RESUMO
We induced systemic sclerosis (SSc)-like disease in both wild-type and Dnase1l3-deficient mice using two distinct approaches involving bleomycin and hypochlorous acid injections. Our observations revealed that the deficiency in DNASE1L3 did not affect tissue fibrosis or inflammation caused by these treatments. Despite the association of single nucleotide polymorphisms in humans with SSc pathogenesis, our study demonstrates that DNASE1L3 is dispensable in two inducible murine models of SSc-like pathogenesis.
Assuntos
Bleomicina , Modelos Animais de Doenças , Endodesoxirribonucleases , Camundongos Knockout , Escleroderma Sistêmico , Animais , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/patologia , Escleroderma Sistêmico/imunologia , Camundongos , Endodesoxirribonucleases/deficiência , Endodesoxirribonucleases/genética , Humanos , Ácido Hipocloroso , Fibrose , Camundongos Endogâmicos C57BLRESUMO
Dendritic cells (DCs) are antigen-presenting cells (APCs) that shape innate and adaptive immunity. There are multiple subsets of DCs distinguished according to their phenotype and functional specialization. DCs are present in lymphoid organs and across multiple tissues. However, their frequency and numbers at these sites are very low making their functional study difficult. Multiple protocols have been developed to generate DCs in vitro from bone marrow progenitors, but they do not fully recapitulate DC complexity found in vivo. Therefore, directly amplifying endogenous DCs in vivo appears as an option to overcome this specific caveat. In this chapter, we describe a protocol to amplify murine DCs in vivo by the injection of a B16 melanoma cell line expressing the trophic factor FMS-like tyrosine kinase 3 ligand (Flt3L). We have also compared two methods of magnetic sorting of amplified DCs, both giving high yields of total murine DCs, but different representation of the main DC subsets found in vivo.
Assuntos
Células Dendríticas , Proteínas de Membrana , Camundongos , Animais , Proteínas de Membrana/metabolismo , Baço/metabolismoRESUMO
Chronic lymphocytic leukemia (CLL) is characterized by an expansion of mature B cells in the bone marrow, peripheral lymphoid organs, and blood. CD4 T helper (Th) lymphocytes significantly contribute to the physiopathology of CLL, but the subset(s) of Th cell involved in CLL pathogenesis is (are) still under debate. In this study, we performed flow cytometry analysis of the circulatory T cells of untreated CLL patients and observed an increase in follicular helper T cells (Tfh), which is a subset of T cells specialized in B cell help. Elevated numbers of Tfh cells correlated with disease severity as measured by the Binet staging system. Tfh from CLL patients were activated and skewed toward a Th1 profile as evidenced by their PD-1+IL-21+IFNγ+ phenotype and their CXCR3+CCR6- chemokine receptor profile. Tfh efficiently enhanced B-CLL survival and proliferation through IL-21 but independently of IFNγ. Finally, we observed an inverse correlation between the Tfh1 and IgA and IgG serum levels in patients, suggesting a role for this Tfh subset in the immune dysfunction associated with CLL. Altogether, our data highlight an impairment in circulatory Tfh subsets in CLL patients and their critical role in CLL physiopathology.
