RESUMO
Agammaglobulinemia is the most profound primary antibody deficiency that can occur due to an early termination of B-cell development. We here investigated 3 novel patients, including the first known adult, from unrelated families with agammaglobulinemia, recurrent infections, and hypertrophic cardiomyopathy (HCM). Two of them also presented with intermittent or severe chronic neutropenia. We identified homozygous or compound-heterozygous variants in the gene for folliculin interacting protein 1 (FNIP1), leading to loss of the FNIP1 protein. B-cell metabolism, including mitochondrial numbers and activity and phosphatidylinositol 3-kinase/AKT pathway, was impaired. These defects recapitulated the Fnip1-/- animal model. Moreover, we identified either uniparental disomy or copy-number variants (CNVs) in 2 patients, expanding the variant spectrum of this novel inborn error of immunity. The results indicate that FNIP1 deficiency can be caused by complex genetic mechanisms and support the clinical utility of exome sequencing and CNV analysis in patients with broad phenotypes, including agammaglobulinemia and HCM. FNIP1 deficiency is a novel inborn error of immunity characterized by early and severe B-cell development defect, agammaglobulinemia, variable neutropenia, and HCM. Our findings elucidate a functional and relevant role of FNIP1 in B-cell development and metabolism and potentially neutrophil activity.
Assuntos
Agamaglobulinemia/genética , Linfócitos B/patologia , Cardiomiopatia Hipertrófica/genética , Proteínas de Transporte/genética , Síndromes de Imunodeficiência/genética , Linfopenia/genética , Adulto , Animais , Linfócitos B/metabolismo , Criança , Pré-Escolar , Cromossomos Humanos Par 5/genética , Códon sem Sentido , Consanguinidade , Doença de Crohn/genética , Variações do Número de Cópias de DNA , Deficiências do Desenvolvimento/genética , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Cardiopatias Congênitas/genética , Humanos , Infecções/etiologia , Mutação com Perda de Função , Masculino , Camundongos , Neutropenia/genética , Linhagem , Dissomia Uniparental , Sequenciamento do ExomaRESUMO
Differentially and functionally distinct T cell subsets are involved in the development of complications after allogeneic hematopoietic stem cell transplantation (HSCT), but little is known about factors regulating their recovery after HSCT. In this study, we investigated associations between immune-regulating cytokines, T cell differentiation, and clinical outcomes. We included 80 children undergoing allogeneic HSCT for acute leukemia using bone marrow or peripheral blood stem cells grafted from a matched sibling or unrelated donor. Cytokines (IL-7, IL-15, IL-18, SCF, IL-6, IL-2, and TNF-α) and active anti-thymocyte globulin (ATG) levels were longitudinally measured along with extended T cell phenotyping. The cytokine profiles showed a temporary rise in IL-7 and IL-15 during lymphopenia, which was strongly dependent on exposure to active ATG. High levels of IL-7 and IL-15 from graft infusion to day +30 were predictive of slower T cell recovery during the first 2 mo post-HSCT; however, because of a major expansion of memory T cell stages, only naive T cells remained decreased after 3 mo (p < 0.05). No differential effect was seen on polarization of CD4+ T cells into Th1, Th2, or Th17 cells or regulatory T cells. Low levels of IL-7 and IL-15 at day +14 were associated with acute graft-versus-host disease grades II-IV in ATG-treated patients (p = 0.0004 and p = 0.0002, respectively). Children with IL-7 levels comparable to healthy controls at day +14 post-HSCT were less likely to develop EBV reactivation posttransplant. These findings suggest that quantification of IL-7 and IL-15 may be useful as biomarkers in assessing the overall T cell depletion and suggest a potential for predicting complications after HSCT.
