RESUMEN
T-cell-recruiting bispecific molecule therapy has yielded promising results in patients with hematologic malignancies; however, resistance and subsequent relapse remains a major challenge. T-cell exhaustion induced by persistent antigen stimulation or tonic receptor signaling has been reported to compromise outcomes of T-cell-based immunotherapies. The impact of continuous exposure to bispecifics on T-cell function, however, remains poorly understood. In relapsed/refractory B-cell precursor acute lymphoblastic leukemia patients, 28-day continuous infusion with the CD19xCD3 bispecific molecule blinatumomab led to declining T-cell function. In an in vitro model system, mimicking 28-day continuous infusion with the half-life-extended CD19xCD3 bispecific AMG 562, we identified hallmark features of exhaustion arising over time. Continuous AMG 562 exposure induced progressive loss of T-cell function (day 7 vs day 28 mean specific lysis: 88.4% vs 8.6%; n = 6; P = .0003). Treatment-free intervals (TFIs), achieved by AMG 562 withdrawal, were identified as a powerful strategy for counteracting exhaustion. TFIs induced strong functional reinvigoration of T cells (continuous vs TFI-specific lysis on day 14: 34.9% vs 93.4%; n = 6; P < .0001) and transcriptional reprogramming. Furthermore, use of a TFI led to improved T-cell expansion and tumor control in vivo. Our data demonstrate the relevance of T-cell exhaustion in bispecific antibody therapy and highlight that T cells can be functionally and transcriptionally rejuvenated with TFIs. In view of the growing number of bispecific molecules being evaluated in clinical trials, our findings emphasize the need to consider and evaluate TFIs in application schedules to improve clinical outcomes.
Asunto(s)
Anticuerpos Biespecíficos , Antineoplásicos , Linfoma de Células B , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Antígenos CD19 , Antineoplásicos/uso terapéutico , Humanos , Inmunoterapia/métodos , Linfoma de Células B/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Linfocitos TRESUMEN
Bispecific T-cell engager (BiTE®) molecules recruit T cells to cancer cells through CD3ε binding, independently of T-cell receptor (TCR) specificity. Whereas physiological T-cell activation is dependent on signal 1 (TCR engagement) and signal 2 (co-stimulation), BiTE molecule-mediated T-cell activation occurs without additional co-stimulation. As co-stimulatory and inhibitory molecules modulate the strength and nature of T-cell responses, we studied the impact of the expression profile of those molecules on target cells for BiTE molecule-mediated T-cell activation in the context of acute myeloid leukemia (AML). Accordingly, we created a novel in vitro model system using murine Ba/F3 cells transduced with human CD33 ± CD86 ± PD-L1. T-cell fitness was assessed by T-cell function assays in co-cultures and immune synapse formation by applying a CD33 BiTE molecule (AMG 330). Using our cell-based model platform, we found that the expression of positive co-stimulatory molecules on target cells markedly enhanced BiTE molecule-mediated T-cell activation. The initiation and stability of the immune synapse between T cells and target cells were significantly increased through the expression of CD86 on target cells. By contrast, the co-inhibitory molecule PD-L1 impaired the stability of BiTE molecule-induced immune synapses and subsequent T-cell responses. We validated our findings in primary T-cell-AML co-cultures, demonstrating a PD-L1-mediated reduction in redirected T-cell activation. The addition of the immunomodulatory drug (IMiD) lenalidomide to co-cultures led to stabilization of immune synapses and improved subsequent T-cell responses. We conclude that target cells modulate CD33 BiTE molecule-dependent T-cell activation and hence, combinatorial strategies might contribute to enhanced efficacy.
