RESUMEN
Metachromatic Leukodystrophy (MLD; MIM# 250100) is a rare inherited lysosomal storage disorder caused by the deficiency of Arylsulfatase A (ARSA). The enzymatic defect results in the accumulation of the ARSA substrate that is particularly relevant in myelin forming cells and leads to progressive dysmyelination and dysfunction of the central and peripheral nervous system. Sulfatide accumulation has also been reported in various visceral organs, although little is known about the potential clinical consequences of such accumulation. Different forms of MLD-associated gallbladder disease have been described, and there is one reported case of an MLD patient presenting with functional consequences of sulfatide accumulation in the kidney. Here we describe a wide cohort of MLD patients in whom a tendency to sub-clinical metabolic acidosis was observed. Furthermore in some of them we report episodes of metabolic acidosis of different grades of severity developed in acute clinical conditions of various origin. Importantly, we finally show how a careful acid-base balance monitoring and prompt correction of imbalances might prevent severe consequences of acidosis.
Asunto(s)
Acidosis/complicaciones , Leucodistrofia Metacromática/complicaciones , Leucodistrofia Metacromática/metabolismo , Monitoreo Fisiológico , Equilibrio Ácido-Base , Desequilibrio Ácido-Base , Acidosis/sangre , Acidosis/prevención & control , Acidosis/orina , Niño , Preescolar , Estudios de Cohortes , Estudios de Seguimiento , Genotipo , Humanos , Lactante , Estudios Retrospectivos , Factores de TiempoAsunto(s)
Alergia e Inmunología/historia , Trastornos Linfoproliferativos/genética , Mutación/genética , Proteína Asociada a la Molécula de Señalización de la Activación Linfocitaria/genética , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria/metabolismo , Linfocitos T/inmunología , Adulto , Animales , Clonación Molecular , Historia del Siglo XX , Humanos , Células Jurkat , Activación de Linfocitos , Trastornos Linfoproliferativos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Alineación de Secuencia , Proteína Asociada a la Molécula de Señalización de la Activación Linfocitaria/metabolismo , Adulto JovenRESUMEN
The immune system is comprised of several CD4(+) T regulatory (Treg) cell types, of which two, the Foxp3(+) Treg and T regulatory type 1 (Tr1) cells, have frequently been associated with transplant tolerance. However, whether and how these two Treg-cell types synergize to promote allograft tolerance remains unknown. We previously developed a mouse model of allogeneic transplantation in which a specific immunomodulatory treatment leads to transplant tolerance through both Foxp3(+) Treg and Tr1 cells. Here, we show that Foxp3(+) Treg cells exert their regulatory function within the allograft and initiate engraftment locally and in a non-antigen (Ag) specific manner. Whereas CD4(+) CD25(-) T cells, which contain Tr1 cells, act from the spleen and are key to the maintenance of long-term tolerance. Importantly, the role of Foxp3(+) Treg and Tr1 cells is not redundant once they are simultaneously expanded/induced in the same host. Moreover, our data show that long-term tolerance induced by Foxp3(+) Treg-cell transfer is sustained by splenic Tr1 cells and functionally moves from the allograft to the spleen.
Asunto(s)
Trasplante de Islotes Pancreáticos , Islotes Pancreáticos/inmunología , Bazo/inmunología , Linfocitos T Reguladores/inmunología , Tolerancia al Trasplante/inmunología , Traslado Adoptivo , Animales , Anticuerpos Monoclonales/inmunología , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Supervivencia de Injerto , Islotes Pancreáticos/metabolismo , Antígenos Comunes de Leucocito/inmunología , Antígenos Comunes de Leucocito/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Bazo/metabolismo , Linfocitos T Reguladores/metabolismo , Trasplante HomólogoRESUMEN
Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency caused by the defective expression of the WAS protein (WASP) in hematopoietic cells. It has been shown that dendritic cells (DCs) are functionally impaired in WAS patients and was(-/-) mice. We have previously demonstrated the efficacy and safety of a murine model of WAS gene therapy (GT), using stem cells transduced with a lentiviral vector (LV). The aim of this study was to investigate whether GT can correct DC defects in was(-/-) mice. As DCs expressing WASP were detected in the secondary lymphoid organs of the treated mice, we tested the in vitro and in vivo function of bone marrow-derived DCs (BMDCs). The BMDCs showed efficient in vitro uptake of latex beads and Salmonella typhimurium. When BMDCs from the treated mice (GT BMDCs) and the was(-/-) mice were injected into wild-type hosts, we found a higher number of cells that had migrated to the draining lymph nodes compared with mice injected with was(-/-) BMDCs. Finally, we found that ovalbumin (OVA)-pulsed GT BMDCs or vaccination of GT mice with anti-DEC205 OVA fusion protein can efficiently induce antigen-specific T-cell activation in vivo. These findings show that WAS GT significantly improves DC function, thus adding new evidence of the preclinical efficacy of LV-mediated WAS GT.
