RESUMEN
OBJECTIVES: NKG2D is an activating receptor expressed by natural killer (NK) and CD8+ T cells and activation intensity varies by NKG2D expression level or nature of its ligand. An NKG2D gene polymorphism determines high (HNK1) or low (LNK1) expression. MICA is the most polymorphic NKG2D ligand and stronger effector cell activation associates with methionine rather than valine at residue 129. We investigated correlation between cord blood (CB) NKG2D and MICA genotypes and haematopoietic stem cell (HSC) transplant outcome. METHODS: We retrospectively studied 267 CB HSC recipients (178 adult and 87 paediatric) who underwent transplant for malignant disease between 2007 and 2018, analysing CB graft DNA for NKG2D and MICA polymorphisms using Sanger sequencing. Multivariate analysis was used to correlate these results with transplant outcomes. RESULTS: In adult patients, LNK1 homozygous CB significantly improved 60-day neutrophil engraftment (hazard ratio (HR) 0.6; 95% confidence interval (CI) 0.4-0.9; p = .003). In paediatrics, HNK1 homozygous CB improved 60-day engraftment (HR 0.4; 95% CI 0.2-0.7; p = .003), as did MICA-129 methionine+ CB grafts (HR 1.7 95% CI 1.1-2.6; p = .02). CONCLUSION: CB NKG2D and MICA genotypes potentially improve CB HSC engraftment. However, results contrast between adult and paediatric recipients and may reflect transplant procedure disparities between cohorts.
Asunto(s)
Trasplante de Células Madre de Sangre del Cordón Umbilical , Antígenos de Histocompatibilidad Clase I , Subfamilia K de Receptores Similares a Lectina de Células NK , Humanos , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Niño , Masculino , Antígenos de Histocompatibilidad Clase I/genética , Adulto , Femenino , Adolescente , Preescolar , Persona de Mediana Edad , Estudios Retrospectivos , Lactante , Genotipo , Trasplante Homólogo , Polimorfismo Genético , Adulto Joven , Resultado del Tratamiento , Anciano , Alelos , Donantes de Tejidos , Neoplasias/genética , Neoplasias/terapia , Supervivencia de Injerto , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/genética , Trasplante de Células Madre Hematopoyéticas/métodosRESUMEN
BACKGROUND: Allogeneic haematopoietic cell transplantation (HCT) is a curative therapy for severe haematological disorders. However, it carries significant risk of morbidity and mortality. To improve patient outcomes, better graft selection strategies are needed, incorporating HLA matching with clinically important graft characteristics. Studies have shown that the cellular content of HCT grafts, specifically higher ratios of T regulatory (Tregs)/T cells, are important factors influencing outcomes when using adult peripheral blood mobilised grafts. So far, no equivalent study exists in umbilical cord blood (CB) transplantation due to the limitations of cryopreserved CB samples. STUDY DESIGN AND METHODS: To establish the most robust and efficient way to measure the Treg content of previously cryopreserved CB units, we compared the enumeration of Treg and CD3+ cells using flow cytometry and an epigenetic, DNA-based methodology. The two methods were assessed for their agreement, consistency and susceptibility to error when enumerating Treg and CD3+ cell numbers in both fresh and cryopreserved CB samples. RESULTS: Epigenetic enumeration gave consistent and comparable results in both fresh and frozen CB samples. By contrast, assessment of Tregs and CD3+ cells by flow cytometry was only possible in fresh samples due to significant cell death following cryopreservation and thawing. CONCLUSION: Epigenetic assessment offers significant advantages over flow cytometry for analysing cryopreserved CB; similar cell numbers were observed both in fresh and frozen samples. Furthermore, multiple epigenetic assessments can be performed from DNA extracted from small cryopreserved CB segments; often the only CB sample available for clinical studies.