Assuntos
Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Interleucina-15/análise , Interleucina-7/análise , Leucemia Mieloide Aguda/terapia , Linfopenia/terapia , Células T de Memória/imunologia , Adolescente , Adulto , Criança , Pré-Escolar , Humanos , Lactente , Interleucina-15/imunologia , Interleucina-7/imunologia , Leucemia Mieloide Aguda/imunologia , Depleção Linfocítica , Linfopenia/imunologia , Pessoa de Meia-Idade , Estudos Retrospectivos , Adulto JovemRESUMO
In the setting of hematopoietic stem cell transplantation (HSCT), Rituximab (RTX) is used for the treatment and prevention of EBV-associated post-transplantation lymphoproliferative disease or autoimmune phenomena such as autoimmune hemolytic anemia (AIHA). Persistent hypogammaglobulinemia and immunoglobulin substitution dependence has been observed in several patients after RTX treatment despite the normalization of total B cell numbers. We aimed to study whether this is a B cell intrinsic phenomenon. We analyzed four patients with different primary diseases who were treated with myeloablative conditioning and matched unrelated donor HSCT who developed persistent hypogammaglobulinemia after receiving RTX treatment. They all received RTX early after HSCT to treat EBV infection or AIHA post-HSCT. All patients showed normalized total B cell numbers but absent to very low IgG positive memory B cells, and three lacked IgA positive memory B cells. All of the patients had full donor chimerism, and none had encountered graft-versus-host disease. Sorted peripheral blood naïve B cells from these patients, when stimulated with CD40L, IL21, IL10 and anti-IgM, demonstrated intact B cell differentiation including the formation of class-switched memory B cells and IgA and IgG production. Peripheral blood T cell numbers including CD4 follicular T-helper (Tfh) cells were all within the normal reference range. In conclusion, in these four HSCT patients, the persistent hypogammaglobulinemia observed after RTX cannot be attributed to an acquired intrinsic B cell problem nor to a reduction in Tfh cell numbers.
Assuntos
Agamaglobulinemia , Transplante de Células-Tronco Hematopoéticas , Humanos , Rituximab/uso terapêutico , Agamaglobulinemia/tratamento farmacológico , Agamaglobulinemia/etiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Imunoglobulina G , Imunoglobulina ARESUMO
The first successful European hematopoietic stem cell transplantation (HSCT) was performed in 1968 as treatment in a newborn with IL2RG deficiency using an HLA-identical sibling donor. Because of declining naive T and natural killer (NK) cells, and persistent human papilloma virus (HPV)-induced warts, the patient received a peripheral stem cell boost at the age of 37 years. NK and T cells were assessed before and up to 14 years after the boost by flow cytometry. The boost induced renewed reconstitution of functional NK cells that were 14 years later enriched for CD56dimCD27+ NK cells. T-cell phenotype and T-cell receptor (TCR) repertoire were simultaneously analyzed by including TCR Vß antibodies in the cytometry panel. Naive T-cell numbers with a diverse TCR Vß repertoire were increased by the boost. Before and after the boost, clonal expansions with a homogeneous TIGIT and PD-1 phenotype were identified in the CD27- and/or CD28- memory population in the patient, but not in the donor. TRB sequencing was applied on sorted T-cell subsets from blood and on T cells from skin biopsies. Abundant circulating CD8 memory clonotypes with a chronic virus-associated CD57+KLRG1+CX3CR1+ phenotype were also present in warts, but not in healthy skin of the patient, suggesting a link with HPV. In conclusion, we demonstrate in this IL2RG-deficient patient functional NK cells, a diverse and lasting naive T-cell compartment, supported by a stem cell boost, and an oligoclonal memory compartment half a century after HSCT.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Infecções por Papillomavirus , Verrugas , Adulto , Antígenos CD28 , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Recém-Nascido , Subunidade gama Comum de Receptores de Interleucina , Células Matadoras Naturais , Receptor de Morte Celular Programada 1 , Receptores de Antígenos de Linfócitos T , Receptores ImunológicosRESUMO
The introduction of single-cell platforms inspired the development of high-dimensional single-cell analysis tools to comprehensively characterize the underlying cellular heterogeneity. Flow cytometry data are traditionally analyzed by (subjective) gating of subpopulations on two-dimensional plots. However, the increasing number of parameters measured by conventional and spectral flow cytometry reinforces the need to apply many of the recently developed tools for single-cell analysis on flow cytometry data, as well. However, the myriads of analysis options offered by the continuously released novel packages can be overwhelming to the immunologist with limited computational background. In this article, we explain the main concepts of such analyses and provide a detailed workflow to illustrate their implications and additional prerequisites when applied on flow cytometry data. Moreover, we provide readily applicable R code covering transformation, normalization, dimensionality reduction, clustering, and pseudotime analysis that can serve as a template for future analyses. We demonstrate the merit of our workflow by reanalyzing a public human dataset. Compared with standard gating, the results of our workflow provide new insights in cellular subsets, alternative classifications, and hypothetical trajectories. Taken together, we present a well-documented workflow, which utilizes existing high-dimensional single-cell analysis tools to reveal cellular heterogeneity and intercellular relationships in flow cytometry data.