Asunto(s)
Células Dendríticas/inmunología , Terapia Genética , Lentivirus/genética , Proteína del Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Animales , Células de la Médula Ósea/inmunología , Movimiento Celular , Células Dendríticas/metabolismo , Humanos , Tejido Linfoide/metabolismo , Ratones , Modelos Genéticos , Fagocitosis , Transducción Genética , Proteína del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
Polymorphisms in the 3' untranslated region (3'UTR) of HLA-G, an important player in immunological tolerance, could be involved in post-transcriptional expression control, and their association with different clinical immune-related conditions including autoimmunity and transplantation is of mounting interest. Most studies have focused on a 14 base pair (bp) insertion/deletion (ins/del), while additional single-nucleotide polymorphisms (SNPs) in the HLA-G 3'UTR have been described but not extensively investigated for their clinical relevance. Here we have comparatively studied the association between 3'UTR haplotypes of HLA-G, or the 14 bp ins/del, with clinical outcome of HLA-identical sibling hematopoietic stem cell transplantation (HSCT) in 147 Middle Eastern beta-thalassemia patients. Sequence based typing of 3'UTR HLA-G polymorphisms in the patients and in 102 healthy Italian blood donors showed strong linkage disequilibrium between the 14 bp ins/del and five 3'UTR SNPs, which together could be arranged into eight distinct haplotypes based on expectation-maximization studies, with four predominant haplotypes (UTRs1-4). After HSCT, we found a moderate though not significant association between the presence of UTR-2 in double dose and protection from acute graft versus host disease (hazard ratio (HR) 0.45, 95% confidence intervals (CI): 0.14-1.45; P = 0.18), an effect that was also seen when the corresponding 14 bp ins/ins genotype was considered alone (HR 0.42, 95% CI: 0.16-1.06; P = 0.07). No association was found with rejection or survival. Taken together, our data show that there is no apparent added value of considering entire 3'UTR HLA-G haplotypes for risk prediction after allogeneic HSCT for beta-thalassemia.
Asunto(s)
Regiones no Traducidas 3'/genética , Enfermedad Injerto contra Huésped/genética , Antígenos HLA-G/genética , Trasplante de Células Madre Hematopoyéticas , Talasemia beta/genética , Regiones no Traducidas 3'/inmunología , Adolescente , Adulto , Estudios de Casos y Controles , Niño , Preescolar , Femenino , Genotipo , Enfermedad Injerto contra Huésped/diagnóstico , Enfermedad Injerto contra Huésped/inmunología , Haplotipos/genética , Haplotipos/inmunología , Humanos , Tolerancia Inmunológica , Italia , Desequilibrio de Ligamiento , Masculino , Mutagénesis Insercional , Polimorfismo Genético , Eliminación de Secuencia , Hermanos , Trasplante Homólogo , Resultado del Tratamiento , Talasemia beta/inmunología , Talasemia beta/terapiaRESUMEN
Immune dysregulation, Polyendocrinopathy, Enteropathy X-linked (IPEX) syndrome is a unique example of primary immunodeficiency characterized by autoimmune manifestations due to defective regulatory T (Treg) cells, in the presence of FOXP3 mutations. However, autoimmune symptoms phenotypically resembling IPEX often occur in the absence of detectable FOXP3 mutations. The cause of this "IPEX-like" syndrome presently remains unclear. To investigate whether a defect in Treg cells sustains the immunological dysregulation in IPEX-like patients, we measured the amount of peripheral Treg cells within the CD3(+) T cells by analysing demethylation of the Treg cell-Specific-Demethylated-Region (TSDR) in the FOXP3 locus and demethylation of the T cell-Specific-Demethylated-Region (TLSDR) in the CD3 locus, highly specific markers for stable Treg cells and overall T cells, respectively. TSDR demethylation analysis, alone or normalized for the total T cells, showed that the amount of peripheral Treg cells in a cohort of IPEX-like patients was significantly reduced, as compared to both healthy subjects and unrelated disease controls. This reduction could not be displayed by flow cytometric analysis, showing highly variable percentages of FOXP3(+) and CD25(+)FOXP3(+) T cells. These data provide evidence that a quantitative defect of Treg cells could be considered a common biological hallmark of IPEX-like syndrome. Since Treg cell suppressive function was not impaired, we propose that this reduction per se could sustain autoimmunity.
