RESUMO
MRL/MpJ-Fas(lpr/lpr)/J (MRL(lpr)) mice develop lupus-like disease manifestations in an IL-21-dependent manner. IL-21 is a pleiotropic cytokine that can influence the activation, differentiation, and expansion of B and T cell effector subsets. Notably, autoreactive CD4(+) T and B cells spontaneously accumulate in MRL(lpr) mice and mediate disease pathogenesis. We sought to identify the particular lymphocyte effector subsets regulated by IL-21 in the context of systemic autoimmunity and, thus, generated MRL(lpr) mice deficient in IL-21R (MRL(lpr).IL-21R(-/-)). Lymphadenopathy and splenomegaly, which are characteristic traits of the MRL(lpr) model were significantly reduced in the absence of IL-21R, suggesting that immune activation was likewise decreased. Indeed, spontaneous germinal center formation and plasma cell accumulation were absent in IL-21R-deficient MRL(lpr) mice. Correspondingly, we observed a significant reduction in autoantibody titers. Activated CD4(+) CD44(+) CD62L(lo) T cells also failed to accumulate, and CD4(+) Th cell differentiation was impaired, as evidenced by a significant reduction in CD4(+) T cells that produced the pronephritogenic cytokine IFN-γ. T extrafollicular helper cells are a recently described subset of activated CD4(+) T cells that function as the primary inducers of autoantibody production in MRL(lpr) mice. Importantly, we demonstrated that T extrafollicular helper cells are dependent on IL-21R for their generation. Together, our data highlighted the novel observation that IL-21 is a critical regulator of multiple pathogenic B and T cell effector subsets in MRL(lpr) mice.
Assuntos
Autoimunidade , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Interleucinas/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Ativação Linfocitária , Receptores de Interleucina-21/imunologia , Animais , Autoanticorpos/genética , Autoanticorpos/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Interferon gama/biossíntese , Doenças Linfáticas/genética , Doenças Linfáticas/imunologia , Doenças Linfáticas/patologia , Camundongos , Camundongos Endogâmicos MRL lpr , Camundongos Knockout , Receptores de Interleucina-21/deficiência , Receptores de Interleucina-21/genética , Pele/imunologia , Pele/patologia , Esplenomegalia/genética , Esplenomegalia/imunologia , Esplenomegalia/patologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
BACKGROUND AND AIMS: Anti-tumour necrosis factor [anti-TNF] therapy is widely used for the treatment of inflammatory bowel disease, yet many patients are primary non-responders, failing to respond to induction therapy. We aimed to identify blood gene expression differences between primary responders and primary non-responders to anti-TNF monoclonal antibodies [infliximab and adalimumab], and to predict response status from blood gene expression and clinical data. METHODS: The Personalised Anti-TNF Therapy in Crohn's Disease [PANTS] study is a UK-wide prospective observational cohort study of anti-TNF therapy outcome in anti-TNF-naive Crohn's disease patients [ClinicalTrials.gov identifier: NCT03088449]. Blood gene expression in 324 unique patients was measured by RNA-sequencing at baseline [week 0], and at weeks 14, 30, and 54 after treatment initiation [total sample sizeâ =â 814]. RESULTS: After adjusting for clinical covariates and estimated blood cell composition, baseline expression of major histocompatibility complex, antigen presentation, myeloid cell enriched receptor, and other innate immune gene modules was significantly higher in anti-TNF responders vs non-responders. Expression changes from baseline to week 14 were generally of consistent direction but greater magnitude [i.e. amplified] in responders, but interferon-related genes were upregulated uniquely in non-responders. Expression differences between responders and non-responders observed at week 14 were maintained at weeks 30 and 54. Prediction of response status from baseline clinical data, cell composition, and module expression was poor. CONCLUSIONS: Baseline gene module expression was associated with primary response to anti-TNF therapy in PANTS patients. However, these baseline expression differences did not predict response with sufficient sensitivity for clinical use.
