RESUMEN
Chimeric antigen receptor (CAR) T cell dysfunction is a major barrier to achieving lasting remission in hematologic cancers, especially in chronic lymphocytic leukemia (CLL). We have shown previously that Δ133p53α, an endogenous isoform of the human TP53 gene, decreases in expression with age in human T cells, and that reconstitution of Δ133p53α in poorly functional T cells can rescue proliferation [A. M. Mondal et al., J. Clin. Invest. 123, 5247-5257 (2013)]. Although Δ133p53α lacks a transactivation domain, it can form heterooligomers with full-length p53 and modulate the p53-mediated stress response [I. Horikawa et al., Cell Death Differ. 24, 1017-1028 (2017)]. Here, we show that constitutive expression of Δ133p53α potentiates the anti-tumor activity of CD19-directed CAR T cells and limits dysfunction under conditions of high tumor burden and metabolic stress. We demonstrate that Δ133p53α-expressing CAR T cells exhibit a robust metabolic phenotype, maintaining the ability to execute effector functions and continue proliferating under nutrient-limiting conditions, in part due to upregulation of critical biosynthetic processes and improved mitochondrial function. Importantly, we show that our strategy to constitutively express Δ133p53α improves the anti-tumor efficacy of CAR T cells generated from CLL patients that previously failed CAR T cell therapy. More broadly, our results point to the potential role of the p53-mediated stress response in limiting the prolonged antitumor functions required for complete tumor clearance in patients with high disease burden, suggesting that modulation of the p53 signaling network with Δ133p53α may represent a translationally viable strategy for improving CAR T cell therapy.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Receptores Quiméricos de Antígenos , Humanos , Inmunoterapia Adoptiva/métodos , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/terapia , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Antígenos CD19 , Tratamiento Basado en Trasplante de Células y Tejidos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
Chimeric antigen receptor (CAR)-modified T cells are being investigated in many settings, including classical Hodgkin lymphoma (cHL). The unique biology of cHL, characterized by scant Hodgkin and Reed-Sternberg (HRS) cells within an immunosuppressive tumor microenvironment (TME), may pose challenges for cellular therapies directly targeting antigens expressed on HRS cells. We hypothesized that eradicating CD19+ B cells within the TME and the putative circulating CD19+ HRS clonotypic cells using anti-CD19-directed CAR-modified T cells (CART19) may indirectly affect HRS cells, which do not express CD19. Here we describe our pilot trial using CART19 in patients with relapsed or refractory cHL. To limit potential toxicities, we used nonviral RNA CART19 cells, which are expected to express CAR protein for only a few days, as opposed to CART19 generated by viral vector transduction, which expand in vivo and retain CAR expression. All 5 enrolled patients underwent successful manufacturing of nonviral RNA CART19, and 4 were infused with protocol-specified cell dose. There were no severe toxicities. Responses were seen, but these were transient. To our knowledge, this is the first CART19 clinical trial to use nonviral RNA gene delivery. This trial was registered at www.clinicaltrials.gov as #NCT02277522 (adult) and #NCT02624258 (pediatric).
Asunto(s)
Técnicas de Transferencia de Gen , Enfermedad de Hodgkin/terapia , Inmunoterapia Adoptiva , Receptores Quiméricos de Antígenos , Linfocitos T/metabolismo , Microambiente Tumoral/inmunología , Adulto , Femenino , Enfermedad de Hodgkin/genética , Enfermedad de Hodgkin/inmunología , Humanos , Masculino , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunologíaRESUMEN
Cytokines dimerize cell surface receptors to activate signaling and regulate many facets of the immune response. Many cytokines have pleiotropic effects, inducing a spectrum of redundant and distinct effects on different cell types. This pleiotropy has hampered cytokine-based therapies, and the high doses required for treatment often lead to off-target effects, highlighting the need for a more detailed understanding of the parameters controlling cytokine-induced signaling and bioactivities. Using the prototypical cytokine interleukin-13 (IL-13), we explored the interrelationships between receptor binding and a wide range of downstream cellular responses. We applied structure-based engineering to generate IL-13 variants that covered a spectrum of binding strengths for the receptor subunit IL-13Rα1. Engineered IL-13 variants representing a broad range of affinities for the receptor exhibited similar potencies in stimulating the phosphorylation of STAT6 (signal transducer and activator of transcription 6). Delays in the phosphorylation and nuclear translocation of STAT6 were only apparent for those IL-13 variants with markedly reduced affinities for the receptor. From these data, we developed a mechanistic model that quantitatively reproduced the kinetics of STAT6 phosphorylation for the entire spectrum of binding affinities. Receptor endocytosis played a key role in modulating STAT6 activation, whereas the lifetime of receptor-ligand complexes at the plasma membrane determined the potency of the variant for inducing more distal responses. This complex interrelationship between extracellular ligand binding and receptor function provides the foundation for new mechanism-based strategies that determine the optimal cytokine dose to enhance therapeutic efficacy.
