RESUMEN
Primary immunodeficiencies in the costimulatory molecule CD27 and its ligand, CD70, predispose for pathologies of uncontrolled Epstein-Barr virus (EBV) infection in nearly all affected patients. We demonstrate that both depletion of CD27+ cells and antibody blocking of CD27 interaction with CD70 cause uncontrolled EBV infection in mice with reconstituted human immune system components. While overall CD8+ T-cell expansion and composition are unaltered after antibody blocking of CD27, only some EBV-specific CD8+ T-cell responses, exemplified by early lytic EBV antigen BMLF1-specific CD8+ T cells, are inhibited in their proliferation and killing of EBV-transformed B cells. This suggests that CD27 is not required for all CD8+ T-cell expansions and cytotoxicity but is required for a subset of CD8+ T-cell responses that protect us from EBV pathology.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Inmunidad Celular , Fosfoproteínas/inmunología , Transactivadores/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Animales , Linfocitos B/inmunología , Transformación Celular Viral/genética , Transformación Celular Viral/inmunología , Infecciones por Virus de Epstein-Barr/genética , Herpesvirus Humano 4/genética , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Fosfoproteínas/genética , Transactivadores/genética , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genéticaRESUMEN
Regulatory T cells (Treg) are potent inhibitors of autoreactive T cells. The intracellular transcription factor FoxP3 controls the expression levels of a diverse set of genes and plays a critical role in programming functional Tregs. Although, antigen-specific Tregs are more potent than polyclonal Tregs in treating ongoing autoimmunity, phenotype plasticity associated with loss of FoxP3 expression in Tregs can lead to the conversion into antigen-specific effector T cells which might exacerbate autoimmune pathology. In this study, we designed a retroviral vector driving the expression of FoxP3 and a human HLA-DR-restricted TCR from the same promoter. Transduction of purified human Tregs revealed that all TCR-positive cells had elevated levels of FoxP3 expression, increased CD25 and CTLA4 expression and potent suppressive function. Elevated FoxP3 expression did not impair the in vitro expansion of engineered Tregs. Adoptive transfer into HLA-DR transgenic mice revealed that FoxP3+TCR engineered Tregs showed long-term persistence with stable FoxP3 and TCR expression. In contrast, adoptive transfer of Tregs engineered with TCR only resulted in the accumulation of TCR-positive, FoxP3-negative T cells which displayed antigen-specific effector function when stimulated with the TCR-recognised peptides. Our data indicate that forced expression of FoxP3 can prevent accumulation of antigen-specific effector T cells without impairing the engraftment and persistence of engineered Tregs.
Asunto(s)
Autoinmunidad , Linfocitos T Reguladores , Humanos , Animales , Ratones , Traslado Adoptivo , Ratones Transgénicos , Factores de Transcripción Forkhead/genética , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
Epstein Barr virus (EBV) is one of the most ubiquitous human pathogens in the world, persistently infecting more than 90% of the adult human population. It drives some of the strongest human CD8+ T cell responses, which can be observed during symptomatic primary infection known as infectious mononucleosis (IM). Despite high viral loads and prolonged CD8+ T cell stimulation during IM, EBV enters latency and is under lifelong immune control in most individuals that experience this disease. We investigated whether changes in T cell function, as frequently characterized by PD-1 up-regulation, occur during IM due to the prolonged exposure to high antigen levels. We readily detected the expansion of PD-1 positive CD8+ T cells together with high frequencies of Tim-3, 2B4, and KLRG1 expression during IM and in mice with reconstituted human immune system components (huNSG mice) that had been infected with a high dose of EBV. These PD-1 positive CD8+ T cells, however, retained proliferation, cytokine production, and cytotoxic abilities. Multiple subsets of CD8+ T cells expanded during EBV infection, including PD-1+Tim-3+KLRG1+ cells that express CXCR5 and TCF-1 germinal center homing and memory markers, and may also contain BATF3. Moreover, blocking the PD-1 axis compromised EBV specific immune control and resulted in virus-associated lymphomagenesis. Finally, PD-1+, Tim-3+, and KLRG1+ CD8+ T cell expansion coincided with declining viral loads during low dose EBV infection. These findings suggest that EBV infection primes PD-1 positive CD8+ T cell populations that rely on this receptor axis for the efficient immune control of this ubiquitous human tumor virus.