RESUMO
BACKGROUND AND AIMS: Long-lasting immunological memory is the ultimate goal of vaccination. Homeostatic maintenance of memory CD8 + cytotoxic T cells (MemCD8TCs) is thought to be mediated by IL-15/IL-15R heterodimer (15HD)-expressing myeloid cells. Nonmyeloid hepatic stellate cells (HSCs) also express 15HD, but their role in maintaining MemCD8TC homeostasis is unknown. APPROACH AND RESULTS: We engineered a genetically engineered mouse in which IL-15R complementary DNA (cDNA) had been inserted in-frame with lecithin-retinol acyltransferase gene and bred onto an IL-15R-KO (15R-KO) genetic background (L15R) that expressed IL-15R in HSCs at normal levels, but not in other liver cells. Outside of the liver of L15R mice, IL-15R expression was found in a number of organs, but not in dendritic cells and macrophages. The low IL-15R expression in the bone marrow (BM) of L15R mice was eliminated by the reconstitution of lethally-irradiated L15R mice with 15R-KO BM to generate L15RC mice. Because MemCD8TC maintenance is mediated by 15HD, not empty IL-15R, 15HD content in L15R mice was determined and found for liver, lung, kidney, and heart. L15R and L15RC mice developed and maintained long-lasting, systemic antigen-specific MemCD8TCs that were efficacious against tumor growth and Listeria monocytogenes infection in an antigen-specific manner. Among the four organs with 15HD content, liver-associated MemCD8TCs were different from those found in the lung, kidney, and heart in two ways: (1) they were quantitatively the most numerous, and (2) they appeared uniquely in the form of clusters in a specialized structure, sinusoidal niches of the liver. CONCLUSIONS: The liver, the largest organ of the body, is endowed with the capability of effectuating long-lasting functional cytotoxic T cell memory.
Assuntos
Linfócitos T CD8-Positivos , Células Estreladas do Fígado , Camundongos , Animais , Receptores de Interleucina-15/metabolismo , Memória Imunológica , Fígado , Camundongos Endogâmicos C57BLRESUMO
BCR-mediated tonic signaling is an indispensable requirement for the survival of follicular B (FOB) cells and Burkitt lymphoma (BL) cells. FOB cells of the I-A12% mutant mouse express unfolded protein response and are extremely short lived. Among the myriad molecules activated by unfolded protein response in I-A12% B cells, Xbp1s singularly "hijacked" p110 from p85:p110 heterodimeric PI3K, thereby abating BCR tonic signaling, resulting in their extremely short lifespan. Long-lived normal FOB cells became short lived upon ectopic Xbp1s expression. The proapoptotic Xbp1s role in FOB cells starkly contrasts with its antithetical prosurvival function in plasma cells. Also, tonic signaling and clonal expansion, two important functions mediated by the same BCR, operate in independent and distinct manners. Furthermore, concerning the development of new therapeutic treatment of drug-refractory BL patients, our finding of Xbp1s-mediated rapid death of BL cells brings forth a conceptual advancement based on blocking PI3K heterodimer formation rather than inhibition of PI3K enzyme activity.
Assuntos
Apoptose/fisiologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Proteína 1 de Ligação a X-Box/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Plasmócitos/metabolismo , Plasmócitos/patologia , Transdução de Sinais/fisiologiaRESUMO
BCR engagement leads to activation and clonal expansion of B cells. The I-A12% mutant mouse possesses a branch site point mutation in the H2-Aa gene that causes highly reduced I-Aa protein expression. As I-A is a heterodimer made up of I-Aa and I-Ab, reduced I-Aa results not only in reduced surface I-A expression but also in an excess of unpaired I-Ab. B cells that develop in I-A12% mice proliferated in response to LPS stimulation but failed to do so upon BCR stimulation. Developing I-A12% B cells were engaged in unfolded protein response due to an excess of unpaired I-Ab. BCR responsiveness was restored by transduced I-Aa expression and by BiP, the unfolded protein response sensor. Reducing the load of unpaired I-Ab also restored BCR responsiveness of I-A12% B cells. Mef2c protein, a transcription factor required for BCR-stimulated proliferation, was missing in I-A12% B cells, and that transduced Mef2c expression restored BCR responsiveness. Mef2c protein appeared in I-A12% B cells after addition of proteasome inhibitors. Mef2c degradation was mediated by Skp2 E3 ligase, and that knockdown of Skp2 mRNA in I-A12% B cells restored BCR responsiveness. Our results point to a generalized incompatibility between BCR responsiveness and increased Skp2 stability. They also imply the existence of regulatory mechanisms other than Ig gene rearrangement that govern Mef2c turnover in a specific, exquisite, and dynamic fashion.
