RESUMEN
Cell therapies such as tumor-infiltrating lymphocyte (TIL) therapy have shown promise in the treatment of patients with refractory solid tumors, with improvement in response rates and durability of responses nevertheless sought. To identify targets capable of enhancing the antitumor activity of T cell therapies, large-scale in vitro and in vivo clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens were performed, with the SOCS1 gene identified as a top T cell-enhancing target. In murine CD8+ T cell-therapy models, SOCS1 served as a critical checkpoint in restraining the accumulation of central memory T cells in lymphoid organs as well as intermediate (Texint) and effector (Texeff) exhausted T cell subsets derived from progenitor exhausted T cells (Texprog) in tumors. A comprehensive CRISPR tiling screen of the SOCS1-coding region identified sgRNAs targeting the SH2 domain of SOCS1 as the most potent, with an sgRNA with minimal off-target cut sites used to manufacture KSQ-001, an engineered TIL therapy with SOCS1 inactivated by CRISPR/Cas9. KSQ-001 possessed increased responsiveness to cytokine signals and enhanced in vivo antitumor function in mouse models. These data demonstrate the use of CRISPR/Cas9 screens in the rational design of T cell therapies.
Asunto(s)
Sistemas CRISPR-Cas , Neoplasias , Humanos , Animales , Ratones , ARN Guía de Sistemas CRISPR-Cas , Linfocitos Infiltrantes de Tumor , Inmunoterapia Adoptiva , Neoplasias/genética , Edición Génica , Proteína 1 Supresora de la Señalización de Citocinas/genéticaRESUMEN
Inhibitors of Bruton's tyrosine kinase (BTK) possess much promise for the treatment of oncologic and autoimmune indications. However, our current knowledge of the role of BTK in immune competence has been gathered in the context of genetic inactivation of btk in both mice and man. Using the novel BTK inhibitor PF-303, we model the clinical phenotype of BTK inhibition by systematically examining the impact of PF-303 on the mature immune system in mice. We implicate BTK in tonic BCR signaling, demonstrate dependence of the T3 B cell subset and IgM surface expression on BTK activity, and find that B1 cells survive and function independently of BTK. Although BTK inhibition does not impact humoral memory survival, Ag-driven clonal expansion of memory B cells and Ab-secreting cell generation are inhibited. These data define the role of BTK in the mature immune system and mechanistically predict the clinical phenotype of chronic BTK inhibition.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Inmunidad Humoral/fisiología , Memoria Inmunológica/fisiología , Modelos Inmunológicos , Proteínas Tirosina Quinasas/inmunología , Agammaglobulinemia Tirosina Quinasa , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Humanos , Inmunoglobulina M/genética , Inmunoglobulina M/inmunología , Ratones , Ratones Endogámicos CBA , Ratones Noqueados , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genéticaRESUMEN
Autoantibody production and immune complex deposition within the kidney promote renal disease in patients with lupus nephritis. Thus, therapeutics that inhibit these pathways may be efficacious in the treatment of systemic lupus erythematosus. Bruton's tyrosine kinase (BTK) is a critical signaling component of both BCR and FcR signaling. We sought to assess the efficacy of inhibiting BTK in the development of lupus-like disease, and in this article describe (R)-5-amino-1-(1-cyanopiperidin-3-yl)-3-(4-[2,4-difluorophenoxy]phenyl)-1H-pyrazole-4-carboxamide (PF-06250112), a novel highly selective and potent BTK inhibitor. We demonstrate in vitro that PF-06250112 inhibits both BCR-mediated signaling and proliferation, as well as FcR-mediated activation. To assess the therapeutic impact of BTK inhibition, we treated aged NZBxW_F1 mice with PF-06250112 and demonstrate that PF-06250112 significantly limits the spontaneous accumulation of splenic germinal center B cells and plasma cells. Correspondingly, anti-dsDNA and autoantibody levels were reduced in a dose-dependent manner. Moreover, administration of PF-06250112 prevented the development of proteinuria and improved glomerular pathology scores in all treatment groups. Strikingly, this therapeutic effect could occur with only a modest reduction observed in anti-dsDNA titers, implying a critical role for BTK signaling in disease pathogenesis beyond inhibition of autoantibody production. We subsequently demonstrate that PF-06250112 prevents proteinuria in an FcR-dependent, Ab-mediated model of glomerulonephritis. Importantly, these results highlight that BTK inhibition potently limits the development of glomerulonephritis by impacting both cell- and effector molecule-mediated pathways. These data provide support for evaluating the efficacy of BTK inhibition in systemic lupus erythematosus patients.