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Linfocitos T Citotóxicos/inmunología , Carga Viral/inmunología , Adulto , Animales , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Estudios de Casos y Controles , Citocinas/metabolismo , Infecciones por Virus de Epstein-Barr/metabolismo , Infecciones por Virus de Epstein-Barr/virología , Perfilación de la Expresión Génica , Humanos , Mediadores de Inflamación/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
Ongoing clinical trials explore T cell receptor (TCR) gene therapy as a treatment option for cancer, but responses in solid tumors are hampered by the immunosuppressive microenvironment. The production of TCR gene-engineered T cells requires full T cell activation in vitro, and it is currently unknown whether in vivo interactions with conventional dendritic cells (cDCs) regulate the accumulation and function of engineered T cells in tumors. Using the B16 melanoma model and the inducible depletion of CD11c+ cells in CD11c.diphtheria toxin receptor (DTR) mice, we analyzed the interaction between tumor-resident cDCs and engineered T cells expressing the melanoma-specific TRP-2 TCR. We found that depletion of CD11c+ cells triggered the recruitment of cross-presenting cDC1 into the tumor and enhanced the accumulation of TCR-engineered T cells. We show that the recruited tumor cDCs present melanoma tumor antigen, leading to enhanced activation of TCR-engineered T cells. In addition, detailed analysis of the tumor myeloid compartment revealed that the depletion of a population of DT-sensitive macrophages can contribute to the accumulation of tumor-infiltrating T cells. Together, these data suggest that the relative frequency of tumor-resident cDCs and macrophages may impact the therapeutic efficacy of TCR gene therapy in solid tumors.
Asunto(s)
Células Dendríticas/metabolismo , Macrófagos/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Animales , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Antígeno CD11c/inmunología , Antígeno CD11c/metabolismo , Factor de Crecimiento Similar a EGF de Unión a Heparina/inmunología , Factor de Crecimiento Similar a EGF de Unión a Heparina/metabolismo , Humanos , Inmunoterapia Adoptiva/métodos , Melanoma Experimental/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
Ag receptors used for cancer immunotherapy are often directed against tumor-associated Ags also expressed in normal tissues. Targeting of such Ags can result in unwanted autoimmune attack of normal tissues or induction of tolerance in therapeutic T cells. We used a murine model to study the phenotype and function of T cells redirected against the murine double minute protein 2 (MDM2), a tumor-associated Ag that shows low expression in many normal tissues. Transfer of MDM2-TCR-engineered T cells into bone marrow chimeric mice revealed that Ag recognition in hematopoietic tissues maintained T cell function, whereas presentation of MDM2 in nonhematopoietic tissues caused reduced effector function. TCR-engineered CD8(+) T cells underwent rapid turnover, downmodulated CD8 expression, and lost cytotoxic function. We found that MDM2-TCR-engineered CD4(+) T cells provided help and restored cytotoxic function of CD8(+) T cells bearing the same TCR. Although the introduction of the CD8 coreceptor enhanced the ability of CD4(+) T cells to recognize MDM2 in vitro, the improved self-antigen recognition abolished their ability to provide helper function in vivo. The data indicate that the same class I-restricted TCR responsible for Ag recognition and tolerance induction in CD8(+) T cells can, in the absence of the CD8 coreceptor, elicit CD4 T cell help and partially reverse tolerance. Thus MHC class I-restricted CD4(+) T cells may enhance the efficacy of therapeutic TCR-engineered CD8(+) T cells and can be readily generated with the same TCR.
