RESUMEN
Unlike genomic alterations, gene expression profiles have not been widely used to refine cancer therapies. We analyzed transcriptional changes in acute myeloid leukemia (AML) cell lines in response to standard first-line AML drugs cytarabine and daunorubicin by means of RNA sequencing. Those changes were highly cell- and treatment-specific. By comparing the changes unique to treatment-sensitive and treatment-resistant AML cells, we enriched for treatment-relevant genes. Those genes were associated with drug response-specific pathways, including calcium ion-dependent exocytosis and chromatin remodeling. Pharmacological mimicking of those changes using EGFR and MEK inhibitors enhanced the response to daunorubicin with minimum standalone cytotoxicity. The synergistic response was observed even in the cell lines beyond those used for the discovery, including a primary AML sample. Additionally, publicly available cytotoxicity data confirmed the synergistic effect of EGFR inhibitors in combination with daunorubicin in all 60 investigated cancer cell lines. In conclusion, we demonstrate the utility of treatment-evoked gene expression changes to formulate rational drug combinations. This approach could improve the standard AML therapy, especially in older patients.
Asunto(s)
Leucemia Mieloide Aguda , Humanos , Anciano , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Daunorrubicina/farmacología , Línea Celular , Ensamble y Desensamble de Cromatina , Receptores ErbBRESUMEN
Accumulating evidence suggests that both the nature of oncogenic lesions and the cell-of-origin can strongly influence cancer histopathology, tumor aggressiveness and response to therapy. Although oncogenic Kras expression and loss of Trp53 tumor suppressor gene function have been demonstrated to initiate murine lung adenocarcinomas (LUADs) in alveolar type II (AT2) cells, clear evidence that Club cells, representing the second major subset of lung epithelial cells, can also act as cells-of-origin for LUAD is lacking. Equally, the exact anatomic location of Club cells that are susceptible to Kras transformation and the resulting tumor histotype remains to be established. Here, we provide definitive evidence for Club cells as progenitors for LUAD. Using in vivo lineage tracing, we find that a subset of Kras12V -expressing and Trp53-deficient Club cells act as precursors for LUAD and we define the stepwise trajectory of Club cell-initiated tumors leading to lineage marker conversion and aggressive LUAD. Our results establish Club cells as cells-of-origin for LUAD and demonstrate that Club cell-initiated tumors have the potential to develop aggressive LUAD.
Asunto(s)
Adenocarcinoma/genética , Transformación Celular Neoplásica/genética , Células Epiteliales/metabolismo , Genes ras/genética , Neoplasias Pulmonares/genética , Mutación , Proteína p53 Supresora de Tumor/genética , Adenocarcinoma/metabolismo , Animales , Transformación Celular Neoplásica/metabolismo , Progresión de la Enfermedad , Células Epiteliales/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína p53 Supresora de Tumor/deficienciaRESUMEN
In the last decade there has been a rapid expansion in clinical trials using mesenchymal stromal cells (MSCs) from a variety of tissues. However, despite similarities in morphology, immunophenotype, and differentiation behavior in vitro, MSCs sourced from distinct tissues do not necessarily have equivalent biological properties. We performed a genome-wide methylation, transcription, and in vivo evaluation of MSCs from human bone marrow (BM), white adipose tissue, umbilical cord, and skin cultured in humanized media. Surprisingly, only BM-derived MSCs spontaneously formed a BM cavity through a vascularized cartilage intermediate in vivo that was progressively replaced by hematopoietic tissue and bone. Only BM-derived MSCs exhibited a chondrogenic transcriptional program with hypomethylation and increased expression of RUNX3, RUNX2, BGLAP, MMP13, and ITGA10 consistent with a latent and primed skeletal developmental potential. The humanized MSC-derived microenvironment permitted homing and maintenance of long-term murine SLAM(+) hematopoietic stem cells (HSCs), as well as human CD34(+)/CD38(-)/CD90(+)/CD45RA(+) HSCs after cord blood transplantation. These studies underscore the profound differences in developmental potential between MSC sources independent of donor age, with implications for their clinical use. We also demonstrate a tractable human niche model for studying homing and engraftment of human hematopoietic cells in normal and neoplastic states.