Asunto(s)
Linfocitos B/inmunología , Glomerulonefritis/inmunología , Lupus Eritematoso Sistémico/inmunología , Piperidinas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/uso terapéutico , Agammaglobulinemia Tirosina Quinasa , Animales , Linfocitos B/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Centro Germinal/citología , Glomerulonefritis/metabolismo , Glomerulonefritis/prevención & control , Riñón/inmunología , Lupus Eritematoso Sistémico/metabolismo , Lupus Eritematoso Sistémico/prevención & control , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos NZB , Piperidinas/farmacología , Células Plasmáticas/efectos de los fármacos , Células Plasmáticas/inmunología , Proteínas Tirosina Quinasas/metabolismo , Pirazoles/farmacología , Receptores Fc , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
B cells and plasma cells possess distinct RNA processing environments that respectively promote the expression of membrane-associated Ig by B cells versus the secretion of Ig by plasma cells. Through a combination of transcriptional profiling and screening using a lentiviral short-hairpin RNA interference library, we show that both the splicing factor hnRNPLL and the transcription elongation factor ELL2 modulate the ratio of secreted versus membrane-encoding Ighg2b transcripts in MPC11 plasmacytoma cell lines. hnRNPLL and ELL2 are both highly expressed in primary plasma cells relative to B cells, but hnRNPLL binds Ighg2b mRNA transcripts and promotes an increase in levels of the membrane-encoding Ighg2b isoform at the expense of the secreted Ighg2b isoform, whereas ELL2 counteracts this effect and drives Ig secretion by increasing the frequency of the secreted Ighg2b isoform. As in T cells, hnRNPLL also alters the splicing pattern of mRNA encoding the adhesion receptor CD44, promoting exon inclusion, and decreasing the overall level of CD44 expression. Further characterization of ELL2-dependent transcription by RNA-Seq revealed that â¼12% of transcripts expressed by plasma cells were differentially processed because of the activities of ELL2, including B-cell maturation antigen BCMA, a receptor with a defined role in plasma cell survival. Taken together, our data identify hnRNPLL and ELL2 as regulators of pre-mRNA processing in plasma cells.
Asunto(s)
Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Cadenas Pesadas de Inmunoglobulina/metabolismo , Células Plasmáticas/fisiología , ARN Mensajero/fisiología , Factores de Elongación Transcripcional/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Citometría de Flujo , Receptores de Hialuranos/metabolismo , Cadenas Pesadas de Inmunoglobulina/genética , Inmunoprecipitación , Ratones , Ratones Endogámicos BALB C , Análisis por Micromatrices , Células Plasmáticas/metabolismo , Isoformas de Proteínas/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ARNRESUMEN
The hypothesis that bystander inflammatory signals promote memory B cell (B(MEM)) self-renewal and differentiation in an antigen-independent manner is critically evaluated herein. To comprehensively address this hypothesis, a detailed analysis is presented examining the response profiles of B-2 lineage B220(+)IgG(+) B(MEM) toward cognate protein antigen in comparison to bystander inflammatory signals. After in vivo antigen encounter, quiescent B(MEM) clonally expand. Surprisingly, proliferating B(MEM) do not acquire germinal center (GC) B cell markers before generating daughter B(MEM) and differentiating into plasma cells or form structurally identifiable GCs. In striking contrast to cognate antigen, inflammatory stimuli, including Toll-like receptor agonists or bystander T cell activation, fail to induce even low levels of B(MEM) proliferation or differentiation in vivo. Under the extreme conditions of adjuvanted protein vaccination or acute viral infection, no detectable bystander proliferation or differentiation of B(MEM) occurred. The absence of a B(MEM) response to nonspecific inflammatory signals clearly shows that B(MEM) proliferation and differentiation is a process tightly controlled by the availability of cognate antigen.