Assuntos
Doença de Crohn , Humanos , Doença de Crohn/tratamento farmacológico , Doença de Crohn/genética , Redes Reguladoras de Genes , Inibidores do Fator de Necrose Tumoral/uso terapêutico , Estudos Prospectivos , Imunoterapia , Fator de Necrose Tumoral alfaRESUMO
The glucocorticoid-induced TNFR-related (GITR) protein is a coactivating receptor that is constitutively expressed on Treg cells and induced on activated T cells. To better under-stand the role of long-term GITR signaling, we generated a mouse that constitutively expresses GITR ligand (GITRL) on APCs that mimics the physiological distribution of GITRL in vivo. Despite a five-fold expansion of the Treg-cell pool, there is increased activation and depletion of naive T cells in the transgenic (Tg) mice, suggesting that the increased number of Treg cells cannot fully suppress T-cell activation. Interestingly, GITRL Tg mice have multiorgan lymphocytic infiltrates yet display no overt autoimmunity, indicating the existence of a compensatory immunoregulatory mechanism(s). In the spleens and tissue infiltrates ofGITRL Tg mice, we found increased numbers of Foxp3(-) IL-10-producing type 1 regulatory T (Tr-1)-like cells that suppress naïve T-cell proliferation in an IL-10-dependent fashion. Increased IL-27 production from Tg APCs and activation of c-Maf in the Tr1-like cells suggest a possible mechanism for their induction. Our results demonstrate that enhanced GITR/GITRL interactions have a pleiotropic role on the regulation of T-cell responses, which includes promoting the differentiation of Tr-1-like cells, which contribute to the maintenance of peripheral T-cell tolerance.
Assuntos
Proteína Relacionada a TNFR Induzida por Glucocorticoide/fisiologia , Interleucina-10/biossíntese , Interleucinas/biossíntese , Linfócitos T Reguladores/fisiologia , Fatores de Necrose Tumoral/fisiologia , Animais , Autoimunidade , Fatores de Transcrição Forkhead/análise , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Development of long-term humoral immunity, characterized by the formation of long-lived plasma cells (PCs) in the bone marrow and memory B cells, is a critical component of protective immunity to pathogens, and as such it is the major goal of vaccination. However, the mechanisms involved in the generation of long-term humoral immunity remain poorly understood. In this study, we used IL-21R-deficient (IL-21R.KO) mice to examine the role of the IL-21 pathway in the development of the B cell memory response. Primary IgG serum Ab responses to the T cell-dependent Ag 4-hydroxy-3-nitrophenylacetyl (NP) hapten conjugated to chicken γ globulin were delayed in IL-21R.KO mice, but reached normal titers within 3 to 4 wk of immunization. IL-21R.KO mice formed germinal centers and generated normal numbers of PCs in their bone marrow. Additionally, memory B cell formation was similar in wild-type and IL-21R.KO mice. However, NP-specific memory B cells and PCs failed to expand following secondary immunization of IL-21R.KO mice, and consequently, secondary IgG Ab responses to NP hapten conjugated to chicken γ globulin were significantly impaired. These results identify the IL-21 pathway as a critical component of the memory B cell response.
Assuntos
Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Diferenciação Celular/imunologia , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Memória Imunológica , Receptores de Interleucina-21/fisiologia , Animais , Antígenos de Superfície/biossíntese , Proteínas Reguladoras de Apoptose/biossíntese , Subpopulações de Linfócitos B/citologia , Diferenciação Celular/genética , Galinhas/imunologia , Células Dendríticas Foliculares/imunologia , Células Dendríticas Foliculares/metabolismo , Centro Germinativo/citologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Haptenos/administração & dosagem , Haptenos/imunologia , Imunização Secundária , Memória Imunológica/genética , Memória Imunológica/imunologia , Antígenos Comuns de Leucócito/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitrofenóis/administração & dosagem , Nitrofenóis/imunologia , Fenilacetatos/administração & dosagem , Fenilacetatos/imunologia , Receptor de Morte Celular Programada 1 , Receptores CXCR5/biossíntese , Receptores de Interleucina-21/deficiência , Receptores de Interleucina-21/genética , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , gama-Globulinas/administração & dosagem , gama-Globulinas/imunologiaRESUMO
