RESUMO
An imbalance between proinflammatory and regulatory processes underlies autoimmune disease pathogenesis. We have shown that acute relapses of multiple sclerosis are characterized by a deficit in the immune suppressive ability of CD8+ T cells. These cells play an important immune regulatory role, mediated in part through cytotoxicity (perforin [PRF]/granzyme [GZM]) and IFNγ secretion. In this study, we further investigated the importance of IFNγ-, GZMB-, PRF1-, and LYST-associated pathways in CD8+ T cell-mediated suppression. Using the CRISPR-Cas9 ribonucleoprotein transfection system, we first optimized efficient gene knockout while maintaining high viability in primary bulk human CD8+ T cells. Knockout was confirmed through quantitative real-time PCR assays in all cases, combined with flow cytometry where appropriate, as well as confirmation of insertions and/or deletions at genomic target sites. We observed that the knockout of IFNγ, GZMB, PRF1, or LYST, but not the knockout of IL4 or IL5, resulted in significantly diminished in vitro suppressive ability in these cells. Collectively, these results reveal a pivotal role for these pathways in CD8+ T cell-mediated immune suppression and provide important insights into the biology of human CD8+ T cell-mediated suppression that could be targeted for immunotherapeutic intervention.
Assuntos
Linfócitos T CD8-Positivos , Granzimas , Interferon gama , Perforina , Humanos , Linfócitos T CD8-Positivos/imunologia , Interferon gama/imunologia , Interferon gama/metabolismo , Perforina/genética , Perforina/metabolismo , Granzimas/metabolismo , Granzimas/genética , Sistemas CRISPR-Cas , Esclerose Múltipla/imunologia , Esclerose Múltipla/genética , Técnicas de Inativação de Genes , Células CultivadasRESUMO
The pathogenic role B cells play in multiple sclerosis is underscored by the success of B cell depletion therapies. Yet, it remains unclear how B cells contribute to disease, although it is increasingly accepted that mechanisms beyond Ab production are involved. Better understanding of pathogenic interactions between B cells and autoreactive CD4 T cells will be critical for novel therapeutics. To focus the investigation on B cell:CD4 T cell interactions in vivo and in vitro, we previously developed a B cell-dependent, Ab-independent experimental autoimmune encephalomyelitis (EAE) mouse model driven by a peptide encompassing the extracellular domains of myelin proteolipid protein (PLPECD). In this study, we demonstrate that B cell depletion significantly inhibited PLPECD-induced EAE disease, blunted PLPECD-elicited delayed-type hypersensitivity reactions in vivo, and reduced CD4 T cell activation, proliferation, and proinflammatory cytokine production. Further, PLPECD-reactive CD4 T cells sourced from B cell-depleted donor mice failed to transfer EAE to naive recipients. Importantly, we identified B cell-mediated Ag presentation as the critical mechanism explaining B cell dependence in PLPECD-induced EAE, where bone marrow chimeric mice harboring a B cell-restricted MHC class II deficiency failed to develop EAE. B cells were ultimately observed to restimulate significantly higher Ag-specific proliferation from PLP178-191-reactive CD4 T cells compared with dendritic cells when provided PLPECD peptide in head-to-head cultures. We therefore conclude that PLPECD-induced EAE features a required pathogenic B cell-mediated Ag presentation function, providing for investigable B cell:CD4 T cell interactions in the context of autoimmune demyelinating disease.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Camundongos , Animais , Apresentação de Antígeno , Glicoproteína Mielina-Oligodendrócito , Linfócitos T CD4-Positivos , Proteína Proteolipídica de Mielina , Anticorpos/metabolismoRESUMO
OBJECTIVES: We evaluated and compared the peripheral blood findings in patients with acute COVID-19 vs other viral respiratory infections. METHODS: We retrospectively reviewed peripheral blood counts and smear morphology in patients with a positive viral respiratory panel (VRP) or SARS-CoV-2 test. RESULTS: A total of 97 peripheral blood samples (COVID-19 infection, 53; VRP positive, 44) from 50 patients (mean [SD] age, 45.8 [20.8] years; females 52%) were reviewed. There were no statistically significant differences in the demographic characteristics between the 2 groups. The most common peripheral blood abnormalities were anemia, thrombocytopenia, absolute lymphopenia, and reactive lymphocytes. The following peripheral blood findings were significantly associated with other viral respiratory infections compared with COVID-19 infection: low red blood cell count, low hematocrit, high mean corpuscular volume, thrombocytopenia, low mean platelet volume, high red cell distribution width, band neutrophilia, and toxic granulation in neutrophils. CONCLUSIONS: Our study showed that there are several peripheral blood count and morphologic abnormalities seen in patients with COVID-19, but most of these findings lack specificity as they are also seen in the other viral respiratory infections.