Asunto(s)
Receptores de Citocinas/metabolismo , Fenómenos Biofísicos , Línea Celular , Cristalografía por Rayos X , Endocitosis , Variación Genética , Células HeLa , Humanos , Interleucina-13/química , Interleucina-13/genética , Interleucina-13/metabolismo , Subunidad alfa1 del Receptor de Interleucina-13/química , Subunidad alfa1 del Receptor de Interleucina-13/genética , Subunidad alfa1 del Receptor de Interleucina-13/metabolismo , Subunidad alfa del Receptor de Interleucina-4/química , Subunidad alfa del Receptor de Interleucina-4/genética , Subunidad alfa del Receptor de Interleucina-4/metabolismo , Ligandos , Modelos Biológicos , Modelos Moleculares , Fosforilación , Unión Proteica , Ingeniería de Proteínas , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Receptores de Citocinas/química , Receptores de Citocinas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factor de Transcripción STAT6/metabolismo , Transducción de SeñalRESUMEN
Two critical functions of dendritic cells (DC) are to activate and functionally polarize T cells. Activated T cells can, in turn, influence DC maturation, although their effect on de novo DC development is poorly understood. Here we report that activation of T cells in mice, with either an anti-CD3 antibody or super antigen, drives the rapid formation of CD209(+)CD11b(+)CD11c(+) MHC II(+) DC from monocytic precursors (Mo-DC). GM-CSF is produced by T cells following activation, but surprisingly, it is not required for the formation of CD209(+) Mo-DC. CD40L, however, is critical for the full induction of Mo-DC following T cell activation. T cell induced CD209(+) Mo-DC are comparable to conventional CD209(-) DC in their ability to stimulate T cell proliferation. However, in contrast to conventional CD209(-) DC, CD209(+) Mo-DC fail to effectively polarize T cells, as indicated by a paucity of T cell cytokine production. The inability of CD209(+) Mo-DC to polarize T cells is partly explained by increased expression of PDL-2, since blockade of this molecule restores some polarizing capacity to the Mo-DC. These findings expand the range of signals capable of driving Mo-DC differentiation in vivo beyond exogenous microbial factors to include endogenous factors produced following T cell activation.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Lectinas Tipo C/metabolismo , Activación de Linfocitos/inmunología , Proteína 2 Ligando de Muerte Celular Programada 1/metabolismo , Receptores de Superficie Celular/metabolismo , Subgrupos de Linfocitos T/inmunología , Animales , Ligando de CD40/metabolismo , Diferenciación Celular/inmunología , Movimiento Celular , Células Dendríticas/inmunología , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Ganglios Linfáticos/inmunología , Ratones , Ratones Noqueados , Monocitos/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
In chronically inflamed tissues, such as those affected by autoimmune disease, activated Th cells often colocalize with monocytes. We investigate in this study how murine Th cells influence the phenotype and function of monocytes. The data demonstrate that Th1, Th2, and Th17 subsets promote the differentiation of autologous monocytes into MHC class II(+), CD11b(+), CD11c(+) DC that we call DCTh. Although all Th subsets induce the formation of DCTh, activated Th17 cells uniquely promote the formation of IL-12/IL-23-producing DCTh (DCTh17) that can polarize both naive and Th17 cells to a Th1 phenotype. In the inflamed CNS of mice with Th17-mediated experimental autoimmune encephalomyelitis, Th cells colocalize with DC, as well as monocytes, and the Th cells obtained from these lesions drive the formation of DCTh that are phenotypically indistinguishable from DCTh17 and polarize naive T cells toward a Th1 phenotype. These results suggest that DCTh17 are critical in the interplay of Th17- and Th1-mediated responses and may explain the previous finding that IL-17-secreting Th cells become IFN-γ-secreting Th1 cells in experimental autoimmune encephalomyelitis and other autoimmune disorders.