BACKGROUND: Janus kinase (JAK) 1 inhibitor upadacitinib and IL-23 inhibitor risankizumab are efficacious in inflammatory bowel disease (IBD) patients who are antitumor necrosis factor (anti-TNF)-α inadequate responders (TNF-IRs). We aimed to understand the mechanisms mediating the response of upadacitinib and risankizumab. METHODS: Eight tissue transcriptomic data sets from IBD patients treated with anti-TNF-α therapies along with single-cell RNAseq data from ulcerative colitis were integrated to identify TNF-IR mechanisms. The RNAseq colon tissue data from clinical studies of TNF-IR Crohn's disease patients treated with upadacitinib or risankizumab were used to identify TNF-IR mechanisms that were favorably modified by upadacitinib and risankizumab. RESULTS: We found 7 TNF-IR upregulated modules related to innate/adaptive immune responses, interferon signaling, and tissue remodeling and 6 TNF-IR upregulated cell types related to inflammatory fibroblasts, postcapillary venules, inflammatory monocytes, macrophages, dendritic cells, and cycling B cells. Upadacitinib was associated with a significant decrease in the expression of most TNF-IR upregulated modules in JAK1 responders (JAK1-R); in contrast, there was no change in these modules among TNF-IR patients treated with a placebo or among JAK1 inadequate responders (JAK1-IR). In addition, 4 of the 6 TNF-IR upregulated cell types were significantly decreased after upadacitinib treatment in JAK1-R but not among subjects treated with a placebo or among JAK1-IR patients. We observed similar findings from colon biopsy samples from TNF-IR patients treated with risankizumab. CONCLUSIONS: Collectively, these data suggest that upadacitinib and risankizumab affect TNF-IR upregulated mechanisms, which may account for their clinical response among TNF-IR IBD patients.
We identified molecular and cellular mechanisms associated with and potentially mediating the response of upadacitinib and risankizumab for IBD patients that inadequately responded to anti-TNF-α treatment.
Assuntos
Doenças Inflamatórias Intestinais , Inibidores do Fator de Necrose Tumoral , Humanos , Fator de Necrose Tumoral alfaRESUMO
BACKGROUND AND AIMS: This study assessed whether baseline triggering receptor expressed on myeloid cells [TREM-1] whole blood gene expression predicts response to anti-TNF therapy in patients with UC or CD. METHODS: TREM-1 whole blood gene expression was analysed by RNA sequencing [RNA-seq] in patients with moderately to severely active UC or CD treated with adalimumab in the Phase 3 SERENE-UC and SERENE-CD clinical trials. The predictive value of baseline TREM-1 expression was evaluated and compared according to endoscopic and clinical response vs non-response, and remission vs non-remission, at Weeks 8 and 52 [SERENE-UC], and Weeks 12 and 56 [SERENE-CD]. RESULTS: TREM-1 expression was analysed in 95 and 106 patients with UC and CD, respectively, receiving standard-dose adalimumab induction treatment. In SERENE-UC, baseline TREM-1 expression was not predictive of endoscopic response [p=0.48], endoscopic remission [p=0.53], clinical response [p=0.58] or clinical remission [p=0.79] at Week 8, or clinical response [p=0.60] at Week 52. However, an association was observed with endoscopic response [p=0.01], endoscopic remission [p=0.048], and clinical remission [p=0.04997] at Week 52. For SERENE-CD, baseline TREM-1 expression was not predictive of endoscopic response [p=0.56], endoscopic remission [p=0.33], clinical response [p=0.07], clinical remission [p=0.65] at Week 12, or endoscopic response [p=0.61], endoscopic remission [p=0.51], clinical response [p=0.62] or clinical remission [p=0.97] at Week 56. CONCLUSIONS: Baseline TREM-1 gene expression did not uniformly predict adalimumab response in SERENE clinical trials. Further research is needed to identify potential blood-based biomarkers predictive of response to anti-TNF therapy in patients with IBD.