Asunto(s)
Trasplante de Células Madre de Sangre del Cordón Umbilical/métodos , Metilación de ADN , Sangre Fetal/citología , Citometría de Flujo/métodos , Factores de Transcripción Forkhead/genética , Linfocitos T Reguladores/metabolismo , Conservación de la Sangre/métodos , Trasplante de Células Madre de Sangre del Cordón Umbilical/normas , Criopreservación/métodos , Sangre Fetal/trasplante , Factores de Transcripción Forkhead/metabolismo , HumanosRESUMEN
Regulatory T cells (Tregs) are CD4+ T cells that are key players of immune tolerance. They are powerful suppressor cells, able to impact the function of numerous immune cells, including key effectors of inflammation such as effector T cells. For this reason, Tregs are an ideal candidate for the development of cell therapy approaches to modulate immune responses. Treg therapy has shown promising results so far, providing key knowledge on the conditions in which these cells can provide protection and demonstrating that they could be an alternative to current pharmacological immunosuppressive therapies. However, a more comprehensive understanding of their characteristics, isolation, activation, and expansion is needed to be able design cost effective therapies. Here, we review the practicalities of making Tregs a viable cell therapy, in particular, discussing the challenges faced in isolating and manufacturing Tregs and defining what are the most appropriate applications for this new therapy.
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Inmunoterapia/métodos , Linfocitos T Reguladores/inmunología , Humanos , Tolerancia Inmunológica/inmunologíaRESUMEN
Highly polymorphic major histocompatibility complex (MHC) molecules are at the heart of adaptive immune responses, playing crucial roles in many kinds of disease and in vaccination. We report that breadth of peptide presentation and level of cell surface expression of class I molecules are inversely correlated in both chickens and humans. This relationship correlates with protective responses against infectious pathogens including Marek's disease virus leading to lethal tumours in chickens and human immunodeficiency virus infection progressing to AIDS in humans. We propose that differences in peptide binding repertoire define two groups of MHC class I molecules strategically evolved as generalists and specialists for different modes of pathogen resistance. We suggest that differences in cell surface expression level ensure the development of optimal peripheral T cell responses. The inverse relationship of peptide repertoire and expression is evidently a fundamental property of MHC molecules, with ramifications extending beyond immunology and medicine to evolutionary biology and conservation.
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Inmunidad Adaptativa , Herpesvirus Gallináceo 2/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Enfermedad de Marek/inmunología , Péptidos/inmunología , Síndrome de Inmunodeficiencia Adquirida/inmunología , Síndrome de Inmunodeficiencia Adquirida/virología , Alelos , Secuencia de Aminoácidos , Animales , Presentación de Antígeno , Sitios de Unión , Línea Celular , Pollos , Cristalografía por Rayos X , Regulación de la Expresión Génica , VIH-1/inmunología , Haplotipos , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/genética , Enfermedad de Marek/virología , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
Allogeneic hematopoietic stem cell therapy (HSCT) remains one of the few curative treatments for high-risk hematological malignancies (high-risk leukemia, myelodysplastic syndromes, advanced myeloproliferative disorders, high-risk lymphomas, and multiple myeloma) and is currently applied in more than 15,000 patients per year in Europe. Following HSCT, patients experience a period of reconstitution of the immune system, which seems to be highly dependent on conditioning, immunosuppression regimes, and the level of adverse events the patients experience. During this reconstitution period, the patient is immune compromised and susceptible to opportunistic infections and disease relapse. Consequently, a large number of clinical studies have been devoted to monitoring the recovery of the immune system following HSCT in the hopes of determining which cellular subsets are indicative of a favorable outcome. In this chapter we review the methods that have been employed to monitor the immune reconstitution and what clinical observations have been made. Of particular interest is the regulatory T cell (Treg) subset, which has been associated with tolerance and has been the subject of recent clinical trials as a possible cellular therapy for rejection reactions. Finally we will detail a proposed methodology for the flow cytometric assessment of cellular reconstitution post-HSCT.