Asunto(s)
Linaje de la Célula , Epigénesis Genética , Células Madre Hematopoyéticas/citología , Células Madre Mesenquimatosas/citología , Nicho de Células Madre , Western Blotting , Células de la Médula Ósea/citología , Diferenciación Celular/fisiología , Condrogénesis/fisiología , Citometría de Flujo , Humanos , Osteogénesis/fisiologíaRESUMEN
Playing a central role in both innate and adaptive immunity, CD4(+) T cells are a key target for genetic modifications in basic research and immunotherapy. In this article, we describe novel lentiviral vectors (CD4-LV) that have been rendered selective for human or simian CD4(+) cells by surface engineering. When applied to PBMCs, CD4-LV transduced CD4(+) but not CD4(-) cells. Notably, also unstimulated T cells were stably genetically modified. Upon systemic or intrasplenic administration into mice reconstituted with human PBMCs or hematopoietic stem cells, reporter gene expression was predominantly detected in lymphoid organs. Evaluation of GFP expression in organ-derived cells and blood by flow cytometry demonstrated exclusive gene transfer into CD4(+) human lymphocytes. In bone marrow and spleen, memory T cells were preferentially hit. Toward therapeutic applications, we also show that CD4-LV can be used for HIV gene therapy, as well as for tumor therapy, by delivering chimeric Ag receptors. The potential for in vivo delivery of the FOXP3 gene was also demonstrated, making CD4-LV a powerful tool for inducible regulatory T cell generation. In summary, our work demonstrates the exclusive gene transfer into a T cell subset upon systemic vector administration opening an avenue toward novel strategies in immunotherapy.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Vectores Genéticos/genética , Lentivirus/genética , Transducción Genética , Animales , Médula Ósea/metabolismo , Línea Celular Tumoral , Trasplante de Células/métodos , Células Cultivadas , Citometría de Flujo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Terapia Genética/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Inmunoterapia Adoptiva/métodos , Leucocitos Mononucleares/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Bazo/metabolismo , Timo/metabolismo , Trasplante HeterólogoRESUMEN
Adoptive transfer of donor-derived cytolytic T-lymphocytes (CTL) has evolved as a promising strategy to improve graft-versus-leukemia (GvL) effects in allogeneic hematopoietic stem-cell transplantation. However, durable clinical responses are often hampered by limited capability of transferred T cells to establish effective and sustained antitumor immunity in vivo. We therefore analyzed GvL responses of acute myeloid leukemia (AML)-reactive CD8(+) CTL with central and effector memory phenotype in a new allogeneic donor-patient specific humanized mouse model. CTL lines and clones obtained upon stimulation of naive CD45RA(+) donor CD8(+) T cells with either single HLA antigen-mismatched or HLA-matched primary AML blasts, respectively, elicited strong leukemia reactivity during cytokine-optimized short to intermediate (i.e., 2-8 weeks) culture periods. Single doses of CTL were intravenously infused into NOD/scidIL2Rcg(null) mice when engraftment with patient AML reached bone marrow infiltration of 1-5%, clinically defining minimal residual disease status. This treatment resulted in complete regression of HLA-mismatched and strong reduction of HLA-matched AML infiltration, respectively. Most importantly, mice receiving AML-reactive CTL showed significantly prolonged survival. Transferred CTL were detectable in murine bone marrow and spleen and demonstrated sustained AML-reactivity ex vivo. Moreover, injections with human IL-15 clearly promoted CTL persistence. In summary, we show that naive donor-derived CD8(+) CTL effectively combat patient AML blasts in immunodeficient mice. The donor-patient specific humanized mouse model appears suitable to evaluate therapeutic efficacy of AML-reactive CTL before adoptive transfer into patients. It may further help to identify powerful leukemia rejection antigens and T-cell receptors for redirecting immunity to leukemias even in a patient-individualized manner.