Assuntos
COVID-19 , Leucopenia , Trombocitopenia , Feminino , Humanos , Pessoa de Meia-Idade , SARS-CoV-2 , COVID-19/diagnóstico , Estudos Retrospectivos , Contagem de Células Sanguíneas , Trombocitopenia/diagnósticoRESUMO
CD4+ T-helper 17 (Th17) T cells are a key population in protective immunity during infection and in self-tolerance/autoimmunity. Through the secretion of IL-17, Th17 cells act in promotion of inflammation and are thus a major potential therapeutic target in autoimmune disorders. Recent reports have brought to light that the IL-17 family cytokines, IL-17A, IL-17F and IL-17AF, can directly act on CD4+ T-cells, both in murine and human systems, inducing functional changes in these cells. Here we show that this action is preferentially targeted toward naïve, but not memory, CD4+ T-cells. Naïve cells showed transcriptome changes as early as 48 hours post-IL-17 exposure, whereas memory cells remained unaffected as late as 7 days. These functional differences occurred despite similar IL-17 receptor expression on these subsets and were maintained in co-culture/transwell systems, with each subset maintaining its functional response to IL-17. Importantly, there were differences in downstream transcriptional signaling by the three IL-17 cytokines, with the IL-17AF heterodimer conferring both the greatest transcriptional change and most altered functional consequences. Detailed transcriptome analysis provides important insights into the genes and pathways that are modulated as a result of IL-17-mediated signaling and may serve as targets of future therapies.
Assuntos
Doenças Autoimunes , Interleucina-17 , Camundongos , Humanos , Animais , Interleucina-17/metabolismo , Citocinas/metabolismo , Linfócitos T CD4-Positivos , Células Th17 , Inflamação/metabolismo , Doenças Autoimunes/metabolismoRESUMO
OBJECTIVE: Despite successful endovascular therapy, a proportion of stroke patients exhibit long-term functional decline, regardless of the cortical reperfusion. Our objective was to evaluate the early activation of the adaptive immune response and its impact on neurological recovery in patients with large vessel occlusion (LVO). METHODS: Nineteen (13 females, 6 males) patients with acute LVO were enrolled in a single-arm prospective cohort study. During endovascular therapy (EVT), blood samples were collected from pre and post-occlusion, distal femoral artery, and median cubital vein (controls). Cytokines, chemokines, cellular and functional profiles were evaluated with immediate and follow-up clinical and radiographic parameters, including cognitive performance and functional recovery. RESULTS: In the hyperacute phase (within hours), adaptive immune activation was observed in the post-occlusion intra-arterial environment (post). Ischemic vascular tissue had a significant increase in T-cell-related cytokines, including IFN-γ and MMP-9, while GM-CSF, IL-17, TNF-α, IL-6, MIP-1a, and MIP-1b were decreased. Cellularity analysis revealed an increase in inflammatory IL-17+ and GM-CSF+ helper T-cells, while natural killer (NK), monocytes and B-cells were decreased. A correlation was observed between hypoperfused tissue, infarct volume, inflammatory helper, and cytotoxic T-cells. Moreover, helper and cytotoxic T-cells were also significantly increased in patients with improved motor function at 3 months. INTERPRETATION: We provide evidence of the activation of the inflammatory adaptive immune response during the hyperacute phase and the association of pro-inflammatory cytokines with greater ischemic tissue and worsening recovery after successful reperfusion. Further characterization of these immune pathways is warranted to test selective immunomodulators during the early stages of stroke rehabilitation.