Assuntos
Linfócitos B/imunologia , Ativação Linfocitária/imunologia , Proteínas Quinases Associadas a Fase S/metabolismo , Resposta a Proteínas não Dobradas/imunologia , Animais , Linfócitos B/citologia , Diferenciação Celular/imunologia , Proliferação de Células/fisiologia , Regulação da Expressão Gênica/imunologia , Fatores de Transcrição MEF2/imunologia , Fatores de Transcrição MEF2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteólise , Receptores de Antígenos de Linfócitos B/imunologia , Proteínas Quinases Associadas a Fase S/imunologiaRESUMO
The development and activation of MHC class II (MHC-II)-restricted CD4+ T cells are distinct immunological processes that are strictly MHC-II-dependent. To address their relative dependence on MHC-II, we established a novel ENU-induced mutant mouse on the C57BL/6 background, named I-A12%, with â¼8-fold reduced I-A expression on the surface of B cells, dendritic cells, cortical thymic epithelial cells, and medullary thymic epithelial cells. I-A100% and I-A12% mice are highly similar with respect to the numbers of double-positive thymocytes, CD4+CD8- T cells, regulatory T cells, CD4+ T cell marker expression, lifespan, and Th/regulatory T cell function. Despite the demonstration of functional intrathymic negative selection in I-A12% mice, transfer of I-A12% CD25-CD4+ T cells into RAG-knockout hosts revealed increased autoaggression activity against the liver. Compared to I-A100% mice, infection of I-A12% mice with graded doses of Listeria monotcytogenes or influenza virus revealed comparable and significantly reduced generation of Ag-specific CD4+ T cells at high and low infection doses, respectively. A significantly weakened Ag-specific recall cytokine production response was also found for I-A12% mice previously infected with a relative low dose of L. monocytogenes CD44hiCD4+ T cells from I-A100% and I-A12% mice previously infected with a relatively high L. monocytogenes dose displayed highly similar Ag-specific multicytokine production profiles. In contrast, polyclonal activation of endogenous memory-like I-A12% CD44hiCD4+ T cells revealed highly elevated production of multiple cytokines. Our results demonstrate that there exist distinct thresholds for different MHC-II-dependent immunological processes. The I-A12% mutant mouse model we describe in the present study is a valuable tool for investigations on the quantitative cause-effect relationship in MHC-II-dependent normal and autoimmune responses.
Assuntos
Citocinas/biossíntese , Genes MHC da Classe II , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Timo/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/genética , Citocinas/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Memória Imunológica , Listeria monocytogenes/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Orthomyxoviridae/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T Reguladores/imunologia , Timo/anatomia & histologia , Timo/citologiaRESUMO
UNLABELLED: Like poliovirus infection, severe infection with enterovirus 71 (EV71) can cause neuropathology. Unlike poliovirus, EV71 is often associated with hand-foot-and-mouth disease (HFMD). Here we established three mouse models for experimental infection with the same clinical isolate of EV71. The NOD/SCID mouse model is unique for the development of skin rash, an HFMD-like symptom. While the NOD/SCID mice developed limb paralysis and death at near-100% efficiency, the gamma interferon receptor knockout (ifngr KO) and stat-1 knockout mice exhibited paralysis and death rates near 78% and 30%, respectively. Productive infection with EV71 depends on the viral dose, host age, and inoculation route. Levels of infectious EV71, and levels of VP1-specific RNA and protein in muscle, brain, and spinal cord, were compared side by side between the NOD/SCID and stat-1 knockout models before, during, and after disease onset. Spleen fibrosis and muscle degeneration are common in the NOD/SCID and stat-1 knockout models. The main differences between these two models include their disease manifestations and cytokine/chemokine profiles. The pathology of the NOD/SCID model includes (i) inflammation and expression of viral VP1 antigen in muscle, (ii) increased neutrophil levels and decreased eosinophil and lymphocyte levels, and (iii) hair loss and skin rash. The characteristic pathology of the stat-1 knockout model includes (i) a strong tropism of EV71 for the central nervous system, (ii) detection of VP1 protein