Assuntos
Leucemia Linfocítica Crônica de Células B , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Linfócitos T Auxiliares-Indutores , Linfócitos B , Linfócitos T CD4-Positivos/patologia , Proliferação de CélulasRESUMO
BACKGROUND: The reported association of mTOR-inhibitor (mTORi) treatment with a lower incidence of cytomegalovirus (CMV) infection in kidney transplant recipients (KTR) who are CMV seropositive (R+) remains unexplained. METHODS: The incidence of CMV infection and T-cell profile was compared between KTRs treated with mTORis and mycophenolic acid (MPA), and in vitro mTORi effects on T-cell phenotype and functions were analyzed. RESULTS: In KTRs who were R+ and treated with MPA, both αß and γδ T cells displayed a more dysfunctional phenotype (PD-1+, CD85j+) at day 0 of transplantation in the 16 KTRs with severe CMV infection, as compared with the 17 KTRs without or with spontaneously resolving CMV infection. In patients treated with mTORis (n=27), the proportion of PD-1+ and CD85j+ αß and γδ T cells decreased, when compared with patients treated with MPA (n=44), as did the frequency and severity of CMV infections. mTORi treatment also led to higher proportions of late-differentiated and cytotoxic γδ T cells and IFNγ-producing and cytotoxic αß T cells. In vitro, mTORis increased proliferation, viability, and CMV-induced IFNγ production of T cells and decreased PD-1 and CD85j expression in T cells, which shifted the T cells to a more efficient EOMESlow Hobithigh profile. In γδ T cells, the mTORi effect was related to increased TCR signaling. CONCLUSION: Severe CMV replication is associated with a dysfunctional T-cell profile and mTORis improve T-cell fitness along with better control of CMV. A dysfunctional T-cell phenotype could serve as a new biomarker to predict post-transplantation infection and to stratify patients who should benefit from mTORi treatment. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Proportion of CMV Seropositive Kidney Transplant Recipients Who Will Develop a CMV Infection When Treated With an Immunosuppressive Regimen Including Everolimus and Reduced Dose of Cyclosporine Versus an Immunosuppressive Regimen With Mycophenolic Acid and Standard Dose of Cyclosporine A (EVERCMV), NCT02328963.
Assuntos
Infecções por Citomegalovirus/prevenção & controle , Transplante de Rim/efeitos adversos , Inibidores de MTOR/uso terapêutico , Subpopulações de Linfócitos T/efeitos dos fármacos , Idoso , Antibacterianos/uso terapêutico , Antígenos CD/metabolismo , Técnicas de Cultura de Células , Infecções por Citomegalovirus/epidemiologia , Infecções por Citomegalovirus/patologia , Feminino , Humanos , Receptor B1 de Leucócitos Semelhante a Imunoglobulina/metabolismo , Masculino , Pessoa de Meia-Idade , Ácido Micofenólico/uso terapêutico , Receptor de Morte Celular Programada 1/metabolismo , Subpopulações de Linfócitos T/metabolismoRESUMO
Common variable immunodeficiency is the most common clinical primary immunodeficiency in adults. Its hallmarks are hypogammaglobulinemia and compromised B-cell differentiation into memory or antibody-secreting cells leading to recurrent infections. This disease is heterogeneous, with some patients harboring multiple complications such as lymphoproliferative disorders, autoimmune manifestations, or granulomatous inflammation. The mechanisms leading to these complications remain elusive despite numerous associations found in the literature. For instance, although described as a B cell intrinsic disease, numerous abnormalities have been reported in other immune cell compartments. Here, we tuned our attention to follicular helper T cells, a CD4+ T cell population specialized in B cell help, considering the recent publications showing an involvement of these cells in CVID pathogenesis.
Assuntos
Imunodeficiência de Variável Comum/complicações , Imunodeficiência de Variável Comum/imunologia , Células T Auxiliares Foliculares/imunologia , Adulto , Animais , Linfócitos B/imunologia , Criança , Pré-Escolar , Imunodeficiência de Variável Comum/classificação , Humanos , Ativação Linfocitária/imunologia , CamundongosRESUMO
Malaria is an infectious disease caused by the protozoan parasite Plasmodium sp, the most lethal being Plasmodium falciparum. Clinical malaria is associated with the asexual replication cycle of Plasmodium parasites inside the red blood cells (RBCs) and a dysregulated immune response. Although the mechanisms of immune responses to blood-or liver-stage parasites have been extensively studied, this has not led to satisfactory leads for vaccine design. Among innate immune cells responding to infection are the non-conventional gamma-delta T-cells. The Vγ9Vδ2 T-cell subset, found only in primates, is activated in response to non-peptidic phosphoantigens produced by stressed mammalian cells or by microorganisms such as Mycobacteria, E.coli, and Plasmodium. The potential protective role of Vγ9Vδ2 T-cells against infections and cancer progression is of current research interest. Vγ9Vδ2 T-cells have been shown to play a role in the early control of P. falciparum parasitemia and to influence malaria adaptive immunity via cytokine release and antigen presentation. They are activated and expanded during a primary P. falciparum infection in response to malaria phosphoantigens and their activity is modulated upon subsequent infections. Here, we review the wide range of functions by which Vγ9Vδ2 T-cells could both contribute to and protect from malaria pathology, with a particular focus on their ability to induce both innate and adaptive responses. We discuss how the multifunctional roles of these T-cells could open new perspectives on gamma-delta T-cell-based interventions to prevent or cure malaria.