Asunto(s)
Antígenos de Neoplasias/inmunología , Autoantígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Tolerancia Inmunológica , Receptores de Antígenos de Linfocitos T/genética , Traslado Adoptivo , Animales , Comunicación Celular , Citotoxicidad Inmunológica , Expresión Génica , Inmunofenotipificación , Ratones , Ratones Transgénicos , Fenotipo , Proteínas Proto-Oncogénicas c-mdm2/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción GenéticaRESUMEN
Due to the lack of specificity for tumor antigens, allogeneic T-cell therapy is associated with graft-versus-host disease. Enhancing the anti-tumor specificity while reducing the graft-versus-host disease risk of allogeneic T cells has remained a research focus. In this study, we demonstrate that the introduction of 'dominant' T-cell receptors into primary murine T cells can suppress the expression of endogenous T-cell receptors in a large proportion of the gene-modified T cells. Adoptive transfer of allogeneic T cells expressing a 'dominant' T-cell receptor significantly reduced the graft-versus-host toxicity in recipient mice. Using two bone marrow transplant models, enhanced anti-tumor activity was observed in the presence of reduced graft-versus-host disease. However, although transfer of T-cell receptor gene-modified allogeneic T cells resulted in the elimination of antigen-positive tumor cells and improved the survival of treated mice, it was associated with accumulation of T cells expressing endogenous T-cell receptors and the development of delayed graft-versus-host disease. The in-vivo deletion of the engineered T cells, mediated by endogenous mouse mammary tumor virus MTV8 and MTV9, abolished graft-versus-host disease while retaining significant anti-tumor activity of adoptively transferred T cells. Together, this study shows that the in-vitro selection of allogeneic T cells expressing high levels of a 'dominant' T-cell receptor can lower acute graft-versus-host disease and enhance anti-tumor activity of adoptive cell therapy, while the in-vivo outgrowth of T cells expressing endogenous T-cell receptors remains a risk factor for the delayed onset of graft-versus-host disease.
Asunto(s)
Enfermedad Injerto contra Huésped/prevención & control , Inmunoterapia Adoptiva/métodos , Virus del Tumor Mamario del Ratón/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/trasplante , Animales , Trasplante de Médula Ósea/métodos , Línea Celular Tumoral , Femenino , Expresión Génica , Genes Dominantes , Vectores Genéticos/inmunología , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/patología , Humanos , Depleción Linfocítica/métodos , Virus del Tumor Mamario del Ratón/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Receptores de Antígenos de Linfocitos T/genética , Análisis de Supervivencia , Linfocitos T/citología , Linfocitos T/inmunología , Transgenes , Trasplante Homólogo , Irradiación Corporal TotalRESUMEN
The expression levels of TCRs on the surface of human T cells define the avidity of TCR-HLA/peptide interactions. In this study, we have explored which components of the TCR-CD3 complex are involved in determining the surface expression levels of TCRs in primary human T cells. The results show that there is a surplus of endogenous TCR α/ß chains that can be mobilised by providing T cells with additional CD3γ,δ,ε,ζ chains, which leads to a 5-fold increase in TCR α/ß surface expression. The analysis of individual CD3 chains revealed that provision of additional ζ chain alone was sufficient to achieve a 3-fold increase in endogenous TCR expression. Similarly, CD3ζ also limits the expression levels of exogenous TCRs transduced into primary human T cells. Interestingly, transduction with TCR plus CD3ζ not only increased surface expression of the introduced TCR, but it also reduced mispairing with endogenous TCR chains, resulting in improved antigen-specific function. TCR reconstitution experiments in HEK293T cells that do not express endogenous TCR or CD3 showed that TCRα/ß and all four CD3 chains were required for optimal surface expression, while in the absence of CD3ζ the TCR expression was reduced by 50%. Together, the data show that CD3ζ is a key regulator of TCR expression levels in human T cells, and that gene transfer of exogenous TCR plus CD3ζ improved TCR surface expression, reduced TCR mispairing and increased antigen-specific function.
Asunto(s)
Complejo CD3 , Humanos , Complejo CD3/inmunología , Complejo CD3/metabolismo , Complejo CD3/genética , Células HEK293 , Linfocitos T/inmunología , Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Activación de Linfocitos/inmunología , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunología , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Complejo Receptor-CD3 del Antígeno de Linfocito T/genéticaRESUMEN
The function of T-cell receptor (TCR) gene modified T cells is dependent on efficient surface expression of the introduced TCR α/ß heterodimer. We tested whether endogenous CD3 chains are rate-limiting for TCR expression and antigen-specific T-cell function. We show that co-transfer of CD3 and TCR genes into primary murine T cells enhanced TCR expression and antigen-specific T-cell function in vitro. Peptide titration experiments showed that T cells expressing introduced CD3 and TCR genes recognized lower concentration of antigen than T cells expressing TCR only. In vivo imaging revealed that TCR+CD3 gene modified T cells infiltrated tumors faster and in larger numbers, which resulted in more rapid tumor elimination compared with T cells modified by TCR only. After tumor clearance, TCR+CD3 engineered T cells persisted in larger numbers than TCR-only T cells and mounted a more effective memory response when rechallenged with antigen. The data demonstrate that provision of additional CD3 molecules is an effective strategy to enhance the avidity, anti-tumor activity and functional memory formation of TCR gene modified T cells in vivo.