Asunto(s)
Inmunoterapia Adoptiva , Leucemia Mieloide Aguda/terapia , Linfocitos T Citotóxicos/inmunología , Animales , Efecto Injerto vs Leucemia , Antígenos HLA-B/inmunología , Prueba de Histocompatibilidad , Humanos , Leucemia Mieloide Aguda/inmunología , Ratones , Neoplasia Residual , Medicina de PrecisiónRESUMEN
Therapeutic natural killer (NK)-cell-mediated alloreactivity toward acute myeloid leukemia has largely been attributed to mismatches between killer immunoglobulin-like receptors (KIRs) on NK cells and their ligands, HLA class I molecules, on target cells. While adult acute B-cell precursor leukemia (BCP-ALL) appears to be resistant to NK-cell-mediated lysis, recent data indicate that pediatric BCP-ALL might yet be a target of NK cells. In this study, we demonstrate in a donor-patient-specific NOD.Cg-Prkdc(scid) IL2rg(tmWjl)/Sz (NSG) xenotransplantation model that NK cells mediate considerable alloreactivity toward pediatric BCP-ALL in vivo. Notably, both adoptively transferred mature KIR(+) NK cells and immature KIR(-) NK cells arising early posttransplantation in humanized NSG mice exerted substantial antileukemic activity. Low-dose and long-term treatment of humanized NSG mice with the DNA-demethylating agent 5-aza-cytidine distinctly enhanced the antitumor response, interestingly without inducing common inhibitory KIR expression but rather by promoting the differentiation of various NK-cell precursor subsets. Collectively, these data indicate that the future design of innovative therapy protocols should consider further exploitation of NK-cell-mediated immune responses for poor prognosis pediatric BCP-ALL patients.
Asunto(s)
Antineoplásicos/química , Trasplante de Células Madre Hematopoyéticas , Células Asesinas Naturales/citología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Receptores KIR/metabolismo , Animales , Azacitidina/química , Niño , Citocinas/metabolismo , Citotoxicidad Inmunológica/inmunología , Metilación de ADN , Modelos Animales de Enfermedad , Genotipo , Efecto Injerto vs Leucemia , Humanos , Interleucina-2/genética , Ratones , Ratones Endogámicos NOD , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Pronóstico , Trasplante HeterólogoRESUMEN
Although immortalized cell lines have been extensively used to optimize treatment strategies in cancer, the usefulness of such in vitro systems to recapitulate primary disease is limited. Therefore, the design of in vivo models ideally utilizing patient-derived material is of critical importance. In this regard, NOD.Cg-Prkdc(scid) IL2rg(tmWjl) /Sz (NSG) mice have been reported to provide superior engraftment rates. However, limited data exist on the validity of such a model to constitute a surrogate marker for clinical parameters. We studied primary and serial engraftment on more than 200 NSG mice with 54 primary pediatric B cell precursor acute lymphatic leukemia (B-ALL), myeloid leukemia (AML) and T cell leukemia (T-ALL) samples, characterized the leukemogenic profile and correlated engraftment kinetics with clinical outcome. Median time to engraftment was 7-10 weeks and 90% of the mice engrafted. Male recipients conferred significantly higher engraftment levels than female recipients (p ≤ 0.004). PCR-based minimal residual disease marker expression and fluorescence in situ hybridization confirmed the presence of patient-specific genetic aberrations in mice. Transcriptome cluster analysis of genes known to be important in the leukemogenesis of all three diseases revealed that well-known tumor-regulating genes were expressed to a comparable extent in mice and men. The extent of engraftment and overall survival of NSG mice highly correlated with the individual prognosis of B-ALL, AML and T-ALL patients. Thus, we propose an in vivo model that provides a valuable preclinical tool to explore the heterogeneity of leukemic disease and exploit patient-tailored leukemia-targeting strategies within multivariate analyses.