Asunto(s)
Anticuerpos Biespecíficos , Leucemia Mieloide Aguda , Animales , Humanos , Ratones , Antígeno B7-H1/metabolismo , Proteínas de Punto de Control Inmunitario/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Lectina 3 Similar a Ig de Unión al Ácido Siálico/metabolismo , Linfocitos TRESUMEN
Spondyloarthritis (SpA) is a chronic inflammatory disease that is tightly linked to HLA-B*27 but the pathophysiological basis of this link is still unknown. It is discussed whether either the instability of HLA-B*27 molecules triggers predominantly innate immune reactions or yet unknown antigenic peptides presented by HLA-B*27 induce adaptive autoimmune reactions by CD8+ T cells. To analyze the pathogenesis of SpA, we here investigated the T cell receptor (TCR) usage and whole transcriptomes of CD8+ single cells from synovial fluid of HLA-B*27-positive SpA patients and HLA-B*27-negative controls. In HLA-B*27-positive patients, we confirmed preferential expression of several TCR ß-chain families, found even more restricted usage of particular TCR α-chains, assigned matching TCR αß-chain pairs with homologous CDR3-sequences, and detected identical TCR-chains in different patients. Gene expression analyses by single cell mRNAseq revealed that genes specific for the tissue resident memory phenotype, exhaustion, and apoptosis were particularly highly expressed in expanded clonotypes from HLA-B*27-positive SpA patients. Together, several independent lines of evidence argue in favor of an (auto)antigenic peptide related pathogenesis.
Asunto(s)
Linfocitos T CD8-positivos , Antígenos HLA-BRESUMEN
NF-κB inducing kinase (NIK) is the key protein of the non-canonical NF-κB pathway and is important for the development of lymph nodes and other secondary immune organs. We elucidated the specific role of NIK in T cells using T-cell specific NIK-deficient (NIKΔT) mice. Despite showing normal development of lymphoid organs, NIKΔT mice were resistant to induction of CNS autoimmunity. T cells from NIKΔT mice were deficient in late priming, failed to up-regulate T-bet and to transmigrate into the CNS. Proteomic analysis of activated NIK-/- T cells showed de-regulated expression of proteins involved in the formation of the immunological synapse: in particular, proteins involved in cytoskeleton dynamics. In line with this we found that NIK-deficient T cells were hampered in phosphorylation of Zap70, LAT, AKT, ERK1/2 and PLCγ upon TCR engagement. Hence, our data disclose a hitherto unknown function of NIK in T-cell priming and differentiation.
Asunto(s)
Actinas/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Activación de Linfocitos , Proteínas Serina-Treonina Quinasas/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Actinas/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/patología , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/inmunología , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/inmunología , Glicoproteína Mielina-Oligodendrócito/administración & dosificación , Fragmentos de Péptidos/administración & dosificación , Fosfolipasa C gamma/genética , Fosfolipasa C gamma/inmunología , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Cultivo Primario de Células , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , Receptores de Antígenos de Linfocitos T/genética , Transducción de Señal , Bazo/inmunología , Bazo/patología , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/inmunología , Linfocitos T/patología , Proteína Tirosina Quinasa ZAP-70/genética , Proteína Tirosina Quinasa ZAP-70/inmunología , Quinasa de Factor Nuclear kappa BRESUMEN
The small adaptor protein growth factor receptor-bound protein 2 (Grb2) modulates and integrates signals from receptors on cellular surfaces in inner signaling pathways. In murine T cells, Grb2 is crucial for amplification of TCR signaling. T cell-specific Grb2(fl/fl) Lckcre(tg) Grb2-deficient mice show reduced T cell numbers due to impaired negative and positive selection. In this study, we found that T cell numbers in Grb2(fl/fl) CD4cre(tg) mice were normal in the thymus and were only slightly affected in the periphery. Ex vivo analysis of CD4(+) Th cell populations revealed an increased amount of Th1 cells within the CD4(+) population of Grb2(fl/fl) CD4cre(tg) mice. Additionally, Grb2-deficient T cells showed a greater potential to differentiate into Th17 cells in vitro. To test whether these changes in Th cell differentiation potential rendered Grb2(fl/fl) CD4cre(tg) mice more prone to inflammatory diseases, we used the murine Th1 cell- and Th17 cell-driven model of experimental autoimmune encephalomyelitis (EAE). In contrast to our expectations, Grb2(fl/fl) CD4cre(tg) mice developed a milder form of EAE. The impaired EAE disease can be explained by the reduced proliferation rate of Grb2-deficient CD4(+) T cells upon stimulation with IL-2 or upon activation by allogeneic dendritic cells, because the activation of T cells by dendritic cells and the subsequent T cell proliferation are known to be crucial factors for the induction of EAE. In summary, Grb2-deficient T cells show defects in T cell development, increased Th1 and Th17 cell differentiation capacities, and impaired proliferation after activation by dendritic cells, which likely reduce the clinical symptoms of EAE.