Asunto(s)
Complejo CD3/fisiología , Genes Codificadores de los Receptores de Linfocitos T/genética , Terapia Genética , Animales , Complejo CD3/genética , Complejo CD3/metabolismo , Células Cultivadas , Regulación hacia Abajo , Femenino , Terapia Genética/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Linfoma de Células T/genética , Linfoma de Células T/patología , Linfoma de Células T/terapia , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Resultado del TratamientoRESUMEN
Recently, vaccines against the Wilms Tumor antigen 1 (WT1) have been tested in cancer patients. However, it is currently not known whether physiologic levels of WT1 expression in stem and progenitor cells of normal tissue result in the deletion or tolerance induction of WT1-specific T cells. Here, we used an human leukocyte antigen-transgenic murine model to study the fate of human leukocyte antigen class-I restricted, WT1-specific T cells in the thymus and in the periphery. Thymocytes expressing a WT1-specific T-cell receptor derived from high avidity human CD8 T cells were positively selected into the single-positive CD8 population. In the periphery, T cells specific for the WT1 antigen differentiated into CD44-high memory phenotype cells, whereas T cells specific for a non-self-viral antigen retained a CD44(low) naive phenotype. Only the WT1-specific T cells, but not the virus-specific T cells, displayed rapid antigen-specific effector function without prior vaccination. Despite long-term persistence of WT1-specific memory T cells, the animals did not develop autoimmunity, and the function of hematopoietic stem and progenitor cells was unimpaired. This is the first demonstration that specificity for a tumor-associated self-antigen may drive differentiation of functionally competent memory T cells.
Asunto(s)
Linfocitos T/citología , Linfocitos T/inmunología , Timo/inmunología , Proteínas WT1/inmunología , Animales , Línea Celular , Expresión Génica , Técnicas de Transferencia de Gen , Humanos , Memoria Inmunológica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Madre/inmunología , Células Madre/metabolismo , Vacunación , Proteínas WT1/genética , Tumor de Wilms/inmunologíaRESUMEN
Regulatory T cells (Tregs) can suppress a wide range of immune cells, making them an ideal candidate for the treatment of autoimmunity. The potential clinical translation of targeted therapy with antigen-specific Tregs is hampered by the difficulties of isolating rare specificities from the natural polyclonal T cell repertoire. Moreover, the initiating antigen is often unknown in autoimmune disease. Here we tested the ability of antigen-specific Tregs generated by retroviral gene transfer to ameliorate arthritis through linked suppression and therefore without cognate recognition of the disease-initiating antigen. We explored two distinct strategies: T cell receptor (TCR) gene transfer into purified CD4+CD25+ T cells was used to redirect the specificity of naturally occurring Tregs; and co-transfer of FoxP3 and TCR genes served to convert conventional CD4(+) T cells into antigen-specific regulators. Following adoptive transfer into recipient mice, the gene-modified T cells engrafted efficiently and retained TCR and FoxP3 expression. Using an established arthritis model, we demonstrate antigen-driven accumulation of the gene modified T cells at the site of joint inflammation, which resulted in a local reduction in the number of inflammatory Th17 cells and a significant decrease in arthritic bone destruction. Together, we describe a robust strategy to rapidly generate antigen-specific regulatory T cells capable of highly targeted inhibition of tissue damage in the absence of systemic immune suppression. This opens the possibility to target Tregs to tissue-specific antigens for the treatment of autoimmune tissue damage without the knowledge of the disease-causing autoantigens recognized by pathogenic T cells.