Asunto(s)
Metilación de ADN , Factores de Transcripción Forkhead/genética , Poliendocrinopatías Autoinmunes/genética , Poliendocrinopatías Autoinmunes/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Adolescente , Adulto , Complejo CD3/inmunología , Complejo CD3/metabolismo , Niño , Preescolar , Estudios de Cohortes , Femenino , Citometría de Flujo , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Enfermedades Genéticas Ligadas al Cromosoma X/inmunología , Humanos , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunología , Lactante , Masculino , Síndrome , Adulto JovenRESUMEN
Active suppression by T regulatory (Tr) cells plays an important role in the downregulation of T cell responses to foreign and self-antigens. Mouse CD4(+) Tr cells that express CD25 possess remarkable suppressive activity in vitro and in autoimmune disease models in vivo. Thus far, the existence of a similar subset of CD25(+)CD4(+) Tr cells in humans has not been reported. Here we show that human CD25(+)CD4(+) Tr cells isolated from peripheral blood failed to proliferate and displayed reduced expression of CD40 ligand (CD40L), in response to T cell receptor-mediated polyclonal activation, but strongly upregulated cytotoxic T lymphocyte-associated antigen (CTLA)-4. Human CD25(+)CD4(+) Tr cells also did not proliferate in response to allogeneic antigen-presenting cells, but they produced interleukin (IL)-10, transforming growth factor (TGF)-beta, low levels of interferon (IFN)-gamma, and no IL-4 or IL-2. Importantly, CD25(+)CD4(+) Tr cells strongly inhibited the proliferative responses of both naive and memory CD4(+) T cells to alloantigens, but neither IL-10, TGF-beta, nor CTLA-4 seemed to be directly required for their suppressive effects. CD25(+)CD4(+) Tr cells could be expanded in vitro in the presence of IL-2 and allogeneic feeder cells and maintained their suppressive capacities. These findings that CD25(+)CD4(+) Tr cells with immunosuppressive effects can be isolated from peripheral blood and expanded in vitro without loss of function represent a major advance towards the therapeutic use of these cells in T cell-mediated diseases.
Asunto(s)
Antígenos CD4/análisis , Inmunoconjugados , Activación de Linfocitos , Receptores de Interleucina-2/análisis , Linfocitos T Reguladores/fisiología , Linfocitos T/inmunología , Abatacept , Antígenos CD , Antígenos de Diferenciación/análisis , Antígeno CTLA-4 , Línea Celular , Citocinas/biosíntesis , Humanos , Inmunofenotipificación , Interleucina-10/biosíntesis , Isoantígenos/inmunología , Factor de Crecimiento Transformador beta/biosíntesisRESUMEN
To study the role of thymic education on the development of the human T cell repertoire, SCID-hu mice were constructed with fetal liver and fetal thymus obtained from the same or two different donors. These animals were studied between 7 and 12 mo after transplantation, at which times all thymocytes and peripheral T cells were derived from stem cells of the fetal liver graft. Immunohistology of the thymus grafts demonstrated that thymic epithelial cells were of fetal thymus donor (FTD) origin. Dendritic cells and macrophages of fetal liver donor (FLD) origin were abundantly present in the medullary and cortico-medullary areas. Thymocytes of SCID-hu mice transplanted with liver and thymus of two different donors (FLDA/FTDB animals) were nonresponsive to Epstein-Barr virus-transformed B cell lines (B-LCL) established from both the FLDA and FTDB, but proliferated vigorously when stimulated with third-party allogeneic B-LCL. Mixing experiments showed that the nonresponsiveness to FTDB was not due to suppression. Limiting dilution analysis revealed that T cells reacting with the human histocompatibility leukocyte antigens (HLA) of the FLD were undetectable in the CD8+ T cell population and barely measurable in the CD4+ subset. On the other hand, CD4+ and CD8+ T cells reactive to the HLA antigens of the FTD were readily detectable. These results indicate that FLD-reactive cells were clonally deleted, whereas FTD-reactive cells were not. However, the frequencies of FTD-reactive T cells were consistently twofold lower than those of T cells specific for any third-party B-LCL. In addition, the cytotoxic activity and interleukin 2 production by FTD-specific T cells were lower compared with that of third-party-reactive T cell clones, suggesting that FTD-specific cells are anergic. These data demonstrate that T cells become tolerant to autologous and allogeneic HLA antigens expressed in the thymus via two different mechanisms: hematopoietic cells present in the thymus induce tolerance to "self"-antigens by clonal deletion, whereas thymic epithelial cells induce tolerance by clonal energy and possibly deletion of high affinity clones.