RESUMO
BACKGROUND AND AIMS: Anti-TNF treatment failure in patients with inflammatory bowel disease (IBD) is common and frequently related to low drug concentrations. In order to identify patients who may benefit from dose optimisation at the outset of anti-TNF therapy, we sought to define epigenetic biomarkers in whole blood at baseline associated with anti-TNF drug concentrations at week 14. METHODS: DNA methylation from 1,104 whole blood samples from 385 patients in the Personalised Anti-TNF Therapy in Crohn's disease (PANTS) study were assessed using the Illumina EPIC Beadchip (v1.0) at baseline, weeks 14, 30 and 54. We compared DNA methylation profiles in anti-TNF-treated patients who experienced primary non-response at week 14 and if they were assessed at subsequent time points, were not in remission at week 30 or 54 (infliximab n = 99, adalimumab n = 94), with patients who responded at week 14 and when assessed at subsequent time points, were in remission at week 30 or 54 (infliximab n = 99, adalimumab n = 93). RESULTS: Overall, between baseline and week 14, we observed 4,999 differentially methylated probes (DMPs) annotated to 2376 genes following anti-TNF treatment. Pathway analysis identified 108 significant gene ontology terms enriched in biological processes related to immune system processes and responses.Epigenome-wide association (EWAS) analysis identified 323 DMPs annotated to 210 genes at baseline associated with higher anti-TNF drug concentrations at week 14. Of these, 125 DMPs demonstrated shared associations with other common traits (proportion of shared CpGs compared to DMPs) including body mass index (23.2%), followed by CRP (11.5%), smoking (7.4%), alcohol consumption per day (7.1%) and IBD type (6.8%). EWAS of primary non-response to anti-TNF identified 20 DMPs that were associated with both anti-TNF drug concentration and primary non-response to anti-TNF with a strong correlation of the coefficients (Spearman's rho = -0.94, p < 0.001). CONCLUSION: Baseline DNA methylation profiles may be used as a predictor for anti-TNF drug concentration at week 14 to identify patients who may benefit from dose optimisation at the outset of anti-TNF therapy.
RESUMO
B cells generated in the bone marrow of adult mice enter the periphery as transitional B cells and subsequently differentiate into one of two phenotypically and functionally distinct subsets, marginal zone (MZ) or follicular (Fo) B cells. Recent reports indicate, however, that in response to environmental cues, such as lymphopenia, mature Fo B cells can change to display phenotypic markers characteristic of MZ B cells. Previously, we found that splenic B cells transferred to SCID mice responded to polyoma virus (PyV) infection with T cell-independent (TI) IgM and IgG secretion, reducing the viral load and protecting mice from the lethal effect of the infection. The contribution of MZ and Fo B cell subsets to this antiviral TI-2 response, however, has not been addressed. In this study, we show that both sort-purified MZ and Fo B cells generate protective TI Ab responses to PyV infection when transferred into SCID mice. Moreover, the transferred Fo B cells in the spleens of the PyV-infected SCID mice change phenotype, with many of them displaying MZ B cell characteristics. These findings demonstrate the plasticity of the B cell subsets in virus-infected hosts and show for the first time that B cells derived exclusively from Fo B cells can effectively function in antiviral TI-2 responses.
Assuntos
Anticorpos Antivirais/biossíntese , Subpopulações de Linfócitos B/transplante , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/virologia , Doença Aguda , Transferência Adotiva , Animais , Antígenos T-Independentes/imunologia , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/virologia , Células Clonais , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Imunofenotipagem , Linfoma de Zona Marginal Tipo Células B , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Infecções por Polyomavirus/imunologia , Infecções por Polyomavirus/mortalidade , Infecções por Polyomavirus/prevenção & controle , Baço/citologia , Baço/imunologia , Baço/transplante , Análise de Sobrevida , Subpopulações de Linfócitos T/metabolismoRESUMO
BACKGROUND: Janus kinase (JAK) inhibition shows promise for treatment of patients with moderate to severe Crohn's disease. We aimed to provide mechanistic insights into the JAK1-selective inhibitor upadacitinib through a transcriptomics substudy on biopsies from patients with Crohn's disease from CELEST. METHODS: Seventy-four patients consented to this optional substudy. Ileal and colonic biopsies were collected during endoscopy at screening and week 12 or 16. RNA isolated from 226 samples was analyzed by RNAseq, with additional qPCR analysis. Additional biopsies from patients with Crohn's disease receiving anti-tumor necrosis factor (anti-TNF; nâ =â 34) and healthy controls (nâ =â 10) were used for qPCR. Single-cell RNAseq public profiles were used to evaluate treatment effects on specific cellular subsets, associations with endoscopic improvement, and indirect comparisons with the anti-TNF-treated cohort. RESULTS: In involved areas of mucosa with endoscopic remission after upadacitinib treatment, 1156 and 76 protein-coding genes were significantly regulated (false discovery rate < 0.05) at week 12/16 in colonic and ileal biopsies, respectively (60 overlapped), compared with baseline. Upadacitinib did not significantly affect transcriptomes of noninvolved intestinal areas. CELEST patients (mostly anti-TNF-refractory) showed baseline differences in gene expression compared with a separate cohort of biologic-naïve patients. Notably, upadacitinib reversed overexpression of inflammatory fibroblast and interferon-γ effector signature markers. CONCLUSIONS: Upadacitinib modulates inflammatory pathways in mucosal lesions of patients with anti-TNF-refractory Crohn's disease, including inflammatory fibroblast and interferon-γ-expressing cytotoxic T cell compartments. This substudy is the first to describe the molecular response to JAK1 inhibition in inflammatory bowel disease and differential effects relative to anti-TNF treatment. (Clinical trial identifier: NCT02365649).