Asunto(s)
Leucemia Mieloide , Trasplante de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras , Animales , Niño , Preescolar , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunofenotipificación , Lactante , Subunidad alfa del Receptor de Interleucina-2/deficiencia , Subunidad alfa del Receptor de Interleucina-2/genética , Leucemia Mieloide/genética , Leucemia Mieloide/inmunología , Leucemia Mieloide/mortalidad , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Pronóstico , Análisis de Supervivencia , Transcriptoma , Trasplante HeterólogoRESUMEN
Epidermal Langerhans cells (LC) are potent APCs surveying the skin. They are crucial regulators of T cell activation in the context of inflammatory skin disease and graft-versus-host disease (GVHD). In contrast to other dendritic cell subtypes, murine LC are able to reconstitute after local depletion without the need of peripheral blood-derived precursors. In this study, we introduce an experimental model of human skin grafted to NOD-SCID IL2Rγ(null) mice. In this model, we demonstrate that xenografting leads to the transient loss of LC from the human skin grafts. Despite the lack of a human hematopoietic system, human LC repopulated the xenografts 6 to 9 wk after transplantation. By staining of LC with the proliferation marker Ki67, we show that one third of the replenishing LC exhibit proliferative activity in vivo. We further used the skin xenograft as an in vivo model for human GVHD. HLA-disparate third-party T cells stimulated with skin donor-derived dendritic cells were injected intravenously into NOD-SCID IL2Rγ(null) mice that had been transplanted with human skin. The application of alloreactive T cells led to erythema and was associated with histological signs of GVHD limited to the transplanted human skin. The inflammation also led to the depletion of LC from the epidermis. In summary, we provide evidence that human LC are able to repopulate the skin independent of blood-derived precursor cells and that this at least partly relates to their proliferative capacity. Our data also propose xeno-transplantation of human skin as a model system for studying the role of skin dendritic cells in the efferent arm of GVHD.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Epidermis/inmunología , Células de Langerhans/inmunología , Activación de Linfocitos/inmunología , Trasplante de Piel/inmunología , Trasplante Heterólogo/inmunología , Animales , Linfocitos T CD8-positivos/patología , Linfocitos T CD8-positivos/trasplante , Muerte Celular/inmunología , Muerte Celular/efectos de la radiación , Diferenciación Celular/inmunología , Diferenciación Celular/efectos de la radiación , Movimiento Celular/inmunología , Movimiento Celular/efectos de la radiación , Proliferación Celular/efectos de la radiación , Células Cultivadas , Modelos Animales de Enfermedad , Epidermis/patología , Epidermis/trasplante , Femenino , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/patología , Humanos , Células de Langerhans/patología , Células de Langerhans/trasplante , Activación de Linfocitos/efectos de la radiación , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Trasplante de Piel/patología , Trasplante Heterólogo/patologíaRESUMEN
Mycoplasma infection leads to false and non-reproducible scientific data and poses a risk to human health. Despite strict guidelines calling for regular mycoplasma screening, there is no universal and widely established standard procedure. Here, we describe a reliable and cost-effective PCR method that establishes a universal protocol for mycoplasma testing. The applied strategy utilizes ultra-conserved eukaryotic and mycoplasma sequence primers covering by design 92% of all species in the six orders of the class Mollicutes within the phylum Mycoplasmatota and is applicable to mammalian and many non-mammalian cell types. This method can stratify mycoplasma screening and is suitable as a common standard for routine mycoplasma testing.
RESUMEN
Allogeneic hematopoietic stem cell transplantation represents the most effective form of immunotherapy for chemorefractory diseases. However, animal models have been missing that allow evaluation of donor-patient-specific graft-versus-leukemia effects. Thus, we sought to establish a patient-tailored humanized mouse model that would result in long-term engraftment of various lymphocytic lineages and would serve as a donor-specific surrogate. Following transfer of donor-derived peripheral blood stem cells into NOD/SCID/IL-2Rgamma(null) (NSG) mice with supplementation of human IL-7, we could demonstrate robust engraftment and multilineage differentiation comparable to earlier studies using cord blood stem cells. Phenotypical and functional analyses of lymphoid lineages revealed that >20 wk posthematopoietic stem cell transplantation, the majority of T lymphocytes consisted of memory-type CD4(+) T cells capable of inducing specific immune functions, whereas CD8(+) T cells were only present in low numbers. Analysis of NSG-derived NK cells revealed the expression of constitutively activated CD56(bright)CD16(-) killer Ig-like receptor(negative) NK cells that exhibited functional impairments. Thus, the data presented in this study demonstrate that humanized NSG mice can be successfully used to develop a xenotransplantation model that might allow patient-tailored treatment strategies in the future, but also highlight the need to improve this model, for example, by coadministration of differentiation-promoting cytokines and induction of human MHC molecules to complement existing deficiencies in NK and CD8(+) T cell development.
