RESUMEN
Chimeric antigen receptor (CAR) T cells have revolutionized treatment of B cell malignancies. However, enhancing the efficacy of engineered T cells without compromising their safety is warranted. The estrogen receptor-binding fragment-associated antigen 9 (EBAG9) inhibits release of cytolytic enzymes from cytotoxic T lymphocytes. Here, we examined the potency of EBAG9 silencing for the improvement of adoptive T cell therapy. MicroRNA (miRNA)-mediated EBAG9 downregulation in transplanted cytolytic CD8+ T cells (CTLs) from immunized mice improved their cytolytic competence in a tumor model. In tolerant female recipient mice that received organ transplants, a minor histocompatibility antigen was turned into a rejection antigen by Ebag9 deletion, indicating an immune checkpoint function for EBAG9. Considerably fewer EBAG9-silenced human CAR T cells were needed for tumor growth control in a xenotransplantation model. Transcriptome profiling did not reveal additional risks regarding genotoxicity or aberrant differentiation. A single-step retrovirus transduction process links CAR or TCR expression with miRNA-mediated EBAG9 downregulation. Despite higher cytolytic efficacy, release of cytokines associated with cytokine release syndrome remains unaffected. Collectively, EBAG9 silencing enhances effector capacity of TCR- and CAR-engineered T cells, results in improved tumor eradication, facilitates efficient manufacturing, and decreases the therapeutic dose.
Asunto(s)
Antígenos de Neoplasias , Inmunoterapia Adoptiva , Neoplasias , Animales , Femenino , Humanos , Ratones , MicroARNs/genética , Neoplasias/terapia , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T Citotóxicos , Silenciador del Gen , Proteínas de Punto de Control Inmunitario , Antígenos de Neoplasias/genéticaRESUMEN
The testis is the second most frequent extramedullary site of relapse in pediatric acute lymphoblastic leukemia (ALL). The mechanism for B-cell (B) ALL cell migration towards and survival within the testis remains elusive. Here, we identified CXCL12-CXCR4 as the leading signaling axis for B-ALL cell migration and survival in the testicular leukemic niche. We combined analysis of primary human ALL with a novel patient-derived xenograft (PDX)-ALL mouse model with testicular involvement. Prerequisites for leukemic cell infiltration in the testis were prepubertal age of the recipient mice, high surface expression of CXCR4 on PDX-ALL cells, and CXCL12 secretion from the testicular stroma. Analysis of primary pediatric patient samples revealed that CXCR4 was the only chemokine receptor being robustly expressed on B-ALL cells both at the time of diagnosis and relapse. In affected patient testes, leukemic cells localized within the interstitial space in close proximity to testicular macrophages. Mouse macrophages isolated from affected testes, in the PDX model, revealed a macrophage polarization towards a M2-like phenotype in the presence of ALL cells. Therapeutically, blockade of CXCR4-mediated functions using an anti-CXCR4 antibody treatment completely abolished testicular infiltration of PDX-ALL cells and strongly impaired the overall development of leukemia. Collectively, we identified a prepubertal condition together with high CXCR4 expression as factors affecting the leukemia permissive testicular microenvironment. We propose CXCR4 as a promising target for therapeutic prevention of testicular relapses in childhood B-ALL. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras , Testículo , Animales , Movimiento Celular , Quimiocina CXCL12/metabolismo , Niño , Humanos , Masculino , Ratones , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Receptores CXCR4/metabolismo , Recurrencia , Transducción de Señal , Testículo/química , Testículo/metabolismo , Testículo/patología , Microambiente TumoralRESUMEN
The advent of CAR T cells targeting CD19 or BCMA on B cell neoplasm demonstrated remarkable efficacy, but rapid relapses and primary refractoriness remains challenging. A leading cause of CAR T cell failure is their lack of expansion and limited persistence. Long-lived, self-renewing multipotent T memory stem cells (TSCM) and T central memory cells (TCM) likely sustain superior tumor regression, but their low frequencies in blood from cancer patients impose a major hurdle for clinical CAR T production. We designed a clinically compliant protocol for generating BCMA CAR T cells starting with increased TSCM/TCM cell input. A CliniMACS Prodigy process was combined with flow cytometry-based enrichment of CD62L+CD95+ T cells. Although starting with only 15% of standard T cell input, the selected TSCM/TCM material was efficiently activated and transduced with a BCMA CAR-encoding retrovirus. Cultivation in the presence of IL-7/IL-15 enabled the harvest of CAR T cells containing an increased CD4+ TSCM fraction and 70% TSCM cells amongst CD8+. Strong cell proliferation yielded cell numbers sufficient for clinical application, while effector functions were maintained. Together, adaptation of a standard CliniMACS Prodigy protocol to low input numbers resulted in efficient retroviral transduction with a high CAR T cell yield.