Assuntos
Doença de Crohn , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Inibidores de Janus Quinases , Doença de Crohn/tratamento farmacológico , Endoscopia Gastrointestinal , Humanos , Interferon gama , Mucosa Intestinal/efeitos dos fármacos , Janus Quinase 1/antagonistas & inibidores , Inibidores de Janus Quinases/uso terapêutico , Inibidores do Fator de Necrose TumoralRESUMO
BACKGROUND: Anti-IL-21R antibodies are potential therapeutics for the treatment of autoimmune diseases. This study evaluated correlations between the pharmacodynamic (PD) activity, pharmacokinetics, and anti-product antibody responses of human anti-IL-21R antibodies Ab-01 and Ab-02 following IV administration to cynomolgus monkeys. METHODS: The PD assay was based on the ability of recombinant human IL-21 (rhuIL-21) to induce expression of the IL-2RA gene in cynomolgus monkey whole blood samples ex vivo. Monkeys screened for responsiveness to rhuIL-21 stimulation using the PD assay, were given a single 10 mg/kg IV dosage of Ab-01, Ab-02, or a control antibody (3/group), and blood samples were evaluated for PD activity (inhibition of IL-2RA expression) for up to 148 days. Anti-IL-21R antibody concentrations and anti-product antibody responses were measured in serum using immunoassays and flow cytometry. RESULTS: Following IV administration of Ab-01 and Ab-02 to cynomolgus monkeys, PD activity was observed as early as 5 minutes (first time point sampled). This PD activity had good correlation with the serum concentrations and anti-product antibody responses throughout the study. The mean terminal half-life (t1/2) was approximately 10.6 and 2.3 days for Ab-01 and Ab-02, respectively. PD activity was lost at approximately 5-13 weeks for Ab-01 and at approximately 2 weeks for Ab-02, when serum concentrations were relatively low. The estimated minimum concentrations needed to maintain PD activity were approximately 4-6 nM for Ab-01 and approximately 2.5 nM for Ab-02, and were consistent with the respective KD values for binding to human IL-21R. For Ab-01, there was noticeable inter-animal variability in t1/2 values (approximately 6-14 days) and the resulting PD profiles, which correlated with the onset of anti-product antibody formation. While all three Ab-01-dosed animals were positive for anti-Ab-01 antibodies, only one monkey (with the shortest t1/2 and the earliest loss of PD activity) had evidence of neutralizing anti-Ab-01 antibodies. All three Ab-02-dosed monkeys developed neutralizing anti-Ab-02 antibodies. CONCLUSIONS: For anti-IL-21R antibodies Ab-01 and Ab-02, there was good correlation between PD activity and PK profiles following IV administration to cynomolgus monkeys. Compared with Ab-01, Ab-02 was eliminated markedly faster from the circulation, which correlated with a shorter duration of PD activity.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/farmacocinética , Formação de Anticorpos/imunologia , Macaca fascicularis/imunologia , Receptores de Interleucina-21/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/imunologia , Formação de Anticorpos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoensaio , Injeções Intravenosas , Macaca fascicularis/sangue , Masculino , Proteínas Recombinantes/imunologiaRESUMO
Using phage display, we generated a panel of optimized neutralizing antibodies against the human and mouse receptors for interleukin 21 (IL-21), a cytokine that is implicated in the pathogenesis of many types of autoimmune disease. Two antibodies, Ab-01 and Ab-02, which differed by only four amino acids in V(L) CDR3, showed potent inhibition of human and mouse IL-21R in cell-based assays and were evaluated for their pharmacological and pharmacodynamic properties. Ab-01, but not Ab-02, significantly reduced a biomarker of disease (anti-dsDNA antibodies) and IgG deposits in the kidney in the MRL-Fas(lpr) mouse model of lupus, suggesting that anti-IL-21R antibodies may prove useful in the treatment of lupus. Ab-01 also had a consistently higher exposure (AUC(0-infinity)) than Ab-02 following a single dose in rodents or cynomolgus monkeys (2-3-fold or 4-7-fold, respectively). Our data demonstrate that small differences in CDR3 sequences of optimized antibodies can lead to profound differences in in vitro and in vivo properties, including differences in pharmacological activity and pharmacokinetic profiles. The lack of persistent activity of Ab-02 in the MRL-Fas(lpr) mouse lupus model may have been a consequence of faster elimination, reduced potency in blocking the effects of mouse IL-21R, and more potent/earlier onset of the anti-product response relative to Ab-01.