in the Purkinje layer of cerebellar cortex, pons, brain stem, and spinal cord, (iii) amplification of microglial cells, and (iv) dystrophy of intestinal villi. Our comparative studies on these new models with oral or intraperitoneal (i.p.) infection underscored the contribution of host immunity, including the gamma interferon receptor, to EV71 pathogenesis. IMPORTANCE: In the past decade, enterovirus 71 (EV71) has emerged as a major threat to public health in the Asia-Pacific region. Disease manifestations include subclinical infection, common-cold-like syndromes, hand-foot-and-mouth disease (HFMD), uncomplicated brain stem encephalitis, severe dysregulation of the autonomic nerve system, fatal pulmonary edema, and cardiopulmonary collapse. To date, no effective vaccine or treatment is available. A user-friendly and widely accessible animal model for researching EV71 infection and pathogenesis is urgently needed by the global community, both in academia and in industry.
Assuntos
Modelos Animais de Doenças , Enterovirus Humano A/crescimento & desenvolvimento , Doença de Mão, Pé e Boca/patologia , Doença de Mão, Pé e Boca/virologia , Animais , Encéfalo/virologia , Citocinas/sangue , Fibrose/patologia , Leucócitos/imunologia , Camundongos Knockout , Camundongos SCID , Músculos/patologia , Músculos/virologia , Medula Espinal/virologia , Baço/patologia , Análise de Sobrevida , Carga ViralRESUMO
Rag-1-knockout (KO) mice are highly resistant to Listeria monocytogenes infection. The role played by the many Rag-1-dependent lymphocyte lineages was studied using a genetic approach in which each Rag-1-dependent lymphocyte lineage was eliminated one at a time. Only B cell-deficient Igh-KO mice displayed reduced bacterial load and improved survival upon Listeria infection. Listeria infection of Rag-1-KO and Il-10-KO hosts that had been adoptively transferred with wild-type marginal zone B (MZB) cells, but not follicular B cells, resulted in heightened bacterial load and increased Il-10 production in the spleen, but not the liver. This MZB cell-dependent increase in bacterial load was eliminated by anti-Il-10 mAb. In addition, Listeria infection of MZB cell-deficient Rbpj-cKO mice showed decreased bacterial load and increased survival. Whereas multiple cell types have been shown to be capable of Il-10 production, our results indicate that the MZB cell is the most dominant and relevant Il-10 source in the context of Listeria susceptibility. In marked contrast to the generally protective nature of MZB cells in defending against pathogenic infection, our results demonstrate that MZB cells play a detrimental role in Listeria infection and possibly other infections as well.
Assuntos
Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/microbiologia , Interleucina-10/metabolismo , Listeriose/imunologia , Baço/imunologia , Baço/microbiologia , Animais , Subpopulações de Linfócitos B/transplante , Carga Bacteriana/genética , Carga Bacteriana/imunologia , Sobrevivência Celular/imunologia , Interleucina-10/biossíntese , Interleucina-10/fisiologia , Listeriose/genética , Listeriose/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Baço/transplante , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
Specific anti-CD3 treatment is deemed to be a promising therapy for allograft rejection and type 1 diabetes (T1D). Fc receptor (FcR) reduced-binding antibodies, by avoiding adverse effects of Fc and FcR interaction, have good therapeutic potential. We generated a trivalent anti-mouse-CD3 Collabody, h145CSA, by using a triplex-forming collagen-like peptide (Gly-Pro-Pro)10 to drive the trimerization of the Fab fragments. Exposure to h145CSA, but not its bivalent counterparts 145-2C11 and h145chIgGAA (FcR reduced-binding format), upregulates FasL expression on Th1 cells and causes Th1 cell apoptosis. Administration of h145CSA invokes minimal mitogenic effects in mice. The ability of multiple dosing of h145CSA to induce splenic CD4+ T-cell depletion is comparable to bivalent antibodies but is characterized by more rapid CD4+ T-cell recovery kinetics. h145CSA is more potent than h145chIgGAA in inducing long-lasting remission in recent-onset diabetic NOD mice. Its therapeutic effect is accompanied by a significantly lower percentage of CD4+IFNγ+ T cells and a higher Treg/Th1 ratio in pancreatic and mesenteric lymph nodes. The results of our study demonstrate that trivalent non-Fc anti-CD3 Collabody has the potential to be used in the treatment of T1D.
