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
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
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
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 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
Regulatory T-cells (Tregs) are vital for maintaining immunological self-tolerance, and the transcription factor FOXP3 is considered critical for their development and function. Peripheral Treg induction may significantly contribute to the total Treg pool in healthy adults, and this pathway may be enhanced in thymic-deficient conditions like multiple sclerosis (MS). Here, we evaluated iTreg formation from memory versus naïve CD4(+)CD25(-) T-cell precursors. We report the novel finding that memory T-cells readily expressed CD25 and FOXP3, and demonstrated significantly greater suppressive function. Additionally, the CD25(-)FOXP3(-) fraction of stimulated memory T-cells also displayed robust suppression not observed in naïve counterparts or ex vivo resting (CD25(-)) T-cells. This regulatory population was present in both healthy subjects and clinically-quiescent MS patients, but was specifically deficient during disease exacerbation. These studies indicate that iTreg development and function are precursor dependent. Furthermore, MS quiescence appears to correlate with restoration of suppressive function in memory-derived CD4(+)CD25(-)FOXP3(-) iTregs.
Assuntos
Antígenos CD4/imunologia , Fatores de Transcrição Forkhead/imunologia , Memória Imunológica , Subunidade alfa de Receptor de Interleucina-2/imunologia , Esclerose Múltipla/imunologia , Linfócitos T Reguladores/imunologia , Doença Aguda , Adulto , Anticorpos/farmacologia , Antígenos CD4/genética , Estudos de Casos e Controles , Diferenciação Celular , Fatores de Transcrição Forkhead/deficiência , Fatores de Transcrição Forkhead/genética , Expressão Gênica , Humanos , Tolerância Imunológica , Subunidade alfa de Receptor de Interleucina-2/deficiência , Subunidade alfa de Receptor de Interleucina-2/genética , Ativação Linfocitária/efeitos dos fármacos , Esclerose Múltipla/genética , Esclerose Múltipla/patologia , Cultura Primária de Células , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/patologiaRESUMO
Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the CNS, and CD8 T cells are the predominant T cell population in MS lesions. Given that transfer of CNS-specific CD8 T cells results in an attenuated clinical demyelinating disease in C57BL/6 mice with immunization-induced experimental autoimmune encephalomyelitis (EAE), we investigated the cellular targets and mechanisms of autoreactive regulatory CD8 T cells. In this study we report that myelin oligodendrocyte glycoprotein peptide (MOG35-55)-induced CD8 T cells could also attenuate adoptively transferred, CD4 T cell-mediated EAE. Whereas CD8(-/-) mice exhibited more severe EAE associated with increased autoreactivity and inflammatory cytokine production by myelin-specific CD4 T cells, this was reversed by adoptive transfer of MOG-specific CD8 T cells. These autoregulatory CD8 T cells required in vivo MHC class Ia (K(b)D(b)) presentation. Interestingly, MOG-specific CD8 T cells could also suppress adoptively induced disease using wild-type MOG35-55-specific CD4 T cells transferred into K(b)D(b-/-) recipient mice, suggesting direct targeting of encephalitogenic CD4 T cells. In vivo trafficking analysis revealed that autoregulatory CD8 T cells are dependent on neuroinflammation for CNS infiltration, and their suppression/cytotoxicity of MOG-specific CD4 T cells is observed both in the periphery and in the CNS. These studies provide important insights into the mechanism of disease suppression mediated by autoreactive CD8 T cells in EAE.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/prevenção & controle , Transferência Adotiva , Sequência de Aminoácidos , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Doenças Autoimunes/prevenção & controle , Linfócitos T CD8-Positivos/transplante , Células Cultivadas , Citotoxicidade Imunológica , Encefalomielite Autoimune Experimental/patologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência MolecularRESUMO
Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS). Although its etiology remains unknown, pathogenic T cells are thought to underlie MS immune pathology. We recently showed that MS patients harbor CNS-specific CD8+ Tregs that are deficient during disease relapse. We now demonstrate that CNS-specific CD8+ Tregs were cytolytic and could eliminate pathogenic CD4+ T cells. These CD8+ Tregs were present primarily in terminally differentiated (CD27-, CD45RO-) subset and their suppression was IFNγ, perforin and granzyme B-dependent. Interestingly, MS patients with acute relapse displayed a significant loss in terminally differentiated CD8+ T cells, with a concurrent loss in expression of perforin and granzyme B. Pre-treatment of exacerbation-derived CD8+ T cells with IL-12 significantly restored suppressive capability of these cells through upregulation of granzyme B. Our studies uncover immune-suppressive mechanisms of CNS-specific CD8+ Tregs, and may contribute to design of novel immune therapies for MS.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Esclerose Múltipla/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Diferenciação Celular/imunologia , Progressão da Doença , Feminino , Granzimas/biossíntese , Granzimas/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Interferon gama/imunologia , Interleucina-12/farmacologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/patologia , Perforina/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Regulação para Cima , Adulto JovemRESUMO