Assuntos
Isquemia Encefálica , Disfunção Cognitiva , Transtornos das Habilidades Motoras , Feminino , Humanos , Masculino , Citocinas , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Imunidade , Interleucina-17 , Estudos Prospectivos , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/imunologia , Acidente Vascular Cerebral/terapia , Isquemia Encefálica/complicações , Isquemia Encefálica/imunologia , Isquemia Encefálica/terapia , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/imunologia , Transtornos das Habilidades Motoras/etiologia , Transtornos das Habilidades Motoras/imunologia , Doenças Neuroinflamatórias/imunologiaRESUMO
Intravascular (IV) perfusion of tissue fixative is commonly used in the field of neuroscience as the central nervous system tissues are exquisitely sensitive to handling and fixation artifacts which can affect downstream microscopic analysis. Both 10% neutral-buffered formalin (NBF) and 4% paraformaldehyde (PFA) are used, although IV perfusion with PFA is most commonly referenced. The study objective was to compare the severity of handling and fixation artifacts, semiquantitative scores of inflammatory and neurodegenerative changes, and quantitative immunohistochemistry following terminal IV perfusion of mice with either 10% NBF or 4% PFA in a model of experimental autoimmune encephalitis (EAE). The study included 24 mice; 12 were control animals not immunized and an additional 12 were immunized with PLP139-151 subcutaneously, harvested at day 20, and fixed in the same fashion. Equal numbers (4 per group) were perfused with 10% NBF or 4% PFA, and 4 were immersion-fixed in 10% NBF. NBF-perfused mice had less severe dark neuron artifact than PFA-perfused mice (P < .001). Immersion-fixed animals had significantly higher scores for oligodendrocyte halos, dark neuron artifact, and perivascular clefts than perfusion-fixed animals. Histopathology scores in EAE mice for inflammation, demyelination, and necrosis did not differ among fixation methods. Also, no significant differences in quantitative immunohistochemistry for CD3 and Iba-1 were observed in immunized animals regardless of the method of fixation. These findings indicate that IV perfusion of mice with 10% NBF and 4% PFA are similar and adequate fixation techniques in this model.
Assuntos
Encefalomielite Autoimune Experimental , Doenças dos Roedores , Animais , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/veterinária , Fixadores , Formaldeído , Imuno-Histoquímica , Camundongos , Perfusão/veterinária , Polímeros , Fixação de Tecidos/métodos , Fixação de Tecidos/veterináriaRESUMO
Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the CNS. We have previously demonstrated that CNS-specific CD8 T cells possess a disease-suppressive function in MS and variations of its animal model, experimental autoimmune encephalomyelitis (EAE), including the highly clinically relevant relapsing-remitting EAE disease course. Regulatory CD8 T cell subsets have been identified in EAE and other autoimmune diseases, but studies vary in defining phenotypic properties of these cells. In relapsing-remitting EAE, PLP178-191 CD8 T cells suppress disease, whereas PLP139-151 CD8 T cells lack this function. In this study, we used this model to delineate the unique phenotypic properties of CNS-specific regulatory PLP178-191 CD8 T cells versus nonregulatory PLP139-151 or OVA323-339 CD8 T cells. Using multiparametric flow cytometric analyses of phenotypic marker expression, we identified a CXCR3+ subpopulation among activated regulatory CD8 T cells, relative to nonregulatory counterparts. This subset exhibited increased degranulation and IFN-γ and IL-10 coproduction. A similar subset was also identified in C57BL/6 mice within autoregulatory PLP178-191 CD8 T cells but not within nonregulatory OVA323-339 CD8 T cells. This disease-suppressing CD8 T cell subpopulation provides better insights into functional regulatory mechanisms, and targeted enhancement of this subset could represent a novel immunotherapeutic approach for MS.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Encefalomielite Autoimune Experimental/imunologia , Esclerose Múltipla/imunologia , Bainha de Mielina/patologia , Subpopulações de Linfócitos T/imunologia , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/transplante , Separação Celular , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/patologia , Feminino , Citometria de Fluxo , Humanos , Interferon gama/metabolismo , Interleucina-10/metabolismo , Camundongos , Esclerose Múltipla/patologia , Bainha de Mielina/imunologia , Receptores CXCR3/metabolismo , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/transplanteRESUMO
OBJECTIVES: We sought to investigate the clinical utility of flow cytometry (FC) and fluorescence in situ hybridization (FISH) in the workup of myeloma. METHODS: We retrospectively reviewed the reports of bone marrow biopsies received for myeloma evaluation between October 2015 and January 2019. RESULTS: A total of 1,708 biopsy specimens from 469 myeloma patients (mean age, 64.5 years [SD, 9.3]; female, 41.4%) were reviewed. Both FC and FISH had comparable detection rates at the time of initial diagnosis (97.6% vs 98.8%) and for follow-up cases (28.6% vs 28.2%). FC and FISH results were concordant in 98.8% of the initial diagnosis cases and 89.6% of the follow-up cases. The FISH-positive (FISH+)/FC-negative (FC-) discordance and FISH-/FC+ discordance occurred among 81 (5.0%) and 87 (5.4%) follow-up cases. In comparison with all concordant cases, FISH+/FC- discordant cases were more likely to have received treatment with daratumumab (P < .05). CONCLUSIONS: Plasma cell-enriched FISH and FC have comparable abnormal plasma cell detection rates, and approximately 10% of the follow-up cases have discordant FISH and FC results in which residual disease is detected by only one of these modalities. FISH testing should be considered for cases with negative FC, especially in patients who have received treatment with daratumumab or in cases in which there is concern about specimen adequacy.