Asunto(s)
Células Madre Hematopoyéticas/inmunología , Tolerancia Inmunológica , Linfocitos T/fisiología , Timo/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Quimera , Relación Dosis-Respuesta Inmunológica , Epitelio/inmunología , Antígenos HLA-D/inmunología , Humanos , Interleucina-2/biosíntesis , Hígado/embriología , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones SCID , Linfocitos T/citología , Linfocitos T Citotóxicos/inmunología , Timo/citología , Timo/embriologíaRESUMEN
Human CD4+ T cells, activated by allogeneic monocytes in a primary mixed lymphocyte reaction in the presence of exogenous interleukin (IL) 10, specifically failed to proliferate after restimulation with the same alloantigens. A comparable state of T cell unresponsiveness could be induced by activation of CD4+ T cells by cross-linked anti-CD3 monoclonal antibodies (mAbs) in the presence of exogenous IL-10. The anergic T cells failed to produce IL-2, IL-5, IL-10, interferon gamma, tumor necrosis factor alpha, and granulocyte/macrophage colony-stimulating factor. The IL-10-induced anergic state was long-lasting. T cell anergy could not be reversed after restimulation of the cells with anti-CD3 and anti-CD28 mAbs, although CD3 and CD28 expression was normal. In addition, restimulation of anergized T cells with anti-CD3 mAbs induced normal Ca2+ fluxes and resulted in increased CD3, CD28, and class II major histocompatibility complex expression, indicating that calcineurin-mediated signaling occurs in these anergic cells. However, the expression of the IL-2 receptor alpha chain was not upregulated, which may account for the failure of exogenous IL-2 to reverse the anergic state. Interestingly, anergic T cells and their nonanergic counterparts showed comparable levels of proliferation and cytokine production after activation with phorbol myristate acetate and Ca2+ ionophore, indicating that a direct activation of a protein kinase C-dependent pathway can overcome the tolerizing effect of IL-10. Taken together, these data demonstrate that IL-10 induces T cell anergy and therefore may play an important role in the induction and maintenance of antigen-specific T cell tolerance.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Anergia Clonal , Interleucina-10/fisiología , Antígenos CD28/inmunología , Calcio/fisiología , Células Cultivadas , Citocinas/metabolismo , Humanos , Interleucina-2/inmunología , Prueba de Cultivo Mixto de Linfocitos , Monocitos/inmunología , Receptores de Antígenos de Linfocitos T/fisiología , Receptores de Interleucina-2/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
The phenotypic similarities between natural killer (NK) and T cells have led to the hypothesis that these distinctive lymphocyte subsets may be developmentally related and thus may share a common progenitor (Lanier, L. L., H. Spits, and J. H. Phillips, 1992. Immunol. Today. 13:392; Rodewald, H.-R., P. Moingeon, J. L. Lurich, C. Dosiou, P. Lopez, and E. L. Reinherz. 1992. Cell. 69:139). In this report, we have investigated the potential of human CD34+ triple negative thymocytes ([TN] CD3-, CD4-, CD8-) to generate both T cells and NK cells in murine fetal thymic organ cultures (mFTOC) and in vitro clonogenic assays. CD34+ TN thymocytes, the majority of which express prominent cytoplasmic CD3 epsilon (cytoCD3 epsilon) protein, can be divided into high (CD34Bright) and low (CD34Dim) surface expressing populations. CD34Bright TN thymocytes were capable of differentiating into T and NK cells when transferred into mFTOC, and demonstrated high NK cell clonogenic capabilities when cultured in interleukin (IL)-2, IL-7, and stem cell factor (SCF). Likewise, CD34Bright TN thymocyte clones after 5 d in culture were capable of generating NK and T cells when transferred into mFTOC but demonstrated clonogenic NK cell differentiation capabilities when maintained in culture with IL-2. CD34Dim TN thymocytes, however, possessed only T cell differentiation capabilities in mFTOC but were not expandable in clonogenic conditions containing IL-2, IL-7, and SCF. No significant differentiation of other cell lineage was detected in either mFTOC or in clonogenic assays from CD34+ TN thymocytes. These results represent the first definitive evidence of a common T/NK cell progenitor in the human fetal thymus and delineate the point in thymocyte differentiation where T and NK cells diverge.