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Citometría de Flujo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Animales , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Humanos , InmunofenotipificaciónRESUMEN
Human umbilical cord blood (hUCB) has been proposed to contain not only haematopoietic stem cells, but also a rare pluripotent embryonic-like stem cell (ELSc) population that is negative for hematopoietic markers (Lin(-)CD45(-)) and expresses markers typical of pluripotent cells. The aim of this work was to isolate, characterise and expand this ELSc fraction from hUCB, as it may provide a valuable cell source for regenerative medicine applications. We found that we could indeed isolate a Lin(-)CD45(-) population of small cells (3-10 µm diameter) with a high nucleus to cytoplasm ratio that expressed the stem cell markers CD34 and CXCR4. However, in contrast to some previous reports, this fraction was not positive for CD133. Furthermore, although these cells expressed transcripts typical of pluripotent cells, such as SOX2, OCT3/4, and NANOG, they were not able to proliferate in any of the culture media known to support stem cell growth that we tested. Further analysis of the Lin(-)CD45(-) population by flow cytometry showed the presence of a Lin(-)CD45(-)Nestin(+) population that were also positive for CD34 (20%) but negative for CXCR4. These data suggest that the Lin(-)CD45(-) stem cell fraction present in the cord blood represents a small heterogeneous population with phenotypic characteristics of stem cells, including a Lin(-)CD45(-)Nestin(+) population not previously described. This study also suggests that heterogeneity within the Lin(-)CD45(-) cell fraction is the likely explanation for differences in the hUCB cell populations described by different groups that were isolated using different methods. These populations have been widely called "embryonic-like stem cell" on the basis of their phenotypical similarity to embryonic stem cells. However, the fact they do not seem to be able to self-renew casts some doubt on their identity, and warns against defining them as "embryonic-like stem cell" at this stage.
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Células Madre Embrionarias/fisiología , Sangre Fetal/fisiología , Antígenos Comunes de Leucocito/metabolismo , Biomarcadores/metabolismo , Diferenciación Celular/fisiología , Núcleo Celular/metabolismo , Núcleo Celular/fisiología , Proliferación Celular , Separación Celular/métodos , Células Cultivadas , Citoplasma/metabolismo , Citoplasma/fisiología , Células Madre Embrionarias/metabolismo , Sangre Fetal/metabolismo , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/fisiología , Humanos , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/fisiología , Receptores CXCR4/metabolismo , Medicina Regenerativa/métodosRESUMEN
The assessment of nonviable haematopoietic cells by Annexin V staining method in flow cytometry has recently been published by Duggleby et al. Resulting in a better correlation with the observed colony formation in methylcellulose assays than the standard ISHAGE protocol, it presents a promising method to predict cord blood potency. Herein, we applied this method for examining the parameters during processing which potentially could affect cord blood viability. We could verify that the current standards regarding time and temperature are sufficient, since no significant difference was observed within 48 hours or in storage at 4°C up to 26°C. However, the addition of DMSO for cryopreservation alone leads to an inevitable increase in nonviable haematopoietic stem cells from initially 14.8% ± 4.3% to at least 30.6% ± 5.5%. Furthermore, CFU-assays with varied seeding density were performed in order to evaluate the applicability as a quantitative method. The results revealed that only in a narrow range reproducible clonogenic efficiency (ClonE) could be assessed, giving at least a semiquantitative estimation. We conclude that both Annexin V staining method and CFU-assays with defined seeding density are reliable means leading to a better prediction of the final potency. Especially Annexin V, due to its fast readout, is a practical tool for examining and optimising specific steps in processing, while CFU-assays add a functional confirmation.
RESUMEN
BACKGROUND: Nonviable CD34+ cells are commonly assessed by standard flow cytometry using the nuclear stain 7-aminoactinomycin D (7AAD). 7AAD, however, only detects necrotic and late apoptotic cells, not earlier apoptosis, which engraft poorly in animal models of cord blood (cord) transplantation. The standard method, therefore, may overestimate engraftment potency of cord units under certain conditions. STUDY DESIGN AND METHODS: To detect apoptotic events, costaining with 7AAD and annexin V (AnnV), in parallel with the quantitative, standard enumeration, was used. Cord units were assessed before and after cryopreservation using both staining methods and colony-forming units (CFU) to determine if graft potency can be predicted using a "functional flow cytometry" approach. RESULTS: Significant numbers of CD34+ AnnV+ events were found within the 7AAD-gated population. Nonapoptotic cell dose (CD34+ AnnV-) correlated well with CFUs in both a small-scale (n = 10) and a large-scale banking study (n = 107). Finally, following samples postthaw with time showed increasing numbers of apoptotic CD34+ cells and consequently the AnnV assessed dose was better at predicting the CFU compared with just the standard enumeration. CONCLUSION: Defining the apoptotic population of CD34+ cells improved the prediction of CFU, making this method a rapid test of potency for assessment of cord units for clinical use.