Assuntos
Citometria de Fluxo/métodos , Hibridização in Situ Fluorescente/métodos , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/patologia , Idoso , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Estudos de Coortes , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/tratamento farmacológico , Neoplasia Residual/diagnóstico , Estudos RetrospectivosRESUMO
Gut microbiota has emerged as an important environmental factor in the pathobiology of multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS). Both genetic and environmental factors have been shown to play an important role in MS. Among genetic factors, the human leukocyte antigen (HLA) class II allele such as HLA-DR2, DR3, DR4, DQ6, and DQ8 show the association with the MS. We have previously used transgenic mice expressing MS susceptible HLA class II allele such as HLA-DR2, DR3, DQ6, and DQ8 to validate significance of HLA alleles in MS. Although environmental factors contribute to 2/3 of MS risk, less is known about them. Gut microbiota is emerging as an imporatnt environmental factor in MS pathogenesis. We and others have shown that MS patients have distinct gut microbiota compared to healthy control (HC) with a lower abundance of Prevotella. Additionally, the abundance of Prevotella increased in patients receiving disease-modifying therapies (DMTs) such as Copaxone and/or Interferon-beta (IFNß). We have previously identified a specific strain of Prevotella (Prevotella histicola), which can suppress experimental autoimmune encephalomyelitis (EAE) disease in HLA-DR3.DQ8 transgenic mice. Since Interferon-ß-1b [IFNß (Betaseron)] is a major DMTs used in MS patients, we hypothesized that treatment with the combination of P. histicola and IFNß would have an additive effect on the disease suppression. We observed that treatment with P. histicola suppressed disease as effectively as IFNß. Surprisingly, the combination of P. histicola and IFNß was not more effective than either treatment alone. P. histicola alone or in combination with IFNß increased the frequency and number of CD4+FoxP3+ regulatory T cells in the gut-associated lymphoid tissue (GALT). Treatment with P. histicola alone, IFNß alone, and in the combination decreased frequency of pro-inflammatory IFN-γ and IL17-producing CD4+ T cells in the CNS. Additionally, P. histicola alone or IFNß alone or the combination treatments decreased CNS pathology, characterized by reduced microglia and astrocytic activation. In conclusion, our study indicates that the human gut commensal P. histicola can suppress disease as effectively as commonly used MS drug IFNß and may provide an alternative treatment option for MS patients.
Assuntos
Anti-Inflamatórios/farmacologia , Encefalomielite Autoimune Experimental/prevenção & controle , Microbioma Gastrointestinal , Interferon beta/farmacologia , Intestinos/microbiologia , Prevotella/fisiologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/imunologia , Astrócitos/metabolismo , Astrócitos/microbiologia , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/microbiologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/microbiologia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Cadeias beta de HLA-DQ/genética , Cadeias HLA-DRB1/genética , Humanos , Interferon gama/metabolismo , Interleucina-17/metabolismo , Tecido Linfoide/efeitos dos fármacos , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Tecido Linfoide/microbiologia , Masculino , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/imunologia , Microglia/metabolismo , Microglia/microbiologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/microbiologiaRESUMO
Autoimmune diseases are characterized by regulatory deficit in both the CD4+ and CD8+ T-cell compartments. We have shown that CD8+ T-cells associated with acute relapse of multiple sclerosis are significantly deficient in their immune suppressive ability. We hypothesized that distinct CD8+ cytotoxic T-cell (Tc) lineages, determined by cytokine milieu during naïve T-cell differentiation, may harbor differential ability to suppress effector CD4+ T-cells. We differentiated purified human naïve CD8+ T-cells in vitro toward Tc0 (media control), Tc1 and Tc17 lineages. Using in vitro flow cytometric suppression assays, we observed that Tc0 and Tc17 cells had similar suppressive ability. In contrast, Tc1 cells showed significant loss of suppressive ability against ex vivo CD4+ T-cells and in vitro-differentiated Th0, Th1 and Th17 cells. Of note, Tc1 cells were also suboptimal in suppressing CD4-induced acute xenogeneic graft versus host disease (xGVHD) in vivo. Tc subtypes derived under various cytokine combinations revealed that IL-12-containing conditions resulted in less suppressive cells exhibiting dysregulated cytotoxic degranulation. RNA sequencing transcriptome analyses indicated differential regulation of inflammatory genes and enrichment in GM-CSF-associated pathways. These studies provide insights into the role of T-cell differentiation in CD8 suppressive biology and may reveal therapeutically targetable pathways to reverse suppressive deficit during immune-mediated disease.