Asunto(s)
Artritis Reumatoide/inmunología , Artritis Reumatoide/terapia , Inmunoterapia Adoptiva/métodos , Linfocitos T Reguladores/inmunología , Traslado Adoptivo , Animales , Autoantígenos/inmunología , Citometría de Flujo , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Ratones , Ratones Endogámicos C57BL , Retroviridae , Especificidad del Receptor de Antígeno de Linfocitos T/genéticaRESUMEN
This review presents key advances in combining T cell receptor (TCR) gene transfer to redirect T-cell specificity with gene engineering in order to enhance cancer-protective immune function. We discuss how emerging insights might be applied to CD4+ T cells. Although much attention has been paid to the role of CD8+ cytotoxic T cells in tumour protection, we provide convincing evidence that CD4+ helper T cells play a critical role in cancer immune responses in animal models and also in patients. We demonstrate that genetic engineering technologies provide exciting opportunities to extend the specificity range of CD4+ T cells from MHC class-II-presented epitopes to include peptides presented by MHC class I molecules. Functional enhancement of tumour immunity can improve the sensitivity of T cells to cancer antigens, promote survival in a hostile tumour microenvironment, boost cancer-protective effector mechanisms and enable the formation of T-cell memory. Engineered cancer-specific CD4+ T cells may contribute to protective immunity by a direct pathway involving cancer cell killing, and by an indirect pathway that boosts the function, persistence and memory formation of CD8+ T cells.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Ingeniería Celular/métodos , Neoplasias/inmunología , Animales , Citocinas/metabolismo , Humanos , Memoria Inmunológica , Transducción de SeñalRESUMEN
We explored whether engineering of T cell specificity and effector function improves immunotherapy of solid tumors. Although IL-12 can enhance cancer immunity, a strategy of safe IL-12 delivery without toxicity is currently lacking. We engineered T cells to express IL-12 controlled by the NFAT promoter responsive to TCR stimulation, or by the Tet-On promoter responsive to doxycycline. In vivo, NFAT-engineered T cells caused lethal toxicity, while Tet-engineered T cells were safe in the absence of doxycycline. Combining gene transfer of the melanoma-specific TRP2-TCR with Tet-IL-12 engineering revealed that temporal induction of IL-12 was essential to inhibit the growth of B16F10 melanoma tumors. Induced IL-12 increased the number of tumor-infiltrating T cells and also prevented the down-modulation of the TRP2-TCR and the associated up-regulation of the PD1 marker that was observed in the absence of IL-12. In addition, temporal induction of IL-12 expression also increased the number of plasmacytoid DC in the tumor micro-environment. We show that repeated induction of IL-12 can be used to enhance control of tumor growth without encountering systemic toxicity. The observation that TCR engineering combined with Tet-regulated IL-12 expression can achieve tumor immunity without the side effects that are usually associated with the in vivo use of IL-12 warrants translation of this concept into the clinic.
RESUMEN
TCR-gene-transfer is an efficient strategy to produce therapeutic T cells of defined antigen specificity. However, there are substantial variations in the cell surface expression levels of human TCRs, which can impair the function of engineered T cells. Here we demonstrate that substitutions of 3 amino acid residues in the framework of the TCR variable domains consistently increase the expression of human TCRs on the surface of engineered T cells.The modified TCRs mediate enhanced T cell proliferation, cytokine production and cytotoxicity, while reducing the peptide concentration required for triggering effector function up to 3000-fold. Adoptive transfer experiments in mice show that modified TCRs control tumor growth more efficiently than wild-type TCRs. Our data indicate that simple variable domain modifications at a distance from the antigen-binding loops lead to increased TCR expression and improved effector function. This finding provides a generic platform to optimize the efficacy of TCR gene therapy in humans.