Asunto(s)
Antígenos CD34/biosíntesis , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Diferenciación Celular/inmunología , Trasplante de Células Madre Hematopoyéticas/métodos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Células Asesinas Naturales/inmunología , Trasplante Heterólogo/inmunología , Adulto , Animales , Muerte Celular/genética , Muerte Celular/inmunología , Linaje de la Célula/inmunología , Efecto Injerto vs Leucemia/inmunología , Humanos , Subunidad gamma Común de Receptores de Interleucina/genética , Células K562 , Células Asesinas Naturales/patología , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Trasplante Heterólogo/métodos , Trasplante Heterólogo/patologíaRESUMEN
BACKGROUND: HLA mismatch antigens are major targets of alloreactive T cells in HLA-incompatible stem-cell transplantation, which can trigger severe graft-versus-host disease and reduce survival in transplant recipients. Our objective was to identify T-cell subsets with reduced in vitro reactivity to allogeneic HLA antigens. DESIGN AND METHODS: We sorted CD4 and CD8 T-cell subsets from peripheral blood by flow cytometry according to their expression of naive and memory markers CD45RA, CD45RO, CD62L, and CCR7. Subsets were defined by a single marker to facilitate future establishment of a clinical-grade procedure for reducing alloreactive T-cell precursors and graft-versus-host disease. T cells were stimulated in mixed lymphocyte reactions against HLA-deficient K562 cells transfected with single HLA-A/-B/-C/-DR/-DQ mismatch alleles. Alloreactivity was measured by interferon-γ spot production and cell proliferation. RESULTS: We observed that allogeneic HLA-reactivity was preferentially derived from subsets enriched for naïve T cells rather than memory T cells in healthy donors, irrespective of the HLA mismatch allele. This separation was most efficient if CD45RA (versus other markers) was used for sorting. The numbers of allogeneic HLA-reactive effector cells were in median 7.2-fold and 16.6-fold lower in CD45RA(neg) memory CD8 and CD4 T cells than in entire CD8 and CD4 T cells, respectively. In contrast, proliferation of memory T cells in response to allogeneic HLA was more variably reduced (CD8) or equivalent (CD4) when compared to that of naïve T cells. We also demonstrated in HLA-matched donor-patient pairs that leukemia-reactive CD8 cytotoxic T-lymphocytes were mainly derived from subsets enriched for naïve T cells compared to memory T cells. CONCLUSIONS: Memory T-cell subsets of most healthy individuals showed decreased allogeneic HLA-reactivity, but lacked significant anti-leukemia responses in vitro. The clinical use of memory or naïve-depleted T cells might be beneficial for HLA-mismatched patients at high risk of graft-versus-host disease and low risk of leukemia relapse. Preferred allografts are those which contain leukemia-reactive memory T cells. Alternatively, replenishment with leukemia-reactive T cells isolated from naïve subsets is desirable.
Asunto(s)
Memoria Inmunológica , Inmunoterapia , Leucemia/inmunología , Células Precursoras de Linfocitos T/metabolismo , Linfocitos T/inmunología , Linfocitos T/trasplante , Donantes de Tejidos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Enfermedad Injerto contra Huésped/inmunología , Antígenos HLA/inmunología , Humanos , Células K562 , Receptores CCR7/inmunología , Receptores CCR7/metabolismo , Trasplante HomólogoRESUMEN
OBJECTIVE: Current in vitro techniques for isolating leukemia-reactive cytotoxic T lymphocytes (CTLs) from healthy donors are of relatively low efficiency and yield responder populations with unknown biological significance. This study aimed at the development of a more reliable approach, allowing generation and expansion of acute myeloid leukemia (AML)-reactive CTLs using primary in vitro stimulation. MATERIALS AND METHODS: We established allogeneic mini-mixed lymphocyte-leukemia cultures (mini-MLLCs) by stimulating donor CD8(+) T cells with human leukocyte antigen (HLA) class I-matched AML blasts in microtiter plates. Before culture, CD8(+) T cells were separated into CD62L((high)+) and CD62L((low)+/neg) subsets enriched for naive/central memory and effector memory cells, respectively. RESULTS: In eight different related and unrelated donor/AML pairs, numerous CTL populations were isolated that specifically lysed myeloid leukemias in association with various HLA-A, -B, or -C alleles. These CTLs expressed T-cell receptors of single Vbeta-chain families, indicating their clonal origin. The majority of CTL clones were obtained from mini-MLLCs initiated with CD62L((high)+) cells. Using antigen-specific stimulation, multiple CTL populations were amplified to 10(8)-10(10) cells within 6 to 8 weeks. Three of four representative CTL clones were capable of completely preventing engraftment of human primary AML blasts in nonobese diabetic/severe combined immune deficient IL2Rgamma(null) mice. CONCLUSION: The mini-MLLC approach allows the efficient in vitro expansion of AML-reactive CTL clones from CD8(+)CD62L((high)+) precursors of healthy donors. These CTLs can inhibit leukemia engraftment in immunodeficient mice, suggesting their potential biological relevance.
