RESUMO
Recent advancements in T cell immunotherapy suggest that T cells engineered with high-affinity TCR can offer better tumor regression. However, whether a high-affinity TCR alone is sufficient to control tumor growth, or the T cell subset bearing the TCR is also important remains unclear. Using the human tyrosinase epitope-reactive, CD8-independent, high-affinity TCR isolated from MHC class I-restricted CD4(+) T cells obtained from tumor-infiltrating lymphocytes (TIL) of a metastatic melanoma patient, we developed a novel TCR transgenic mouse with a C57BL/6 background. This HLA-A2-restricted TCR was positively selected on both CD4(+) and CD8(+) single-positive cells. However, when the TCR transgenic mouse was developed with a HLA-A2 background, the transgenic TCR was primarily expressed by CD3(+)CD4(-)CD8(-) double-negative T cells. TIL 1383I TCR transgenic CD4(+), CD8(+), and CD4(-)CD8(-) T cells were functional and retained the ability to control tumor growth without the need for vaccination or cytokine support in vivo. Furthermore, the HLA-A2(+)/human tyrosinase TCR double-transgenic mice developed spontaneous hair depigmentation and had visual defects that progressed with age. Our data show that the expression of the high-affinity TIL 1383I TCR alone in CD3(+) T cells is sufficient to control the growth of murine and human melanoma, and the presence or absence of CD4 and CD8 coreceptors had little effect on its functional capacity.
Assuntos
Autoimunidade , Imunoterapia Adotiva/métodos , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Complexo CD3/imunologia , Citometria de Fluxo , Antígeno HLA-A2/imunologia , Humanos , Linfócitos do Interstício Tumoral/imunologia , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Persistence of effector cytotoxic T lymphocytes (CTLs) during an immunological response is critical for successfully controlling a viral infection or tumor growth. Various cytokines are known to play an important part in regulating the immune response. The IL-2 family of cytokines that includes IL-2 and IL-15 are known to function as growth and survival factors for antigen-experienced T cells. IL-2 and IL-15 possess similar properties, including the ability to induce T cell proliferation. Whereas long-term IL-2 exposure has been shown to promote apoptosis and limit CD8(+) memory T cell survival and proliferation, it is widely believed that IL-15 can inhibit apoptosis and helps maintain a memory CD8(+) T-cell population. However, mechanisms for superior outcomes for IL-15 as compared to IL-2 are still under investigation. Our data shows that human T cells cultured in the presence of IL-15 exhibit increased expression of anti-oxidant molecules glutathione reductase (GSR), thioredoxin reductase 1 (TXNDR1), peroxiredoxin (PRDX) and superoxide dismutase (SOD). An increased expression of cell-surface thiols, intracellular glutathione, and thioredoxins was also noted in IL-15 cultured T cells. Additionally, IL-15 cultured T cells showed an increase in cytolytic effector molecules. Apart from increased level of Granzyme A and Granzyme B, IL-15 cultured T cells exhibited increased accumulation of reactive oxygen (ROS) and reactive nitrogen species (RNS) as compared to IL-2 cultured T cells. Overall, this study suggests that T cells cultured in IL-15 show increased persistence not only due to levels of anti-apoptotic proteins, but also due to increased anti-oxidant levels, which is complimented by increased cytolytic effector functions.
Assuntos
Antioxidantes/metabolismo , Imunidade Inata/imunologia , Interleucina-15/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Apoptose/imunologia , Proliferação de Células , Células Cultivadas , Humanos , Peróxido de Hidrogênio/metabolismo , Interleucina-15/imunologia , Interleucina-2/imunologia , Potencial da Membrana Mitocondrial , Óxido Nítrico/metabolismo , Oxidantes/metabolismo , Estresse Oxidativo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
The activating NK cell receptor DNAX accessory molecule-1 (DNAM-1) contributes to tumor immune surveillance and plays a crucial role in NK cell-mediated recognition of several types of human tumors, including ovarian carcinoma. Here, we have analyzed the receptor repertoire and functional integrity of NK cells in peritoneal effusions from patients with ovarian carcinoma. Relative to autologous peripheral blood NK cells, tumor-associated NK cells expressed reduced levels of the DNAM-1, 2B4, and CD16 receptors and were hyporesponsive to HLA class I-deficient K562 cells and to coactivation via DNAM-1 and 2B4. Moreover, tumor-associated NK cells were also refractory to CD16 receptor stimulation, resulting in diminished Ab-dependent cellular cytotoxicity against autologous tumor cells. Coincubation of NK cells with ovarian carcinoma cells expressing the DNAM-1 ligand CD155 led to reduction of DNAM-1 expression. Therefore, NK cell-mediated rejection of ovarian carcinoma may be limited by perturbed DNAM-1 expression on tumor-associated NK cells induced by chronic ligand exposure. Thus, these data support the notion that tumor-induced alterations of activating NK cell receptor expression may hamper immune surveillance and promote tumor progression.