Asunto(s)
Autoinmunidad/inmunología , Células Dendríticas/inmunología , Inflamación/inmunología , Células TH1/inmunología , Células Th17/inmunología , Animales , Antígeno CD11b/metabolismo , Antígeno CD11c/metabolismo , Diferenciación Celular , Movimiento Celular , Polaridad Celular , Células Cultivadas , Sistema Nervioso Central/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Antígenos de Histocompatibilidad Clase II/metabolismo , Interferón gamma/metabolismo , Interleucina-12/biosíntesis , Interleucina-17/metabolismo , Interleucina-23/biosíntesis , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/inmunologíaRESUMEN
BACKGROUND: Adoptive cell transfer of tumor infiltrating lymphocytes has shown clinical efficacy in the treatment of melanoma and is now also being explored in other tumor types. Generation of sufficient numbers of effector T cells requires extensive ex vivo expansion, often at the cost of T cell differentiation and potency. For the past 20 years, IL-2 has been the key cytokine applied in the expansion of TIL for ACT. However, the use of IL-2 has also led to collateral expansion of regulatory T cells (Tregs) and progressive T cell differentiation, factors known to limit in vivo persistence and activity of transferred TIL. The use of alternative T cell growth factors is therefore warranted. Here, we have compared the effects of IL-2, -15 and -21 cytokines on the expansion and activation of TIL from single-cell suspensions of non-small cell lung cancer, ovarian cancer and melanoma. METHODS: We applied the K562-based artificial APC (aAPC) platform for the direct and rapid expansion of tumor infiltrating lymphocytes isolated from primary cancer specimens. These aAPC were engineered to express the Fc-γ receptor CD32 (for anti-CD3 antibody binding), the co-stimulatory molecule 4-1BBL, and to secrete either IL-2, IL-15 or IL-21 cytokine. RESULTS: Although IL-2 aAPC induced the greatest overall TIL expansion, IL-21 aAPC induced superior expansion of CD8+ T cells with a CD27+ CD28+ "young" phenotype and superior functional cytotoxic effector characteristics, without collateral expansion of Tregs. CONCLUSION: Our data rationalize the clinical application of IL-21-secreting aAPC as a standardized cell-based platform in the expansion of "young" effector TIL for ACT.
Asunto(s)
Antígenos CD28/metabolismo , Interleucinas/metabolismo , Linfocitos Infiltrantes de Tumor/citología , Linfocitos T Reguladores/citología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Biopsia , Linfocitos T CD8-positivos/citología , Proliferación Celular , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Células K562 , Neoplasias/metabolismo , Fenotipo , Receptores de IgG/metabolismoRESUMEN
Cytokines dimerize their receptors, with the binding of the 'second chain' triggering signaling. In the interleukin (IL)-4 and IL-13 system, different cell types express varying numbers of alternative second receptor chains (γc or IL-13Rα1), forming functionally distinct type I or type II complexes. We manipulated the affinity and specificity of second chain recruitment by human IL-4. A type I receptor-selective IL-4 'superkine' with 3,700-fold higher affinity for γc was three- to ten-fold more potent than wild-type IL-4. Conversely, a variant with high affinity for IL-13Rα1 more potently activated cells expressing the type II receptor and induced differentiation of dendritic cells from monocytes, implicating the type II receptor in this process. Superkines showed signaling advantages on cells with lower second chain numbers. Comparative transcriptional analysis reveals that the superkines induce largely redundant gene expression profiles. Variable second chain numbers can be exploited to redirect cytokines toward distinct cell subsets and elicit new actions, potentially improving the selectivity of cytokine therapy.
