RESUMO
Chimeric antigen receptor (CAR)-T cell therapies reprogram T cells to engage and eliminate cancer cells. Patients' T cells are transduced in vitro using lentiviral or retroviral vectors containing a CAR transgene. Following infusion, CAR-T cells expand in vivo and may persist in the peripheral blood and bone marrow for years. Therefore, monitoring in vivo copies of the CAR transgene requires highly sensitive, validated analytical methods. Herein, we describe the validation of a qPCR assay to detect tisagenlecleucel transgene in patient samples. The limit of detection and lower limit of quantitation were 3.1 and 10 copies/200 ng genomic DNA, respectively, equivalent to â¼50 copies/µg genomic DNA and in alignment with US Food and Drug Administration guidance on bioanalytical method validation. The assay allowed quantitation of the tisagenlecleucel transgene over a wide dynamic range with a high degree of linearity, that is, 101-106 copies/200 ng genomic DNA (R2 ≥ 0.9988). Coefficients of variation of measured transgene copies ranged from 0.2% to 12.8%. A droplet digital PCR assay was performed as a method of validation and showed a strong correlation with the qPCR assay (R2 = 0.9980, p < 0.0001). This qPCR assay is being utilized to monitor tisagenlecleucel expansion and persistence in clinical trials.
RESUMO
Exceptional clinical responses produced by the first chimeric antigen receptor T [CAR-T] cell therapies, and their entry into commercial markets prompted a logarithmic increase in the number of next generation CAR-T clinical trials. As a result, there is a growing interest in understanding the analytical approaches utilized for reliable monitoring of these "living" drugs, and the challenges encountered during their clinical development. Multiparametric flow cytometry (MFC) assays have played a crucial role in understanding the phenotype and function of first approved CAR-T therapies. Herein, three main areas for monitoring CAR-T therapies in clinical trials are discussed: (1) analytical considerations critical for development of MFC assays for the reliable enumeration of CAR-T levels, (2) operational challenges associated with clinical trial sampling and transportation, and (3) differential cellular kinetics observed by MFC and qPCR analyses and their relationship with efficacy (measurable residual disease levels). Initial experiences described here may enable design of fit-for-purpose tools and help to more rapidly advance the development of next generation CAR-T therapies.
Assuntos
Citometria de Fluxo , Imunoterapia Adotiva , Ensaios Clínicos como Assunto , Humanos , Cinética , Receptores de Antígenos Quiméricos , Linfócitos TRESUMO
Effective immune responses depend upon appropriate T cell differentiation in accord with the nature of an infectious agent, and the contingency of differentiation depends minimally on TCR, coreceptor, and cytokine signals. In this reverse genetic study, we show that the MAPK Erk2 is not essential for T cell proliferation in the presence of optimum costimulation. Instead, it has opposite effects on T-bet and Gata3 expression and, hence, on Th1 and Th2 differentiation. Alternatively, in the presence of TGF-ß, the Erk pathway suppresses a large program of gene expression, effectively limiting the differentiation of Foxp3(+) regulatory T cells. In the latter case, the mechanisms involved include suppression of Gata3 and Foxp3, induction of Tbx21, phosphorylation of Smad2,3, and possibly suppression of Socs2, a positive inducer of Stat5 signaling. Consequently, loss of Erk2 severely impeded Th1 differentiation while enhancing the development of Foxp3(+)-induced T regulatory cells. Selected profiles of gene expression under multiple conditions of T cell activation illustrate the opposing consequences of Erk pathway signaling.