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Interleucina-12/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular , Feminino , Doença Enxerto-Hospedeiro/imunologia , Humanos , Tolerância Imunológica , CamundongosRESUMO
Factors regulating self-antigen directed immune-responses in autoimmunity are poorly understood. Signal regulatory protein gamma (SIRPγ) is a human T-cell specific protein with genetic variants associated with type 1 diabetes (T1D). SIRPγ's function in the immune system remains unclear. We show that T1D and relapsing remitting multiple sclerosis (RRMS) subjects have significantly greater frequency of rs2281808 T genetic variant, that correlates with reduced SIRPγ-expression in T-cells. Importantly, reduced SIRPγ-expression in RRMS and T1D subjects was not restricted to T variant, suggesting SIRPγ-expression is also regulated by disease specific factors in autoimmunity. Interestingly, increased frequencies of SIRPγlow T-cells in RRMS and T1D positively correlated with proinflammatory molecules from T-cells. Finally, we show that SIRPγlow T-cells have enhanced pathogenecity in vivo in a GVHD model. These findings suggest that decreased-SIRPγ expression, either determined by genetic variants or through peripherally acquired processes, may have a mechanistic link to autoimmunity through induction of hyperactive T-cells.
Assuntos
Antígenos de Diferenciação/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Regulação da Expressão Gênica , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Receptores Imunológicos/genética , Linfócitos T/metabolismo , Adulto , Alelos , Animais , Autoimunidade , Estudos de Casos e Controles , Feminino , Genótipo , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Recidiva , Linfócitos T/imunologia , Adulto JovemRESUMO
Cerebral vasospasm (VSP) is a common phenomenon after aneurysmal subarachnoid hemorrhage (aSAH) and contributes to neurocognitive decline. The natural history of the pro-inflammatory immune response after aSAH has not been prospectively studied in human cerebrospinal fluid (CSF). In this pilot study, we aimed to identify specific immune mediators of VSP after aSAH. Peripheral blood (PB) and CSF samples from patients with aSAH were prospectively collected at different time-points after hemorrhage: days 0-1 (acute); days 2-4 (pre-VSP); days 5-9 (VSP) and days 10 + (post-VSP peak). Presence and severity of VSP was assessed with computed tomography angiography/perfusion imaging and clinical examination. Cytokine and immune mediators' levels were quantified using ELISA. Innate and adaptive immune cells were characterized by flow cytometry, and cell counts at different time-points were compared with ANOVA. Confocal immunostaining was used to determine the presence of specific immune cell populations detected in flow cytometry. Thirteen patients/aneurysms were included. Five (38.5%) patients developed VSP after a mean of 6.8 days from hemorrhage. Flow cytometry demonstrated decreased numbers of CD45+ cells during the acute phase in PB of aSAH patients compared with healthy controls. In CSF of VSP patients, NK cells (CD3-CD161 +) were increased during the acute phase and progressively declined, whereas CD8+CD161+ lymphocytes significantly increased at days 5-9. Microglia cells (CD45dimCD11b +) increased over time after SAH. This increase was particularly significant in patients with VSP. Levels of VEGF and MMP-9 were consistently higher in VSP patients, with the highest difference occurring at the acute phase. Confocal immunostaining demonstrated the presence of CD8+CD161+ lymphocytes in the arterial wall of two unruptured intracranial aneurysms. In this preliminary study, human CSF showed active presence of innate and adaptive immune cells after aSAH. CD8+CD161+ lymphocytes may have an important role in the inflammatory response after aneurysmal rupture and were identified in the aneurysmal wall of unruptured brain aneurysms. Microglia activation occurs 6 + days after aSAH.