RESUMEN
Chimeric-antigen-receptor (CAR)-T-cell therapy is already widely used to treat patients who are relapsed or refractory to chemotherapy, antibodies, or stem-cell transplantation. Multiple myeloma still constitutes an incurable disease. CAR-T-cell therapy that targets BCMA (B-cell maturation antigen) is currently revolutionizing the treatment of those patients. To monitor and improve treatment outcomes, methods to detect CAR-T cells in human peripheral blood are highly desirable. In this study, three different detection reagents for staining BCMA-CAR-T cells by flow cytometry were compared. Moreover, a quantitative polymerase chain reaction (qPCR) to detect BCMA-CAR-T cells was established. By applying a cell-titration experiment of BCMA-CAR-T cells, both methods were compared head-to-head. In flow-cytometric analysis, the detection reagents used in this study could all detect BCMA-CAR-T cells at a similar level. The results of false-positive background staining differed as follows (standard deviation): the BCMA-detection reagent used on the control revealed a background staining of 0.04% (±0.02%), for the PE-labeled human BCMA peptide it was 0.25% (±0.06%) and for the polyclonal anti-human IgG antibody it was 7.2% (±9.2%). The ability to detect BCMA-CAR-T cells down to a concentration of 0.4% was similar for qPCR and flow cytometry. The qPCR could detect even lower concentrations (0.02-0.01%). In summary, BCMA-CAR-T-cell monitoring can be reliably performed by both flow cytometry and qPCR. In flow cytometry, reagents with low background staining should be preferred.
Asunto(s)
Antígeno de Maduración de Linfocitos B/metabolismo , Citometría de Flujo , Reacción en Cadena de la Polimerasa , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T/metabolismo , Antígeno de Maduración de Linfocitos B/genética , Biomarcadores , Citometría de Flujo/métodos , Citometría de Flujo/normas , Humanos , Inmunofenotipificación , Inmunoterapia Adoptiva/métodos , Inmunoterapia Adoptiva/normas , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/normas , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Quiméricos de Antígenos/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Linfocitos T/inmunologíaRESUMEN
CAR-T cell therapy targeting CD19 demonstrated strong activity against advanced B cell leukemia, however shows less efficacy against lymphoma with nodal dissemination. To target both B cell Non-Hodgkin's lymphoma (B-NHLs) and follicular T helper (Tfh) cells in the tumor microenvironment (TME), we apply here a chimeric antigen receptor (CAR) that recognizes human CXCR5 with high avidity. CXCR5, physiologically expressed on mature B and Tfh cells, is also highly expressed on nodal B-NHLs. Anti-CXCR5 CAR-T cells eradicate B-NHL cells and lymphoma-supportive Tfh cells more potently than CD19 CAR-T cells in vitro, and they efficiently inhibit lymphoma growth in a murine xenograft model. Administration of anti-murine CXCR5 CAR-T cells in syngeneic mice specifically depletes endogenous and malignant B and Tfh cells without unexpected on-target/off-tumor effects. Collectively, anti-CXCR5 CAR-T cells provide a promising treatment strategy for nodal B-NHLs through the simultaneous elimination of lymphoma B cells and Tfh cells of the tumor-supporting TME.