Assuntos
Diabetes Mellitus Tipo 1 , Células Th1 , Camundongos , Animais , Camundongos Endogâmicos NOD , Diabetes Mellitus Tipo 1/tratamento farmacológico , Causalidade , Anticorpos , ApoptoseRESUMO
Upon adoptive transfer into histocompatible mice, naive CD8(+) T cells stimulated ex vivo by TCR+IL-4 turn into long-lived functional memory cells. The liver contains a large number of so formed memory CD8(+) T cells, referred to as liver memory T cells (T(lm)) in the form of cell clusters. The CD62L(low) expression and nonlymphoid tissue distribution of T(lm) cells are similar to effector memory (T(em)) cells, yet their deficient cytotoxicity and IFN-γ inducibility are unlike T(em) cells. Adoptive transfer of admixtures of TCR+IL-4-activated Vß8(+) and Vß5(+) CD8(+) T cells into congenic hosts reveals T(lm) clusters that are composed of all Vß5(+) or Vß8(+), not mixed Vß5(+)/Vß8(+) cells, indicating that T(lm) clusters are formed by clonal expansion. Clonally expanded CD8(+) T cell clusters are also seen in the liver of Listeria monocytogenes-immune mice. T(lm) clusters closely associate with hepatic stellate cells and their formation is IL-15/IL-15R-dependent. CD62L(low) T(LM) cells can home to the liver and secondary lymphoid tissues, remain CD62L(low), or acquire central memory (T(cm))-characteristic CD62L(hi) expression. Our findings show the liver as a major site of CD8(+) memory T cell growth and that T(lm) cells contribute to the pool of peripheral memory cells. These previously unappreciated T(lm) characteristics indicate the inadequacy of the current T(em)/T(cm) classification scheme and help ongoing efforts aimed at establishing a unifying memory T cell development pathway. Lastly, our finding of T(lm) clusters suggests caution against interpreting focal lymphocyte infiltration in clinical settings as pathology and not normal physiology.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Memória Imunológica/imunologia , Interleucina-4/imunologia , Fígado/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/metabolismo , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Memória Imunológica/genética , Interleucina-4/genética , Interleucina-4/metabolismo , Selectina L/biossíntese , Selectina L/genética , Selectina L/imunologia , Listeria monocytogenes/imunologia , Listeria monocytogenes/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Knockout , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismoRESUMO
We have previously revealed the protective role of CD8(+) T cells in host defense against Histoplasma capsulatum in animals with CD4(+) T cell deficiency and demonstrated that sensitized CD8(+) T cells are restimulated in vitro by dendritic cells that have ingested apoptotic macrophage-associated Histoplasma antigen. Here we show that immunization with apoptotic phagocytes containing heat-killed Histoplasma efficiently activated functional CD8(+) T cells whose contribution was equal to that of CD4(+) T cells in protection against Histoplasma challenge. Inhibition of macrophage apoptosis due to inducible nitric oxide synthase (iNOS) deficiency or by caspase inhibitor treatment dampened the CD8(+) T cell but not the CD4(+) T cell response to pulmonary Histoplasma infection. In mice subcutaneously immunized with viable Histoplasma yeasts whose CD8(+) T cells are protective against Histoplasma challenge, there was heavy granulocyte and macrophage infiltration and the infiltrating cells became apoptotic. In mice subcutaneously immunized with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled apoptotic macrophages containing heat-killed Histoplasma, the CFSE-labeled macrophage material was found to localize within dendritic cells in the draining lymph node. Moreover, depleting dendritic cells in immunized CD11c-DTR mice significantly reduced CD8(+) T cell activation. Taken together, our results revealed that phagocyte apoptosis in the Histoplasma-infected host is associated with CD8(+) T cell activation and that immunization with apoptotic phagocytes containing heat-killed Histoplasma efficiently evokes a protective CD8(+) T cell response. These results suggest that employing apoptotic phagocytes as antigen donor cells is a viable approach for the development of efficacious vaccines to elicit strong CD8(+) T cell as well as CD4(+) T cell responses to Histoplasma infection.