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Anexina A5/metabolismo , Apoptosis , Trasplante de Células Madre de Sangre del Cordón Umbilical/métodos , Citometría de Flujo/métodos , Células Madre Hematopoyéticas/citología , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Recuento de Células , Trasplante de Células Madre de Sangre del Cordón Umbilical/normas , Dactinomicina/análogos & derivados , Sangre Fetal/citología , Citometría de Flujo/normas , Colorantes Fluorescentes , Células Madre Hematopoyéticas/metabolismo , Humanos , Valor Predictivo de las PruebasRESUMEN
Natural regulatory T cells (Tregs), characterized as CD4 CD25high Foxp3+, have been described as paramount contributors in immuno-regulation and self-tolerance. CD4 and CD25 have been the main markers used for their isolation, resulting in cells with potent suppressive properties. Nevertheless, low purity and yield continue to be an issue when attempting thorough characterizations and/or up scaling to bigger models and for clinical trials. Here we present a single-step methodology optimized for cord blood CD25+ isolation, using magnetic microbeads that achieves a reproducible purity of 89% for CD4 CD25high CD127low. These cells showed a more consistent suppressive effect in mixed lymphocyte cultures. In addition, the proportion of contaminating effector T cells was < 9% whilst the yield of Tregs was doubled compared to the standard protocol. Gating on CD4 CD25high CD127low populations post isolation showed better correlation with suppressive efficacy compared to CD4 CD25+ gate. These data should facilitate the clinical scale-up of this procedure to obtain consistent Tregs for clinical application and research.
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Sangre Fetal/citología , Separación Inmunomagnética/métodos , Linfocitos T Reguladores/citología , Antígenos CD4/inmunología , Sangre Fetal/inmunología , Citometría de Flujo/métodos , Factores de Transcripción Forkhead/inmunología , Humanos , Subunidad alfa del Receptor de Interleucina-2/inmunología , Subunidad alfa del Receptor de Interleucina-7/inmunología , Linfocitos T Reguladores/inmunologíaRESUMEN
Because antigen-specific cells are the central coordinators of the immune response to infectious organisms, and the principal effector cells in autoimmune disease, there are many circumstances in which investigators may wish to examine the T-cell responses to particular antigens. This chapter outlines techniques for assessing the responses of polyclonal populations of T-lymphocytes by measuring a variety of outputs each of which gives different kinds of information about the response. The outputs discussed are proliferation and cytokine production, with methods for measuring cytokine secretion by the whole population together with techniques for making an estimate of the numbers of cells producing a cytokine in response to antigen, and examining the phenotype of the responsive cells. In many cases detailed information about responses to particular antigens requires the isolation and characterization of antigen-responsive T-cell clones, and this is also described together with methods of identifying unknown antigens by screening recombinant expression libraries. Lastly, because the techniques differ in many respects, methods for isolating antigen-specific CD8+ T-cells, particularly those which recognize bacteria, are also included.
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Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Separación Celular/métodos , Animales , Antígenos/genética , Bacterias/inmunología , Bacterias/patogenicidad , Linfocitos T CD8-positivos/citología , Células Cultivadas , Citocinas/inmunología , Citometría de Flujo , Humanos , FenotipoRESUMEN
It is clear that regulatory T cells (Treg) have an important role in preventing autoimmunity and modulating responses to pathogens. Full characterization of Treg cell function in human patients would be greatly facilitated by practical methods for expanding Treg in vitro. Methods for expansion have been reported but whether expression of surface and intracellular markers associated with freshly isolated Treg following expansion correlates with the maintenance of function is unclear. Our aim was to investigate the various methods of expansion and to correlate regulatory activity with expression of these markers. We show that, of the markers associated with freshly isolated Treg, only CD27 expression correlated with regulatory activity and could be used to isolate cells with regulatory activity from lines expanded from CD4+ CD25+ cells. Also, cells expressing high levels of the transcription factor forkhead box P3 (Foxp3) were confined to the CD27+ population within these lines. Expression of CD27 by cells in lines expanded from CD4+ CD25- cells varied depending on the stimulus used for expansion, but these lines did not have significant regulatory activity even when the CD27+ cells were tested. Analysis of synovial CD4+ CD25+ cells from reactive arthritis patients revealed that they were predominantly CD27 positive. This also applied to CD25(high) and CD25(intermediate) CD4+ cells, despite their reported different abilities to regulate. We conclude that, whilst CD27 is useful for identifying Treg in the cell lines obtained after expansion of CD4+ CD25+ cells, its expression may not reliably identify the Treg cell population in other T-cell populations such as those found in joints.