Asunto(s)
Citocinas/fisiología , Interleucina-4/análogos & derivados , Interleucina-4/farmacología , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Células Dendríticas/efectos de los fármacos , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Factores Inmunológicos/farmacología , Interleucina-4/química , Mutación/fisiología , Fenotipo , Fosforilación , Ingeniería de Proteínas , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Interleucina-4/efectos de los fármacos , Factor de Transcripción STAT6/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
CD14 is a monocytic differentiation antigen that regulates innate immune responses to pathogens. Here, we show that murine Cd14 SNPs regulate the length of Cd14 mRNA and CD14 protein translation efficiency, and consequently the basal level of soluble CD14 (sCD14) and type I IFN production by murine macrophages. This has substantial downstream consequences for the innate immune response; the level of expression of at least 40 IFN-responsive murine genes was altered by this mechanism. We also observed that there was substantial variation in the length of human CD14 mRNAs and in their translation efficiency. sCD14 increased cytokine production by human dendritic cells (DCs), and sCD14-primed DCs augmented human CD4T cell proliferation. These findings may provide a mechanism for exploring the complex relationship between CD14 SNPs, serum sCD14 levels, and susceptibility to human infectious and allergic diseases.
Asunto(s)
Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Receptores de Lipopolisacáridos/genética , Polimorfismo de Nucleótido Simple , Animales , Linfocitos B/inmunología , Western Blotting , Linfocitos T CD4-Positivos/inmunología , Citocinas/biosíntesis , Células Dendríticas/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunidad Innata/inmunología , Prueba de Cultivo Mixto de Linfocitos , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Monocytes and T helper (T(H)) cells rapidly infiltrate inflamed tissues where monocytes differentiate into inflammatory dendritic cells (DCs) through undefined mechanisms. Our studies indicate that T(H) cells frequently interact with monocytes in inflamed skin and elicit the differentiation of specialized DC subsets characteristic of these lesions. In psoriasis lesions, T(H)1 and T(H)17 cells interact with monocytes and instruct these cells to differentiate into T(H)1- and T(H)17-promoting DCs, respectively. Correspondingly, in acute atopic dermatitis, T(H)2 cells interact with monocytes and elicit the formation of T(H)2-promoting DCs. DC formation requires GM-CSF and cell contact, whereas T(H) subset specific cytokines dictate DC function and the expression of DC subset specific surface molecules. Moreover, the phenotypes of T cell-induced DC subsets are maintained after subsequent stimulation with a panel of TLR agonists, suggesting that T(H)-derived signals outweigh downstream TLR signals in their influence on DC function. These findings indicate that T(H) cells govern the formation and function of specialized DC subsets.
Asunto(s)
Citocinas/biosíntesis , Células Dendríticas/inmunología , Monocitos/inmunología , Psoriasis/metabolismo , Transducción de Señal , Piel/metabolismo , Células TH1/inmunología , Células Th17/inmunología , Células Th2/inmunología , Antígenos CD/análisis , Antígenos CD/biosíntesis , Comunicación Celular/inmunología , Diferenciación Celular/inmunología , Técnicas de Cocultivo , Citocinas/inmunología , Células Dendríticas/citología , Células Dendríticas/metabolismo , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Humanos , Inmunohistoquímica , Inmunofenotipificación , Activación de Linfocitos , Monocitos/citología , Monocitos/metabolismo , Psoriasis/inmunología , Psoriasis/patología , Transducción de Señal/inmunología , Piel/inmunología , Piel/patología , Células TH1/citología , Células TH1/metabolismo , Células Th17/citología , Células Th17/metabolismo , Células Th2/citología , Células Th2/metabolismo , Receptores Toll-Like/inmunologíaRESUMEN
BACKGROUND: Development of a standardized platform for the rapid expansion of tumor-infiltrating lymphocytes (TILs) with anti-tumor function from patients with limited TIL numbers or tumor tissues challenges their clinical application. METHODS: To facilitate adoptive immunotherapy, we applied genetically-engineered K562 cell-based artificial antigen presenting cells (aAPCs) for the direct and rapid expansion of TILs isolated from primary cancer specimens. RESULTS: TILs outgrown in IL-2 undergo rapid, CD28-independent expansion in response to aAPC stimulation that requires provision of exogenous IL-2 cytokine support. aAPCs induce numerical expansion of TILs that is statistically similar to an established rapid expansion method at a 100-fold lower feeder cell to TIL ratio, and greater than those achievable using anti-CD3/CD28 activation beads or extended IL-2 culture. aAPC-expanded TILs undergo numerical expansion of tumor antigen-specific cells, remain amenable to secondary aAPC-based expansion, and have low CD4/CD8 ratios and FOXP3+ CD4+ cell frequencies. TILs can also be expanded directly from fresh enzyme-digested tumor specimens when pulsed with aAPCs. These "young" TILs are tumor-reactive, positively skewed in CD8+ lymphocyte composition, CD28 and CD27 expression, and contain fewer FOXP3+ T cells compared to parallel IL-2 cultures. CONCLUSION: Genetically-enhanced aAPCs represent a standardized, "off-the-shelf" platform for the direct ex vivo expansion of TILs of suitable number, phenotype and function for use in adoptive immunotherapy.