Assuntos
Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/imunologia , Polaridade Celular/imunologia , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Subpopulações de Linfócitos T/enzimologia , Subpopulações de Linfócitos T/imunologia , Animais , Linfócitos T CD4-Positivos/virologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Polaridade Celular/genética , Proliferação de Células , Células Cultivadas , Fatores de Transcrição Forkhead/biossíntese , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteína Quinase 1 Ativada por Mitógeno/deficiência , Proteína Quinase 1 Ativada por Mitógeno/genética , Subpopulações de Linfócitos T/virologia , Linfócitos T Reguladores/enzimologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/virologia , Células Th1/enzimologia , Células Th1/imunologia , Células Th1/virologia , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
Cell populations are regulated in size by at least two forms of apoptosis. More recently, necroptosis, a parallel, nonapoptotic pathway of cell death, has been described, and this pathway is invoked in the absence of caspase 8. In caspase 8-deficient T cells, necroptosis occurs as the result of antigen receptor-mediated activation. Here, through a genetic analysis, we show that necroptosis in caspase 8-deficient T cells is related neither to the programmed necrosis as defined by the requirement for mitochondrial cyclophilin D nor to autophagy as defined by the requirement for autophagy-related protein 7. Rather, survival of caspase 8-defective T cells can be completely rescued by loss of receptor-interacting serine-threonine kinase (Ripk) 3. Additionally, complementation of a T cell-specific caspase 8 deficiency with a loss of Ripk3 gives rise to lymphoproliferative disease reminiscent of lpr or gld mice. In conjunction with previous work, we conclude that necroptosis in antigen-stimulated caspase 8-deficient T cells is the result of a novel Ripk1- and Ripk3-mediated pathway of cell death.
Assuntos
Apoptose , Caspase 8/fisiologia , Linfócitos T/imunologia , Animais , Apoptose/imunologia , Autofagia , Proteína 7 Relacionada à Autofagia , Sobrevivência Celular , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/fisiologia , Necrose/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologiaRESUMO
Foxo transcription factors integrate extrinsic signals to regulate cell division, differentiation and survival, and specific functions of lymphoid and myeloid cells. Here, we showed the absence of Foxo1 severely curtailed the development of Foxp3(+) regulatory T (Treg) cells and those that developed were nonfunctional in vivo. The loss of function included diminished CTLA-4 receptor expression as the Ctla4 gene was a direct target of Foxo1. T cell-specific loss of Foxo1 resulted in exocrine pancreatitis, hind limb paralysis, multiorgan lymphocyte infiltration, anti-nuclear antibodies and expanded germinal centers. Foxo-mediated control over Treg cell specification was further revealed by the inability of TGF-ß cytokine to suppress T-bet transcription factor in the absence of Foxo1, resulting in IFN-γ secretion. In addition, the absence of Foxo3 exacerbated the effects of the loss of Foxo1. Thus, Foxo transcription factors guide the contingencies of T cell differentiation and the specific functions of effector cell populations.
Assuntos
Antígenos CD/biossíntese , Fatores de Transcrição Forkhead/metabolismo , Proteínas com Domínio T/metabolismo , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismo , Animais , Antígenos CD/genética , Autoimunidade/genética , Antígeno CTLA-4 , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Regulação da Expressão Gênica/imunologia , Tolerância Imunológica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas com Domínio T/genética , Proteínas com Domínio T/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Equilíbrio Th1-Th2 , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Programmed cell death (PCD) occurs widely in species from every kingdom of life. It has been shown to be an integral aspect of development in multicellular organisms, and it is an essential component of the immune response to infectious agents. An analysis of the phylogenetic origin of PCD now shows that it evolved independently several times, and it is fundamental to basic cellular physiology. Undoubtedly, PCD pervades all life at every scale of analysis. These considerations provide a backdrop for understanding the complexity of intertwined, but independent, cell death programs that operate within the immune system. In particular, the contributions of apoptosis, autophagy, and necrosis in the resolution of an immune response are considered.
Assuntos
Apoptose/imunologia , Autofagia/imunologia , Imunidade/imunologia , Transdução de Sinais/imunologia , Animais , Caspase 8/metabolismo , Sobrevivência Celular/imunologia , Humanos , Necrose/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Signals initiated through the TCR during development can result in either survival and differentiation or cell death. High affinity signals that induce death elicit a robust yet transient activation of signaling pathways, including Erk, whereas low affinity ligands, which promote survival, generate a gradual and weaker activation of the same pathways. It was recently demonstrated that Erk localizes to distinct cellular locations in response to high and low affinity ligands. Although a requirement for Erk in positive selection is well established, its role in negative selection is controversial and, thus, the importance of Erk relocalization during development is not understood. In this study, we examined the role of Erk in negative selection using mice that are genetically deficient in both Erk1 and Erk2 in T cells. Results from three different models reveal that thymocyte deletion remains intact in the absence of Erk.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Linfócitos T/imunologia , Timo/imunologia , Animais , Deleção Clonal , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Técnicas de Cultura de Órgãos , Linfócitos T/enzimologia , Timo/enzimologiaRESUMO
Foxo transcription factors regulate cell cycle progression, cell survival and DNA-repair pathways. Here we demonstrate that deficiency in Foxo3 resulted in greater expansion of T cell populations after viral infection. This exaggerated expansion was not T cell intrinsic. Instead, it was caused by the enhanced capacity of Foxo3-deficient dendritic cells to sustain T cell viability by producing more interleukin 6. Stimulation of dendritic cells mediated by the coinhibitory molecule CTLA-4 induced nuclear localization of Foxo3, which in turn inhibited the production of interleukin 6 and tumor necrosis factor. Thus, Foxo3 acts to constrain the production of key inflammatory cytokines by dendritic cells and to control T cell survival.