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Proteína Adaptadora GRB2/genética , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Células Th17/citología , Células Th17/inmunología , Animales , Citocinas/metabolismo , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/mortalidad , Activación de Linfocitos/inmunología , Recuento de Linfocitos , Ratones , Ratones Noqueados , Ratones Transgénicos , Subgrupos de Linfocitos T/metabolismo , Células Th17/metabolismoAsunto(s)
Encefalomielitis Autoinmune Experimental , Proteoglicanos/deficiencia , Células TH1/patología , Células Th17/patología , Animales , Antígenos/genética , Antígenos CD/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Transgénicos , Glicoproteína Mielina-Oligodendrócito/inmunología , Glicoproteína Mielina-Oligodendrócito/toxicidad , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/toxicidad , Toxina del Pertussis/toxicidad , Proteoglicanos/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismoRESUMEN
The function of regulatory T (Treg) cells depends on lipid oxidation. However, the molecular mechanism by which Treg cells maintain lipid metabolism after activation remains elusive. Liver kinase B1 (LKB1) acts as a coordinator by linking cellular metabolism to substrate AMP-activated protein kinase (AMPK). We show that deletion of LKB1 in Treg cells exhibited reduced suppressive activity and developed fatal autoimmune inflammation. Mechanistically, LKB1 induced activation of the mevalonate pathway by upregulating mevalonate genes, which was essential for Treg cell functional competency and stability by inducing Treg cell proliferation and suppressing interferon-gamma and interleukin-17A expression independently of AMPK. Furthermore, LKB1 was found to regulate intracellular cholesterol homeostasis and to promote the mevalonate pathway. In agreement, mevalonate and its metabolite geranylgeranyl pyrophosphate inhibited conversion of Treg cells and enhanced survival of LKB1-deficient Treg mice. Thus, LKB1 is a key regulator of lipid metabolism in Treg cells, involved in optimal programming of suppressive activity, immune homeostasis, and tolerance.
Asunto(s)
Ácido Mevalónico/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Linfocitos T Reguladores/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Enfermedades Autoinmunes/terapia , Proliferación Celular , Colesterol/metabolismo , Femenino , Factores de Transcripción Forkhead/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Hidroximetilglutaril-CoA Reductasas/deficiencia , Hidroximetilglutaril-CoA Reductasas/genética , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Metabolismo de los Lípidos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfatos de Poliisoprenilo/uso terapéutico , Proteínas Serina-Treonina Quinasas/genética , Factor de Transcripción STAT5/metabolismo , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/trasplanteRESUMEN
Statins are a well-established family of drugs that lower cholesterol levels via the competitive inhibition of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). In addition, the pleiotropic anti-inflammatory effects of statins on T cells make them attractive as therapeutic drugs in T-cell-driven autoimmune disorders. Since statins do not exclusively target HMGCR and thus might have varying effects on different cell types, we generated a new mouse strain allowing for the tissue-specific deletion of HMGCR. Deletion of HMGCR expression in T cells led to a severe decrease in their numbers with the remaining cells displaying an activated phenotype, with an increased proportion of regulatory T cells (Tregs) in particular. However, deletion of HMGCR specifically in Tregs resulted in severe autoimmunity, suggesting that this enzyme is also essential for the maintenance of Tregs. We were able to prevent the death of HMGCR-deficient lymphocytes by the addition of either the direct metabolite of HMGCR, namely mevalonate, or the downstream metabolite geranylgeranyl pyrophosphate, which is essential for protein prenylation. However, the addition of cholesterol, which is the final product of the mevalonate pathway, did not inhibit cell death, indicating that protein prenylation rather than the cholesterol biosynthesis pathway is indispensible for T-cell survival.