Assuntos
Citometria de Fluxo , Software , Animais , HumanosRESUMO
The immunosuppressive character of head and neck cancers may explain the relatively low response rates to antibody therapy targeting a tumor antigen, such as cetuximab, and anti-PD-1 checkpoint inhibition. Immunostimulatory agents that overcome tumor-derived inhibitory signals could augment therapeutic efficacy, thereby enhancing tumor elimination and improving patient survival. Here, we demonstrate that cetuximab treatment combined with immunostimulatory agonists for Toll-like receptor (TLR) 2 induces profound immune responses. Natural killer (NK) cells, isolated from healthy individuals or patients with head and neck cancer, harbored enhanced cytotoxic capacity and increased tumor-killing potential in vitro. Additionally, combination treatment increased the release of several pro-inflammatory cytokines and chemokines by NK cells. Tumor-bearing mice that received cetuximab and the TLR2 ligand Pam3CSK4 showed increased infiltration of immune cells into the tumors compared to mice that received cetuximab monotherapy, resulting in a significant delay in tumor growth or even complete tumor regression. Moreover, combination treatment resulted in improved overall survival in vivo. In conclusion, combining tumor-targeting antibody-based immunotherapy with TLR stimulation represents a promising treatment strategy to improve the clinical outcomes of cancer patients. This treatment could well be applied together with other therapeutic strategies such as anti-PD-(L)1 checkpoint inhibition to further overcome immunosuppression.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos/imunologia , Neoplasias de Cabeça e Pescoço/terapia , Células Matadoras Naturais/imunologia , Receptor 2 Toll-Like/agonistas , Animais , Linhagem Celular Tumoral , Cetuximab/farmacologia , Cetuximab/uso terapêutico , Citocinas/metabolismo , Quimioterapia Combinada , Feminino , Humanos , Imunoterapia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Lipopeptídeos/farmacologia , Lipopeptídeos/uso terapêutico , Camundongos , Camundongos Nus , Receptores de IgG/agonistas , Receptores de IgG/metabolismo , Receptor 2 Toll-Like/metabolismo , Transplante HeterólogoRESUMO
Facioscapulohumeral dystrophy (FSHD) is associated with somatic chromatin relaxation of the D4Z4 repeat array and derepression of the D4Z4-encoded DUX4 retrogene coding for a germline transcription factor. Somatic DUX4 derepression is caused either by a 1-10 unit repeat-array contraction (FSHD1) or by mutations in SMCHD1, which encodes a chromatin repressor that binds to D4Z4 (FSHD2). Here, we show that heterozygous mutations in DNA methyltransferase 3B (DNMT3B) are a likely cause of D4Z4 derepression associated with low levels of DUX4 expression from the D4Z4 repeat and increased penetrance of FSHD. Recessive mutations in DNMT3B were previously shown to cause immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome. This study suggests that transcription of DUX4 in somatic cells is modified by variations in its epigenetic state and provides a basis for understanding the reduced penetrance of FSHD within families.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Repressão Epigenética/genética , Distrofia Muscular Facioescapuloumeral/genética , Mutação/genética , Penetrância , Sequências de Repetição em Tandem/genética , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Criança , Pré-Escolar , Cromatina/genética , DNA (Citosina-5-)-Metiltransferases/química , Metilação de DNA , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Linhagem , Conformação Proteica , Homologia de Sequência de Aminoácidos , DNA Metiltransferase 3BRESUMO
Autoimmune or alloimmune cytopenia (AIC) is a known rare complication of hematopoietic stem cell transplantation (SCT). AIC after SCT is considered difficult to treat and is associated with high morbidity and mortality. In this retrospective study in pediatric patients we evaluated incidence, outcome, potential risk factors, and current treatment strategies. A nested matched case-control study was performed to search for biomarkers associated with AIC. Of 531 consecutive SCTs at our center between 2000 and 2016, 26 were complicated by the development of AIC (cumulative incidence, 5.0%) after a median of 5 months post-SCT. Autoimmune hemolytic anemia was the most common AIC with 12 patients (46%). We identified nonmalignant disease, alemtuzumab serotherapy pre-SCT, and cytomegalovirus (CMV) reactivation as independently associated risk factors. The cytokine profile of patients at the time of AIC diagnosis appeared to skew toward a more pronounced Th 2 response compared with control subjects at the corresponding time point post-SCT. Corticosteroids and intravenous immunoglobulin as first-line treatment or a wait-and-see approach led to resolution of AIC in 35% of cases. Addition of step-up therapies rituximab (n = 15), bortezomib (n = 7), or sirolimus (n = 3) was associated with AIC resolution in 40%, 57%, and 100% of cases, respectively. In summary, we identified CMV reactivation post-SCT as a new clinical risk factor for the development of AIC in children. The cytokine profile during AIC appears to favor a Th 2 response. Rituximab, bortezomib, and sirolimus are promising step-up treatment modalities.