Assuntos
Células Dendríticas/imunologia , Fatores de Transcrição Forkhead/imunologia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Animais , Apresentação de Antígeno/imunologia , Antígenos CD/imunologia , Antígenos CD/metabolismo , Infecções por Arenaviridae/imunologia , Western Blotting , Antígeno CTLA-4 , Células Dendríticas/metabolismo , Citometria de Fluxo , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Congênicos , Camundongos Transgênicos , Transporte Proteico/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
T cells enigmatically require caspase-8, an inducer of apoptosis, for antigen-driven expansion and effective antiviral responses, and yet the pathways responsible for this effect have been elusive. A defect in caspase-8 expression does not affect progression through the cell cycle but causes an abnormally high rate of cell death that is distinct from apoptosis and does not involve a loss of NFkappaB activation. Instead, antigen or mitogen activated Casp8-deficient T cells exhibit an alternative type of cell death similar to programmed necrosis that depends on receptor interacting protein (Ripk1). The selective genetic ablation of caspase-8, NFkappaB, and Ripk1, reveals two forms of cell death that can regulate virus-specific T cell expansion.
Assuntos
Caspase 8/imunologia , NF-kappa B/imunologia , Necrose/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Linfócitos T/fisiologia , Transferência Adotiva , Animais , Caspase 8/genética , Cruzamentos Genéticos , Citometria de Fluxo , Inativação Gênica , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Linfócitos T/virologiaRESUMO
The NF-kappaB signaling system has important and distinct roles in determining cell fate decisions, such as cell proliferation and cell death. Specifically, recent evidence indicates that NF-B regulates several types of programmed cell death, such as apoptosis, necroptosis, necrosis, as well as cellular senescence, but its precise role in these is not fully understood. Distinguishing these cell fates experimentally is therefore important, and several techniques are available to researchers. We summarize experimental strategies and protocols that reveal changes in nuclear morphology and cell shrinkage, exposure of phosphatidylserine, compromised membrane integrity, DNA fragmentation, and altered mitochondrial membrane potential. Together, these may discriminate distinct cell death pathways and lead to a better understanding of the underlying regulatory mechanisms.
Assuntos
Apoptose/fisiologia , NF-kappa B/fisiologia , Animais , Ciclo Celular , Membrana Celular/metabolismo , Sobrevivência Celular , Senescência Celular/fisiologia , DNA/análise , DNA/metabolismo , Fragmentação do DNA , Marcação In Situ das Extremidades Cortadas , Potencial da Membrana Mitocondrial , Microscopia Eletrônica , Fosfatidilserinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Necroptosis is a cellular mechanism of necrotic cell death induced by apoptotic stimuli in the form of death domain receptor engagement by their respective ligands under conditions where apoptotic execution is prevented. Although it occurs under regulated conditions, necroptotic cell death is characterized by the same morphological features as unregulated necrotic death. Here we report that necrostatin-1, a previously identified small-molecule inhibitor of necroptosis, is a selective allosteric inhibitor of the death domain receptor-associated adaptor kinase RIP1 in vitro. We show that RIP1 is the primary cellular target responsible for the antinecroptosis activity of necrostatin-1. In addition, we show that two other necrostatins, necrostatin-3 and necrostatin-5, also target the RIP1 kinase step in the necroptosis pathway, but through mechanisms distinct from that of necrostatin-1. Overall, our data establish necrostatins as the first-in-class inhibitors of RIP1 kinase, the key upstream kinase involved in the activation of necroptosis.