Asunto(s)
Linfocitos T CD8-positivos/citología , Subunidad gamma Común de Receptores de Interleucina/genética , Selectina L/biosíntesis , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/prevención & control , Linfocitos T Citotóxicos/trasplante , Alelos , Animales , Linfocitos T CD8-positivos/metabolismo , Células Cultivadas , Células Clonales , Pruebas Inmunológicas de Citotoxicidad , Genes MHC Clase I , Humanos , Leucemia Mieloide Aguda/sangre , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Trasplante de Neoplasias , Estándares de Referencia , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
In allogeneic hematopoietic stem cell transplantation (AHSCT) graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect are closely but not invariably linked. Thus, harnessing donor lymphocyte mediated GVL immunity and separating it from GVHD is of particular interest. Based on results obtained in murine models we have explored the CD95-mediated activation-induced cell death (AICD) strategy to selectively deplete alloreactivity in human donor T lymphocytes in vitro. Following stimulation of CD3(+) T cells isolated from HLA-A* 0201-positive donors with HLA or minor histocompatibility antigen mismatched hematopoietic or nonhematopoietic cells in the presence of agonistic anti-CD95 antibody, we achieved efficient and selective allodepletion across major and minor histocompatibility mismatched barriers. Residual alloreactivity was in the range of 10% and 25% using hematopoietic cells and primary keratinocytes as alloantigen-presenting cells, respectively. CD8(+) T cells specific for HLA-A * 0201-associated cytomegalovirus (CMV), Epstein-Barr virus (EBV), and Wilms tumor 1 peptide epitopes were retained at significant numbers within the allodepleted donor lymphocyte subsets. Additionally, CD4(+) FoxP3(+) regulatory T cells persisted after the allodepletion procedure. Our results show that AICD induced by an agonistic anti-CD95 antibody might be useful to generate allodepleted donor lymphocyte products with preserved beneficial immune functions for patients undergoing AHSCT.
Asunto(s)
Efecto Injerto vs Leucemia/inmunología , Trasplante de Células Madre Hematopoyéticas , Depleción Linfocítica/métodos , Transfusión de Linfocitos/métodos , Receptor fas/inmunología , Apoptosis , Línea Celular Tumoral , Humanos , Trasplante Homólogo/inmunologíaRESUMEN
Current methods for the detection and isolation of antigen-specific CD4(+) and CD8(+) T cells require the availability of peptide/MHC multimers or are restricted to cells that produce cytokines after antigen contact. Here we show that de novo cell surface expression of the TNF-receptor family member CD137 (4-1BB) identifies recently activated, but not resting, human CD4(+) and CD8(+) memory T cells. Maximum CD137 expression level is uniformly observed in both T-cell subsets at 24h after stimulation with antigen. In experiments with CMV and EBV-reactive T cells, we confirmed the specificity of CD137 expression by co-staining with peptide/HLA tetramers. Substantial proportions of CD137(+) T cells did not produce IFN-gamma, suggesting that CD137 detects a broader repertoire of antigen-specific T cells. Activated CD137(+) T cells could be easily purified by MACS and expanded in vitro thereafter. This CD137-based enrichment method was capable of isolating 2-fold higher numbers of anti-viral CD4(+) and CD8(+) T cells compared to the IFN-gamma secretion assay. In conclusion, antigen-triggered CD137 expression allows the rapid detection and sorting of virus-reactive CD4(+) and CD8(+) T cells. The CD137 assay is most attractive for the simultaneous targeting of anti-viral T helper and effector cells in monitoring studies and adoptive immunotherapy trials.