Assuntos
Anticorpos Anti-Idiotípicos/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Lúpus Eritematoso Sistêmico/terapia , Receptores de Interleucina-21/antagonistas & inibidores , Animais , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Anticorpos Neutralizantes/imunologia , Células Cultivadas , Regiões Determinantes de Complementaridade/genética , Modelos Animais de Doenças , Feminino , Humanos , Injeções Intraperitoneais , Injeções Intravenosas , Injeções Subcutâneas , Interleucinas/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos MRL lpr , Camundongos Endogâmicos , Ratos , Ratos Sprague-Dawley , Receptores de Interleucina-21/imunologiaRESUMO
We have examined processes leading to the spontaneous development of autoimmune inflammatory arthritis in transgenic mice containing CD4+ T cells targeted to a nominal Ag (hemagglutinin (HA)) and coexpressing HA driven by a MHC class II promoter. Despite being subjected to multiple tolerance mechanisms, autoreactive CD4+ T cells accumulate in the periphery of these mice and promote systemic proinflammatory cytokine production. The majority of mice spontaneously develop inflammatory arthritis, which is accompanied by an enhanced regional immune response in lymph nodes draining major joints. Arthritis development is accompanied by systemic B cell activation; however, neither B cells nor Ab is required for arthritis development, since disease develops in a B cell-deficient background. Moreover, arthritis also develops in a recombinase activating gene-deficient background, indicating that the disease process is driven by CD4+ T cells recognizing the neo-self HA Ag. These findings show that autoreactive CD4+ T cells recognizing a single self-Ag, expressed by systemically distributed APCs, can induce arthritis via a mechanism that is independent of their ability to provide help for autoantibody production.
Assuntos
Apresentação de Antígeno , Artrite/imunologia , Autoantígenos/imunologia , Doenças Autoimunes/imunologia , Linfócitos T CD4-Positivos/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Linfócitos B/imunologia , Hemaglutininas/genética , Hemaglutininas/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Camundongos , Camundongos Transgênicos , Peptídeos/imunologia , Regiões Promotoras Genéticas , Linfócitos T Reguladores/imunologiaRESUMO
Although somatically mutated autoantibodies are characteristic of many autoimmune diseases, the processes that can lead to their development remain poorly understood. We have examined the formation of autoreactive memory B cells in PevHA mice, which express the influenza virus PR8 hemagglutinin (HA) as a transgenic membrane bound neo-self-Ag. Using a virus immunization strategy, we show that PR8 HA-specific memory B cell formation can occur in PevHA mice, even though a major subset of PR8 HA-specific B cells is negatively selected from the primary repertoire. Moreover, PR8 HA-specific memory B cells develop spontaneously in TS1 x PevHA mice, which coexpress a transgenic PR8 HA-specific TCR and contain a high frequency of HA-specific CD4(+) T cells. Notably, autoreactive memory B cell formation occurred in TS1 x PevHA mice even though approximately half of the HA-specific CD4(+) T cells were CD25(+)Foxp3(+) cells that could significantly attenuate, but did not completely abolish HA-specific autoantibody production in an adoptive transfer setting. The findings provide evidence that a high frequency of autoreactive CD4(+) T cells can be sufficient to promote autoreactive memory B cell formation in the absence of signals provided by overt immunization or infection and despite the presence of abundant autoantigen-specific CD4(+)CD25(+)Foxp3(+) regulatory T cells.