The autoimmune disease systemic lupus erythematosus is characterized by loss of tolerance to nuclear Ags and a heightened inflammatory environment, which together result in end organ damage. Lyn-deficient mice, a model of systemic lupus erythematosus, lack an inhibitor of B-cell and myeloid cell activation. This results in B-cell hyper-responsiveness, plasma cell accumulation, autoantibodies, and glomerulonephritis (GN). IL-21 is associated with autoimmunity in mice and humans and promotes B-cell differentiation and class switching. Here, we explore the role of IL-21 in the autoimmune phenotypes of lyn(-/-) mice. We find that IL-21 mRNA is reduced in the spleens of lyn(-/-) IL-6(-/-) and lyn(-/-) Btk(lo) mice, neither of which produce pathogenic autoantibodies or develop significant GN. While IL-21 is dispensable for plasma cell accumulation and IgM autoantibodies in lyn(-/-) mice, it is required for anti-DNA IgG antibodies and some aspects of T-cell activation. Surprisingly, GN still develops in lyn(-/-) IL-21(-/-) mice. This likely results from the presence of IgG autoantibodies against a limited set of non-DNA Ags. These studies identify a specific role for IL-21 in the class switching of anti-DNA B cells and demonstrate that neither IL-21 nor anti-DNA IgG is required for kidney damage in lyn(-/-) mice.
Assuntos
Anticorpos Antinucleares/imunologia , DNA/imunologia , Imunoglobulina G/imunologia , Interleucinas/imunologia , Rim/imunologia , Quinases da Família src/genética , Tirosina Quinase da Agamaglobulinemia , Animais , Autoanticorpos/imunologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , DNA/genética , Glomerulonefrite/genética , Glomerulonefrite/imunologia , Glomerulonefrite/metabolismo , Interleucina-6/deficiência , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-6/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Rim/metabolismo , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Mieloides/imunologia , Células Mieloides/metabolismo , Plasmócitos/imunologia , Plasmócitos/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Baço/imunologia , Baço/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Quinases da Família src/imunologia , Quinases da Família src/metabolismoRESUMO
Daily administration of FDA-approved glatiramer acetate (GA) has beneficial effects on clinical course of relapsing remitting multiple sclerosis (RRMS). Although mechanisms of GA-action have been widely investigated and partially understood, immediate immune dynamics following GA-therapy are unknown. In the present study, we characterized the immediate effects of GA on phenotype, quantity and function of immune cells in MS patients. Prominent changes in immune cells were detected within 4-12h post-first GA-injection. T-cell modulation included significantly decreased CD4/CD8 ratio, perturbed homeostasis of predominantly CD8+ T-cells, significant enhancement in CD8+ T-cell mediated suppression and inhibitory potential of induced CD4-suppressors. Changes in APC were restricted to monocytes and included reduced stimulatory capacity in MLR and significantly increased IL-10 and TNF-α production. Our study provides the first evidence that GA treatment induces rapid immunologic changes within hours of first dose. Interestingly, these responses are not only restricted to innate immune cells but also include complex modulation of T-cell functionality.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Fatores Imunológicos/uso terapêutico , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Peptídeos/uso terapêutico , Adulto , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Feminino , Acetato de Glatiramer , Humanos , Interleucina-10/imunologia , Teste de Cultura Mista de Linfócitos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/imunologia , Fator de Necrose Tumoral alfa/imunologia , Adulto JovemRESUMO
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
FMS-like tyrosine kinase 3 (FLT3) mutation in T lymphoblastic leukemia/lymphoma (T-LL) is rare (â¼4%) and reported only in cases with CD117 expression. This study aimed to identify the immunophenotypic features that may predict FLT3 mutations. We report 3 (43%) of 7 CD117(+) T-LL cases harboring FLT3-internal tandem duplication mutation. Compared with 4 FLT3-unmutated cases, all 3 FLT3-mutated cases had a distinct immunophenotype (CD1a(-)/CD2(+)/CD7(+)/CD34(+)/CD117(uniform+)/Tdt(+)) corresponding to the stage of earliest thymic T-cell progenitors possessing myeloid lineage potential. Indeed, all FLT3-mutated T-LL cases expressed myeloperoxidase on a very small subset of blasts and, thus, may be further considered a mixed phenotype acute leukemia, T/myeloid, by the 2008 World Health Organization classification scheme. We conclude that this unique immunophenotype (CD1a(-)/CD2(+)/CD7(+)/CD34(+)/CD117(+)/Tdt(+)) is a better predictor of FLT3 mutation than sole CD117 expression.