Assuntos
Apoptose/fisiologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Fúngicas/imunologia , Histoplasma/imunologia , Histoplasmose/imunologia , Animais , Antígeno CD11c/metabolismo , Linfócitos T CD4-Positivos/fisiologia , Regulação da Expressão Gênica/fisiologia , Histoplasmose/prevenção & controle , Imunização , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismoRESUMO
CD28 is required for T cell activation as well as the generation of CD4+Foxp3+ Treg. It is unclear, however, how CD28 costimulation affects the development of CD8+ T cell suppressive function. Here, by use of Hepa1.6.gp33 in vitro killing assay and B16.gp33 tumor mouse model we demonstrate that CD28 engagement during TCR ligation prevents CD8+ T cells from becoming suppressive. Interestingly, our results showed that ectonucleotidase CD73 expression on CD8+ T cells is upregulated in the absence of CD28 costimulation. In both murine and human tumor-bearing hosts, CD73 is upregulated on CD28-CD8+ T cells that infiltrate the solid tumor. UPLC-MS/MS analysis revealed that CD8+ T cells activation without CD28 costimulation produces elevated levels of adenosine and that CD73 mediates its production. Adenosine receptor antagonists block CD73-mediated suppression. Our data support the notion that CD28 costimulation inhibits CD73 upregulation and thereby prevents CD8+ T cells from becoming suppressive. This study uncovers a previously unidentified role for CD28 costimulation in CD8+ T cell activation and suggests that the CD28 costimulatory pathway can be a potential target for cancer immunotherapy.
Assuntos
5'-Nucleotidase/metabolismo , Antígenos CD28/metabolismo , Linfócitos T CD8-Positivos/imunologia , Ativação Linfocitária/imunologia , Melanoma Experimental/imunologia , 5'-Nucleotidase/genética , Animais , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Foreign Ag-specific TCR-transgenic (Tg) mice contain a small fraction of T cells bearing the endogenous Vbeta and Valpha chains as well as a population expressing an intermediate level of Tg TCR. Importantly, these minor nonclonotypic populations contain > or = 99% of the CD4(+)Foxp3(+) regulatory T cells (Treg) and, despite low overall Treg expression, peripheral tolerance is maintained. In the OT-II TCR (OVA-specific, Vbeta5(high)Valpha2(high)) Tg scurfy (Sf) mice (OT-II Sf) that lack Treg, nonclonotypic T cells markedly expanded in the periphery but not in the thymus. Expanded T cells expressed memory/effector phenotype and were enriched in blood and inflamed lungs. In contrast, Vbeta5(high)Valpha2(high) clonotypic T cells were not expanded, displayed the naive phenotype, and found mainly in the lymph nodes. Importantly, Vbeta5(neg) T cells were able to transfer multiorgan inflammation in Rag1(-/-) recipients. T cells bearing dual TCR (dual Vbeta or dual Valpha) were demonstrated frequently in the Vbeta5(int) and Valpha2(int) populations. Our study demonstrated that in the absence of Treg, the lack of peripheral expansion of clonotypic T cells is due to the absence of its high-affinity Ag OVA. Thus, the rapid expansion of nonclonotypic T cells in OT-II Sf mice must require Ag (self and foreign) with sufficient affinity. Our study has implications with respect to the roles of Ag and dual TCR in the selection and regulation of Treg and Treg-controlled Ag-dependent T cell expansion in TCR Tg and TCR Tg Sf mice, respectively.