Asunto(s)
Hidroximetilglutaril-CoA Reductasas/metabolismo , Prenilación de Proteína , Linfocitos T/citología , Linfocitos T/enzimología , Animales , Recuento de Células , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Eliminación de Gen , Hidroximetilglutaril-CoA Reductasas/deficiencia , Integrasas/metabolismo , Activación de Linfocitos/efectos de los fármacos , Ácido Mevalónico/análogos & derivados , Ácido Mevalónico/farmacología , Ratones Endogámicos C57BL , Fenotipo , Fosfatos de Poliisoprenilo/farmacología , Prenilación de Proteína/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/enzimologíaRESUMEN
Arrival of encephalitogenic T cells at inflammatory foci represents a critical step in development of experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. EBI2 and its ligand, 7α,25-OHC, direct immune cell localization in secondary lymphoid organs. CH25H and CYP7B1 hydroxylate cholesterol to 7α,25-OHC. During EAE, we found increased expression of CH25H by microglia and CYP7B1 by CNS-infiltrating immune cells elevating the ligand concentration in the CNS. Two critical pro-inflammatory cytokines, interleukin-23 (IL-23) and interleukin-1 beta (IL-1ß), maintained expression of EBI2 in differentiating Th17 cells. In line with this, EBI2 enhanced early migration of encephalitogenic T cells into the CNS in a transfer EAE model. Nonetheless, EBI2 was dispensable in active EAE. Human Th17 cells do also express EBI2, and EBI2 expressing cells are abundant within multiple sclerosis (MS) white matter lesions. These findings implicate EBI2 as a mediator of CNS autoimmunity and describe mechanistically its contribution to the migration of autoreactive T cells into inflamed organs.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/fisiología , Movimiento Celular/fisiología , Sistema Nervioso Central/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Esclerosis Múltiple/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Autoinmunidad/fisiología , Sistema Nervioso Central/fisiología , Familia 7 del Citocromo P450/metabolismo , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/patología , Femenino , Interleucina-1beta/metabolismo , Interleucina-23/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Esteroide Hidroxilasas/metabolismo , Células Th17/metabolismo , Células Th17/fisiologíaRESUMEN
Effector functions of inflammatory IL-17-producing Th (Th17) cells have been linked to autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS). However, what determines Th17 cell encephalitogenicity is still unresolved. Here, we show that after EAE induction, mice deficient for the NF-κB regulator MALT1 (Malt1-/- mice) exhibit strong lymphocytic infiltration in the CNS, but do not develop any clinical signs of EAE. Loss of Malt1 interfered with expression of the Th17 effector cytokines IL-17 and GM-CSF both in vitro and in vivo. In line with their impaired GM-CSF secretion, Malt1-/- Th cells failed to recruit myeloid cells to the CNS to sustain neuroinflammation, whereas autoreactive WT Th cells successfully induced EAE in Malt1-/- hosts. In contrast, Malt1 deficiency did not affect Th1 cells. Despite their significantly decreased secretion of Th17 effector cytokines, Malt1-/- Th17 cells showed normal expression of lineage-specific transcription factors. Malt1-/- Th cells failed to cleave RelB, a suppressor of canonical NF-κB, and exhibited altered cellular localization of this protein. Our results indicate that MALT1 is a central, cell-intrinsic factor that determines the encephalitogenic potential of inflammatory Th17 cells in vivo.