Asunto(s)
Células Madre Hematopoyéticas , Células Asesinas Naturales , Linfocitos T/inmunología , Timo/citología , Animales , Antígenos CD/inmunología , Antígenos CD34 , Diferenciación Celular , Separación Celular , Citometría de Flujo , Células Madre Hematopoyéticas/inmunología , Humanos , Interleucina-2/inmunología , Células Asesinas Naturales/inmunología , Ratones , Fenotipo , Timo/embriologíaRESUMEN
Injecting human peripheral blood mononuclear cells into severe combined immunodeficient (SCID) mice results in long-term engraftment of human lymphocytes, of which > 98% are phenotypically mature, activated T cells. Here we have characterized the human T cells that populate such hu-PBL-SCID chimeras. We report that these human T cells do not mobilize Ca2+ after CD3 stimulation, i.e., their T cell receptor (TCR)-mediated signal transduction is deficient. Chimera-derived human T cells do not secrete lymphokines or undergo blastogenesis after CD3 stimulation, but proliferate in response to interleukin 2 (IL-2), defining the chimera derived human T cells as anergic. Anergy was seen in both the CD4+ and the CD8+ subpopulations. We established human T cell lines from chimeras. These T cells retained their anergic state for 1-2 mo in culture, after which they simultaneously regained the ability to mobilize Ca2+, secrete lymphokines, and to undergo blastogenesis following stimulation via the TCR. Once regaining proliferative responsiveness to CD3 stimulation, these CD4+ T cell lines displayed anti-SCID mouse reactivity and showed no specificity for recall antigens. All CD3-responsive CD4+ T cell clones obtained from such lines were SCID mouse specific, recognizing native major histocompatibility complex class II products on the murine cells. In contrast, chimera-derived human CD8+ cell lines and clones did not display detectable anti-mouse reactivity. The data show that the human T cell system in long term hu-PBL-SCID chimeras is nonfunctional due to both anergy and the limitation of the CD4+ repertoire to xenoreactive clones. The data suggest that long-term hu-PBL-SCID chimerism represents an atypical graft-versus-host reaction in which the human effector T cells become anergic in the murine environment.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Quimera/inmunología , Ratones SCID/inmunología , Adulto , Animales , Complejo CD3/inmunología , Linfocitos T CD8-positivos/inmunología , Calcio/metabolismo , Línea Celular , Citocinas/biosíntesis , Femenino , Reacción Injerto-Huésped , Humanos , Interleucina-2/farmacología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana EdadRESUMEN
T cell clones of donor origin that specifically react with recipient cells were obtained from a SCID patient successfully reconstituted by allogeneic fetal liver and thymus transplantation performed 10 yr ago. The majority of these clones displayed both cytotoxic and proliferative responses towards PBL and an EBV-transformed B cell line derived from the patient. In addition, these T cell clones had proliferative and cytotoxic responses towards the parental PBL, EBV cell lines, and PHA blasts. Blocking studies with anti-class I and anti-class II HLA mAbs indicated that the activity of the CD4+ T cell clones was specifically directed against class II HLA antigens of the recipient. On the other hand, the cytotoxic and proliferative responses of the CD8+ T cell clones were specific for class I HLA antigens which are ubiquitously expressed on the recipient cells. Thus, the establishment of transplantation tolerance observed in this stable human chimera is not due to the elimination of host-reactive T cells from the repertoire and suggests the presence of a peripheral autoregulatory suppressor mechanism.
Asunto(s)
Antígenos HLA/inmunología , Tolerancia Inmunológica , Linfocitos T/inmunología , Niño , Quimera , Citotoxicidad Inmunológica , Supervivencia de Injerto , Antígenos HLA-D/inmunología , Humanos , Síndromes de Inmunodeficiencia/terapia , Hígado/embriología , Trasplante de Hígado , Activación de Linfocitos , Masculino , Linfocitos T/trasplante , Timo/embriología , Timo/trasplanteRESUMEN
Tetanus toxin (TT)-specific T cell clones of donor origin were obtained from a patient with severe combined immunodeficiency (SCID) successfully reconstituted by transplantation of allogeneic fetal liver and thymus cells from two different donors performed 10 yr ago. A series of these clones recognized TT in the context of "allo" class II HLA determinants expressed by recipient APC. The restriction element of two T cell clones with the HLA phenotype of the first donor (HLA-DR1,8) and one T cell clone with the HLA phenotype of the second transplant (HLA-DR3,9) was HLA-DR4 of the recipient, whereas other T cell clones derived from the second transplant recognized TT in the context of HLA-DR5 of the recipient's APC. These latter T cell clones were not able to proliferate in response to TT when autologous APC were used. These data demonstrate that recipient and donor cells having different HLA phenotypes could cooperate across the allogeneic barrier and that MHC restriction of antigen (Ag) recognition is independent from the MHC genotype of the T cells but is influenced by the environment in which the T cells mature. We also isolated T cell clones that were able to recognize processed TT presented by all allogeneic EBV cell lines tested, indicating that the Ag specificity of these clones was not restricted by a particular class II MHC molecule. The Ag-specific proliferative response of one of these clones could be blocked by anti-class II MHC mAbs. These results demonstrate that in addition to Ag recognition in the context of specific class II MHC Ags, other types of Ag-specific responses may occur in this human chimera. It is not clear whether this "allo" plus Ag recognition is the result of education of transplanted fetal cells in the host thymus. Taking into consideration our previous findings indicating that alloreactive T cell clones specific for the recipient cells could be isolated in vitro from the PBL of the same patient, our data suggest that the mechanism for deletion of self-reactive clones and the generation of MHC-restricted responses are different.