Assuntos
Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Transferência Adotiva , Animais , Citometria de Fluxo , Tolerância Imunológica/imunologia , Masculino , Camundongos , Camundongos Transgênicos , Ovalbumina/imunologia , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T Reguladores/imunologiaRESUMO
We hypothesize that regulatory T-cell (Treg)-deficient strains have an altered TCR repertoire in part due to the expansion of autoimmune repertoire by self-antigen. We compared the Vbeta family expression profile between B6 and Treg-lacking B6.Cg-Foxp3(sf)(/Y) (B6.sf) mice using fluorescent anti-Vbeta mAbs and observed no changes. However, while the spectratypes of 20 Vbeta families among B6 mice were highly similar, the Vbeta family spectratypes of B6.sf mice were remarkably different from B6 mice and from each other. Significant spectratype changes in many Vbeta families were also observed in Treg-deficient IL-2 knockout (KO) and IL-2Ralpha KO mice. Such changes were not observed with anti-CD3 mAb-treated B6 mice or B6 CD4+CD25- T cells. TCR transgenic (OT-II.sf) mice displayed dramatic reduction of clonotypic TCR with concomitant increase in T cells bearing non-transgenic Vbeta and Valpha families, including T cells with dual receptors expressing reduced levels of transgenic Valpha and endogenous Valpha. Collectively, the data demonstrate that Treg deficiency allows polyclonal expansion of T cells in a stochastic manner, resulting in widespread changes in the TCR repertoire.
Assuntos
Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Citometria de Fluxo , Masculino , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Processos EstocásticosRESUMO
Marginal zone B (MZ B) cells can rapidly produce antibody in response to infection with blood-borne encapsulated pathogens. Although TLR-mediated activation of MZ B is known to trigger humoral immune response, the signal cascade directing this response remains undefined. Here, we demonstrate that STAT1 plays an essential role in TLR-mediated antibody response of MZ B cells. Further, the TLR-induced IgM response is impaired in a type I and type II IFN-independent manner. Although activation, proliferation, and apoptosis are not affected, both differentiation into plasma cells and IgM production are impaired in Stat1-/- MZ B cells. Interestingly, STAT1 directly regulates the expression of Prdm1 (encodes BLIMP-1) by binding to its promoter, and Prdm1 expression is reduced in Stat1-/- MZ B cells. Restoration of BLIMP-1 to cells rescues TLR-induced IgM response. Moreover, Stat1-/- mice are more susceptible to S. pneumoniae infection, which can be rescued by the serum of bacteria-primed WT mice. The increased susceptibility to S. pneumoniae infection in Stat1-/- mice is also intrinsic to STAT1 requirement in MZ B cells. Collectively, these results define a differential regulation of TLR-mediated activation and differentiation of MZ B cells by STAT1 and reveal a STAT1-dependent, but IFN-independent, antibody response during infection and inflammation.
Assuntos
Linfócitos B/imunologia , Patógenos Transmitidos pelo Sangue , Diferenciação Celular/imunologia , Infecções Pneumocócicas/imunologia , Fator de Transcrição STAT1/imunologia , Streptococcus pneumoniae/imunologia , Animais , Linfócitos B/patologia , Diferenciação Celular/genética , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interferons/genética , Interferons/imunologia , Camundongos , Camundongos Knockout , Infecções Pneumocócicas/genética , Infecções Pneumocócicas/patologia , Fator 1 de Ligação ao Domínio I Regulador Positivo , Fator de Transcrição STAT1/genética , Fatores de Transcrição/genética , Fatores de Transcrição/imunologiaRESUMO
In a routine phenotype-driven screen, we identified a point mutation in exon 7 of the IL-15 gene in Pedigree 191 (deficient memory (DM)) of N-ethyl-N-nitrosourea mutagenized mice. The DM epidermis expressed an alternatively spliced IL-15 mRNA isoform, IL-15ΔE7, and a wild-type (WT) IL-15 isoform at comparable levels. Mechanical stimulation of DM skin or DM skin graft transplanted onto the WT host resulted in reduced keratinocyte activation and inhibition of neutrophil infiltration into the dermis, demonstrating that DM keratinocytes produced less inflammatory response to external stimulation. Ectopic expression of IL-15ΔE7 in WT skin prevented abrasion-induced epidermal thickening, blocked the accumulation of nuclear antigen Ki67(+) cells in the basal and the suprabasal cell layers, increased loricrin expression, and also increased keratinocyte CXCL1 and G-CSF production. IL-15ΔE7 also profoundly blocked neutrophil infiltration in SDS- or immiquimod (IMQ)-treated WT skin. Recombinant IL-15ΔE7 failed to activate STAT-5 and its downstream target bcl-2 expression. Our study points to IL-15ΔE7 as a potential therapeutic agent for treating neutrophilia-associated inflammatory skin disorders.