Assuntos
Aneurisma Roto/complicações , Imunidade , Hemorragia Subaracnóidea/etiologia , Hemorragia Subaracnóidea/metabolismo , Imunidade Adaptativa , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Citocinas/metabolismo , Feminino , Humanos , Imunidade Inata , Imuno-Histoquímica , Mediadores da Inflamação , Masculino , Pessoa de Meia-Idade , Hemorragia Subaracnóidea/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Vasoespasmo Intracraniano/etiologia , Vasoespasmo Intracraniano/metabolismoRESUMO
Untoward effector CD4+ T cell responses are kept in check by immune regulatory mechanisms mediated by CD4+ and CD8+ T cells. CD4+ T helper 17 (Th17) cells, characterized by IL-17 production, play important roles in the pathogenesis of autoimmune diseases (such as arthritis, multiple sclerosis, psoriasis, inflammatory bowel disease, among others) and in the host response to infection and cancer. Here, we demonstrate that human CD4+ T cells cells exposed to a Th17-differentiating milieu are significantly more resistant to immune suppression by CD8+ T cells compared to control Th0 cells. This resistance is mediated, in part, through the action of IL-17A, IL-17F, and IL-17AF heterodimer through their receptors (IL-17RA and IL-17RC) on CD4+ T cells themselves, but not through their action on CD8+ T cells or APC. We further show that IL-17 can directly act on non-Th17 effector CD4+ T cells to induce suppressive resistance, and this resistance can be reversed by blockade of IL-1ß, IL-6, or STAT3. These studies reveal a role for IL-17 cytokines in mediating CD4-intrinsic immune resistance. The pathways induced in this process may serve as a critical target for future investigation and immunotherapeutic intervention.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Tolerância Imunológica/imunologia , Interleucina-17/imunologia , Células Th17/imunologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular , Humanos , Interleucina-17/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Subpopulações de Linfócitos T/imunologiaRESUMO
Investigating the complex cellular interplay controlling immunopathogenic and immunoregulatory responses is critical for understanding multiple sclerosis (MS) and for developing successful immunotherapies. Our group has demonstrated that CNS myelin-specific CD8 T cells unexpectedly harbor immune regulatory capacity in both mouse and human. In particular, PLP178-191-specific CD8 T cells (PLP-CD8) robustly suppress the MS mouse model experimental autoimmune encephalomyelitis. We have recently shown that this depends on PLP-CD8 elaborating IFN-γ and perforin in a coordinated suppression program over time. However, the cellular target and downstream effects of CD8 T cell-derived IFN-γ remains poorly understood. In this study, we show that although wild-type (WT) PLP-CD8 were robustly suppressive in IFN-γR-deficient mice, IFN-γR-deficient PLP-CD8 exhibited suboptimal suppression in WT mice. Compared with WT counterparts, IFN-γR-deficient PLP-CD8 were defective in suppressing disease in IFN-γ-deficient recipients, a scenario in which the only IFN-γ available to WT PLP-CD8 is that which they produce themselves. Further, we found that IFN-γR-deficient PLP-CD8 exhibited altered granzyme/IFN-γ profiles, altered migration in recipients, and deficits in killing capacity in vivo. Collectively, this work suggests that IFN-γ responsiveness allows myelin-specific CD8 T cells to optimally perform autoregulatory function in vivo. These insights may help elucidate future adoptive immunotherapeutic approaches for MS patients.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Sistema Nervoso Central/imunologia , Encefalomielite Autoimune Experimental/imunologia , Interferon gama/metabolismo , Esclerose Múltipla/imunologia , Animais , Autoantígenos/imunologia , Autoimunidade , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Tolerância Imunológica , Interferon gama/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Proteolipídica de Mielina/imunologia , Bainha de Mielina/imunologia , Fragmentos de Peptídeos/imunologia , Receptores de Interferon/genética , Receptores de Interferon/metabolismo , Receptor de Interferon gamaRESUMO
Therapeutic success of B cell-targeting approaches in multiple sclerosis (MS) has intensified research into the pathogenic and regulatory roles these cells play in demyelinating disease. Dissecting the function of B cells in the MS mouse model experimental autoimmune encephalomyelitis (EAE) is largely confined to induction with either the myelin oligodendrocyte glycoprotein epitope MOG35-55 or the full-length recombinant human MOG protein, the latter representing the most-used B cell-dependent EAE model. There is a clear need to investigate B cell function in additional myelin antigen contexts. Unlike MOG35-55, where lack of B cells yields more severe disease, we show here that the immunodominant myelin proteolipid protein epitope (PLP178-191) elicited identical EAE in WT and µMT mice, suggesting an absence of B cell engagement by this peptide. We hypothesized that a longer PLP antigen may better engage B cells and designed a peptide encompassing the extracellular domains (ECD) of PLP. We demonstrate here that PLPECD-immunized B cell-deficient mice failed to exhibit EAE. In contrast, PLPECD induced EAE not only in WT mice, but in B cell-sufficient mice incapable of secreting antibodies, suggesting a predominant antigen presentation role. These results establish a novel, efficient B cell-dependent EAE model.