Asunto(s)
Quimera , Antígenos HLA/inmunología , Linfocitos T/inmunología , Anticuerpos Monoclonales , Línea Celular , Células Clonales , Epítopos , Antígenos de Histocompatibilidad Clase II/inmunología , Prueba de Histocompatibilidad , Humanos , Síndromes de Inmunodeficiencia/inmunología , Trasplante de Hígado , Activación de Linfocitos , Toxina Tetánica/inmunología , Timo/trasplanteRESUMEN
In this article, we report that the human fetal thymus contains CD34bright cells (< 0.01% of total thymocytes) with a phenotype that resembles that of multipotent hematopoietic progenitors in the fetal bone marrow. CD34bright thymocytes were CD33-/dull and were negative for CD38, CD2, and CD5 as well as for the lineage markers CD3, CD4, and CD8 (T cells), CD19 and CD20 (B cells), CD56 (NK cells), glycophorin (erythrocytes), and CD14 (monocytes). In addition, total CD34+ lineage negative (lin-) thymocytes contained a low number of primitive myeloid progenitor cells, thus suggesting that the different hematopoietic lineages present in the thymus may be derived from primitive hematopoietic progenitor cells seeding the thymus. To investigate whether the thymus is permissive for the development of non-T cells, human fetal organ culture (FTOC) assays were performed by microinjecting sorted CD34+lin- fetal liver cells into fragments of HLA-mismatched fetal thymus. Sequential phenotypic analysis of the FTOC-derived progeny of CD34+lin- cells indicated that the differentiation into T cells was preceded by a wave of myeloid differentiation into CD14+CD11b+CD4dull cells. Donor-derived B cells (CD19+CD20+) were also generated, which produced immunoglobulins (IgG and IgM) when cultured under appropriate conditions, as well as functional CD56+CD3- NK cells, which efficiently killed K562 target cells in cytotoxicity assays. These results demonstrate that the microinjection of fetal liver hematopoietic progenitors into fetal thymic organ fragments results in multilineage differentiation in vitro.
Asunto(s)
Antígenos CD/análisis , Feto/inmunología , Células Madre Hematopoyéticas/inmunología , Linfocitos T/inmunología , Timo/citología , Antígenos CD34 , Linfocitos B/fisiología , Diferenciación Celular , Células Cultivadas , Femenino , Humanos , Hígado/inmunología , Técnicas de Cultivo de Órganos , EmbarazoRESUMEN
Implantation of pieces of human fetal liver and thymus into SCID mice results in the development of a human thymus-like organ, in which sustained lymphopoiesis is reproducibly observed. In this model, T cell development can be experimentally manipulated. To study the influence of thymic selection on the development of the human T cell repertoire, the T cell receptor (TCR) V beta gene repertoire of double-positive (CD4+CD8+) and single-positive (CD4+CD8- and CD4-CD8+) T cells was analyzed in the SCID-hu thymus using a multiprobe ribonuclease protection assay. TCR diversity in double-positive SCID-hu thymocytes was found to be comparable with that present in the thymus of the fetal liver donor, did not change with time, and was independent of the origin of the thymus donor. Thymic selection in SCID-hu thymus induces changes in V beta usage by the single-positive CD4+ or CD8+ T cells comparable with those previously reported for single-positive cells present in a normal human thymus. Finally, significant differences were observed in the V beta usage by CD4 or CD8 single-positive T cells that matured from genetically identical stem cells in different thymic environments. Collectively, these data suggest: first, that the generation of TCR diversity at the double-positive stage is determined by the genotype of the stem cells; and second, that polymorphic determinants expressed by thymic epithelium measurably influence the V beta repertoire of mature single-positive T cells.