Asunto(s)
Antígenos/inmunología , Ingeniería Celular , Genes Codificadores de los Receptores de Linfocitos T/genética , Genes Codificadores de los Receptores de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Línea Celular Tumoral , Proliferación Celular , Citocinas/metabolismo , Expresión Génica , Terapia Genética , Humanos , Lectinas Tipo C/metabolismo , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Modelos Moleculares , Dominios Proteicos , Ingeniería de Proteínas , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunologíaRESUMEN
T-cell-based antigen-specific immunotherapy targeting tumour-associated antigens offers the potential for cancer immunotherapy. However, the majority of identified tumour-associated antigens are also expressed at low levels in normal tissues and mechanisms of tolerance induction are likely to affect the quality of T-cell responses to such antigens. In this study a T-cell receptor transgenic model was developed to determine the magnitude of T-cell tolerance to the tumour-associated antigen murine double minute-2 (MDM2), a widely expressed protein that is found at elevated levels in many tumours. The analysis of transgenic mice showed that thymic deletion was responsible for purging large numbers of MDM2-specific T cells from the repertoire. However, some T cells with specificity for MDM2 were able to escape thymic deletion and persisted in the peripheral T-cell pool. Functional analysis revealed that these T cells displayed defects in antigen-driven expansion. This functional impairment of the MDM2-specific T cells was maintained following adoptive transfer of the T cells into hosts that were unable to present the T-cell-receptor-recognized antigen. This study demonstrates that thymic deletion and the functional impairment of T cells present in the periphery both operate to establish T-cell tolerance to the tumour-associated antigen MDM2. Furthermore, the tolerant phenotype was stable and did not require continuous MDM2 peptide presentation in normal tissues.
Asunto(s)
Proteínas Proto-Oncogénicas c-mdm2/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Animales , Presentación de Antígeno/inmunología , Antígenos de Neoplasias/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Supresión Clonal/inmunología , Tolerancia Inmunológica , Ratones , Ratones Transgénicos , Bazo/inmunología , Timo/inmunologíaRESUMEN
A key predictor for the success of gene-modified T cell therapies for cancer is the persistence of transferred cells in the patient. The propensity of less differentiated memory T cells to expand and survive efficiently has therefore made them attractive candidates for clinical application. We hypothesized that redirecting T cells to specialized niches in the BM that support memory differentiation would confer increased therapeutic efficacy. We show that overexpression of chemokine receptor CXCR4 in CD8+ T cells (TCXCR4) enhanced their migration toward vascular-associated CXCL12+ cells in the BM and increased their local engraftment. Increased access of TCXCR4 to the BM microenvironment induced IL-15-dependent homeostatic expansion and promoted the differentiation of memory precursor-like cells with low expression of programmed death-1, resistance to apoptosis, and a heightened capacity to generate polyfunctional cytokine-producing effector cells. Following transfer to lymphoma-bearing mice, TCXCR4 showed a greater capacity for effector expansion and better tumor protection, the latter being independent of changes in trafficking to the tumor bed or local out-competition of regulatory T cells. Thus, redirected homing of T cells to the BM confers increased memory differentiation and antitumor immunity, suggesting an innovative solution to increase the persistence and functions of therapeutic T cells.
Asunto(s)
Médula Ósea/inmunología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Movimiento Celular/inmunología , Memoria Inmunológica , Neoplasias/inmunología , Linfocitos T Reguladores/inmunología , Animales , Médula Ósea/patología , Linfocitos T CD8-positivos/patología , Línea Celular Tumoral , Quimiocina CXCL12/genética , Quimiocina CXCL12/inmunología , Humanos , Interleucina-15/genética , Interleucina-15/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neoplasias/genética , Neoplasias/patología , Neoplasias/terapia , Receptores CXCR4/genética , Receptores CXCR4/inmunología , Linfocitos T Reguladores/patologíaRESUMEN
PURPOSE: The Wilms' tumor antigen (WT1) is overexpressed in approximately 90% of breast tumors and, thus, is a potential target antigen for the immunotherapy of breast cancer. We have tested the working hypotheses that WT1 can be immunogenic in patients with breast cancer and can stimulate CTL of sufficient avidity to kill tumor cells. EXPERIMENTAL DESIGN: Paired tumor-draining lymph node and peripheral blood samples were analyzed from five HLA-A2-positive patients with stage I/II breast cancer. Fluorescent HLA-A*0201/WT1 tetramers were used to quantify WT1-specific CTL and the functional capacity of the CTL was assessed using cytotoxicity assays and intracellular cytokine staining. RESULTS: WT1 tetramer-binding T cells expanded from all lymph node samples but none of the corresponding peripheral blood samples. Functional assays were carried out on T cells from the patient who had yielded the highest frequency of HLA-A*0201/WT1 tetramer-positive cells. The cytotoxicity assays showed WT1 peptide--specific killing activity of the CTL, whereas intracellular cytokine staining confirmed that the tetramer--positive T cells produced IFN-gamma after stimulation with WT1 peptide. These WT1-specific T cells killed HLA-A2-positive breast cancer cell lines treated with IFN-gamma but no killing was observed with untreated tumor cells. CONCLUSIONS: These results show that WT1-specific CTL can be expanded from the tumor-draining lymph nodes of breast cancer patients and that they can display peptide-specific effector function. However, the CTL only killed IFN-gamma-treated tumor targets expressing high levels of HLA-A2 and not tumor cells with low HLA expression. This suggests that induction of autologous WT1-specific CTL may offer only limited tumor protection and that strategies that allow a high level of peptide/MHC complex presentation and/or improve CTL avidity may be required.