Asunto(s)
Linfocitos B/inmunología , Linfoma no Hodgkin/inmunología , Neoplasias/inmunología , Receptores CXCR5/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T/inmunología , Animales , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Células HEK293 , Células Hep G2 , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
B lymphocytes can suppress immunity through interleukin (IL)-10 production in infectious, autoimmune, and malignant diseases. Here, we have identified a natural plasma cell subset that distinctively expresses the inhibitory receptor LAG-3 and mediates this function in vivo. These plasma cells also express the inhibitory receptors CD200, PD-L1, and PD-L2. They develop from various B cell subsets in a B cell receptor (BCR)-dependent manner independently of microbiota in naive mice. After challenge they upregulate IL-10 expression via a Toll-like receptor-driven mechanism within hours and without proliferating. This function is associated with a unique transcriptome and epigenome, including the lowest amount of DNA methylation at the Il10 locus compared to other B cell subsets. Their augmented accumulation in naive mutant mice with increased BCR signaling correlates with the inhibition of memory T cell formation and vaccine efficacy after challenge. These natural regulatory plasma cells may be of broad relevance for disease intervention.
Asunto(s)
Antígenos CD/genética , Expresión Génica , Interleucina-10/biosíntesis , Células Plasmáticas/inmunología , Animales , Antígenos CD/inmunología , Subgrupos de Linfocitos B/inmunología , Epigénesis Genética , Femenino , Perfilación de la Expresión Génica , Interleucina-10/genética , Activación de Linfocitos , Masculino , Ratones , Células Plasmáticas/fisiología , Receptores de Antígenos de Linfocitos B/metabolismo , Salmonelosis Animal/inmunología , Transducción de Señal , Linfocitos T/inmunología , Receptores Toll-Like/metabolismo , Regulación hacia Arriba/genética , Vacunas/inmunología , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
Regulatory T cells (Tregs) play an important role in the pathogenesis of HIV-1 infection and they frequently express the chemokine receptor CCR5. We therefore investigated whether antiretroviral treatment with the CCR5 antagonist Maraviroc affected Tregs in chronically HIV-1-infected individuals. HIV-1-infected patients with high viral loads had elevated frequencies of activated Tregs in the peripheral blood compared with healthy controls. In patients successfully treated with antiretroviral drugs (undetectable viral loads), the frequency and the activation status of Tregs were comparable with healthy controls without any specific effect related to the treatment with Maraviroc. These results indicate that the control of viral replication in general rather than a direct binding of Maraviroc to CCR5-positive Tregs influences Treg responses in successfully treated chronically HIV-1-infected individuals.
Asunto(s)
Antagonistas de los Receptores CCR5/uso terapéutico , Ciclohexanos/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , VIH-1/inmunología , Receptores CCR5/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Triazoles/uso terapéutico , Replicación Viral/efectos de los fármacos , Femenino , Infecciones por VIH/virología , Humanos , Masculino , Maraviroc , Receptores CCR5/metabolismo , Linfocitos T Reguladores/efectos de los fármacos , Carga Viral/efectos de los fármacosRESUMEN
It is still unclear whether expanded and activated regulatory T cells (Tregs) in chronic viral infections can influence primary immune responses against superinfections with unrelated viruses. Expanded Tregs found in the spleens of chronically Friend virus (FV)-infected mice decreased murine cytomegalovirus (mCMV)-specific CD8(+) T cell responses during acute mCMV superinfection. This suppression of mCMV-specific T cell immunity was found only in organs with FV-induced Treg expansion. Surprisingly, acute mCMV infection itself did not expand or activate Tregs.