Assuntos
Linfócitos B/fisiologia , Linfócitos T CD4-Positivos/fisiologia , Memória Imunológica , Sequência de Aminoácidos , Animais , Autoanticorpos/biossíntese , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Dados de Sequência Molecular , Linfócitos T Reguladores/fisiologiaRESUMO
Development of long-term humoral immunity is a major goal of vaccination, but the mechanisms involved in the formation of long-term Ab responses are still being determined. In this study, we identify a previously unknown requirement for MyD88, an adaptor molecule that mediates signals at most TLRs, for the generation of long-term humoral immunity during live virus infection. Polyoma virus-infected MyD88 knockout mice generated strong acute T cell-dependent antiviral IgM and IgG responses and developed germinal centers. Activation-induced cytidine deaminase, an enzyme required for isotype switching and somatic hypermutation, was also induced in germinal center B cells, similar to wild-type mice. However, MyD88 knockout mice failed to develop bone marrow plasma cells and did not maintain long-term serum antiviral Ab responses. The isotype distribution of antiviral IgG responses was also altered; serum IgG2a and IgG2b levels were diminished, whereas IgG1 responses were not affected. The requirement for MyD88 for the formation of long-term humoral immunity to polyoma virus was intrinsic to B cells and was independent of IL-1R and IL-18R, cytokine receptors that also signal through MyD88. Our findings show that MyD88-dependent signaling pathways in B cells are essential for effectively generating long-term Ab responses and implicate a role for TLR in the formation of long-term humoral immunity.
Assuntos
Anticorpos Antivirais/sangue , Fator 88 de Diferenciação Mieloide/fisiologia , Infecções por Polyomavirus/imunologia , Animais , Linfócitos B/imunologia , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina-1/fisiologia , Receptores de Interleucina-18/fisiologia , Transdução de Sinais , Linfócitos T/imunologia , Receptores Toll-Like/fisiologia , Carga ViralRESUMO
Hypergammaglobulinemia and production of autoantibodies occur during many viral infections, and studies have suggested that viral antigen-presenting B cells may become polyclonally activated by CD4 T cells in vivo in the absence of viral engagement of the BCR. However, we have reported that CD4 cells in lymphocytic choriomengitis virus (LCMV)-infected mice kill adoptively transferred B cells coated with LCMV class II peptides. We report here that most of the surviving naïve B cells presenting class II MHC peptides undergo an extensive differentiation process involving both proliferation and secretion of antibodies. Both events require CD4 cells and CD40/CD40L interactions but not MyD88-dependent signaling within the B cells. B cells taken from immunologically tolerant donor LCMV-carrier mice with high LCMV antigen load became activated following adoptive transfer into LCMV-infected hosts, suggesting that B cells present sufficient antigen for this process during a viral infection. No division or activation of B cells was detected at all in virus-infected hosts in the absence of cognate CD4 T cells and class II antigen. This approach, therefore, formally demonstrates and quantifies a virus-induced polyclonal proliferation and differentiation of B cells, which, due to their high proportion, would mostly have BCR not specific for the virus.
Assuntos
Apresentação de Antígeno , Antígenos Virais/metabolismo , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/virologia , Ativação Linfocitária/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Receptores de Antígenos de Linfócitos B/fisiologia , Sequência de Aminoácidos , Animais , Células Produtoras de Anticorpos/citologia , Células Produtoras de Anticorpos/imunologia , Células Produtoras de Anticorpos/metabolismo , Células Produtoras de Anticorpos/virologia , Antígenos Virais/imunologia , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/metabolismo , Diferenciação Celular/imunologia , Células Clonais , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência MolecularRESUMO
This study describes a method for increasing the immunogenicity of influenza virus vaccines by exploiting the natural anti-Gal antibody to effectively target vaccines to antigen-presenting cells (APC). This method is based on enzymatic engineering of carbohydrate chains on virus envelope hemagglutinin to carry the alpha-Gal epitope (Gal alpha 1-3Gal beta 1-4GlcNAc-R). This epitope interacts with anti-Gal, the most abundant antibody in humans (1% of immunoglobulins). Influenza virus vaccine expressing alpha-Gal epitopes is opsonized in situ by anti-Gal immunoglobulin G. The Fc portion of opsonizing anti-Gal interacts with Fc gamma receptors on APC and induces effective uptake of the vaccine virus by APC. APC internalizes the opsonized virus to transport it to draining lymph nodes for stimulation of influenza virus-specific T cells, thereby eliciting a protective