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Células Artificiales/citología , Técnicas de Cultivo de Célula/métodos , Ingeniería Genética , Linfocitos Infiltrantes de Tumor/citología , Células Presentadoras de Antígenos/citología , Células Presentadoras de Antígenos/efectos de los fármacos , Antígenos de Neoplasias/inmunología , Células Artificiales/efectos de los fármacos , Células Artificiales/inmunología , Antígenos CD28/metabolismo , Proliferación Celular/efectos de los fármacos , Epítopos/inmunología , Humanos , Interleucina-2/farmacología , Subgrupos Linfocitarios/efectos de los fármacos , Subgrupos Linfocitarios/inmunología , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , FenotipoRESUMEN
Human T helper 17 (T(H)17) cells regulate host defense, autoimmunity, and tumor immunity. Although cytokines that control human T(H)17 cell development have been identified, the costimulatory molecules important for T(H)17 cell generation are unknown. Here, we found that the inducible costimulator (ICOS) was critical for the differentiation and expansion of human T(H)17 cells. Human cord blood contained a subset of CD161(+)CD4(+) T cells that were recent emigrants from the thymus, expressed ICOS constitutively, and were imprinted as T(H)17 cells through ICOS signaling. ICOS stimulation induced c-MAF, RORC2, and T-bet expression in these cells, leading to increased secretion of interleukin-21 (IL-21), IL-17, and interferon-γ (IFN-γ) compared with cells stimulated with CD28. Conversely, CD28 ligation abrogated ICOS costimulation, dampening RORC2 expression while promoting the expression of the aryl hydrocarbon receptor, which led to reduced secretion of IL-17 and enhanced production of IL-22 compared with cells stimulated with ICOS. Moreover, ICOS promoted the robust expansion of IL-17(+)IFN-γ(+) human T cells, and the antitumor activity of these cells after adoptive transfer into mice bearing large human tumors was superior to that of cells expanded with CD28. The therapeutic effectiveness of ICOS-expanded cells was associated with enhanced functionality and engraftment in vivo. These findings reveal a vital role for ICOS signaling in the generation and maintenance of human T(H)17 cells and suggest that components of this pathway could be therapeutically targeted to treat cancer or chronic infection and, conversely, that interruption of this pathway may have utility in multiple sclerosis and other autoimmune syndromes. These findings have provided the rationale for designing new clinical trials for tumor immunotherapy.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/inmunología , Diferenciación Celular/inmunología , Activación de Linfocitos/inmunología , Transducción de Señal/inmunología , Células Th17 , Animales , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos CD28/genética , Antígenos CD28/inmunología , Linfocitos T CD4-Positivos/inmunología , Sangre Fetal/citología , Humanos , Inmunoterapia/métodos , Proteína Coestimuladora de Linfocitos T Inducibles , Interferón gamma/genética , Interferón gamma/inmunología , Interleucinas/genética , Interleucinas/inmunología , Ratones , Subfamilia B de Receptores Similares a Lectina de Células NK/genética , Subfamilia B de Receptores Similares a Lectina de Células NK/inmunología , Neoplasias/inmunología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/inmunología , Proteínas Proto-Oncogénicas c-maf/genética , Proteínas Proto-Oncogénicas c-maf/inmunología , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/inmunología , Subgrupos de Linfocitos T/inmunología , Células Th17/inmunología , Células Th17/fisiologíaRESUMEN