Asunto(s)
Virus de la Leucemia Murina de Friend/inmunología , Infecciones por Herpesviridae/inmunología , Tolerancia Inmunológica , Muromegalovirus/inmunología , Infecciones por Retroviridae/inmunología , Sobreinfección/inmunología , Linfocitos T Reguladores/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Leucemia Experimental/complicaciones , Leucemia Experimental/inmunología , Masculino , Ratones Endogámicos C57BL , Infecciones por Retroviridae/complicaciones , Bazo/inmunología , Infecciones Tumorales por Virus/complicaciones , Infecciones Tumorales por Virus/inmunologíaRESUMEN
Vß5(+) regulatory T cells (Tregs), which are specific for a mouse endogenous retroviral superantigen, become activated and proliferate in response to Friend virus (FV) infection. We previously reported that FV-induced expansion of this Treg subset was dependent on CD8(+) T cells and TNF-α, but independent of IL-2. We now show that the inflammatory milieu associated with FV infection is not necessary for induction of Vß5(+) Treg expansion. Rather, it is the presence of activated CD8(+) T cells that is critical for their expansion. The data indicate that the mechanism involves signaling between the membrane-bound form of TNF-α on activated CD8(+) T cells and TNFR2 on Tregs. CD8(+) T cells expressing membrane-bound TNF-α but no soluble TNF-α remained competent to induce strong Vß5(+) Treg expansion in vivo. In addition, Vß5(+) Tregs expressing only TNFR2 but no TNFR1 were still responsive to expansion. Finally, treatment of naive mice with soluble TNF-α did not induce Vß5(+) Treg expansion, but treatment with a TNFR2-specific agonist did. These results reveal a new mechanism of intercellular communication between activated CD8(+) T cell effectors and Tregs that results in the activation and expansion of a Treg subset that subsequently suppresses CD8(+) T cell functions.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/biosíntesis , Receptores Tipo II del Factor de Necrosis Tumoral/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Proteínas Portadoras/genética , Femenino , Virus de la Leucemia Murina de Friend/inmunología , Leucemia Experimental/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Tipo I de Factores de Necrosis Tumoral , Receptores Tipo II del Factor de Necrosis Tumoral/agonistas , Infecciones por Retroviridae/inmunología , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunología , Infecciones Tumorales por Virus/inmunologíaRESUMEN
Formation of immunological synapses (IS) between dendritic cells (DCs) and conventional CD4(+) T cells (Tcon) is critical for productive immune responses. However, when DCs are HIV-infected such synapses are critical to establish HIV infection. As regulatory T cells (Treg) control DC-Tcon interactions, we inquired whether Treg might interfere with DC to Tcon HIV infection. We developed a model, using monocyte-derived DC infected with R5-HIV, and cultured with Tcon in the presence or absence of autologous Treg, using the physiological ratio of 1 Treg for 10 Tcon. Cultures containing Treg significantly decreased HIV infection in DC:T cell clusters. Notably, Treg appear to have an effect on the quality of the IS, as Treg decreased actin polymerization and DC maturation. Importantly, Treg decreased the trafficking of HIV punctate to the IS. Further, CD152 and cyclic adenosine monophosphate were critical Treg effector molecules, as their individual or simultaneous blockade abolished Treg activity, however no additive effect was found. Together, these data suggest that Treg can reduce HIV dissemination, which may be beneficial to the host in the early stages of infection.