Asunto(s)
Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Timo/inmunología , Alelos , Animales , Antígenos CD4/análisis , Antígenos CD8/análisis , Trasplante de Tejido Fetal/inmunología , Humanos , Trasplante de Hígado/inmunología , Ratones , Ratones SCID , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Timo/trasplante , Transcripción GenéticaRESUMEN
In the present study, it is demonstrated that cloned surface IgM-positive human B cells can be induced to proliferate and to switch with high frequencies to IgG4 and IgE production after a contact-mediated signal provided by T cell clones and interleukin 4 (IL-4). This T cell signal is antigen nonspecific and is provided by activated CD4+ cells, whereas activated CD8+ or resting CD4+ T cell clones are ineffective. 15-35% of the B cell clones cultured with cloned CD4+ T cells and IL-4 produced antibodies; 35-45% of those wells in which antibodies were produced contained IgE and IgG4. In addition to B cell clones that produced IgG4 or IgE only, B cell clones producing multiple isotypes were observed. Simultaneous production of IgG4 and IgE, IgM, IgE, and IgM, or IgG4 and IgE was detected, suggesting that during clonal expansion switching might occur in successive steps from IgM to IgG4 and IgE. In addition, production of only IgM, IgG4, and IgE during clonal expansion indicates that this isotype switching is directed by the way a B cell is stimulated and that it is not a stochastic process.
Asunto(s)
Linfocitos B/inmunología , Interleucina-4/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/inmunología , Anticuerpos Monoclonales , Antígenos CD/análisis , Antígenos CD4/análisis , Células Clonales , Humanos , Inmunoglobulina E/biosíntesis , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/clasificación , Inmunoglobulina M/análisis , Inmunoglobulina M/biosíntesis , Proteínas Recombinantes/farmacología , Transducción de SeñalRESUMEN
The ability to analyze T cell receptor (TCR) thymic repertoire shaping in humans by self and foreign ligands is hampered by the lack of suitable models. We recently documented that the mouse severe combined immunodeficiency (SCID)-human fetal liver/thymus model recapitulates the TCR V beta gene repertoire of human thymocytes. Here, we show that an exogenous superantigen, staphylococcal enterotoxin B, administered to such mice induces clonal deletions in both CD4+8- and CD8+4- cells involving the same human V beta clones that are selected in vitro by this toxin. This model, therefore, may allow comprehensive studies into the effects of microbial and other agents on human T cell thymic selection processes in a biologically relevant setting.
Asunto(s)
Enterotoxinas/farmacología , Hígado/citología , Linfocitos T/citología , Timo/citología , Animales , Diferenciación Celular/efectos de los fármacos , Células Clonales , Humanos , Hígado/embriología , Hígado/inmunología , Ratones , Ratones SCID , Modelos Biológicos , Receptores de Antígenos de Linfocitos T alfa-beta/biosíntesis , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Staphylococcus aureus , Linfocitos T/inmunología , Timo/embriología , Timo/inmunología , Trasplante de TejidosRESUMEN
Interleukin 10 (IL-10) and viral IL-10 (v-IL-10) strongly reduced antigen-specific proliferation of human T cells and CD4+ T cell clones when monocytes were used as antigen-presenting cells. In contrast, IL-10 and v-IL-10 did not affect the proliferative responses to antigens presented by autologous Epstein-Barr virus-lymphoblastoid cell line (EBV-LCL). Inhibition of antigen-specific T cell responses was associated with downregulation of constitutive, as well as interferon gamma- or IL-4-induced, class II MHC expression on monocytes by IL-10 and v-IL-10, resulting in the reduction in antigen-presenting capacity of these cells. In contrast, IL-10 and v-IL-10 had no effect on class II major histocompatibility complex (MHC) expression on EBV-LCL. The reduced antigen-presenting capacity of monocytes correlated with a decreased capacity to mobilize intracellular Ca2+ in the responder T cell clones. The diminished antigen-presenting capacities of monocytes were not due to inhibitory effects of IL-10 and v-IL-10 on antigen processing, since the proliferative T cell responses to antigenic peptides, which did not require processing, were equally well inhibited. Furthermore, the inhibitory effects of IL-10 and v-IL-10 on antigen-specific proliferative T cell responses could not be neutralized by exogenous IL-2 or IL-4. Although IL-10 and v-IL-10 suppressed IL-1 alpha, IL-1 beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 production by monocytes, it was excluded that these cytokines played a role in antigen-specific T cell proliferation, since normal antigen-specific responses were observed in the presence of neutralizing anti-IL-1, -IL-6, and -TNF-alpha mAbs. Furthermore, addition of saturating concentrations of IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha to the cultures had no effect on the reduced proliferative T cell responses in the presence of IL-10, or v-IL-10. Collectively, our data indicate that IL-10 and v-IL-10 can completely prevent antigen-specific T cell proliferation by inhibition of the antigen-presenting capacity of monocytes through downregulation of class II MHC antigens on monocytes.