Assuntos
Imunidade Adaptativa/imunologia , Imunidade Inata/imunologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Apresentação de Antígeno/imunologia , Antígenos de Protozoários/imunologia , Eritrócitos/imunologia , HumanosRESUMO
During Plasmodium falciparum infections, erythrocyte-stage parasites inhibit dendritic cell maturation and function, compromising effective antimalarial adaptive immunity. Human Vγ9Vδ2 T cells can act in vitro as antigen-presenting cells (APCs) and induce αß T-cell activation. However, the relevance of this activity in vivo has remained elusive. Because Vγ9Vδ2 T cells are activated during the early immune response against P. falciparum infection, we investigated whether they could contribute to the instruction of adaptive immune responses toward malaria parasites. In P. falciparum-infected patients, Vγ9Vδ2 T cells presented increased surface expression of APC-associated markers HLA-DR and CD86. In response to infected red blood cells in vitro, Vγ9Vδ2 T cells upregulated surface expression of HLA-DR, HLA-ABC, CD40, CD80, CD83, and CD86, induced naive αß T-cell responses, and cross- presented soluble prototypical protein to antigen-specific CD8+ T cells. Our findings qualify Vγ9Vδ2 T cells as alternative APCs, which could be harnessed for therapeutic interventions and vaccine design.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Ativação Linfocitária/imunologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Linfócitos T/imunologia , Apresentação de Antígeno/imunologia , Células Apresentadoras de Antígenos/química , Humanos , Fenótipo , Linfócitos T/químicaRESUMO
Human γδ T cells contribute to tissue homeostasis under normal conditions and participate in lymphoid stress surveillance against infection and tumors. However, the molecular mechanisms underlying the recognition of complex cell stress signatures by γδ T cells are still unclear. Tumor cells and human cytomegalovirus (HCMV)-infected cells are known targets of γδ T cells. We show here that many tumor and CMV-infected cells express caspase-1 inflammasomes and release interleukin (IL)-18. Engagement of the T-cell receptor (TCR) on Vδ2neg γδ T cells controlled the direct innate immune sensing of IL-18 that enhanced cytotoxicity and interferon gamma (IFNγ) production. This TCR-dependent sensitization to IL-18 was mediated by the upregulation of the innate IL-18 receptor ß chain (IL-18Rß) expression. These findings shed light on inflammasomes as a unified stress signal of tumor and infected cells to alert γδ T cells. Moreover, uncovering the TCR-mediated sensitization of γδ T cells to inflammatory mediators establishes a molecular link between the innate and adaptive immune functions of γδ T cells that could fine tune the commitment of antigen-experienced γδ T cells to inflammatory responses.
RESUMO
Malaria induces potent activation and expansion of the Vγ9Vδ2 subpopulation of γδT cells, which inhibit the Plasmodium falciparum blood cycle through soluble cytotoxic mediators, abrogating merozoite invasion capacity. Intraerythrocytic stages efficiently trigger Vγ9Vδ2 T-cell activation and degranulation through poorly understood mechanisms. P. falciparum blood-stage extracts are known to contain phosphoantigens able to stimulate Vγ9Vδ2 T cells, but how these are presented by intact infected red blood cells (iRBCs) remains elusive. Here we show that, unlike activation by phosphoantigen-expressing cells, Vγ9Vδ2 T-cell activation by intact iRBCs is independent of butyrophilin expression by the iRBC, and contact with an intact iRBC is not required. Moreover, blood-stage culture supernatants proved to be as potent activators of Vγ9Vδ2 T cells as iRBCs. Bioactivity in the microenvironment is attributable to phosphoantigens, as it is dependent on the parasite DOXP pathway, on Vγ9Vδ2 TCR signaling, and on butyrophilin expression by Vγ9Vδ2 T cells. Kinetic studies showed that the phosphoantigens were released at the end of the intraerythrocytic cycle at the time of parasite egress. We document exquisite sensitivity of Vγ9Vδ2 T cells, which respond to a few thousand parasites. These data unravel a novel framework, whereby release of phosphoantigens into the extracellular milieu by sequestered parasites likely promotes activation of distant Vγ9Vδ2 T cells that in turn exert remote antiparasitic functions.