Asunto(s)
Antígenos/inmunología , Linfocitos B/inmunología , Efecto Espectador/inmunología , Diferenciación Celular/inmunología , Memoria Inmunológica/inmunología , Inflamación/inmunología , Animales , Antígenos/metabolismo , Linfocitos B/citología , Bromodesoxiuridina , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Ratones , Ratones Endogámicos BALB C , Microscopía FluorescenteRESUMEN
SUMMARY: During the generation of a successful adaptive immune response, multiple molecular signals are required. A primary signal is the binding of cognate antigen to an antigen receptor expressed by T and B lymphocytes. Multiple secondary signals involve the engagement of costimulatory molecules expressed by T and B lymphocytes with their respective ligands. Because of its essential role in immunity, one of the best characterized of the costimulatory molecules is the receptor CD40. This receptor, a member of the tumor necrosis factor receptor family, is expressed by B cells, professional antigen-presenting cells, as well as non-immune cells and tumors. CD40 binds its ligand CD40L, which is transiently expressed on T cells and other non-immune cells under inflammatory conditions. A wide spectrum of molecular and cellular processes is regulated by CD40 engagement including the initiation and progression of cellular and humoral adaptive immunity. In this review, we describe the downstream signaling pathways initiated by CD40 and overview how CD40 engagement or antagonism modulates humoral and cellular immunity. Lastly, we discuss the role of CD40 as a target in harnessing anti-tumor immunity. This review underscores the essential role CD40 plays in adaptive immunity.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Linfocitos B/inmunología , Antígenos CD40/inmunología , Ligando de CD40/inmunología , Linfocitos T/inmunología , Animales , Células Presentadoras de Antígenos/metabolismo , Linfocitos B/metabolismo , Antígenos CD40/metabolismo , Ligando de CD40/metabolismo , Humanos , Inmunidad Celular , Inmunoterapia , Activación de Linfocitos/inmunología , Neoplasias/inmunología , Neoplasias/terapia , Transducción de Señal/inmunología , Linfocitos T/metabolismo , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/inmunología , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismoAsunto(s)
Antineoplásicos/farmacología , Factores de Transcripción Forkhead/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T/inmunología , Factor de Crecimiento Transformador beta/efectos de los fármacos , Tretinoina/farmacología , Animales , Ratones , Linfocitos T Reguladores/efectos de los fármacosRESUMEN
On occasion, emerging scientific fields intersect and great discoveries result. In the last decade, the discovery of regulatory T cells (T(reg)) in immunity has revolutionized our understanding of how the immune system is controlled. Intersecting the rapidly emerging field of T(reg) function, has been the discovery that retinoic acid (RA) controls both the homing and differentiation of T(reg). Instantly, the wealth and breadth of knowledge of the molecular basis for RA action, its receptors, and how it controls cellular differentiation can and will be exploited to understand its profound effects on T(reg). Historically, vitamin A deprivation and repletion and RA agonists have been shown to profoundly affect immunity. Now these findings can be interpreted in light of the revelations that RA controls leukocyte homing and T(reg) function.
Asunto(s)
Linfocitos T Reguladores/inmunología , Tretinoina/fisiología , Animales , Diferenciación Celular , Humanos , Sistema Inmunológico/fisiología , Proteínas de Unión al Retinol/inmunología , Proteínas de Unión al Retinol/metabolismo , Transducción de Señal , Linfocitos T Reguladores/metabolismo , Tretinoina/metabolismoRESUMEN
Memory B (B(MEM)) cells and long-lived bone marrow plasma cells (BM-PCs) persist within local environmental survival niches that afford cellular longevity. However, the factors supporting B(MEM) cell survival within the secondary lymphoid organs and allowing BM-PC persistence in the bone marrow remain poorly characterized. We report herein that long-lived B(MEM) cell survival and function are completely independent of BAFF (B cell-activating factor of the TNF family) or APRIL (a proliferation-inducing ligand). Thus, B(MEM) cells represent the only mature B2 lineage subset whose survival is independent of these ligands. We have previously shown that the TNFR family member receptor BCMA (B cell maturation Ag) is a critical survival receptor for BM-PC survival in vivo. We identify in this study the ligands critical for BM-PC survival and show that either BAFF or APRIL supports the survival of BM-PCs in vivo. These data define the BAFF/APRIL-dependent and -independent components of long-lived humoral immunity.
Asunto(s)
Factor Activador de Células B/fisiología , Subgrupos de Linfocitos B/inmunología , Memoria Inmunológica , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/fisiología , Animales , Formación de Anticuerpos/genética , Factor Activador de Células B/metabolismo , Receptor del Factor Activador de Células B/biosíntesis , Receptor del Factor Activador de Células B/genética , Receptor del Factor Activador de Células B/metabolismo , Antígeno de Maduración de Linfocitos B/biosíntesis , Antígeno de Maduración de Linfocitos B/genética , Antígeno de Maduración de Linfocitos B/metabolismo , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Memoria Inmunológica/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Células Plasmáticas/citología , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Proteína Activadora Transmembrana y Interactiva del CAML/biosíntesis , Proteína Activadora Transmembrana y Interactiva del CAML/genética , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/deficiencia , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genéticaRESUMEN
We demonstrate that all-trans retinoic acid (RA) induces FoxP3(+) adaptive T regulatory cells (A-Tregs) to acquire a gut-homing phenotype (alpha 4 beta 7(+) CC chemokine receptor 9(+)) and the capacity to home to the lamina propria of the small intestine. Under conditions that favor the differentiation of A-Tregs (transforming growth factor-beta1 and interleukin 2) in vitro, the inclusion of RA induces nearly all activated CD4(+) T cells to express FoxP3 and greatly increases the accumulation of these cells. In the absence of RA, A-Treg differentiation is abruptly impaired by proficient antigen presenting cells or through direct co-stimulation. In the presence of RA, A-Treg generation occurs even in the presence of high levels of co-stimulation, with RA attenuating co-stimulation from interfering from FoxP3 induction. The recognition that RA induces gut imprinting, together with our finding that it enhances A-Treg conversion, differentiation, and expansion, indicates that RA production in vivo may drive both the imprinting and A-Treg development in the face of overt inflammation.