Mesothelin is a cell-surface molecule over-expressed on a large fraction of carcinomas, and thus is an attractive target of immunotherapy. A molecularly targeted therapy for these cancers was created by engineering T cells to express a chimeric receptor with high affinity for human mesothelin. Lentiviral vectors were used to express a single-chain variable fragment that binds mesothelin and that is fused to signaling domains derived from T-cell receptor zeta, CD28, and CD137 (4-1BB). When stimulated by mesothelin, lentivirally transduced T cells were induced to proliferate, express the antiapoptotic gene Bcl-X(L), and secrete multiple cytokines, all features characteristic of central memory T cells. When transferred intratumorally or intravenously into NOD/scid/IL2rgamma(-/-) mice engrafted with large pre-established tumors, the engineered T cells reduced the tumor burden, and in some cases resulted in complete eradication of the tumors at low effector-to-target ratios. Incorporation of the CD137 signaling domain specifically reprogrammed cells for multifunctional cytokine secretion and enhanced persistence of T cells. These findings have important implications for adoptive immunotherapy of cancer, especially in the context of poorly immunogenic tumors. Genetically redirected T cells have promise of targeting T lymphocytes to tumor antigens, confer resistance to the tumor microenvironment, and providing immunosurveillance.
Asunto(s)
Antígenos CD28/inmunología , Antígenos CD28/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Animales , Antígenos CD28/genética , Línea Celular Tumoral , Humanos , Mesotelina , Ratones , Transducción de Señal/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
HIV's considerable capacity to vary its HLA-I-restricted peptide antigens allows it to escape from host cytotoxic T lymphocytes (CTLs). Nevertheless, therapeutics able to target HLA-I-associated antigens, with specificity for the spectrum of preferred CTL escape mutants, could prove effective. Here we use phage display to isolate and enhance a T-cell antigen receptor (TCR) originating from a CTL line derived from an infected person and specific for the immunodominant HLA-A(*)02-restricted, HIVgag-specific peptide SLYNTVATL (SL9). High-affinity (K(D) < 400 pM) TCRs were produced that bound with a half-life in excess of 2.5 h, retained specificity, targeted HIV-infected cells and recognized all common escape variants of this epitope. CD8 T cells transduced with this supraphysiologic TCR produced a greater range of soluble factors and more interleukin-2 than those transduced with natural SL9-specific TCR, and they effectively controlled wild-type and mutant strains of HIV at effector-to-target ratios that could be achieved by T-cell therapy.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , VIH-1/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Secuencia de Aminoácidos , Células Cultivadas , Productos del Gen gag/química , Productos del Gen gag/inmunología , Humanos , Mutación/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Unión Proteica , SolubilidadRESUMEN
Many recent advances in basic cell biology and immunology are a harbinger of progress in adoptive cell therapy (ACT) including (1) the finding that host lymphodepletion enhances engraftment and efficacy, (2) the recognition that in vitro T cell functions may not correlate with in vivo efficacy, and (3) the development of advanced ex vivo culture methods to expand lymphocytes to therapeutically effective numbers. In this article, we focus on the development of artificial antigen presenting cells (aAPCs) in our laboratory and their applicability to augment ACT protocols. We also describe how aAPCs can be used to broaden ACT to treat patients with a wide variety of cancers, chronic infectious diseases, and autoimmune manifestations.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Inmunoterapia Adoptiva/métodos , Linfocitos T/trasplante , Animales , Humanos , Células K562 , Activación de Linfocitos/inmunología , Neoplasias/inmunología , Neoplasias/patología , Neoplasias/terapia , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/trasplante , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
The costimulatory requirements required for peripheral blood T regulatory cells (Tregs) are unclear. Using cell-based artificial APCs we found that CD28 but not ICOS, OX40, 4-1BB, CD27, or CD40 ligand costimulation maintained high levels of Foxp3 expression and in vitro suppressive function. Only CD28 costimulation in the presence of rapamycin consistently generated Tregs that consistently suppressed xenogeneic graft-vs-host disease in immunodeficient mice. Restimulation of Tregs after 8-12 days of culture with CD28 costimulation in the presence of rapamycin resulted in >1000-fold expansion of Tregs in <3 wk. Next, we determined whether other costimulatory pathways could augment the replicative potential of CD28-costimulated Tregs. We observed that while OX40 costimulation augmented the proliferative capacity of CD28-costimulated Tregs, Foxp3 expression and suppressive function were diminished. These studies indicate that the costimulatory requirements for expanding Tregs differ from those for T effector cells and, furthermore, they extend findings from mouse Tregs to demonstrate that human postthymic Tregs require CD28 costimulation to expand and maintain potent suppressive function in vivo.