Assuntos
Antígenos de Diferenciação de Linfócitos T/metabolismo , Carcinoma/imunologia , Células Matadoras Naturais/imunologia , Neoplasias Ovarianas/imunologia , Receptores Virais/metabolismo , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/imunologia , Líquido Ascítico/imunologia , Líquido Ascítico/metabolismo , Carcinoma/metabolismo , Feminino , Proteínas Ligadas por GPI , Humanos , Células Matadoras Naturais/metabolismo , Neoplasias Ovarianas/metabolismo , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Receptores Virais/imunologia , Família de Moléculas de Sinalização da Ativação LinfocitáriaRESUMO
NK cells are promising effectors for tumor adoptive immunotherapy, particularly when considering the targeting of MHC class I low or negative tumors. Yet, NK cells cannot respond to many tumors, which is particularly the case for nonhematopoietic tumors such as carcinomas or melanoma even when these cells lose MHC class I surface expression. Therefore, we targeted primary human NK cells by gene transfer of an activating chimeric receptor specific for HER-2, which is frequently overexpressed on carcinomas. We found that these targeted NK cells were specifically activated upon recognition of all evaluated HER-2 positive tumor cells, including autologous targets, as indicated by high levels of cytokine secretion as well as degranulation. The magnitude of this specific response correlated with the level of HER-2 expression on the tumor cells. Finally, these receptor transduced NK cells, but not their mock transduced counterpart, efficiently eradicated tumor cells in RAG2 knockout mice as visualized by in vivo imaging. Taken together, these results indicate that the expression of this activating receptor overrides inhibitory signals in primary human NK cells and directs them specifically toward HER-2 expressing tumor cells both in vitro and in vivo.
Assuntos
Carcinoma/imunologia , Carcinoma/terapia , Imunoterapia Adotiva/métodos , Células Matadoras Naturais/imunologia , Receptor ErbB-2/imunologia , Animais , Proteínas de Ligação a DNA/genética , Engenharia Genética/métodos , Vetores Genéticos/genética , Humanos , Luciferases , Camundongos , Camundongos Knockout , Receptor ErbB-2/metabolismoRESUMO
Objective clinical responses can be achieved in melanoma patients by infusion of T cell receptor (TCR) gene transduced T cells. Although promising, the therapy is still largely ineffective, as most patients did not benefit from treatment. That only a minority of the infused T cells were genetically modified and that these were extensively expanded ex vivo may have prevented their efficacy. We developed novel and generally applicable retroviral vectors that allow rapid and efficient selection of T cells transduced with human TCRs. These vectors encode two TCR chains and a truncated CD34 molecule (CD34t) in a single mRNA transcript. Transduced T cells were characterized and the effects of CD34-based enrichment of redirected T cells were evaluated. Both CD8(+) and CD4(+) T cells could be transduced and efficiently co-expressed all introduced transgenes on their surface. Importantly, more than fivefold enrichment of both the frequency of transduced cells and the specific anti-tumor reactivity of the effector population could be achieved by magnetic beads-based enrichment procedures readily available for clinical grade hematopoietic stem cell isolation. This CD34-based enrichment technology will improve the feasibility of adoptive transfer of clinically relevant effectors. In addition to their enhanced tumor recognition, the enriched redirected T cells may also show superior reactivity and persistence in vivo due to the high purity of transduced cells and the shortened ex vivo culture.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer , Imunoterapia Adotiva , Melanoma/imunologia , Melanoma/terapia , Retroviridae , Antígenos CD34/genética , Antígenos CD34/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/transplante , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/transplante , Genes Codificadores dos Receptores de Linfócitos T/genética , Engenharia Genética , Humanos , Separação Imunomagnética , Melanoma/genética , Melanoma/patologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Transdução GenéticaRESUMO