Assuntos
Antígenos de Protozoários/imunologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Subpopulações de Linfócitos T/imunologia , Antígenos de Protozoários/metabolismo , Eritrócitos/parasitologia , Humanos , Ativação Linfocitária , Malária Falciparum/parasitologia , Merozoítos/crescimento & desenvolvimento , Merozoítos/imunologia , Merozoítos/fisiologia , Fosforilação , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/fisiologia , Proteínas de Protozoários/metabolismo , Subpopulações de Linfócitos T/parasitologiaRESUMO
BACKGROUND: Severe malarial anaemia (SMA) is a major life-threatening complication of paediatric malaria. Protracted production of pro-inflammatory cytokines promoting erythrophagocytosis and depressing erythropoiesis is thought to play an important role in SMA, which is characterized by a high TNF/IL-10 ratio. Whether this TNF/IL-10 imbalance results from an intrinsic incapacity of SMA patients to produce IL-10 or from an IL-10 unresponsiveness to infection is unknown. Monocytes and T cells are recognized as the main sources of TNF and IL-10 in vivo, but little is known about the activation status of those cells in SMA patients. METHODS: The IL-10 and TNF production capacity and the activation phenotype of monocytes and T cells were compared in samples collected from 332 Ghanaian children with non-overlapping SMA (n = 108), cerebral malaria (CM) (n = 144) or uncomplicated malaria (UM) (n = 80) syndromes. Activation status of monocytes and T cells was ascertained by measuring HLA-DR+ and/or CD69+ surface expression by flow cytometry. The TNF and IL-10 production was assessed in a whole-blood assay after or not stimulation with lipopolysaccharide (LPS) or phytohaemaglutinin (PHA) used as surrogate of unspecific monocyte and T cell stimulant. The number of circulating pigmented monocytes was also determined. RESULTS: Monocytes and T cells from SMA and CM patients showed similar activation profiles with a comparable decreased HLA-DR expression on monocytes and increased frequency of CD69+ and HLA-DR+ T cells. In contrast, the acute-phase IL-10 production was markedly decreased in SMA compared to CM (P = .003) and UM (P = .004). Although in SMA the IL-10 response to LPS-stimulation was larger in amplitude than in CM (P = .0082), the absolute levels of IL-10 reached were lower (P = .013). Both the amplitude and levels of TNF produced in response to LPS-stimulation were larger in SMA than CM (P = .019). In response to PHA-stimulation, absolute levels of IL-10 produced in SMA were lower than in CM (P = .005) contrasting with TNF levels, which were higher (P = .001). CONCLUSIONS: These data reveal that SMA patients have the potential to mount efficient IL-10 responses and that the TNF/IL-10 imbalance may reflect a specific monocyte and T cell programming/polarization pattern in response to infection.
Assuntos
Interleucina-10/metabolismo , Malária/imunologia , Malária/patologia , Fator de Necrose Tumoral alfa/metabolismo , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Antígenos HLA-DR/análise , Humanos , Lactente , Lectinas Tipo C/análise , Ativação Linfocitária , Masculino , Monócitos/química , Monócitos/imunologia , Linfócitos T/química , Linfócitos T/imunologiaRESUMO
The control of Plasmodium falciparum erythrocytic parasite density is essential for protection against malaria, because it prevents pathogenesis and progression toward severe disease. P falciparum blood-stage parasite cultures are inhibited by human Vγ9Vδ2 γδ T cells, but the underlying mechanism remains poorly understood. Here, we show that both intraerythrocytic parasites and the extracellular red blood cell-invasive merozoites specifically activate Vγ9Vδ2 T cells in a γδ T cell receptor-dependent manner and trigger their degranulation. In contrast, the γδ T cell-mediated antiparasitic activity only targets the extracellular merozoites. Using perforin-deficient and granulysin-silenced T-cell lines, we demonstrate that granulysin is essential for the in vitro antiplasmodial process, whereas perforin is dispensable. Patients infected with P falciparum exhibited elevated granulysin plasma levels associated with high levels of granulysin-expressing Vδ2(+) T cells endowed with parasite-specific degranulation capacity. This indicates in vivo activation of Vγ9Vδ2 T cells along with granulysin triggering and discharge during primary acute falciparum malaria. Altogether, this work identifies Vγ9Vδ2 T cells as unconventional immune effectors targeting the red blood cell-invasive extracellular P falciparum merozoites and opens novel perspectives for immune interventions harnessing the antiparasitic activity of Vγ9Vδ2 T cells to control parasite density in malaria patients.