Asunto(s)
Antígenos CD28/metabolismo , Activación de Linfocitos/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Traslado Adoptivo , Animales , Antígenos CD28/fisiología , Técnicas de Cultivo de Célula , Femenino , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/terapia , Humanos , Células K562 , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Transducción de Señal/inmunología , Linfocitos T Reguladores/trasplante , Timo/citología , Timo/inmunología , Timo/metabolismoRESUMEN
Previously, we showed that human umbilical cord blood (UCB) regulatory T cells (Tregs) could be expanded approximately 100-fold using anti-CD3/28 monoclonal antibody (mAb)-coated beads to provide T-cell receptor and costimulatory signals. Because Treg numbers from a single UCB unit are limited, we explored the use of cell-based artificial antigen-presenting cells (aAPCs) preloaded with anti-CD3/28 mAbs to achieve higher levels of Treg expansion. Compared with beads, aAPCs had similar expansion properties while significantly increasing transforming growth factor beta (TGF-beta) secretion and the potency of Treg suppressor function. aAPCs modified to coexpress OX40L or 4-1BBL expanded UCB Tregs to a significantly greater extent than bead- or nonmodified aAPC cultures, reaching mean expansion levels exceeding 1250-fold. Despite the high expansion and in contrast to studies using other Treg sources, neither OX40 nor 4-1BB signaling of UCB Tregs reduced in vitro suppression. UCB Tregs expanded with 4-1BBL expressing aAPCs had decreased levels of proapoptotic bim. UCB Tregs expanded with nonmodified or modified aAPCs versus beads resulted in higher survival associated with increased Treg persistence in a xeno-geneic graft-versus-host disease lethality model. These data offer a novel approach for UCB Treg expansion using aAPCs, including those coexpressing OX40L or 4-1BBL.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Sangre Fetal/citología , Receptores OX40/inmunología , Linfocitos T Reguladores/citología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Ligando 4-1BB/metabolismo , Animales , Células Presentadoras de Antígenos/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Sangre Fetal/efectos de los fármacos , Enfermedad Injerto contra Huésped/inmunología , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Microesferas , Proteínas Proto-Oncogénicas/metabolismo , Sirolimus/farmacología , Análisis de Supervivencia , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunologíaRESUMEN
Artificial APCs (aAPCs) genetically modified to express selective costimulatory molecules provide a reproducible, cost-effective, and convenient method for polyclonal and Ag-specific expansion of human T cells for adoptive immunotherapy. Among the variety of aAPCs that have been studied, acellular beads expressing anti-CD3/anti-CD28 efficiently expand CD4+ cells, but not CD8+ T cells. Cell-based aAPCs can effectively expand cytolytic CD8+ cells, but optimal costimulatory signals have not been defined. 4-1BB, a costimulatory molecule expressed by a minority of resting CD8+ T cells, is transiently up-regulated by all CD8+ T cells following activation. We compared expansion of human cytolytic CD8+ T cells using cell-based aAPCs providing costimulation via 4-1BB vs CD28. Whereas anti-CD3/anti-CD28 aAPCs mostly expand naive cells, anti-CD3/4-1BBL aAPCs preferentially expand memory cells, resulting in superior enrichment of Ag-reactive T cells which recognize previously primed Ags and efficient expansion of electronically sorted CD8+ populations reactive toward viral or self-Ags. Using HLA-A2-Fc fusion proteins linked to 4-1BBL aAPCs, 3-log expansion of Ag-specific CD8+ CTL was induced over 14 days, whereas similar Ag-specific CD8+ T cell expansion did not occur using HLA-A2-Fc/anti-CD28 aAPCs. Furthermore, when compared with cytolytic T cells expanded using CD28 costimulation, CTL expanded using 4-1BB costimulation mediate enhanced cytolytic capacity due, in part, to NKG2D up-regulation. These results demonstrate that 4-1BB costimulation is essential for expanding memory CD8+ T cells ex vivo and is superior to CD28 costimulation for generating Ag-specific products for adoptive cell therapy.