Asunto(s)
Genes MHC Clase II/efectos de los fármacos , Interleucina-10/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/inmunología , Anticuerpos Monoclonales , Antígenos CD/inmunología , Calcio/metabolismo , Línea Celular Transformada , Células Cultivadas , Clonación Molecular , Citocinas/biosíntesis , Replicación del ADN/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Herpesvirus Humano 4/genética , Humanos , Monocitos/inmunología , Proteínas Recombinantes/farmacología , Linfocitos T/efectos de los fármacosRESUMEN
Transplantation of HLA mismatched hematopoietic stem cells in patients with severe combined immunodeficiency (SCID) can result in a selective engraftment of T cells of donor origin with complete immunologic reconstitution and in vivo tolerance. The latter may occur in the absence of clonal deletion of donor T lymphocytes able to recognize the host HLA antigens. The activity of these host-reactive T cells is suppressed in vivo, since no graft-vs. -host disease is observed in these human chimeras. Here it is shown that the CD4+ host-reactive T cell clones isolated from a SCID patient transplanted with fetal liver stem cells produce unusually high quantities of interleukin 10 (IL-10) and very low amounts of IL-2 after antigen-specific stimulation in vitro. The specific proliferative responses of the host-reactive T cell clones were considerably enhanced in the presence of neutralizing concentrations of an anti-IL-10 monoclonal antibody, suggesting that high levels of endogenous IL-10 suppress the activity of these cells. These in vitro data correlate with observations made in vivo. Semi-quantitative polymerase chain reaction analysis carried out on freshly isolated peripheral blood mononuclear cells (PBMC) of the patient indicated that the levels of IL-10 messenger RNA (mRNA) expression were strongly enhanced, whereas IL-2 mRNA expression was much lower than that in PBMC of healthy donors. In vivo IL-10 mRNA expression was not only high in the T cells, but also in the non-T cell fraction, indicating that host cells also contributed to the high levels of IL-10 in vivo. Patient-derived monocytes were found to be major IL-10 producers. Although no circulating IL-10 could be detected, freshly isolated monocytes of the patient showed a reduced expression of class II HLA antigens. However, their capacity to stimulate T cells of normal donors in primary mixed lymphocyte cultures was within the normal range. Interestingly, similar high in vivo IL-10 mRNA expressions in the T and non-T cell compartment were also observed in three SCID patients transplanted with fetal liver stem cells and in four SCID patients transplanted with T cell-depleted haploidentical bone marrow stem cells. Taken together, these data indicate that high endogenous IL-10 production is a general phenomenon in SCID patients in whom allogenic stem cell transplantation results in immunologic reconstitution and induction of tolerance. Both donor T cells and host accessory cells contribute to these high levels of IL-10, which would suppress the activity of host-reactive T cell in vivo.
Asunto(s)
Antígenos HLA/inmunología , Trasplante de Células Madre Hematopoyéticas , Tolerancia Inmunológica , Interleucina-10/biosíntesis , Inmunodeficiencia Combinada Grave/inmunología , Secuencia de Bases , Linfocitos T CD4-Positivos/inmunología , División Celular , Células Clonales , ADN , Antígenos HLA-DR/inmunología , Células Madre Hematopoyéticas/inmunología , Humanos , Interleucina-2/biosíntesis , Hígado/citología , Masculino , Datos de Secuencia Molecular , Monocitos/inmunología , Reacción en Cadena de la Polimerasa , Inmunodeficiencia Combinada Grave/terapiaRESUMEN
Mutations in the tyrosine kinase, Btk, result in a mild immunodeficiency in mice (xid). While B lymphocytes from xid mice do not proliferate to anti-immunoglobulin (Ig), we show here induction of the complete complement of cell cycle regulatory molecules, though the level of induction is about half that detected in normal B cells. Cell cycle analysis reveals that anti-Ig stimulated xid B cells enter S phase, but fail to complete the cell cycle, exhibiting a high rate of apoptosis. This correlated with a decreased ability to induce the anti-apoptosis regulatory protein, Bcl-xL. Ectopic expression of Bcl-xL in xid B cells permitted anti-Ig induced cell cycle progression demonstrating dual requirements for induction of anti-apoptotic proteins plus cell cycle regulatory proteins during antigen receptor mediated proliferation. Furthermore, our results link one of the immunodeficient traits caused by mutant Btk with the failure to properly regulate Bcl-xL.