BACKGROUND: Adjuvant trastuzumab (Herceptin) treatment of breast cancer patients significantly improves their clinical outcome. Vaccination is an attractive alternative approach to provide HER-2/neu (Her2)-specific antibodies and may in addition concomitantly stimulate Her2-reactive T-cells. Here we report the first administration of a Her2-plasmid DNA (pDNA) vaccine in humans. PATIENTS AND METHODS: The vaccine, encoding a full-length signaling-deficient version of the oncogene Her2, was administered together with low doses of GM-CSF and IL-2 to patients with metastatic Her2-expressing breast carcinoma who were also treated with trastuzumab. Six of eight enrolled patients completed all three vaccine cycles. In the remaining two patients treatment was discontinued after one vaccine cycle due to rapid tumor progression or disease-related complications. The primary objective was the evaluation of safety and tolerability of the vaccine regimen. As a secondary objective, treatment-induced Her2-specific immunity was monitored by measuring antibody production as well as T-cell proliferation and cytokine production in response to Her2-derived antigens. RESULTS: No clinical manifestations of acute toxicity, autoimmunity or cardiotoxicity were observed after administration of Her2-pDNA in combination with GM-CSF, IL-2 and trastuzumab. No specific T-cell proliferation following in vitro stimulation of freshly isolated PBMC with recombinant human Her2 protein was induced by the vaccination. Immediately after all three cycles of vaccination no or even decreased CD4+ T-cell responses towards Her2-derived peptide epitopes were observed, but a significant increase of MHC class II restricted T-cell responses to Her2 was detected at long term follow-up. Since concurrent trastuzumab therapy was permitted, lambda-subclass specific ELISAs were performed to specifically measure endogenous antibody production without interference by trastuzumab. Her2-pDNA vaccination induced and boosted Her2-specific antibodies that could be detected for several years after the last vaccine administration in a subgroup of patients. CONCLUSION: This pilot clinical trial demonstrates that Her2-pDNA vaccination in conjunction with GM-CSF and IL-2 administration is safe, well tolerated and can induce long-lasting cellular and humoral immune responses against Her2 in patients with advanced breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , DNA/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Interleucina-2/uso terapêutico , Receptor ErbB-2/imunologia , Vacinação , Adulto , Idoso , Anticorpos Antineoplásicos/imunologia , Neoplasias da Mama/patologia , Vacinas Anticâncer/imunologia , Relação Dose-Resposta a Droga , Feminino , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Imunidade Humoral/imunologia , Pessoa de Meia-Idade , Metástase Neoplásica/tratamento farmacológico , Metástase Neoplásica/imunologia , Projetos Piloto , Plasmídeos/genética , Receptor ErbB-2/genética , Linfócitos T/imunologiaRESUMO
Patients with diseases characterized by chronic inflammation, caused by infection or cancer, have T cells and NK cells with impaired function. The underlying molecular mechanisms are diverse, but one of the major mediators in this immune suppression is oxidative stress caused by activated monocytes, granulocytes, or myeloid-derived suppressor cells. Reactive oxygen species can seriously hamper the efficacy of active immunotherapy and adoptive transfer of T and NK cells into patients. In this study, we have evaluated whether enhanced expression of the antioxidant enzyme catalase in human T cells can protect them against reactive oxygen species. Human CD4(+) and CD8(+) T cells retrovirally transduced with the catalase gene had increased intracellular expression and activity of catalase. Catalase transduction made CD4(+) T cells less sensitive to H(2)O(2)-induced loss-of-function, measured by their cytokine production and ability to expand in vitro following anti-CD3 stimulation. It also enhanced the resistance to oxidative stress-induced cell death after coculture with activated granulocytes, exposure to the oxidized lipid 4-hydroxynonenal, or H(2)O(2). Expression of catalase by CMV-specific CD8(+) T cells saved cells from cell death and improved their capacity to recognize CMV peptide-loaded target cells when exposed to H(2)O(2). These findings indicate that catalase-transduced T cells potentially are more efficacious for the immunotherapy of patients with advanced cancer or chronic viral infections.