Asunto(s)
Antígenos de Neoplasias/inmunología , Ganglios Linfáticos/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas WT1/inmunología , Anciano , Línea Celular Tumoral , Pruebas Inmunológicas de Citotoxicidad , Femenino , Citometría de Flujo , Antígeno HLA-A2/inmunología , Humanos , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
There is evidence showing that high avidity CTLs can be more effective than low avidity CTLs for adoptive tumor immunotherapy. Because many T cell-recognized tumor antigens are nonmutated self-proteins, tolerance mechanisms are likely to render high avidity T cells unresponsive or cause T cell elimination by clonal deletion. We recently used the allo-restricted strategy to circumvent immunologic tolerance to a ubiquitously expressed tumor-associated protein, MDM2, and raised high avidity CTLs in humans and in mice. In this study, we investigated whether high avidity MDM2-specific CTLs can mediate tumor protection without causing damage to normal tissues in mice. Although the CTLs prolonged survival of tumor-bearing mice without causing damage to normal tissues, tumor protection was incomplete. We show that tumor growth occurred despite the continued presence of MDM2-specific CTLs and the continued susceptibility of tumor cells to CTL killing. However, analysis of the CTLs revealed that they had been rendered unresponsive in vivo because they did not produce interferon gamma in response to antigen-specific stimulation. These experiments suggest that induction of unresponsiveness may be an important mechanism limiting the efficacy of adoptive CTL therapy.
Asunto(s)
Inmunoterapia Adoptiva , Neoplasias Experimentales/terapia , Proteínas Nucleares/inmunología , Proteínas Proto-Oncogénicas/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Línea Celular Tumoral , Proteínas de Unión al ADN/fisiología , Interferón gamma/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-mdm2RESUMEN
A key challenge in the field of T-cell immunotherapy for cancer is creating a suitable platform for promoting differentiation of effector cells while at the same time enabling self-renewal needed for long-term memory. Although transfer of less differentiated memory T cells increases efficacy through greater expansion and persistence in vivo, the capacity of such cells to sustain effector functions within immunosuppressive tumor microenvironments may still be limiting. We have therefore directly compared the impact of effector versus memory differentiation of therapeutic T cells in tumor-bearing mice by introducing molecular switches that regulate cell fate decisions via mTOR. Ectopic expression of RAS homolog enriched in brain (RHEB) increased mTORC1 signaling, promoted a switch to aerobic glycolysis, and increased expansion of effector T cells. By rapidly infiltrating tumors, RHEB-transduced T cells significantly reduced the emergence of immunoedited escape variants. In contrast, expression of proline-rich Akt substrate of 40 kDa (PRAS40) inhibited mTORC1, promoted quiescence, and blocked tumor infiltration. Fate mapping studies following transient expression of PRAS40 demonstrated that mTORC1(low) T cells made no contribution to initial tumor control but instead survived to become memory cells proficient in generating recall immunity. Our data support the design of translational strategies for generating heterogeneous T-cell immunity against cancer, with the appropriate balance between promoting effector differentiation and self-renewal. Unlike pharmacologic inhibitors, the genetic approach described here allows for upregulation as well as inhibition of the mTORC1 pathway and is highly selective for the therapeutic T cells without affecting systemic mTORC1 functions.