Asunto(s)
Antígenos Virales/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Citomegalovirus/inmunología , Herpesvirus Humano 4/inmunología , Separación Inmunomagnética/métodos , Fosfoproteínas/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Proteínas de la Matriz Viral/inmunología , Traslado Adoptivo , Antígenos Virales/farmacología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Humanos , Interferón gamma/inmunología , Células K562 , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Fosfoproteínas/farmacología , Proteínas de la Matriz Viral/farmacologíaRESUMEN
Fascinating earlier evidence suggests an intrinsic capacity of human natural killer (NK) cells to acquire adaptive immune features in the context of cytomegalovirus (CMV) infection or pro-inflammatory cytokine stimulation. Since the role of memory NK cells in cancer has so far remained elusive and adoptive NK cell transfer in relapsing pediatric acute B cell precursor leukemia (BCP-ALL) patients awaits improvement, we asked the question whether tumor-priming could promote the generation of memory NK cells with enhanced graft-vs.-leukemia (GvL) reactivity. Here, we provide substantial evidence that priming of naive human NK cells with pediatric acute B cell leukemia or acute myeloid leukemia specimens induces a functional conversion to tumor-induced memory-like (TIML)-NK cells displaying a heightened tumor-specific cytotoxicity and enhanced perforin synthesis. Cell cycles analyses reveal that tumor-priming sustainably alters the balance between NK cell activation and apoptosis in favor of survival. In addition, gene expression patterns differ between TIML- and cytokine-induced memory-like (CIML)-NK cells with the magnitude of regulated genes being distinctly higher in TIML-NK cells. As such, the tumor-induced conversion of NK cells triggers the emergence of a so far unacknowledged NK cell differentiation stage that might promote GvL effects in the context of adoptive cell transfer.
RESUMEN
AIM: T lymphocytes are used as cellular therapeutics in many disease entities including cancer. We investigated the uptake and retention of nanoparticles (NPs) by these nonphagocytic cells. MATERIALS & METHODS: Uptake, release and toxicity of various polymeric NP preparations were analyzed by flow cytometry, confocal laser scanning microscopy and transmission electron microscopy. T-cell effector functions were measured using IFN-γ-ELISPOT and (51)Chromium-release assays. RESULTS: Amino-functionalized NPs were efficiently ingested by antigen-specific T cells without adversely influencing effector functions. NPs were stored in membrane-surrounded vesicles, with major proportions released extracellularly during 24 h. CONCLUSION: Amino-functionalized polymeric NPs are efficiently taken up by human T cells and could be used to design nanocarriers for direct access and manipulation of antigen-specific T cells in vivo.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Portadores de Fármacos/metabolismo , Nanopartículas/metabolismo , Polímeros/metabolismo , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Línea Celular , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Liberación de Fármacos , Endocitosis , Humanos , Ratones Endogámicos C57BL , Microscopía Confocal , Microscopía Electrónica de Transmisión , Nanopartículas/química , Nanopartículas/ultraestructura , Polímeros/químicaRESUMEN
Pandemic and seasonal influenza viruses cause considerable morbidity and mortality in the general human population. Protection from severe disease may result from vaccines that activate antigen-presenting DC for effective stimulation of influenza-specific memory T cells. Special attention is paid to vaccine-induced CD8+ T-cell responses, because they are mainly directed against conserved internal influenza proteins thereby presumably mediating cross-protection against circulating seasonal as well as emerging pandemic virus strains. Our study showed that influenza whole virus vaccines of major seasonal A and B strains activated DC more efficiently than those of pandemic swine-origin H1N1 and pandemic-like avian H5N1 strains. In contrast, influenza split virus vaccines had a low ability to activate DC, regardless which strain was investigated. We also observed that whole virus vaccines stimulated virus-specific CD8+ memory T cells much stronger compared to split virus counterparts, whereas both vaccine formats activated CD4+ Th cell responses similarly. Moreover, our data showed that whole virus vaccine material is delivered into the cytosolic pathway of DC for effective activation of virus-specific CD8+ T cells. We conclude that vaccines against seasonal and pandemic (-like) influenza strains that aim to stimulate cross-reacting CD8+ T cells should include whole virus rather than split virus formulations.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Vacunas contra la Influenza/inmunología , Orthomyxoviridae/inmunología , Adulto , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular , Células Dendríticas/citología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/ultraestructura , Células Dendríticas/virología , Endosomas/metabolismo , Endosomas/virología , Humanos , Memoria Inmunológica , Técnicas In Vitro , Virus de la Influenza A/clasificación , Virus de la Influenza A/genética , Activación de Linfocitos/inmunología , Persona de Mediana Edad , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Adulto JovenRESUMEN