Assuntos
Antioxidantes/metabolismo , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD8-Positivos/enzimologia , Catalase/genética , Estresse Oxidativo/genética , Estresse Oxidativo/imunologia , Transdução Genética , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Catalase/biossíntese , Morte Celular/genética , Morte Celular/imunologia , Linhagem Celular , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Citocinas/biossíntese , Vetores Genéticos , Humanos , Peróxido de Hidrogênio/farmacologia , Espécies Reativas de Oxigênio/efeitos adversos , Espécies Reativas de Oxigênio/metabolismo , Retroviridae/genéticaRESUMO
To study DNA vaccination directed against human HER-2 in the HHD mouse Tg strain, we created a novel HER-2-expressing syngeneic tumor transplantation model. We found that a DNA vaccine encoding the full length HER-2 DNA protected HHD mice from HER-2(+) tumor challenge by a CTL independent mechanism. A more efficient approach to induce HLA-A2 restricted CTLs, through immunization with a multi-epitope DNA vaccine expressing the HLA-A2 restricted HER-2 369-377, 435-443 and 689-697 epitopes, resulted in high numbers of peptide specific T cells but failed to induce tumor protection. Subsequently we discovered that HER-2 transfected tumor cells down-regulated MHC class I antigen expression and exhibited a series of defects in the antigen processing pathway which impaired the capacity to produce and display MHC class I peptide-ligands to specific CTLs. Our data demonstrate that HER-2 transfection is associated with defects in the MHC class I presentation pathway, which may be the underlying mechanism behind the inability of CTLs to recognize tumors in this HLA-A2 transgenic model. As defective MHC class I presentation may be a common characteristic of HER-2 expressing tumors, vaccines targeting HER-2 should aim at inducing an integrated immune response where also CD4(+) T cells and antibodies are important components.
Assuntos
Vacinas Anticâncer/imunologia , Antígeno HLA-A2/genética , Receptor ErbB-2/imunologia , Sarcoma Experimental/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas de DNA/imunologia , Sequência de Aminoácidos , Animais , Apresentação de Antígeno , Sequência de Bases , Epitopos de Linfócito T/imunologia , Genes MHC Classe I , Genes erbB-2 , Antígeno HLA-A2/imunologia , Humanos , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Receptor ErbB-2/genética , Sarcoma Experimental/induzido quimicamente , TransfecçãoRESUMO
Although natural killer (NK) cells are well known for their ability to kill tumors, few studies have addressed the interactions between resting (nonactivated) NK cells and freshly isolated human tumors. Here, we show that human leukocyte antigen class I(low) tumor cells isolated directly from patients with advanced ovarian carcinoma trigger degranulation by resting allogeneic NK cells. This was paralleled by induction of granzyme B and caspase-6 activities in the tumor cells and significant tumor cell lysis. Ovarian carcinoma cells displayed ubiquitous expression of the DNAX accessory molecule-1 (DNAM-1) ligand PVR and sparse/heterogeneous expression of the NKG2D ligands MICA/MICB and ULBP1, ULBP2, and ULBP3. In line with the NK receptor ligand expression profiles, antibody-mediated blockade of activating receptor pathways revealed a dominant role for DNAM-1 and a complementary contribution of NKG2D signaling in tumor cell recognition. These results show that resting NK cells are capable of directly recognizing freshly isolated human tumor cells and identify ovarian carcinoma as a potential target for adoptive NK cell-based immunotherapy.
Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , Células Matadoras Naturais/imunologia , Neoplasias Ovarianas/imunologia , Degranulação Celular/imunologia , Feminino , Granzimas/metabolismo , Humanos , Células K562 , Ligantes , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Neoplasias Ovarianas/enzimologia , Receptores Imunológicos/imunologia , Receptores de Células Matadoras NaturaisRESUMO
BACKGROUND: γ9δ2T cells, which express Vγ9 and Vδ2 chains of the T cell receptor (TCR), mediate cancer immune surveillance by sensing early metabolic changes in malignant leukemic blast and not their healthy hematopoietic stem counterparts via the γ9δ2TCR targeting joined conformational and spatial changes of CD277 at the cell membrane (CD277J). This concept led to the development of next generation CAR-T cells, so-called TEGs: αßT cells Engineered to express a defined γδTCR. The high affinity γ9δ2TCR clone 5 has recently been selected within the TEG format as a clinical candidate (TEG001). However, exploring safety and efficacy against a target, which reflects an early metabolic change in tumor cells, remains challenging given the lack of appropriate tools. Therefore, we tested whether TEG001 is able to eliminate established leukemia in a primary disease model, without harming other parts of the healthy hematopoiesis in vivo. METHODS: Separate sets of NSG mice were respectively injected with primary human acute myeloid leukemia (AML) blasts and cord blood-derived human progenitor cells from healthy donors. These mice were then treated with TEG001 and mock cells. Tumor burden and human cells engraftment were measured in peripheral blood and followed up over time by quantifying for absolute cell number by flow cytometry. Statistical analysis was performed using non-parametric 2-tailed Mann-Whitney t-test. RESULTS: We successfully engrafted primary AML blasts and healthy hematopoietic cells after 6-8 weeks. Here we report that metabolic cancer targeting through TEG001 eradicated established primary leukemic blasts in vivo, while healthy hematopoietic compartments derived from human cord-blood remained unharmed in spite of TEGs persistence up to 50 days after infusion. No additional signs of off-target toxicity were observed in any other tissues. CONCLUSION: Within the limitations of humanized PD-X models, targeting CD277J by TEG001 is safe and efficient. Therefore, we have initiated clinical testing of TEG001 in a phase I first-in-human clinical trial (NTR6541; date of registration 25 July 2017).
Assuntos
Antineoplásicos/farmacologia , Células Sanguíneas/efeitos dos fármacos , Butirofilinas/antagonistas & inibidores , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Animais , Antígenos CD , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Células Sanguíneas/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Células-Tronco Hematopoéticas/metabolismo , Histocitoquímica , Humanos , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Chemotherapy-resistant cancer recurrence is a major cause of mortality. In acute myeloid leukemia (AML), chemorefractory relapses result from the complex interplay between altered genetic, epigenetic and transcriptional states in leukemic cells. Here, we develop an experimental model system using in vitro lineage tracing coupled with exome, transcriptome and in vivo functional readouts to assess the AML population dynamics and associated molecular determinants underpinning chemoresistance development. We find that combining standard chemotherapeutic regimens with low doses of DNA methyltransferase inhibitors (DNMTi, hypomethylating drugs) prevents chemoresistant relapses. Mechanistically, DNMTi suppresses the outgrowth of a pre-determined set of chemoresistant AML clones with stemness properties, instead favoring the expansion of rarer and unfit chemosensitive clones. Importantly, we confirm the capacity of DNMTi combination to suppress stemness-dependent chemoresistance development in xenotransplantation models and primary AML patient samples. Together, these results support the potential of DNMTi combination treatment to circumvent the development of chemorefractory AML relapses.
Assuntos
Metilação de DNA , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Mieloide/genética , Transcriptoma/genética , Doença Aguda , Antibióticos Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Linhagem da Célula/genética , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Decitabina/uso terapêutico , Doxorrubicina/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/patologiaRESUMO
Defective expression of HLA class I molecules is common in tumor cells and may allow escape from CTL-mediated immunity. We here investigate alterations in expression of HLA class I and their underlying molecular mechanisms in ovarian cancer patients. The HLA class I and HLA-A2 expression levels on noncultured tumor cells of 12 patients diagnosed with ovarian carcinoma were investigated by flow cytometry. Molecular analyses of antigen-processing machinery (APM) components were done in metastatic cancer cells, and the HLA genotype was determined in both these and the primary tumor. HER-2/neu-specific immunity was evaluated by enzyme-linked immunospot assays. The metastatic tumor cells from all patients expressed low levels of HLA class I surface antigens. In six of nine HLA-A2+ patients, HLA-A2 expression was heterogeneous with a subpopulation of tumor cells exhibiting decreased or absent HLA-A2 expression. One patient-derived tumor cell line completely lacked HLA-A2 but exhibited constitutive expression of APM components and high HLA class I expression that was further inducible by IFN-gamma treatment. Genotyping showed a haplotype loss in the metastatic tumor cells, whereas tumor tissue microdissected from the primary tumor exhibited an intact HLA gene complex. Interestingly, HLA-A2-restricted HER-2/neu-specific T-cell responses were evident among the lymphocytes of this patient. Abnormalities in HLA class I antigen expression are common features during the progression of ovarian cancer, and haplotype loss was, for the first time, described as an underlying mechanism.