Asunto(s)
Linfocitos T Reguladores/citología , Linfocitos T/metabolismo , Tretinoina/fisiología , Animales , Antígeno B7-1/biosíntesis , Antígeno B7-2/biosíntesis , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular , Impresión Genómica , Intestino Delgado/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Linfocitos T Reguladores/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Three possible effector fates await the naïve follicular B cell following antigen stimulation in thymus-dependent reactions. Short-lived plasma cells produce an initial burst of germline-encoded protective antibodies, and long-lived plasma cells and memory B cells arise from the germinal center and function to enhance and sustain the humoral immune response. The inherent B-cell receptor affinity of naïve follicular B cells and the contribution of other early B-cell signals pre-determines the pattern of transcription factor expression and the differentiation path taken by these cells. High initial B-cell receptor affinity shunts naïve follicular B-cell clones towards the short-lived plasma cell fate, whereas modest-affinity clones are skewed towards a plasma cell fate and low-affinity clones are recruited into the germinal center and are selected for both long-lived plasma cells and memory B cell pathways. In the germinal center reaction, increased levels of the transcription factor interferon regulatory factor-4 drive the molecular program that dictates differentiation into the long-lived plasma cell phenotype but has no impact on the memory B cell compartment. We hypothesize that graded interferon regulatory factor-4 levels driven by signals to B cells, including B-cell receptor signal strength, are responsible for this branch point in the B-cell terminal differentiation pathway.
Asunto(s)
Afinidad de Anticuerpos , Subgrupos de Linfocitos B/inmunología , Linfocitos B/inmunología , Diferenciación Celular/inmunología , Modelos Inmunológicos , Animales , Antígenos/inmunología , Subgrupos de Linfocitos B/citología , Linfocitos B/citología , Antígenos CD40/inmunología , Linaje de la Célula , Humanos , Memoria Inmunológica , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos BRESUMEN
Long-term humoral immunity elicited by pathogens and vaccines alike relies upon the generation of both memory B cells (B(mem)) and long-lived plasma cells (PCs). Virtually all vaccine formulations induce the concomitant emergence of both B(mem) and PCs, suggesting that the emergence of these two differentiated B cells subsets is commonly controlled. Evidence presented shows specific Toll-like receptor (TLR) agonists coupled with soluble protein antigen (sAg) can selectively induce the expansion of antigen specific B(mem) in the absence of PC generation. The co-administration of either TLR 3 or 9 agonists with sAg induced germinal centre (GC) formation, antigen-specific B(mem), but failed to substantively induce the generation of long-lived bone marrow (BM) PCs. Upon re-challenge, high levels of PCs were induced with concomitant high titres of antigen-specific serum IgG. Hence, vaccines can be developed that can prime and protect the host to subsequent infectious agents without initial, high levels of antibody production. Furthermore, these studies suggest that the signals that govern the expansion and differentiation of B(mem) can be uncoupled from those that induce long-lived BM PCs.
Asunto(s)
Linfocitos B/inmunología , Memoria Inmunológica , Receptores Toll-Like/agonistas , Vacunas/inmunología , Animales , Antígenos/inmunología , Epítopos de Linfocito B/inmunología , Adyuvante de Freund/inmunología , Centro Germinal/inmunología , Lípidos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células Plasmáticas/inmunologíaRESUMEN
The diversity of bacteria associated with the deer tick (Ixodes scapularis) was assessed using PCR amplification, cloning, and sequencing of 16S rRNA genes originating from seven ticks collected from Nantucket Island and Wellfleet, Cape Cod, Mass. The majority of sequences obtained originated from gram-negative proteobacteria. Four intracellular bacteria were detected including strains of Ehrlichia, Rickettsia, and Wolbachia and an organism related to intracellular insect symbionts from the Cytophaga-Flavobacterium-Bacteroides group. Several strains of members of the Sphingomonadaceae were also detected in all but one tick. The results provide a view of the diversity of bacteria associated with I. scapularis ticks in the field.