Assuntos
Doenças Autoimunes , Transplante de Células-Tronco Hematopoéticas , Adolescente , Corticosteroides/administração & dosagem , Adulto , Alemtuzumab/administração & dosagem , Aloenxertos , Doenças Autoimunes/etiologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/mortalidade , Doenças Autoimunes/terapia , Bortezomib/administração & dosagem , Criança , Pré-Escolar , Citomegalovirus/imunologia , Infecções por Citomegalovirus/etiologia , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/mortalidade , Infecções por Citomegalovirus/terapia , Feminino , Humanos , Imunoglobulinas Intravenosas/administração & dosagem , Lactente , Recém-Nascido , Masculino , Estudos Retrospectivos , Fatores de Risco , Rituximab/administração & dosagem , Células Th2/imunologiaRESUMO
Knowledge of human NK cells is based primarily on conventional CD56(bright) and CD56(dim) NK cells from blood. However, most cellular immune interactions occur in lymphoid organs. Based on the coexpression of CD69 and CXCR6, we identified a third major NK cell subset in lymphoid tissues. This population represents 30-60% of NK cells in marrow, spleen, and lymph node but is absent from blood. CD69(+)CXCR6(+) lymphoid tissue NK cells have an intermediate expression of CD56 and high expression of NKp46 and ICAM-1. In contrast to circulating NK cells, they have a bimodal expression of the activating receptor DNAX accessory molecule 1. CD69(+)CXCR6(+) NK cells do not express the early markers c-kit and IL-7Rα, nor killer cell Ig-like receptors or other late-differentiation markers. After cytokine stimulation, CD69(+)CXCR6(+) NK cells produce IFN-γ at levels comparable to CD56(dim) NK cells. They constitutively express perforin but require preactivation to express granzyme B and exert cytotoxicity. After hematopoietic stem cell transplantation, CD69(+)CXCR6(+) lymphoid tissue NK cells do not exhibit the hyperexpansion observed for both conventional NK cell populations. CD69(+)CXCR6(+) NK cells constitute a separate NK cell population with a distinct phenotype and function. The identification of this NK cell population in lymphoid tissues provides tools to further evaluate the cellular interactions and role of NK cells in human immunity.
Assuntos
Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos/imunologia , Tecido Linfoide/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Antígeno CD56/metabolismo , Separação Celular , Células Cultivadas , Citometria de Fluxo , Transplante de Células-Tronco Hematopoéticas , Humanos , Imunofenotipagem , Interferon gama/metabolismo , Lectinas Tipo C/metabolismo , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Receptores CXCR6 , Receptores de Quimiocinas/metabolismo , Receptores Virais/metabolismoRESUMO
Elderly with late-onset recurrent respiratory tract infections (RRTI) often have specific anti-polysaccharide antibody deficiency (SPAD). We hypothesized that late-onset RRTI is caused by mild immunodeficiencies, such as SPAD, that remain hidden through adult life. We analyzed seventeen elderly RRTI patients and matched controls. We determined lymphocyte subsets, expression of BAFF receptors, serum immunoglobulins, complement pathways, Pneumovax-23 vaccination response and genetic variations in BAFFR and MBL2. Twelve patients (71%) and ten controls (59%) had SPAD. IgA was lower in patients than in controls, but other parameters did not differ. However, a high percentage of both patients (53%) and controls (65%) were MBL deficient, much more than in the general population. Often, MBL2 secretor genotypes did not match functional deficiency, suggesting that functional MBL deficiency can be an acquired condition. In conclusion, we found SPAD and MBL deficiency in many elderly, and conjecture that at least the latter arises with age.
