RESUMEN
HELLS/LSH (Helicase, Lymphoid Specific) is a SNF2-like chromatin remodelling protein involved in DNA methylation. Its loss-of-function in humans causes humoral immunodeficiency, called ICF4 syndrome (Immunodeficiency, Centromeric Instability, Facial anomalies). Here we show by our newly generated B-cell-specific Hells conditional knockout mouse model that HELLS plays a pivotal role in T-dependent B-cell responses. HELLS deficiency induces accelerated decay of germinal center (GC) B cells and impairs the generation of high affinity memory B cells and circulating antibodies. Mutant GC B cells undergo dramatic DNA hypomethylation and massive de-repression of evolutionary recent retrotransposons, which surprisingly does not directly affect their survival. Instead, they prematurely upregulate either memory B cell markers or the transcription factor ATF4, which is driving an mTORC1-dependent metabolic program typical of plasma cells. Treatment of wild type mice with a DNMT1-specific inhibitor phenocopies the accelerated kinetics, thus pointing towards DNA-methylation maintenance by HELLS being a crucial mechanism to fine-tune the GC transcriptional program and enable long-lasting humoral immunity.
Asunto(s)
Linfocitos B , Metilación de ADN , Animales , Humanos , Ratones , ADN , ADN Helicasas , Centro Germinal , Células PlasmáticasRESUMEN
ÍSince the introduction of new generation pertussis vaccines, resurgence of pertussis has been observed in many developed countries. Former whole-cell pertussis (wP) vaccines are able to protect against disease and transmission but have been replaced in several industrialized countries because of their reactogenicity and adverse effects. Current acellular pertussis (aP) vaccines, made of purified proteins of Bordetella pertussis, are efficient at preventing disease but fail to induce long-term protection from infection. While the systemic and mucosal T cell immunity induced by the 2 types of vaccines has been well described, much less is known concerning B cell responses. Taking advantage of an inducible activation-induced cytidine deaminase fate-mapping mouse model, we compared effector and memory B cells induced by the 2 classes of vaccines and showed that a stronger and broader memory B cell and plasma cell response was achieved by a wP prime. We also observed that homologous or heterologous vaccine combinations that include at least 1 wP administration, even as a booster dose, were sufficient to induce this broad effector response, thus highlighting its dominant imprint on the B cell profile. Finally, we describe the settlement of memory B cell populations in the lung following subcutaneous wP prime vaccination.
Asunto(s)
Subgrupos de Linfocitos B , Tos Ferina , Ratones , Animales , Tos Ferina/prevención & control , Inmunización Secundaria , Vacuna contra la Tos Ferina , Bordetella pertussisRESUMEN
MBCs (MBCs) generated in T-dependent immune responses can persist for a lifetime and rapidly react upon secondary antigen exposure to differentiate into plasma cells (PCs) and/or to improve the affinity of their BCR through new rounds of hypermutation in germinal centers (GCs). The fate of a MBC in secondary immune reactions appears to depend upon multiple parameters, whose understanding is mandatory for the design of efficient vaccine strategies. We followed the behavior of MBCs in recall responses to SRBCs using an inducible AID fate mapping mouse model in which B cells engaged in a germinal center (GC) response are irreversibly labeled upon simultaneous tamoxifen ingestion and immunization. We used different schemes of mouse immunization and tamoxifen feeding in adoptive-transfer experiments of total splenic B cells into congenic mice that have been pre-immunized or not, to assess the contribution of the different effector subsets in a physiological competitive context. We were able to show that naive B cells can differentiate into GC B cells with kinetics similar to MBCs in the presence of previously activated T follicular helper (TFH) cells and a primed microenvironment. We also showed that MBCs are recruited into secondary GCs, together with naive B cells. In contrast, PC differentiation, which dominated secondary MBC responses, was not dependent upon a previous TFH activation. We observed that the presence of persisting germinal centers and circulating antibody levels are key factors determining the germinal center versus plasma cell fate in a recall response. Notably, disruption of persistent germinal center structures by a lymphotoxin beta-receptor fusion protein or a longer timing between the prime and the boost, which correlated with reduced antigen-specific immunoglobulin levels in serum, were two conditions with an opposite impact, respectively inhibiting or promoting a GC fate for MBCs. Altogether, these studies highlight the complexity of recall responses, whose outcome varies according to immunization contexts.