Multiple sclerosis is a demyelinating autoimmune disease of the CNS. Its animal model experimental autoimmune encephalomyelitis is commonly induced by active immunization with myelin antigens. To investigate human immune responses against myelin antigens in vivo we established a new subclinical experimental autoimmune encephalomyelitis model in humanized mice. NOD/Scidγcâ»/â» animals were transferred with peripheral blood mononuclear cells from healthy human donors and immunized with myelin antigens in complete Freund's adjuvant and antigen-pulsed autologous dendritic cells. Human T cells recovered from these animals reacted specifically to the soluble domain of myelin oligodendrocyte glycoprotein and secreted proinflammatory cytokines. Furthermore, immunized animals developed subclinical CNS inflammation with infiltrating CD4⺠and CD8⺠T cells and production of encephalitogenic cytokines. Thus, this model of myelin-induced CNS inflammation by human T cells may allow testing of new human-specific therapeuticals for multiple sclerosis.
Asunto(s)
Antígenos CD/toxicidad , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Glicoproteína Mielina-Oligodendrócito/toxicidad , Animales , Femenino , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
The zebrafish has become a powerful vertebrate model for genetic studies of embryonic development and organogenesis and increasingly for studies in cancer biology. Zebrafish facilitate the performance of reverse and forward genetic approaches, including mutagenesis and small molecule screens. Moreover, several studies report the feasibility of xenotransplanting human cells into zebrafish embryos and adult fish. This model provides a unique opportunity to monitor tumor-induced angiogenesis, invasiveness, and response to a range of treatments in vivo and in real time. Despite the high conservation of gene function between fish and humans, concern remains that potential differences in zebrafish tissue niches and/or missing microenvironmental cues could limit the relevance and translational utility of data obtained from zebrafish human cancer cell xenograft models. Here, we summarize current data on xenotransplantation of human cells into zebrafish, highlighting the advantages and limitations of this model in comparison to classical murine models of xenotransplantation.
Asunto(s)
Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Línea Celular Tumoral , Descubrimiento de Drogas , Humanos , Ratones , Ratones Noqueados , Ratones SCID , Invasividad Neoplásica , Metástasis de la Neoplasia , Trasplante de Neoplasias , Neovascularización Patológica , Especificidad de la Especie , Microambiente Tumoral , Pez CebraRESUMEN
Trials of immune-modulating drugs in septic patients have mostly failed to demonstrate clinical efficacy. Thus, we sought to generate a surrogate model of myelomonocytic lineage differentiation that would potentially allow sepsis induction and preclinical testing of anti-inflammatory drugs. Comparing transplantation of cord blood-derived stem cells in neonatal NOD/SCID/IL2Rγ(null) (neonatal huNSG) mice with transplantation of adult peripheral mobilized stem cells into adult NSG (adult huNSG) recipients, we demonstrate that myelomonocytic lineage differentiation in neonatal huNSG mice is retarded and monocytes are phenotypically immature with respect to HLA-DR expression and the emergence of CD80(+)CD86(+) monocytes. Functionally, neonatal huNSG mice were less sensitive toward interferon-γ-induced upregulation of CD86 and exhibited a reduced T-cell stimulating capacity when compared with adult huNSG mice, whereas the phagocytic activity and the ability for cytokine secretion were mature. However, comparison of these data with data obtained from human neonates indicate that absence of the CD80(+)CD86(+) population and the reduced T-cell stimulating capacity of neonatal huNSG monocytes resemble functional immaturities observed in human neonatal monocytes. Thus, these two mouse models might well serve as 2 independent surrogate models for studying the neonatal myelomonocytic lineage differentiation or for testing the efficacy of immunomodulatory drugs on functionally mature monocytes.