Assuntos
Eritrócitos/imunologia , Plasmodium falciparum/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Animais , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Western Blotting , Células Cultivadas , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Citometria de Fluxo , Interações Hospedeiro-Parasita/imunologia , Humanos , Imunofenotipagem , Estágios do Ciclo de Vida/imunologia , Ativação Linfocitária/imunologia , Proteína 1 de Membrana Associada ao Lisossomo/imunologia , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Malária Falciparum/genética , Malária Falciparum/imunologia , Malária Falciparum/metabolismo , Merozoítos/crescimento & desenvolvimento , Merozoítos/imunologia , Merozoítos/fisiologia , Mutação , Perforina/genética , Perforina/imunologia , Perforina/metabolismo , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/fisiologia , Interferência de RNA , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Esquizontes/crescimento & desenvolvimento , Esquizontes/imunologia , Esquizontes/fisiologia , Linfócitos T/metabolismoRESUMO
gammadelta T cells recognize stress-induced autoantigens and contribute to immunity against infections and cancer. Our previous study revealed that Vdelta2-negative ((neg)) gammadelta T lymphocytes isolated from transplant recipients infected by cytomegalovirus (CMV) killed both CMV-infected cells and HT29 colon cancer cells in vitro. To investigate the antitumor effects of Vdelta2(neg) clones in vivo, we generated hypodermal HT29 tumors in immunodeficient mice. Concomitant injections of Vdelta2(neg)clones, in contrast to Vdelta2(+) cells, prevented the development of HT29 tumors. Vdelta2(neg) clones expressed chemokine C-C motif receptor 3 (CCR3) and migrated in vitro in response to chemokines secreted by HT29 cells, among which were the CCR3 ligands macrophage inflammatory protein-1delta and monocyte chemoattractant protein-4. More importantly, a systemic i.p. treatment with Vdelta2(neg) clones delayed the growth of HT29 s.c. tumors. The effect of in vivo gammadelta T-cell passive immunotherapy on tumor growth could be reverted by addition of a blocking anti-CCR3 antibody. gammadelta T-cell passive immunotherapy was dependent on the cytotoxic activity of the gammadelta effectors toward their targets because Vdelta2(neg) clones were not able to inhibit the growth of A431 hypodermal tumors. Our findings suggest that CMV-specific Vdelta2(neg) cells could target in vivo cancer cells, making them an attractive candidate for antitumor immunotherapy.
Assuntos
Neoplasias do Colo/terapia , Citomegalovirus/imunologia , Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Neoplasias Cutâneas/terapia , Linfócitos T/imunologia , Animais , Células CACO-2 , Quimiocinas/imunologia , Neoplasias do Colo/imunologia , Neoplasias do Colo/virologia , Infecções por Citomegalovirus/imunologia , Células HT29 , Humanos , Camundongos , Receptores CCR3/biossíntese , Receptores CXCR3/biossíntese , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/virologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
T cells are thought to play a critical role in cerebral malaria pathogenesis. However, available evidences are restricted to rodent models in which V beta specific T cell expansion has been associated with neurological syndrome suggesting involvement of superantigens or dominant antigens. Using flow cytometry, we studied the peripheral V beta T cell repertoire of Ghanaian children with cerebral malaria, uncomplicated malaria and asymptomatic control children, to look for either expansion or deletion of specific V beta associated with cerebral malaria. At admission, the general pattern of the repertoire of the patients was very similar, with no major distortion compared to the control group a part a significant increase of the frequency of the V beta 21.3 subset correlating with disease severity and attributed to the CD4 subset. During convalescence very limited fluctuations were observed including a significant decrease of the V beta 21.3 subset and increase of the V beta 20 subset, a subset not detected at admission. The remarkable stability of the V beta repertoire observed in acute malaria either cerebral or uncomplicated argues against the idea that cerebral malaria would result from a T cell-mediated inflammatory shock syndrome driven by some dominant super-antigenic activity(ies). The significance of the reproducible increase of the CD4+V beta 21.3T cell subset deserves further investigations.