RESUMEN
The suppressive capacity of regulatory T cells (Tregs) has been extensively studied and is well established for many diseases. The expansion, accumulation, and activation of Tregs in viral infections are of major interest in order to find ways to alter Treg functions for therapeutic benefit. Tregs are able to dampen effector T cell responses to viral infections and thereby contribute to the establishment of a chronic infection. In the Friend retrovirus (FV) mouse model, Tregs are known to expand in all infected organs. To better understand the characteristics of these Treg populations, their phenotype was analyzed in detail. During acute FV-infection, Tregs became activated in the spleen and bone marrow, as indicated by various T cell activation markers, such as CD43 and CD103. Interestingly, Tregs in the bone marrow, which contains the highest viral loads during acute infection, displayed greater levels of activation than Tregs from the spleen. Treg expansion was driven by proliferation but no FV-specific Tregs could be detected. Activated Tregs in FV-infection did not produce Granzyme B (GzmB) or tumor necrosis factor α (TNFα), which are thought to be a potential mechanism for their suppressive activity. Furthermore, Tregs expressed inhibitory markers, such as TIM3, PD-1 and PD-L1. Blocking TIM3 and PD-L1 with antibodies during chronic FV-infection increased the numbers of activated Tregs. These data may have important implications for the understanding of Treg functions during chronic viral infections.
Asunto(s)
Virus de la Leucemia Murina de Friend/inmunología , Leucemia Experimental/inmunología , Activación de Linfocitos , Infecciones por Retroviridae/inmunología , Linfocitos T Reguladores/inmunología , Infecciones Tumorales por Virus/inmunología , Estructuras Animales/inmunología , Estructuras Animales/virología , Animales , Antígeno B7-H1/análisis , Médula Ósea/inmunología , Médula Ósea/virología , Granzimas/análisis , Receptor 2 Celular del Virus de la Hepatitis A , Inmunofenotipificación , Ratones , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1/análisis , Receptores Virales/análisis , Bazo/inmunología , Bazo/virología , Linfocitos T Reguladores/química , Factor de Necrosis Tumoral alfa/análisisRESUMEN
Friend virus infection of mice induces the expansion and activation of regulatory T cells (Tregs) that dampen acute immune responses and promote the establishment and maintenance of chronic infection. Adoptive transfer experiments and the expression of neuropilin-1 indicate that these cells are predominantly natural Tregs rather than virus-specific conventional CD4(+) T cells that converted into induced Tregs. Analysis of Treg TCR Vß chain usage revealed a broadly distributed polyclonal response with a high proportionate expansion of the Vß5(+) Treg subset, which is known to be responsive to endogenous retrovirus-encoded superantigens. In contrast to the major population of Tregs, the Vß5(+) subset expressed markers of terminally differentiated effector cells, and their expansion was associated with the level of the antiviral CD8(+) T cell response rather than the level of Friend virus infection. Surprisingly, the expansion and accumulation of the Vß5(+) Tregs was IL-2 independent but dependent on TNF-α. These experiments reveal a subset-specific Treg induction by a new pathway.
Asunto(s)
Virus de la Leucemia Murina de Friend/inmunología , Interleucina-2/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Infecciones por Retroviridae/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Femenino , Inmunofenotipificación , Interleucina-2/metabolismo , Ratones , Fenotipo , Linfocitos T Reguladores/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Infecciones Tumorales por Virus/inmunologíaRESUMEN
The innate immune response mediated by cells such as natural killer (NK) cells is critical for the rapid containment of virus replication and spread during acute infection. Here, we show that subtype 11 of the type I interferon (IFN) family greatly potentiates the antiviral activity of NK cells during retroviral infection. Treatment of mice with IFN-α11 during Friend retrovirus infection (FV) significantly reduced viral loads and resulted in long-term protection from virus-induced leukemia. The effect of IFN-α11 on NK cells was direct and signaled through the type I IFN receptor. Furthermore, IFN-α11-mediated activation of NK cells enabled cytolytic killing of FV-infected target cells via the exocytosis pathway. Depletion and adoptive transfer experiments illustrated that NK cells played a major role in successful IFN-α11 therapy. Additional experiments with Mouse Cytomegalovirus infections demonstrated that the therapeutic effect of IFN-α11 is not restricted to retroviruses. The type I IFN subtypes 2 and 5, which bind the same receptor as IFN-α11, did not elicit similar antiviral effects. These results demonstrate a unique and subtype-specific activation of NK cells by IFN-α11.