Assuntos
Processamento Alternativo/fisiologia , Interleucina-15/metabolismo , Queratinócitos/metabolismo , Queratinócitos/patologia , Psoríase/metabolismo , Psoríase/patologia , Estresse Mecânico , Processamento Alternativo/genética , Animais , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Quimiocina CXCL1/metabolismo , Modelos Animais de Doenças , Feminino , Fator Estimulador de Colônias de Granulócitos/metabolismo , Interleucina-15/genética , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/patologia , Mutação Puntual/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Due to a mutation in the Foxp3 transcription factor, Scurfy mice lack regulatory T-cells that maintain self-tolerance of the immune system. They develop multi-organ inflammation (MOI) and die around four weeks old. The affected organs are skin, tail, lungs and liver. In humans, endocrine and gastrointestinal inflammation are also observed, hence the disease is termed IPEX (Immunodysregulation, Polyendocrinopathy, Enteropathy, X-linked) syndrome. The three week period of fatal MOI offers a useful autoimmune model in which the controls by genetics, T-cell subsets, cytokines, and effector mechanisms could be efficiently investigated. In this report, we will review published work, summarize our recent studies of Scurfy double mutants lacking specific autoimmune-related genes, discuss the cellular and cytokine controls by these genes on MOI, the organ-specificities of the MOI controlled by environments, and the effector mechanisms regulated by specific Th cytokines, including several newly identified control mechanisms for organ-specific autoimmune response.
RESUMO
Heterogeneity of cancer stem/progenitor cells that give rise to different forms of cancer has been well demonstrated for leukemia. However, this fundamental concept has yet to be established for solid tumors including breast cancer. In this communication, we analyzed solid tumor cancer stem cell markers in human breast cancer cell lines and primary specimens using flow cytometry. The stem/progenitor cell properties of different marker expressing-cell populations were further assessed by in vitro soft agar colony formation assay and the ability to form tumors in NOD/SCID mice. We found that the expression of stem cell markers varied greatly among breast cancer cell lines. In MDA-MB-231 cells, PROCR and ESA, instead of the widely used breast cancer stem cell markers CD44(+)/CD24(-/low) and ALDH, could be used to highly enrich cancer stem/progenitor cell populations which exhibited the ability to self renew and divide asymmetrically. Furthermore, the PROCR(+)/ESA(+) cells expressed epithelial-mesenchymal transition markers. PROCR could also be used to enrich cells with colony forming ability from MB-361 cells. Moreover, consistent with the marker profiling using cell lines, the expression of stem cell markers differed greatly among primary tumors. There was an association between metastasis status and a high prevalence of certain markers including CD44(+)/CD24(-/low), ESA(+), CD133(+), CXCR4(+) and PROCR(+) in primary tumor cells. Taken together, these results suggest that similar to leukemia, several stem/progenitor cell-like subpopulations can exist in breast cancer.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem da Célula , Citometria de Fluxo/métodos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Ágar , Animais , Antígenos CD/metabolismo , Antígeno CD24/metabolismo , Divisão Celular , Linhagem Celular Tumoral , Separação Celular , Receptor de Proteína C Endotelial , Epitélio/metabolismo , Epitélio/patologia , Feminino , Humanos , Receptores de Hialuronatos/metabolismo , Mesoderma/metabolismo , Mesoderma/patologia , Camundongos , Camundongos SCID , Receptores de Superfície Celular/metabolismo , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
