium-Calmodulin-Dependent Protein Kinase Type 1/biosynthesis , Drug Resistance, Neoplasm , Humans , Mice , Multiple Myeloma/immunology , Multiple Myeloma/therapyABSTRACT
INTRODUCTION: The activating NKG2D receptor plays a central role in the immune recognition and elimination of abnormal self-cells by cytotoxic lymphocytes. NKG2D binding to cell stress-inducible ligands (NKG2DL) up-regulated on cancer cells facilitates their immunorecognition. Yet tumor cells utilize various escape mechanisms to avert NKG2D-based immunosurveillance. Hence, therapeutic strategies targeting the potent NKG2D/NKG2DL axis and such immune escape mechanisms become increasingly attractive in cancer therapy. AREAS COVERED: This perspective provides a brief introduction into the NKG2D/NKG2DL axis and its relevance for cancer immune surveillance. Subsequently, the most advanced therapeutic approaches targeting the NKG2D system are presented focusing on NKG2D-CAR engineered immune cells and antibody-mediated strategies to inhibit NKG2DL shedding by tumors. EXPERT OPINION: Thus far, NKG2D-CAR engineered lymphocytes represent the most advanced therapeutic approach utilizing the NKG2D system. Similarly to other tumor-targeting CAR approaches, NKG2D-CAR cells demonstrate powerful on-target activity, but may also cause off-tumor toxicities or lose efficacy, if NKG2DL expression by tumors is reduced. However, NKG2D-CAR cells also act on the tumor microenvironment curtailing its immunosuppressive properties, thus providing an independent therapeutic benefit. The potency of tumoricidal NKG2D-expressing lymphocytes can be further boosted by enhancing NKG2DL expression through small molecules and therapeutic antibodies inhibiting tumor-associated shedding of NKG2DL.
Subject(s)
Immunotherapy, Adoptive/methods , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Neoplasms/therapy , T-Lymphocytes, Cytotoxic/metabolism , Animals , Combined Modality Therapy/methods , Genetic Therapy/methods , Humans , Immunologic Surveillance/genetics , Immunologic Surveillance/physiology , Ligands , NK Cell Lectin-Like Receptor Subfamily K/genetics , NK Cell Lectin-Like Receptor Subfamily K/immunology , Neoplasms/genetics , Neoplasms/immunology , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/metabolism , T-Lymphocytes, Cytotoxic/transplantation , Tumor Microenvironment/immunologyABSTRACT
The efficacy and safety of Epstein-Barr virus (EBV)-specific antigen peptide-activated cytotoxic T lymphocytes (CTLs) in the treatment of refractory or recurrent angioimmunoblastic T-cell lymphoma (AITL) was determined in this prospective one-arm clinical study. Seven males and two females were enrolled with a median age of 70 years. The tumor stages were all stage III and IV. All patients had group B symptoms and IPI scores of 3 to 5 points. All patients received chemotherapy before CTLs infusion which the median chemotherapy cycle was three. The diseases states before CTLs included five cases of disease progression (PD), two cases of recurrence (R), and two cases with residual lesions after chemotherapy. Eight patients received HLA-haploidentical EBV-specific CTLs, and one patient chose autologous CTLs. The number of transfused cells was 1.67 to 2.38 × 1010 for one course of CTLs therapy. One patient was treated with three courses of CTLs, three patients were treated with two courses of CTLs, and five patients were treated with one course of CTLs. During the infusion, eight patients had fever, one patient had rash, and no graft-vs-host diseases were observed. The EBV-DNA decreased by more than two orders of magnitude in six patients, and the response rate was 66.7%. Two patients of PD status achieved complete remission (CR), one patient of PD status achieved partial remission, two patients with residual lesions after chemotherapy achieved CR, and four patients had no response. The objective remission rate was 55.6%. After the median follow-up of 14.5 months, five patients died, and three patients were completely relieved while one patient was lost during follow-up. The 3-year overall survival was 44.4% and 3-year progression-free survival was 33.3%. EBV-specific antigen peptide-activated CTLs showed positive effect in certain patients with refractory and recurrent AITL with high clinical safety.
Subject(s)
Immunotherapy/methods , Lymphoma, T-Cell/therapy , Neoplasm Recurrence, Local/therapy , Peptide Fragments/immunology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/transplantation , Viral Matrix Proteins/immunology , Adult , Aged , Drug Resistance, Neoplasm , Female , Follow-Up Studies , Humans , Lymphoma, T-Cell/immunology , Lymphoma, T-Cell/pathology , Male , Middle Aged , Neoplasm Recurrence, Local/immunology , Neoplasm Recurrence, Local/pathology , Prognosis , Prospective Studies , Remission Induction , Salvage TherapyABSTRACT
Breast cancer is the most common oncological pathology in women worldwide. Techniques for improving the clinical parameters of patients undergoing combination therapy for breast cancer are currently under development. A type of treatment employing dendritic cells (DCs) and cytotoxic DCinduced antigenspecific T lymphocytes efficiently eliminates residual cancer cells that are the key cause of tumor recurrence and metastasis. In the present study, DCs and activated lymphocytes (treated with IL12 and IL18) were isolated from the peripheral blood of patients with breast cancer, using a lysate of tumor tissue as antigen. The patients received the cells as part of adjuvant or neoadjuvant regimens (stage IV disease or progression). Evaluation of immunity was performed at 3 and 6 months after terminating immunotherapy. Evaluation of the diseasefree period was performed for 3 years after surgery. The use of antigenloaded autologous DCs combined with mononuclear cells with increased cytotoxic activity following Th1 polarization reduced the populations of immunosuppressive cells. The results of the present study demonstrated that the investigated cellular immunotherapy for breast cancer is safe, reduces the risk of relapse and metastasis, and improves immunity by reducing the number of regulatory T cells. Therefore, this therapeutic strategy may represent a novel approach to combating distant metastases of breast cancer.