Assuntos
Linfócitos B/imunologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental , Esclerose Múltipla , Glicoproteína Mielina-Oligodendrócito , Animais , Encefalomielite Autoimune Experimental/imunologia , Epitopos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/etiologia , Esclerose Múltipla/imunologia , Proteína Proteolipídica de Mielina/imunologia , Fragmentos de Peptídeos/imunologia , Domínios ProteicosRESUMO
Multiple sclerosis (MS) is a chronic demyelinating disorder of the central nervous system (CNS). Its corresponding animal model, experimental autoimmune encephalomyelitis (EAE), is widely used to understand disease pathogenesis and test novel therapeutic agents. However, existing methods to score EAE disease severity are subjective and often vary between individual researchers, making it difficult to translate findings across different studies. An enhanced automated method of disease scoring would eliminate subjectivity and reduce operator-dependent errors. Here, we used an Infra-Red Activity Monitoring System (IRAMS) to measure murine locomotor activity as a surrogate measure of disease severity and compared it to standard EAE scoring methods. In mice immunized with CNS-specific myelin antigens, we observed an inverse correlation between disease severity and mouse activity, with the IRAMS showing enhanced disease scoring compared to standard EAE scoring methods. Relative to standard EAE scoring methods, IRAMS showed comparable measurement of disease relapses and remissions in the SJL/J-relapsing-remitting model of EAE, and could comparably assess the therapeutic efficiency of the MS drug, Copaxone (Glatiramer acetate-GA). Thus, the IRAMS is a method to measure disease severity in EAE without subjective bias and is a tool to consistently assess the efficacy of novel therapeutic agents for MS.
Assuntos
Encefalomielite Autoimune Experimental/patologia , Locomoção/fisiologia , Esclerose Múltipla Recidivante-Remitente/patologia , Animais , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/patologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/tratamento farmacológico , Feminino , Acetato de Glatiramer/farmacologia , Humanos , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/patologiaRESUMO
BACKGROUND: Flow cytometry is widely used for minimal residual disease (MRD) detection in plasma cell myeloma (PCM). Recently, an increasing number of studies have demonstrated that polytypic plasma cells (PPCs) display greater immunophenotypic variation than previously appreciated. Our aim was to further characterize the immunophenotype of PPC in this setting. METHODS: PPC in 102 bone marrow specimens (93 MRD-negative post-treatment PCM and 9 negative initial evaluations for plasma cell neoplasm) were evaluated by 10-color flow cytometry. Frequency of CD19, CD27, CD28, CD56, CD117, and CD138 expression was assessed. RESULTS: All cases showed CD27 and CD19 positivity. CD117 was uniformly negative. Percentage of CD138 expression was variable with one negative case (<20% expression). Percentage of CD28 and CD56 expression was variable and included 11 CD28+ cases as well as 38 CD56+ cases. Forty-two percent (43 of 102) cases showed atypical expression of at least one marker with 36 cases (35%) showing atypical expression of a single marker and 7 cases (7%) showing dual atypical marker expression (CD56+/CD28+). CONCLUSIONS: Considerable immunophenotypic variation exists in PPC. The assessment of cytoplasmic light chain distribution, in conjunction with surface marker expression, is recommended to avoid diagnostic inaccuracy in MRD evaluation. © 2019 International Clinical Cytometry Society.