Assuntos
Antígeno HLA-A2/biossíntese , Neoplasias Ovarianas/imunologia , Receptor ErbB-2/imunologia , Idoso , Idoso de 80 Anos ou mais , Feminino , Citometria de Fluxo , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Haplótipos/imunologia , Humanos , Pessoa de Meia-Idade , Metástase Neoplásica , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Receptor ErbB-2/genética , Linfócitos T/imunologiaRESUMO
IFN-gamma regulates the immunogenicity of target cells by increasing their expression of HLA class I molecules. This facilitates the T cell receptor-mediated recognition by CD8(+) T cells but decreases target cell sensitivity to lysis by NK cells due to engagement of inhibitory NK receptors. In this study, short-term tumor cell lines from patients with advanced ovarian carcinomas were established. We demonstrate the paradoxical finding that IFN-gamma treatment of these short-term ovarian carcinoma cell lines (OVACs) resulted in resistance of tumor cells to lysis by peptide- and allospecific CD8(+) T cells. Blocking experiments revealed that this phenomenon was dependent on enhanced inhibitory signalling via CD94/NKG2A receptors expressed on the effector cells. This was associated with increased expression of HLA-E mRNA and HLA-G at the protein level in IFN-gamma-treated OVACs. Furthermore, pulsing of untreated OVACs with the leader sequence peptide of HLA-G protected these cells from lysis by CTLs, thus mimicking the inhibitory effect of IFN-gamma. This study provides evidence that CD94/NKG2A receptors play an important role in regulating T cell activity against tumors and shows that IFN-gamma modulation of target cells may shift the balance of triggering and inhibitory signals to T cells, turning off their cytolytic activity.
Assuntos
Antígenos CD/metabolismo , Interferon gama/farmacologia , Lectinas Tipo C/metabolismo , Neoplasias Ovarianas/imunologia , Receptores Imunológicos/metabolismo , Linfócitos T Citotóxicos/imunologia , Citotoxicidade Imunológica/efeitos dos fármacos , Feminino , Antígenos HLA/metabolismo , Antígenos HLA-A/metabolismo , Antígeno HLA-A11 , Antígeno HLA-A2/metabolismo , Antígenos HLA-G , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Subfamília C de Receptores Semelhantes a Lectina de Células NK , Subfamília D de Receptores Semelhantes a Lectina de Células NK , Sinais Direcionadores de Proteínas , Receptores de Células Matadoras Naturais , Proteínas Recombinantes , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Cancer patients die primarily due to disease recurrence after transient treatment responses. The emergence of therapy-resistant escape variants is fuelled by intra-tumour heterogeneity, underpinned by interference and Darwinian evolution among continuously developing sub-clones in the mutating tumour. Novel cancer cell variants build upon the pre-existing genetic landscape and tumour heterogeneity is often ascribed largely to genetic variability. While mutations are required for cancer development and studies of genetic evolution of tumours have improved our understanding of cancer biology, genetics only represents one dimension of the fitness of each cancer cell. Beyond the mutations, several non-genetic factors also add significant variability, resulting in a complex and highly dynamic tumour cell population that can drive disease under almost any condition. This viewpoint article summarizes the genetic basis of intra-tumour heterogeneity, before dissecting four major interdependent non-genetic factors we think critically contribute to the overall variability of tumour cells in all types of cancer: epigenetic regulation, cellular differentiation hierarchies, gene expression stochasticity and tumour microenvironment. We finally present the relevant technological approaches to address the combined contribution of both genetic and non-genetic factors to intra-tumour heterogeneity, focusing on genomic profiling, cellular lineage tracing and single-cell RNA sequencing technologies. This strategy will ultimately allow dissection of the full range and depth of intra-tumour heterogeneity. We thus believe that understanding how cancer genetics synergize with the emerging non-genetic factors will be key for development of therapies able to tackle tumour escape and thereby improve cancer patient survival.