Asunto(s)
Linfocitos B , Memoria Inmunológica , Animales , Antígenos/metabolismo , Centro Germinal , Ratones , Células Plasmáticas , Tamoxifeno/metabolismoRESUMEN
Klhl6 belongs to the KLHL gene family, which is composed of an N-terminal BTB-POZ domain and four to six Kelch motifs in tandem. Several of these proteins function as adaptors of the Cullin3 E3 ubiquitin ligase complex. In this article, we report that Klhl6 deficiency induces, as previously described, a 2-fold reduction in mature B cells. However, we find that this deficit is centered on the inability of transitional type 1 B cells to survive and to progress toward the transitional type 2 B cell stage, whereas cells that have passed this step generate normal germinal centers (GCs) upon a T-dependent immune challenge. Klhl6-deficient type 1 B cells showed a 2-fold overexpression of genes linked with cell proliferation, including most targets of the anaphase-promoting complex/cyclosome complex, a set of genes whose expression is precisely downmodulated upon culture of splenic transitional B cells in the presence of BAFF. These results thus suggest a delay in the differentiation process of Klhl6-deficient B cells between the immature and transitional stage. We further show, in the BL2 Burkitt's lymphoma cell line, that KLHL6 interacts with Cullin3, but also that it binds to HBXIP/Lamtor5, a protein involved in cell-cycle regulation and cytokinesis. Finally, we report that KLHL6, which is recurrently mutated in B cell lymphomas, is an off-target of the normal somatic hypermutation process taking place in GC B cells in both mice and humans, thus leaving open whether, despite the lack of impact of Klhl6 deficiency on GC B cell expansion, mutants could contribute to the oncogenic process.
Asunto(s)
Linfocitos B/fisiología , Proteínas Portadoras/fisiología , Centro Germinal/citología , Animales , Linfocitos B/inmunología , Linfoma de Burkitt/patología , Proteínas Portadoras/genética , Diferenciación Celular , Línea Celular , Proliferación Celular , Centro Germinal/inmunología , Humanos , Linfoma de Células B/genética , Linfoma de Células B/patología , Ratones , Mutación , Células Precursoras de Linfocitos B/fisiologíaRESUMEN
T cell-dependent immune responses develop soon after birth, whereas it takes 2 yr for humans to develop T cell-independent responses. We used this dissociation to analyze the repertoire diversification of IgM(+)IgD(+)CD27(+) B cells (also known as "IgM memory" B cells), comparing these cells with switched B cells in children <2 yr of age, with the aim of determining whether these two subsets are developmentally related. We show that the repertoire of IgM(+)IgD(+)CD27(+) B cells in the spleen and blood displays no sign of antigen-driven activation and expansion on H-CDR3 spectratyping, despite the many antigenic challenges provided by childhood vaccinations. This repertoire differed markedly from those of switched B cells and splenic germinal center B cells, even at the early stage of differentiation associated with mu heavy chain expression. These data provide evidence for the developmental diversification of IgM(+)IgD(+)CD27(+) B cells, at least in very young children, outside of T cell-dependent and -independent immune responses.
Asunto(s)
Linfocitos B/inmunología , Variación Genética , Inmunoglobulina D/inmunología , Inmunoglobulina M/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Antígenos CD/inmunología , Subgrupos de Linfocitos B/inmunología , Reordenamiento Génico , Humanos , Región Variable de Inmunoglobulina/genética , Cadenas mu de Inmunoglobulina/inmunología , Memoria Inmunológica , Lactante , Activación de Linfocitos , Bazo/inmunología , Linfocitos T/inmunología , Transcripción GenéticaRESUMEN
Interleukin-2 (IL-2) withdrawal is a physiologic process inducing cell death in activated T lymphocytes. Glucocorticoid-induced leucine zipper (GILZ) has recently been identified as a protein modulating T-cell receptor activation by repressing various signaling pathways. We report here that IL-2 deprivation leads to expression of GILZ in T lymphocytes. We then characterized the human gilz promoter and showed that FoxO3 (Forkhead box class O3) binding to the Forkhead responsive elements identified in the promoter is necessary for induction of gilz expression upon IL-2 withdrawal. To assess the functional consequences of this induction, we used 2 strategies, GILZ overexpression and GILZ silencing in murine IL-2-dependent CTLL-2 cells. GILZ overexpression protects CTLL-2 cells from IL-2 withdrawal-induced apoptosis, whereas cell death is accelerated in cells unable to express GILZ. Concomitantly, the expression of Bim is inhibited in GILZ-overexpressing cells and enhanced when GILZ expression is impaired. Furthermore, GILZ inhibits FoxO3 transcriptional activity that leads to inhibition of Bim expression but also to down-regulation of GILZ itself. Therefore, GILZ is a transiently expressed protein induced upon IL-2 withdrawal that protects T cells from the onset of apoptosis.