Asunto(s)
Ligando 4-1BB/metabolismo , Antígenos CD28/inmunología , Antígenos CD28/metabolismo , Inmunoterapia Adoptiva , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Células Presentadoras de Antígenos/metabolismo , Antígenos de Neoplasias/metabolismo , Complejo CD3/inmunología , Proliferación Celular , Células Cultivadas , Antígenos de Histocompatibilidad/inmunología , Humanos , Memoria Inmunológica/inmunología , Antígeno MART-1 , Subfamilia K de Receptores Similares a Lectina de Células NK , Proteínas de Neoplasias/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Fc/inmunología , Receptores Inmunológicos/metabolismo , Receptores de Células Asesinas Naturales , Transducción de Señal , Linfocitos T Citotóxicos/citologíaRESUMEN
An essential function of the immunological synapse (IS) is directed secretion. NK cells are especially adept at this activity, as they direct lytic granules to the synapse for secretion, which enables cytotoxicity and facilitates host defense. This initially requires rearrangement of the actin cytoskeleton and, subsequently, microtubule-dependent trafficking of the lytic granules. As these two steps are sequential, specific linkages between them are likely to serve as critical regulators of cytotoxicity. We studied Cdc42-interacting protein-4 (CIP4), which constitutively interacts with tubulin and microtubules but focuses to the microtubule organizing center (MTOC) after NK cell activation, when it is able to associate with Wiskott-Aldrich syndrome protein (WASp) and the actin filament-rich IS. WASp deficiency, overexpression of CIP4, or parts of CIP4 interfere with this union and block normal CIP4 localization, MTOC polarization to the IS, and cytotoxicity. Reduction of endogenous CIP4 expression using small interfering RNA similarly inhibits MTOC polarization and cytotoxic activity but does not impair actin filament accumulation at the IS, or Cdc42 activation. Thus, CIP4 is an important cytoskeletal adaptor that functions after filamentous actin accumulation and Cdc42 activation to enable MTOC polarization and NK cell cytotoxicity.
Asunto(s)
Actinas/metabolismo , Citosol/inmunología , Citosol/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Línea Celular , Polaridad Celular , Forma de la Célula , Regulación de la Expresión Génica , Humanos , Células Asesinas Naturales/citología , Proteínas Asociadas a Microtúbulos/genética , Centro Organizador de los Microtúbulos/metabolismo , Antígenos de Histocompatibilidad Menor , Unión Proteica , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
To facilitate the therapeutic application of antigen-presenting cells (APCs), we have developed a cell-based artificial APC (aAPC) system by engineering K562 cells with lentiviruses to direct the stable expression and secretion of a variety of co-stimulatory molecules and cytokines. Here we report the use of a combinatorial lentiviral gene transfer approach to achieve long-term stable expression of at least seven genes in the K562 parental cell line. Expression of various combinations of genes on the aAPC enables the precise determination of human T-cell activation requirements, such that aAPCs can be tailored for the optimal propagation of T-cell subsets with specific growth requirements and distinct functions. The aAPCs support ex vivo growth and long-term expansion of functional human CD8 T cells without requiring the addition of exogenous cytokines, in contrast to the use of natural APCs. Distinct populations of T cells can be expanded with aAPCs expressing CD137L (4-1BBL) and/or CD80. Finally, the aAPCs provide an efficient platform to expand genetically modified T cells and to maintain CD28 expression on CD8 T cells. Therefore, K562-based aAPCs have therapeutic potential for adoptive immunotherapies and vaccinations.