Assuntos
Evolução Molecular , Aptidão Genética , Heterogeneidade Genética , Neoplasias/genética , Linhagem da Célula/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Neoplasias/patologia , Microambiente Tumoral/genéticaRESUMO
The perforin (PFN) protein is essential for the elimination of target cells by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. The study of cells releasing PFN has been hampered by a lack of sensitive methods. We therefore produced PFN-reactive monoclonal antibodies (mAb) and developed capture enzyme-linked immunosorbent (ELISA) and enzyme-linked immunospot (ELISpot) assays. Three mAbs were generated and shown to react with unique determinants of PFN. All mAbs recognized intracellular PFN in human peripheral blood mononuclear cell (PBMC) as assessed by flow cytometry and immunohistochemistry. Functional PFN capture ELISA and ELISpot assays were developed utilizing two of the mAbs for capture and the third mAb for detection. When examining PFN release by the YT lymphoma cell line, the ELISpot displayed a greater detection sensitivity than the ELISA. Assessment of PFN release by a CTL clone using ELISpot gave results consistent with a parallel (51)Cr-release cytotoxicity assay. Moreover, PFN release by PBMC could be quantified by ELISpot and ELISA after ex vivo stimulation with defined CTL epitopes from common viruses. These novel immunoassays will be valuable for further investigations of the mechanisms underlying granule-mediated apoptosis. In addition, the capture immunoassays could provide tools for studying CTL responses in infectious and tumor diseases as well as for vaccine development.
Assuntos
Epitopos de Linfócito T/imunologia , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/imunologia , Linfócitos T Citotóxicos/virologia , Vírus/imunologia , Animais , Anticorpos Monoclonais , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Perforina , Proteínas Citotóxicas Formadoras de Poros , Linfócitos T Citotóxicos/química , Linfócitos T Citotóxicos/imunologiaRESUMO
With the promise of T cell-based therapy for cancer finally becoming reality, this Review focuses on the less-studied γδ T cell lineage and its diverse responses to tumours. γδ T cells have well-established protective roles in cancer, largely on the basis of their potent cytotoxicity and interferon-γ production. Besides this, recent studies have revealed a series of tumour-promoting functions that are linked to interleukin-17-producing γδ T cells. Here, we integrate the current knowledge from both human and mouse studies to highlight the potential of γδ T cell modulation to improve cancer immunotherapy.
Assuntos
Receptores de Antígenos de Linfócitos T gama-delta/fisiologia , Linfócitos T/imunologia , Animais , Citotoxicidade Imunológica , Humanos , Interleucina-17/fisiologia , Camundongos , Neoplasias/imunologia , Neoplasias/terapiaRESUMO
NK cells and γδ T lymphocytes display potent cytolytic activity against leukemias and CMV-infected cells and are thus, promising immune effector cells in the context of allo-HSCT. NK cells express HLA class I-specific inhibitory receptors and preferentially kill HLA class I(low) tumors or virus-infected cells. Killing occurs upon engagement of activating NKRs with ligands that are up-regulated on tumors and infected cells. A similar activating receptor/ligand interaction strategy is used by γδ T cells, which in addition, use their TCRs for recognition of phosphorylated antigens and still largely undefined ligands on tumor cells. In the haploidentical allo-HSCT setting, alloreactive NK cells, derived from donor HSCs, can exert potent antileukemia activity and kill residual patient DCs and T cells, thus preventing GvHD and graft rejection. However, generation of KIR(+) alloreactive NK cells from HSCs requires many weeks, during which leukemia relapses, and life-threatening infections may occur. Importantly, mature NK cells and γδ T cells can control certain infectious agents efficiently, in particular, limit CMV reactivation, and infusion of such donor cells at the time of HSCT has been implemented. Development of novel, cell-based immunotherapies, allowing improved trafficking and better targeting, will endow NK cells and γδ T lymphocytes with enhanced anti-tumor activity, also making them key reagents for therapies against solid tumors. The clinical aspects of using NK cells and γδ T lymphocytes against hematological malignancies, including the allo-HSCT context, are reviewed in the related side-by-side paper by Locatelli and colleagues [1].