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Subunidad alfa del Receptor de Interleucina-2/metabolismo , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Artritis Reactiva/inmunología , Biomarcadores/metabolismo , Línea Celular , Proliferación Celular , Separación Celular/métodos , Células Cultivadas , Citometría de Flujo , Factores de Transcripción Forkhead/metabolismo , Humanos , Inmunofenotipificación , Activación de Linfocitos/inmunología , Líquido Sinovial/inmunologíaRESUMEN
Despite substantial advances in our understanding of CD4+ CD25+ regulatory T cells, a possible equivalent regulatory subset within the CD8+ T cell population has received less attention. We now describe novel human CD8+/TCR alphabeta+ T cells that have a regulatory phenotype and function. We expanded and cloned these cells using autologous LPS-activated dendritic cells. The clones were not cytolytic, but responded in an autoreactive HLA class I-restricted fashion, by proliferation and production of IL-4, IL-5, IL-13 and TGFbeta1, but not IFN-gamma. They constitutively expressed CD69 and CD25 as well as molecules associated with CD4+ CD25+ regulatory T cells, including cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and Foxp3. They suppressed IFN-gamma production and proliferation by CD4+ T cells in vitro in a cell contact-dependent manner, which could be blocked using a CTLA-4-specific mAb. They were more readily isolated from patients with ankylosing spondylitis and may therefore be up-regulated in response to inflammation. We suggest that they are the CD8+ counterparts of CD4+ CD25+ regulatory T cells. They resemble recently described CD8+ regulatory cells in the rat that were able to abrogate graft-versus-host disease. Likewise, human HLA-restricted CD8+ regulatory T cells that can be cloned and expanded in vitro may have therapeutic applications.
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Linfocitos T CD8-positivos/inmunología , Activación de Linfocitos/inmunología , Subgrupos de Linfocitos T/inmunología , Antígenos CD , Antígenos de Diferenciación/inmunología , Western Blotting , Antígeno CTLA-4 , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Factores de Transcripción Forkhead/inmunología , Humanos , Fenotipo , Receptores de Interleucina-2/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cross-reactive T cell recognition of self-heat shock proteins (hsp) has been ascribed a regulatory role in inflammatory arthritis in both animal models and human disease. The previous work implies that a repertoire for epitopes in self-hsp60 should exist in normal subjects. Accordingly, we sought to generate self-hsp60-reactive T cell clones from a healthy individual using a highly purified preparation of recombinant human (Hu) hsp60. Epitope mapping using synthetic peptides and truncated constructs indicated that the T cell clones obtained actually recognized hsp60 derived from Escherichia coli. Using a series of alanine-substituted peptides and additional appropriate synthetic peptides, it was demonstrated that the clones maintain self-tolerance because of their sensitivity to an asparagine to aspartic acid sequence difference between E. coli and HuHsp60 in the epitope-containing peptide. In addition, despite substantial conservation of sequence, the homologous peptide from HuHsp60 did not compete with the E. coli-derived peptide for recognition or antagonize responses by acting as an altered peptide ligand. The results suggest that, even when the immune system targets a highly conserved epitope in bacterial hsp60, self-tolerance is maintained. Furthermore, the finding that T cell clones specific for minor contaminant proteins in HuHsp60 preparations can readily be isolated raises the possibility that the HuHsp60 facilitates presentation of antigenic proteins to the immune system.