Assuntos
Malária Cerebral/imunologia , Receptores de Antígenos de Linfócitos T/análise , Subpopulações de Linfócitos T/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Pré-Escolar , Citometria de Fluxo , Gana , Humanos , Lactente , Subpopulações de Linfócitos T/químicaRESUMO
BACKGROUND: Real-time PCR is becoming a common tool for detecting and quantifying expression profiling of selected genes. Cytokines mRNA quantification is widely used in immunological research to dissect the early steps of immune responses or pathophysiological pathways. It is also growing to be of clinical relevancy to immuno-monitoring and evaluation of the disease status of patients. The techniques currently used for "absolute quantification" of cytokine mRNA are based on a DNA standard curve and do not take into account the critical impact of RT efficiency. RESULTS: To overcome this pitfall, we designed a strategy using external RNA as standard in the RT-PCR. Use of synthetic RNA standards, by comparison with the corresponding DNA standard, showed significant variations in the yield of retro-transcription depending the target amplified and the experiment. We then developed primers to be used under one single experimental condition for the specific amplification of human IL-1beta, IL-4, IL-10, IL-12p40, IL-13, IL-15, IL-18, IFN-gamma, MIF, TGF-beta1 and TNF-alpha mRNA. We showed that the beta-2 microglobulin (beta2-MG) gene was suitable for data normalisation since the level of beta2-MG transcripts in naive PBMC varied less than 5 times between individuals and was not affected by LPS or PHA stimulation. The technique, we named CyProQuant-PCR (Cytokine Profiling Quantitative PCR) was validated using a kinetic measurement of cytokine transcripts under in vitro stimulation of human PBMC by lipopolysaccharide (LPS) or Staphylococcus aureus strain Cowan (SAC). Results obtained show that CyProQuant-PCR is powerful enough to precociously detect slight cytokine induction. Finally, having demonstrated the reproducibility of the method, it was applied to malaria patients and asymptomatic controls for the quantification of TGF-beta1 transcripts and showed an increased capacity of cells from malaria patients to accumulate TGF-beta1 mRNA in response to LPS. CONCLUSION: The real-time RT-PCR technique based on a RNA standard curve, CyProQuant-PCR, outlined here, allows for a genuine absolute quantification and a simultaneous analysis of a large panel of human cytokine mRNA. It represents a potent and attractive tool for immunomonitoring, lending itself readily to automation and with a high throughput. This opens the possibility of an easy and reliable cytokine profiling for clinical applications.
Assuntos
Citocinas/genética , Perfilação da Expressão Gênica/métodos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Adulto , Automação , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Criança , DNA/genética , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lipopolissacarídeos/farmacologia , Fatores Inibidores da Migração de Macrófagos/genética , Malária Falciparum/genética , Malária Falciparum/metabolismo , Padrões de Referência , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1 , Fator de Necrose Tumoral alfa/genéticaRESUMO
BACKGROUND: The neotropical primate squirrel monkey is used in many areas of biomedical research including neuroendocrinology, immunology and infectious diseases. However, research has been hampered by the lack of immunological tools for this primate. METHODS: A series of 67 commercially available monoclonal antibodies to human CD antigens or cytokines were tested on Saimiri mononuclear cells and the specificity was assessed by double staining using flow cytometry. RESULTS: Monoclonal antibodies defining the main mononuclear cells subsets (monocytes, B, T, including CD4 and CD8 T cells) as well as activation markers have been identified. The conditions to specifically identify the various cell subsets using two color flow cytometry and establish their relative proportions have been set-up. We also have established normal values of the main circulating mononuclear cell subsets for adult Saimiri sciureus monkeys from the breeding unit of Institut Pasteur in French Guiana. The distribution between spleen, blood and lymph nodes has been compared. CONCLUSIONS: These tools allow documenting the phenotype of most Saimiri mononuclear cell subsets and assessing their activation level. This opens new perspectives for vaccinology and immunopathology research in this experimental non-human primate host, in particular for malaria research.