Assuntos
Envelhecimento/imunologia , Síndromes de Imunodeficiência/imunologia , Infecções Respiratórias/imunologia , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/sangue , Envelhecimento/genética , Receptor do Fator Ativador de Células B/genética , Receptor do Fator Ativador de Células B/imunologia , Diferenciação Celular , Via Alternativa do Complemento , Via Clássica do Complemento , Proteínas do Sistema Complemento/análise , Feminino , Humanos , Imunoglobulinas/sangue , Síndromes de Imunodeficiência/sangue , Síndromes de Imunodeficiência/genética , Linfócitos/citologia , Linfócitos/imunologia , Masculino , Lectina de Ligação a Manose/sangue , Lectina de Ligação a Manose/deficiência , Lectina de Ligação a Manose/genética , Lectina de Ligação a Manose/imunologia , Erros Inatos do Metabolismo/sangue , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/imunologia , Pessoa de Meia-Idade , Vacinas Pneumocócicas/uso terapêutico , Recidiva , Infecções Respiratórias/sangue , Infecções Respiratórias/genética , VacinaçãoRESUMO
The outcome of allogeneic hematopoietic stem cell transplantation (HSCT) is strongly affected by the kinetics of reconstitution of the immune system. This study compared the effects of antithymocyte globulin (ATG) and alemtuzumab on various outcome parameters after HSCT. The study cohort consisted of 148 children, with a median age of 9.6 years (range, .4 to 19.0), who underwent HSCT for malignant and benign hematological disorders in a single HSCT unit. Conditioning included ATG (n = 110) or alemtuzumab (n = 38). Cox proportional hazard regression analysis showed that alemtuzumab significantly delayed the recovery of CD3(+) T cells and CD4(+)as well as CD8(+) T cell subsets (P ≤ .001) and natural killer (NK) cells (P = .008) compared with ATG. In both ATG- and alemtuzumab-treated patients, shorter drug exposure lead to significantly faster recovery of T cells. Alemtuzumab was associated with lower donor chimerism 3 and 6 months after transplantation and a higher risk of disease relapse (P = .001). The overall survival and event-free survival risks were significantly lower for alemtuzumab-treated patients (P = .020 and P < .001, respectively). Patients who received alemtuzumab showed a trend to lower risk of acute graft-versus-host disease, more human adenovirus, and less Epstein-Barr virus reactivations compared with patients who received ATG. These data indicate that children treated with alemtuzumab as part of the conditioning regimen have a slower T cell and NK cell reconstitution compared with those treated with ATG, which compromises the overall and event-free survival. Prolonged length of lympholytic drug exposure delayed the T cell recovery in both ATG- and alemtuzumab-treated patients. Therefore, we recommend detailed pharmacokinetic/pharmacodynamic (PK/PD) analyses in a larger cohort of patients to develop an algorithm aiming at optimization of the serotherapy containing conditioning regimen.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Soro Antilinfocitário/administração & dosagem , Antineoplásicos/administração & dosagem , Transplante de Células-Tronco Hematopoéticas , Recuperação de Função Fisiológica , Condicionamento Pré-Transplante , Adolescente , Adulto , Alemtuzumab , Aloenxertos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/mortalidade , Neoplasias Hematológicas/terapia , Humanos , Lactente , Masculino , Recuperação de Função Fisiológica/efeitos dos fármacos , Recuperação de Função Fisiológica/imunologia , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
Children with advanced stages (relapsed/refractory and stage IV) of rhabdomyosarcoma (RMS) have a poor prognosis despite intensive chemotherapy and autologous stem cell rescue, with 5-year survival rates ranging from 5 to 35 %. Development of new, additional treatment modalities is necessary to improve the survival rate. In this preclinical study, we investigated the potential of resting and cytokine-activated natural killer (NK) cells to lyse RMS cell lines, as well as the pathways involved, to explore the eventual clinical application of (activated) NK cell immunotherapy. RMS cell lines (n = 3 derived from embryonal RMS and n = 2 derived from alveolar RMS) were susceptible to cytolysis mediated by resting NK cells, and this susceptibility was significantly increased using IL-15-activated NK cells. Flow cytometry and cytolytic assays were used to define the activating and inhibitory pathways of NK cells involved in recognizing and lysing RMS cells. NKG2D and DNAM-1 receptor-ligand interactions were essential in cytolysis by resting NK cells, as simultaneous blocking of both pathways resulted in almost complete abrogation of the cytotoxicity. In contrast, combined blocking of DNAM-1 and NKG2D only led to partial reduction of the lytic activity of IL-15-activated NK cells. In this respect, residual lysis was, at least partly, mediated by pathways involving the natural cytotoxicity receptors NKp30 and NKp46. These findings support further exploration of NK cell-based immunotherapy as adjuvant modality in current treatment strategies of RMS.
Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , Citotoxicidade Imunológica , Interleucina-15/imunologia , Células Matadoras Naturais/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Rabdomiossarcoma/terapia , Linhagem Celular Tumoral , Citocinas/imunologia , Antígenos de Histocompatibilidade Classe I/biossíntese , Humanos , Ativação Linfocitária/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Receptor 1 Desencadeador da Citotoxicidade Natural/imunologia , Receptor 3 Desencadeador da Citotoxicidade Natural/imunologia , Rabdomiossarcoma/imunologiaRESUMO
Human cytomegalovirus (CMV) reactivation frequently occurs during the early phase of immune recovery after allogeneic hematopoietic stem cell transplantation (HSCT). Whereas the recovery of virus-specific immunity in the early phase after HSCT is extensively studied, the impact of CMV on the reconstitution and composition of the T cell compartment long-term after HSCT is unknown. We analyzed T cell reconstitution 1 to 2 years after HSCT in 131 pediatric patients. One year after HSCT, patients with early CMV reactivation (n = 46) had 3-fold higher CD8(+) T cell numbers (median, 1323 versus 424 cells/µL; P < .0001) compared with patients without CMV reactivation (n = 85). This effect, caused by a major expansion of CD8(+) effector memory (EM) and end-stage effector (EMRA) T cells, was independent of pretransplantation donor and recipient CMV serostatus and not seen after Epstein-Barr virus or adenovirus reactivations. At 1 and 2 years after HSCT, the absolute numbers of CD8(+) naive and central memory T cells, as well as CD4(+) naive, CM, EM, and EMRA T cells, did not differ between patients with or without CMV reactivation. In the second year after HSCT, a significant contraction of the initially expanded CD8(+) EM and EMRA T cell compartments was observed in patients with early CMV reactivation. In conclusion, CMV reactivation early after pediatric HSCT leaves a specific and dynamic imprint on the size and composition of the CD8(+) T cell compartment without compromising the reconstitution of CD8(+) and CD4(+) naive and central memory T cells pivotal in the response to neo and recall antigens.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citomegalovirus/fisiologia , Transplante de Células-Tronco Hematopoéticas , Herpesvirus Humano 4/fisiologia , Adolescente , Adulto , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/virologia , Criança , Pré-Escolar , Infecções por Citomegalovirus/complicações , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/terapia , Infecções por Citomegalovirus/virologia , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/terapia , Infecções por Vírus Epstein-Barr/virologia , Feminino , Doenças Hematológicas/complicações , Doenças Hematológicas/imunologia , Doenças Hematológicas/terapia , Doenças Hematológicas/virologia , Humanos , Memória Imunológica , Lactente , Estudos Longitudinais , Contagem de Linfócitos , Masculino , Fatores de Tempo , Transplante Homólogo , Ativação ViralRESUMO
Autosomal-recessive immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is mainly characterized by recurrent, often fatal, respiratory and gastrointestinal infections. About 50% of patients carry mutations in the DNA methyltransferase 3B gene (DNMT3B) (ICF1). The remaining patients carry unknown genetic defects (ICF2) but share with ICF1 patients the same immunological and epigenetic features, including hypomethylation of juxtacentromeric repeat sequences. We performed homozygosity mapping in five unrelated ICF2 patients with consanguineous parents and then performed whole-exome sequencing in one of these patients and Sanger sequencing in all to identify mutations in the zinc-finger- and BTB (bric-a-bric, tramtrack, broad complex)-domain-containing 24 (ZBTB24) gene in four consanguineously descended ICF2 patients. Additionally, we found ZBTB24 mutations in an affected sibling pair and in one patient for whom it was not known whether his parents were consanguineous. ZBTB24 belongs to a large family of transcriptional repressors that include members, such as BCL6 and PATZ1, with prominent regulatory roles in hematopoietic development and malignancy. These data thus indicate that ZBTB24 is involved in DNA methylation of juxtacentromeric DNA and in B cell development and/or B and T cell interactions. Because ZBTB24 is a putative DNA-binding protein highly expressed in the lymphoid lineage, we predict that by studying the molecular function of ZBTB24, we will improve our understanding of the molecular pathophysiology of ICF syndrome and of lymphocyte biology in general.