Assuntos
Citometria de Fluxo , Imunofenotipagem , Mieloma Múltiplo/diagnóstico , Plasmócitos/patologia , Antígenos CD/biossíntese , Humanos , Mieloma Múltiplo/metabolismo , Plasmócitos/metabolismoRESUMO
Multiple sclerosis (MS) is a demyelinating disease of the central nervous system. We and others have shown that there is enrichment or depletion of some gut bacteria in MS patients compared to healthy controls (HC), suggesting an important role of the gut bacteria in disease pathogenesis. Thus, specific gut bacteria that are lower in abundance in MS patients could be used as a potential treatment option for this disease. In particular, we and others have shown that MS patients have a lower abundance of Prevotella compared to HC, whereas the abundance of Prevotella is increased in patients that receive disease-modifying therapies such as Copaxone® (Glatiramer acetate-GA). This inverse correlation between the severity of MS disease and the abundance of Prevotella suggests its potential for use as a therapeutic option to treat MS. Notably we have previously identified a specific strain, Prevotella histicola (P. histicola), that suppresses disease in the animal model of MS, experimental autoimmune encephalomyelitis (EAE) compared with sham treatment. In the present study we analyzed whether the disease suppressing effects of P. histicola synergize with those of the disease-modifying drug Copaxone® to more effectively suppress disease compared to either treatment alone. Treatment with P. histicola was as effective in suppressing disease as treatment with Copaxone®, whereas the combination of P. histicola plus Copaxone® was not more effective than either individual treatment. P. histicola-treated mice had an increased frequency and number of CD4+FoxP3+ regulatory T cells in periphery as well as gut and a decreased frequency of pro-inflammatory IFN-γ and IL17-producing CD4 T cells in the CNS, suggesting P. histicola suppresses disease by boosting anti-inflammatory immune responses and inhibiting pro-inflammatory immune responses. In conclusion, our study indicates that the human gut commensal P. histicola can suppress disease as efficiently as Copaxone® and may provide an alternative treatment option for MS patients.
Assuntos
Encefalomielite Autoimune Experimental/terapia , Acetato de Glatiramer/uso terapêutico , Imunossupressores/uso terapêutico , Prevotella , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Encefalomielite Autoimune Experimental/microbiologia , Encefalomielite Autoimune Experimental/patologia , Microbioma Gastrointestinal , Antígeno HLA-DR3/genética , Humanos , Camundongos Transgênicos , Esclerose Múltipla/microbiologia , Esclerose Múltipla/patologia , Esclerose Múltipla/terapia , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologiaRESUMO
Multiple genome-wide association studies have shown that the single-nucleotide polymorphism (SNP) rs2281808 TT variant, present within the signal regulatory protein gamma (SIRPG) gene, is associated with autoimmune diseases, such as type 1 diabetes. SIRPγ is the only SIRP expressed on T cells. The role of SIRPγ in human T-cells or the effect of the TT variant are poorly understood. In this short report, we demonstrate the rather unusual finding that this intronic SNP is associated with a reduction of SIRPγ expression on T cells, both in healthy subjects as well as patients with type 1 diabetes. Using this information, we propose that a simple flow cytometric detection of SIRPγ could be a potential diagnostic testing approach for the presence of SNP in the appropriate clinical context.
Assuntos
Antígenos de Diferenciação/genética , Autoimunidade , Polimorfismo de Nucleotídeo Único , Receptores Imunológicos/genética , Adolescente , Adulto , Idoso , Diabetes Mellitus Tipo 1/genética , Citometria de Fluxo , Genótipo , Humanos , Pessoa de Meia-Idade , Fenótipo , Adulto JovemRESUMO
Multiple Sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS). We have shown that CNS-specific CD8 T cells (CNS-CD8) possess a disease suppressive function in MS and its animal model, experimental autoimmune encephalomyelitis (EAE). Previous studies have focused on the role of these cells predominantly in chronic models of disease, but the majority of MS patients present with a relapsing-remitting disease course. In this study, we evaluated the therapeutic role of CD8 T cells in the context of relapsing-remitting disease (RR-EAE), using SJL mice. We found that PLP178-191- and MBP84-104-CD8 ameliorated disease severity in an antigen-specific manner. In contrast, PLP139-151-CD8 did not suppress disease. PLP178-191-CD8 were able to reduce the number of relapses even when transferred during ongoing disease. We further ascertained that the suppressive subset of CD8 T cells was contained within the CD25+ CD8 T cell compartment post-in vitro activation with PLP178-191. Using Listeria monocytogenes (LM) encoding CNS antigens to preferentially prime suppressive CDS T cells in vivo, we show that LM infection induced disease suppressive CD8 T cells that protected and treated PLP178-191 disease. Importantly, a combination of PLP178-191-CDs transfer boosted by LM-PLP175-194 infection effectively treated ongoing disease induced by a non-cognate peptide (PLP139-151), indicating that this approach could be effective even in the context of epitope spreading. These data support a potential immunotherapeutic strategy using CD8 transfer and/or LM vaccination to boost disease regulatory CD8 T cells.