Assuntos
Mutação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Tirosina Quinase 3 Semelhante a fms/genética , Adolescente , Adulto , Idoso , Criança , Feminino , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células T Precursoras/classificação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Linfócitos T/imunologiaRESUMO
Recent evidence suggests that B- and T-cell interactions may be paramount in relapsing-remitting MS (RRMS) disease pathogenesis. We hypothesized that memory B-cell pools from RRMS patients may specifically harbor a subset of potent neuro-APC that support neuro-Ag reactive T-cell proliferation and cytokine secretion. To test this hypothesis, we compared CD80 and HLA-DR expression, IL-10 and lymphotoxin-α secretion, neuro-Ag binding capacity, and neuro-Ag presentation by memory B cells from RRMS patients to naïve B cells from RRMS patients and to memory and naïve B cells from healthy donors (HD). We identified memory B cells from some RRMS patients that elicited CD4(+) T-cell proliferation and IFN-γ secretion in response to myelin basic protein and myelin oligodendrocyte glycoprotein. Notwithstanding the fact that the phenotypic parameters that promote efficient Ag presentation were observed to be similar between RRMS and HD memory B cells, a corresponding capability to elicit CD4(+) T-cell proliferation in response to myelin basic protein and myelin oligodendrocyte glycoprotein was not observed in HD memory B cells. Our results demonstrate for the first time that the memory B-cell pool in RRMS harbors neuro-Ag specific B cells that can activate T cells.
Assuntos
Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Memória Imunológica/imunologia , Interferon gama/biossíntese , Proteína Básica da Mielina/imunologia , Glicoproteína Associada a Mielina/imunologia , Adulto , Estudos de Coortes , Feminino , Citometria de Fluxo , Humanos , Imunofenotipagem , Interferon gama/sangue , Interferon gama/imunologia , Ativação Linfocitária , Linfotoxina-alfa/imunologia , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/imunologia , Proteínas da Mielina , Glicoproteína Mielina-Oligodendrócito , Adulto JovemRESUMO
Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS). MS is thought to be T-cell-mediated, with prior research predominantly focusing on CD4+ T-cells. There is a high prevalence of CNS-specific CD8+ T-cell responses in MS patients and healthy subjects. However, the role of neuroantigen-specific CD8+ T-cells in MS is poorly understood, with the prevalent notion that these may represent pathogenic T-cells. We show here that healthy subjects and MS patients demonstrate similar magnitudes of CD8+ and CD4+ T-cell responses to various antigenic stimuli. Interestingly, CD8+ T-cells specific for CNS autoantigens, but not those specific for control foreign antigens, exhibit immune regulatory ability, suppressing proliferation of CD4+CD25- T-cells when stimulated by their cognate antigen. While CD8+ T-cell-mediated immune suppression is similar between healthy subjects and clinically quiescent treatment-naïve MS patients, it is significantly deficient during acute exacerbation of MS. Of note, the recovery of neuroantigen-specific CD8+ T-cell suppression correlates with disease recovery post-relapse. These studies reveal a novel immune suppressor function for neuroantigen-specific CD8+ T-cells that is clinically relevant in the maintenance of peripheral tolerance and the intrinsic regulation of MS immune pathology.
Assuntos
Autoantígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Esclerose Múltipla/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , Proliferação de Células , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Feminino , Citometria de Fluxo , Humanos , Subunidade alfa de Receptor de Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/metabolismo , Linfócitos T Reguladores/metabolismo , Adulto JovemRESUMO
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
Primary effusion lymphoma is a human herpes virus 8 (HHV-8)-associated large cell lymphoma of body cavities. Detailed large-scale clinicopathological studies are rarely reported, and the underlying mechanism of lymphomagenesis remains elusive. In the present report, we studied the clinicodemographic, immunophenotypic, and cytomorphological features on a cohort of 12 cases of primary effusion lymphoma. In contrast to HHV-8, which was positive in all nine cases tested (100%), HIV was found in 75% (9/12) of cases, whereas the three HIV-negative cases were either in elderly patients (one with hepatitis C virus infection and one with asbestoses exposure) or in a heart transplantation recipient. By flow cytometry, the antigens expressed in descending order were CD38, CD71, HLA-DR, CD30, and CD45RO. B-cell markers were largely negative. Cytomorphologically, all cases showed atypical to anaplastic morphology. Notch1, a member of transmembrane signal transduction family, was found in six of seven HHV-8-positive cases (86%). In agreement with in vitro studies using human primary effusion lymphoma cell lines, we have found that Notch1 was expressed in the majority of HHV-8-positive primary effusion lymphoma cases, corroborating the notion that Notch1 may have an important role in HHV-8-mediated lymphomagenesis of primary effusion lymphoma.