Assuntos
Centrômero/genética , Metilação de DNA/genética , Proteínas Repressoras/genética , Dedos de Zinco , Adolescente , Adulto , Criança , Pré-Escolar , Análise Mutacional de DNA , Epigenômica , Face/anormalidades , Feminino , Humanos , Síndromes de Imunodeficiência/genética , Masculino , Mutação , Linhagem , Doenças da Imunodeficiência PrimáriaRESUMO
Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for various hematological, immunological and metabolic diseases, replacing the patient's hematopoietic system with donor-derived healthy hematopoietic stem cells. HSCT can be complicated by early and late events related to impaired immunological recovery such as prolonged hypogammaglobulinemia post-HSCT. We present a 16-year-old female patient with sickle-cell disease who underwent HSCT with stem cells from a human leukocyte antigen (HLA) class-II mismatched family donor. While cellular recovery was good post-HSCT, the patient developed mixed chimerism and suffered from cervical lymphadenopathy, recurrent airway infections and cutaneous SLE. She presented with hypogammaglobulinemia and was started on immunoglobulin substitution therapy and antibiotic prophylaxis. B-cell phenotyping showed that she had increased transitional and naïve mature B cells, reduced memory B cells, and diminished marginal zone/natural effector cells. In-depth immunophenotyping and B-cell receptor repertoire sequencing ruled out an intrinsic B-cell defect by expression of activation-induced cytidine deaminase (AID), presence of somatic hypermutations and differentiation into IgG- and IgA-producing plasma cells in vitro. Immunohistochemistry and flow cytometry of lymph node tissue showed a clear block in terminal B-cell differentiation. Chimerism analysis of sorted lymph node populations showed that exclusively patient-derived B cells populated germinal centers, while only a minor fraction of follicular helper T cells was patient-derived. Given this discrepancy, we deduced that the HLA class-II disparity between patient and donor likely hinders terminal B-cell differentiation in the lymph node. This case highlights that studying disturbed cognate T-B interactions in the secondary lymphoid organs can provide unique insights when deciphering prolonged hypogammaglobulinemia post-HSCT.
Assuntos
Agamaglobulinemia , Transplante de Células-Tronco Hematopoéticas , Humanos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Feminino , Agamaglobulinemia/imunologia , Agamaglobulinemia/terapia , Adolescente , Anemia Falciforme/terapia , Anemia Falciforme/imunologia , Linfócitos B/imunologia , Quimeras de Transplante , Antígenos HLA/imunologia , Antígenos HLA/genéticaRESUMO
Osteosarcoma and Ewing's sarcoma tumor cells are susceptible to IL15-induced or antibody-mediated cytolytic activity of NK cells in short-term cytotoxicity assays. When encountering the tumor environment in vivo, NK cells may be in contact with tumor cells for a prolonged time period. We explored whether a prolonged interaction with sarcoma cells can modulate the activation and cytotoxic activity of NK cells. The 40 h coculture of NK cells with sarcoma cells reversibly interfered with the IL15-induced expression of NKG2D, DNAM-1 and NKp30 and inhibited the cytolytic activity of NK cells. The inhibitory effects on receptor expression required physical contact between NK cells and sarcoma cells and were independent of TGF-ß. Five days pre-incubation of NK cells with IL15 prevented the down-regulation of NKG2D and cytolytic activity in subsequent cocultures with sarcoma cells. NK cell FcγRIIIa/CD16 receptor expression and antibody-mediated cytotoxicity were not affected after the coculture. Inhibition of NK cell cytotoxicity was directly linked to the down-regulation of the respective NK cell-activating receptors. Our data demonstrate that the inhibitory effects of sarcoma cells on the cytolytic activity of NK cells do not affect the antibody-dependent cytotoxicity and can be prevented by pre-activation of NK cells with IL15. Thus, the combination of cytokine-activated NK cells and monoclonal antibody therapy may be required to improve tumor targeting and NK cell functionality in the tumor environment.