Assuntos
Imidazóis/metabolismo , Proteínas Quinases/metabolismo , Animais , Apoptose , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Relação Estrutura-AtividadeRESUMO
Caspase-8 is an essential component of death receptor-mediated apoptosis. Along with Fas-associated death domain protein, it is also essential for T cell proliferation in response to antigenic or mitogenic stimuli. To determine whether caspase-8 is also required for B cell proliferation, we generated mice with a B cell-specific Casp8 deficiency. Unlike T cells, caspase-8 was not required for Ag receptor-driven proliferation or Ab formation. Rather, Casp8-deficient B cells failed to proliferate in response to dsRNA and LPS, ligands for TLR3 and TLR4, respectively, but responded normally to the TLR9 agonist CpG DNA. Similarly, Ab production to trinitrophenol-LPS was selectively reduced in B cell-specific Casp8-deficient mice. The activation of NF-kappaB or IFN regulatory factor 3 was found to be unaffected by the loss of caspase-8, implicating it in a novel pathway important for some forms of innate immunity mediated by B cells.
Assuntos
Linfócitos B/imunologia , Caspases/imunologia , Imunidade Inata , Animais , Formação de Anticorpos , Linfócitos B/citologia , Caspase 8 , Caspases/deficiência , Caspases/fisiologia , Proliferação de Células , Camundongos , Camundongos Knockout , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Receptor Toll-Like 9/imunologiaRESUMO
Stimulation of the TCR leads to an oscillatory release of free calcium that activates members of the calcium/calmodulin-dependent protein kinase II (CaMKII) family. The CaMKII molecules have profound and lasting effects on cellular signaling in several cell types, yet the role of CaMKII in T cells is still poorly characterized. In this report we describe a splice variant of CaMKIIbeta, CaMKIIbeta'e, in mouse T cells. We have determined its function, along with that of CaMKIIgamma, by introducing the active and kinase-dead mutants into activated P14 TCR transgenic T cells using retroviral transduction. Active CaMKII enhanced the proliferation and cytotoxic activity of T cells while reducing their IL-2 production. Furthermore, it induced a profound state of unresponsiveness that could be overcome only by prolonged culture in IL-2. These results indicate that members of the CaMKII family play an important role in regulation of CD8 T cell proliferation, cytotoxic effector function, and the response to restimulation.
Assuntos
Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/imunologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Anergia Clonal/imunologia , Ativação Linfocitária/imunologia , Complexos Multienzimáticos/fisiologia , Processamento Alternativo , Animais , Antígenos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/biossíntese , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proliferação de Células , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Anergia Clonal/genética , Citocinas/biossíntese , Citotoxicidade Imunológica/genética , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/fisiologia , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Complexos Multienzimáticos/biossíntese , Complexos Multienzimáticos/genética , Transdução GenéticaRESUMO
LIM domain factors and associated cofactors are important developmental regulators in pattern formation and organogenesis. In addition, overexpression of two LIM-only factors (LMOs) causes acute lymphocytic leukemia. The more recently discovered LMO factor LMO4 is highly expressed in proliferating epithelial cells, and frequently overexpressed in breast carcinoma. Here we show that while LMO4 is expressed throughout mammary gland development, it is dramatically upregulated in mammary epithelial cells during midpregnancy. The LMO coactivator Clim2/Ldb1/NLI showed a similar expression pattern, consistent with the idea that LMO4 and Clim2 act as a complex in mammary epithelial cells. In MCF-7 cells, LMO4 transcripts were upregulated by heregulin, an activator of ErbB receptors that are known to be important in mammary gland development and breast cancer. To test the hypothesis that LMO4 plays roles in mammary gland development, we created an engrailed-LMO4 fusion protein. This fusion protein maintains the ability to interact with Clim2, but acts as a dominant repressor of both basal and activated transcription when recruited to a DNA-regulatory region. When the engrailed-LMO4 fusion protein was expressed under control of the MMTV promoter in transgenic mice, both ductular development in virgin mice and alveolar development in pregnant mice were inhibited. These results suggest that LMO4 plays a role in promoting mammary gland development.