Assuntos
Efeito Enxerto vs Leucemia/imunologia , Transplante de Células-Tronco Hematopoéticas , Células Matadoras Naturais/imunologia , Leucemia/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Aloenxertos , Animais , Antígenos de Histocompatibilidade Classe I , Humanos , Células Matadoras Naturais/patologia , Leucemia/patologia , Leucemia/terapia , Linfócitos T/patologiaRESUMO
Cytotoxic T lymphocytes (CTL) may undergo massive expansion upon appropriate antigenic stimulation. Homeostasis is maintained by a subsequent "contraction" of these cells. Activation-induced cell death (AICD) and programmed cell death prevent the untoward side effects, arising from excessive numbers and prolonged persistence of activated CTL, that occur upon uncontrolled and/or continued expansion. However, effector cell persistence has been identified as a hallmark of successful T-cell-mediated adoptive immunotherapy. Thus, prevention of AICD may be critical to achieve more successful clinical results. We have previously shown that treatment with the c-Jun NH(2)-terminal kinase (JNK) inhibitor SP600125 protects human melanoma epitope Mart-1(27-35)-reactive CTL from apoptotic death upon their reencounter with cognate antigen. However, inhibition of JNK also interferes with the functional ability of the CTL to secrete IFN-gamma. Here, we show that reactive oxygen species (ROS) inhibitors, such as the superoxide dismutase mimetic Mn (III) tetrakis (5, 10, 15, 20-benzoic acid) porphyrin (MnTBAP), efficiently protected Mart-1(27-35)-reactive primary CTL from AICD without impairing their functional capability. MnTBAP prevented the increase in intracellular ROS, mitochondrial membrane collapse, and DNA fragmentation observed in control-treated cells upon cognate antigen encounter. Furthermore, the mechanism of AICD prevention in primary CTL included blockade of JNK activation. Finally, tumor-reactive in vitro expanded tumor infiltrating lymphocytes, which are used clinically in cancer immunotherapy, also benefit from MnTBAP-mediated antioxidant treatment. Thus, modulation of the redox pathway might improve CTL persistence and lead to better clinical results for T cell-based immunotherapies.
Assuntos
Epitopos/imunologia , Proteínas de Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Superóxidos/antagonistas & inibidores , Linfócitos T Citotóxicos/imunologia , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Epitopos de Linfócito T/imunologia , Sequestradores de Radicais Livres/farmacologia , Antígeno HLA-A2/imunologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Melanoma/imunologia , Metaloporfirinas/farmacologia , Orthomyxoviridae/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Superóxidos/imunologia , Superóxidos/metabolismo , Linfócitos T Citotóxicos/efeitos dos fármacos , Proteínas da Matriz Viral/imunologiaRESUMO
Human NK cells can be divided into CD56(dim) and CD56(bright) subsets. These two types of NK cells respond to different types of stimuli, with CD56(dim) NK cells having direct cytotoxic ability and CD56(bright) NK cells having mainly an immunoregulatory function. We show that the CD16+ CD56(dim) NK subset is characterized by sensitivity to cell death induced by activated granulocytes. We identified hydrogen peroxide (H2O2) as the major effector molecule responsible for the cytotoxic effect of granulocytes on CD56(dim) NK cells, because the ability of granulocytes to kill CD56(dim) NK cells was completely abrogated in the presence of the hydrogen peroxide scavenger catalase. When exposing NK cells to H2O2, CD56(dim) cells showed rapid mitochondrial depolarization and down-regulation of activating NKRs, eventually resulting in cell death, whereas CD56(bright) cells remained unaffected. The difference in sensitivity to H2O2 was mirrored by a difference in intracellular oxidation levels between CD56(dim) and CD56(bright) NK cells, and cell lysates from the latter subset possessed a greater ability to block H2O2-mediated oxidation. Our data may explain the preferential accumulation of CD56(bright) NK cells often seen in environments rich in reactive oxygen species, such as at sites of chronic inflammation and in tumors.