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Citotoxicidade Imunológica , Interleucina-15/imunologia , Células Matadoras Naturais/imunologia , Osteossarcoma/imunologia , Sarcoma de Ewing/imunologia , Antígenos de Diferenciação de Linfócitos T/biossíntese , Linhagem Celular Tumoral , Técnicas de Cocultura , Humanos , Células Matadoras Naturais/metabolismo , Ativação Linfocitária , Subfamília K de Receptores Semelhantes a Lectina de Células NK/biossíntese , Receptor 3 Desencadeador da Citotoxicidade Natural/biossíntese , Receptores de IgG/biossíntese , Receptores de Células Matadoras Naturais , Fator de Crescimento Transformador beta/imunologiaRESUMO
BACKGROUND AIMS: Infusion of mesenchymal stromal cells (MSCs) has been reported to be an effective treatment modality for acute graft-versus-host disease, and MSCs have been considered for use in the treatment of patients with autoimmune diseases. Before contemplating clinical studies with MSCs in patients with systemic juvenile idiopathic arthritis (sJIA), the immunomodulatory capacity of MSCs in this setting needs to be explored. A comparative analysis of bone marrow-derived MSCs from children with sJIA and healthy pediatric controls was performed. METHODS: MSCs were successfully expanded from 11 patients with sJIA and 10 controls. The phenotype, differentiation and immunomodulatory capacity of these MSCs were compared. The effect of immunosuppressive drugs on MSC function was also investigated. RESULTS: MSCs from patients with sJIA and controls showed no differences in their suppressive effect using control peripheral blood mononuclear cells. Furthermore, the suppression of the response of peripheral blood mononuclear cells from patients with sJIA by autologous sJIA MSCs and allogeneic control MSCs was comparable. The immunosuppressive effect of both groups of MSCs was diminished in the presence of indomethacin (P < 0.05). MSCs from patients with sJIA and controls suppressed interleukin-2-induced natural killer cell activation to a similar extent. In addition, MSCs of patients with sJIA and controls inhibited the differentiation of monocytes to dendritic cells. CONCLUSIONS: This is the first explorative study in a significant cohort of patients with sJIA to evaluate the effect of MSCs on adaptive and innate immune responses. The comparable immunosuppressive characteristics of MSCs derived from patients with sJIA to age-matched controls support the potential use of patient-derived MSCs in the treatment of sJIA.
Assuntos
Imunidade Adaptativa , Imunidade Inata , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Artrite Juvenil/metabolismo , Artrite Juvenil/fisiopatologia , Criança , Pré-Escolar , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Feminino , Humanos , Terapia de Imunossupressão , Indometacina/administração & dosagem , Lactente , Recém-Nascido , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Masculino , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Human natural killer (NK) cells in lymphoid tissues can be categorized into three subsets: CD56brightCD16+, CD56dimCD16+ and CD69+CXCR6+ lymphoid tissue-resident (lt)NK cells. How the three subsets are functionally and developmentally related is currently unknown. Therefore, we performed single-cell RNA sequencing combined with oligonucleotide-conjugated antibodies against CD56, CXCR6, CD117 and CD34 on fresh bone marrow NK cells. A minor CD56dimGzmK+ subset was identified that shared features with CD56bright and CD56dimGzmK- NK cells based on transcriptome, phenotype (NKG2AhighCD16lowKLRG1highTIGIThigh) and functional analysis in bone marrow and blood, supportive for an intermediate subset. Pseudotime analysis positioned CD56bright, CD56dimGzmK+ and CD56dimGzmK- cells in one differentiation trajectory, while ltNK cells were developmentally separated. Integrative analysis with bone marrow cells from the Human Cell Atlas did not demonstrate a developmental connection between CD34+ progenitor and NK cells, suggesting absence of early NK cell stages in bone marrow. In conclusion, single-cell transcriptomics provide new insights on development and differentiation of human NK cells.