immune response. The efficacy of such an influenza vaccine was demonstrated in alpha 1,3galactosyltransferase (alpha 1,3GT) knockout mice, which produce anti-Gal, using the influenza virus strain A/Puerto Rico/8/34-H1N1 (PR8). Synthesis of alpha-Gal epitopes on carbohydrate chains of PR8 virus (PR8(alpha gal)) was catalyzed by recombinant alpha1,3GT, the glycosylation enzyme that synthesizes alpha-Gal epitopes in cells of nonprimate mammals. Mice immunized with PR8(alpha gal) displayed much higher numbers of PR8-specific CD8(+) and CD4(+) T cells (determined by intracellular cytokine staining and enzyme-linked immunospot assay) and produced anti-PR8 antibodies with much higher titers than mice immunized with PR8 lacking alpha-Gal epitopes. Mice immunized with PR8(alpha gal) also displayed a much higher level of protection than PR8 immunized mice after being challenged with lethal doses of live PR8 virus. We suggest that a similar method for increasing immunogenicity may be applicable to avian influenza vaccines.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Sistemas de Liberação de Medicamentos/métodos , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Trissacarídeos/imunologia , Animais , Anticorpos Antivirais/sangue , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/biossíntese , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Testes de Inibição da Hemaglutinação , Humanos , Vírus da Influenza A Subtipo H1N1/química , Influenza Humana/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de SobrevidaRESUMO
Although many studies have investigated the requirement for CD4(+) T cell help for CD8(+) T cell responses to acute viral infections that are fully resolved, less is known about the role of CD4(+) T cells in maintaining ongoing CD8(+) T cell responses to persistently infecting viruses. Using mouse polyoma virus (PyV), we asked whether CD4(+) T cell help is required to maintain antiviral CD8(+) T cell and humoral responses during acute and persistent phases of infection. Though fully intact during acute infection, the PyV-specific CD8(+) T cell response declined numerically during persistent infection in MHC class II-deficient mice, leaving a small antiviral CD8(+) T cell population that was maintained long term. These unhelped PyV-specific CD8(+) T cells were functionally unimpaired; they retained the potential for robust expansion and cytokine production in response to Ag rechallenge. In addition, although a strong antiviral IgG response was initially elicited by MHC class II-deficient mice, these Ab titers fell, and long-lived PyV-specific Ab-secreting cells were not detected in the bone marrow. Finally, using a minimally myeloablative mixed bone marrow chimerism approach, we demonstrate that recruitment and/or maintenance of new virus-specific CD8(+) T cells during persistent infection is impaired in the absence of MHC class II-restricted T cells. In summary, these studies show that CD4(+) T cells differentially affect CD8(+) T cell responses over the course of a persistent virus infection.
Assuntos
Formação de Anticorpos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Memória Imunológica , Infecções por Polyomavirus/imunologia , Animais , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/virologia , Doença Crônica , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe II/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Polyomavirus/imunologiaRESUMO
Autoreactive B cells are not completely purged from the primary B cell repertoire, and whether they can be prevented from maturation into memory B cells has been uncertain. We show here that a population of B cells that dominates primary immune responses of BALB/c mice to influenza virus A/PR/8/34 hemagglutinin (HA) are negatively selected in transgenic mice expressing PR8 HA as an abundant membrane-bound Ag (HACII mice). However, a separate population of B cells that contains precursors of memory B cells is activated by PR8 virus immunization and is subsequently negatively selected during the formation of the memory response. Negative selection of PR8 HA-specific B cells altered the specificity of the memory B cell response to a mutant virus containing a single amino acid substitution in a B cell epitope. Strikingly, this skewed reactivity resulted from an increase in the formation of memory B cells directed to non-self-epitopes on the mutant virus, which increased 8-fold in HACII mice relative to nontransgenic mice and precisely compensated for the absence of autoreactive PR8 HA-specific memory B cells. Negative selection of PR8 HA-specific B cells was a dominant process, since B cells from HACII mice could induce negative selection of PR8 HA-specific B cells from BALB/c mice. Lastly, HA-specific memory responses were unaffected by self-tolerance in another lineage of HA-transgenic mice (HA104 mice), indicating that the amount and/or cell type in which self-Ags are expressed can determine their ability to prevent autoreactive memory B cell formation.