NKT cells, naïve CD4(+) T cells, and TCR-gammadelta T cells belong to distinct T cell lineages but all express T cell receptors generated through random combinatorial joining of V-(D)-J genes. These distinct lineage T cells also possess the property of promptly activating the IL-4 gene upon T cell receptor stimulation. A comparative accounting of features as they pertain to IL-4 inducibility in these three distinct lineage T cells is provided here.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Interleucina-4/biossíntese , Receptores de Antígenos de Linfócitos T gama-delta/análise , Subpopulações de Linfócitos T/imunologia , Animais , Antígenos/metabolismo , Diferenciação Celular , Linhagem da Célula , Humanos , Interferon gama/metabolismo , Interleucina-4/genética , Células Matadoras Naturais/imunologia , Timo/imunologiaRESUMO
Hemorrhage is a common clinical manifestation in dengue patients. However, the pathogenic mechanism of dengue virus (DV)-induced hemorrhage awaits clarification. We established a mouse model of DV hemorrhage using immunocompetent C57BL/6 mice by injecting DV serotype 2 strain 16681 intradermally. While inoculation of 3 x 10(9) PFU of DV induced systemic hemorrhage in all of the mice by day 3 of infection, one out of three of those injected with 4 x 10(7) to 8 x 10(7) PFU developed hemorrhage in the subcutaneous tissues. The mice that were inoculated with 4 x 10(7) to 8 x 10(7) PFU but that did not develop hemorrhage were used as a basis for comparison to explore the pathogenic mechanism of dengue hemorrhage. The results showed that mice with severe thrombocytopenia manifested signs of vascular leakage and hemorrhage. We observed that high viral titer, macrophage infiltration, and tumor necrosis factor alpha (TNF-alpha) production in the local tissues are three important events that lead to hemorrhage. Immunofluorescence staining revealed that DV targeted both endothelial cells and macrophages. In addition, the production of high levels of TNF-alpha in tissues correlated with endothelial cell apoptosis and hemorrhage. By comparing TNF-alpha(-/-) to IgH(-/-), C5(-/-), and wild-type mice, we found that TNF-alpha was important for the development of hemorrhage. In vitro studies showed that mouse primary microvascular endothelial cells were susceptible to DV but that TNF-alpha enhanced DV-induced apoptosis. Our mouse model illustrated that intradermal inoculation of high titers of DV predisposes endothelial cells to be susceptible to TNF-alpha-induced cell death, which leads to endothelium damage and hemorrhage development. This finding highlights the contribution of the innate immune response to dengue hemorrhage.
Assuntos
Vírus da Dengue/patogenicidade , Modelos Animais de Doenças , Endotélio Vascular/patologia , Endotélio Vascular/virologia , Dengue Grave/virologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Apoptose/fisiologia , Endotélio Vascular/metabolismo , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dengue Grave/metabolismo , Dengue Grave/patologia , Fator de Necrose Tumoral alfa/deficiência , Fator de Necrose Tumoral alfa/genéticaRESUMO
Among Ag-inexperienced naive T cells, the CD1d-restricted NKT cell that uses invariant TCR-alpha-chain is the most widely studied cell capable of prompt IL-4 inducibility. We show in this study that thymus CD161-CD44lowCD4+CD8- T cells promptly produce IL-4 upon TCR stimulation, a response that displays biased Vbeta(2/7/8) and Valpha3.2 TCR usage. The association of Vbeta family bias and IL-4 inducibility in thymus CD161-CD44lowCD4+CD8- T cells is found for B6, B10, BALB/c, CBA, B10.A(4R), and ICR mouse strains. Despite reduced IL-4 inducibility, there is a similarly biased Vbeta(2/7/8) TCR usage by IL-4 inducibility+ spleen CD161-CD44lowCD4+CD8- T cells. Removal of alpha-galacotosylceramide/CD1d-binding cells from CD161-CD44lowCD4+CD8- thymocytes does not significantly affect their IL-4 inducibility. The development of thymus CD161-CD44lowCD4+CD8- T cells endowed with IL-4 inducibility and their associated use of Vbeta(2/7/8) are beta2-microglobulin-, CD1d-, and p59fyn-independent. Thymus CD161-CD44lowCD4+CD8- T cells produce low and no IFN-gamma inducibility in response to TCR stimulation and to IL-12 + IL-18, respectively, and they express diverse complementarity determining region 3 sequences for both TCR-alpha- and -beta-chains. Taken together, these results demonstrate the existence of a NKT cell distinct, TCR-repertoire diverse naive CD4+ T cell subset capable of prompt IL-4 inducibility. This subset has the potential to participate in immune response to a relatively large number of Ags. The more prevalent nature of this unique T cell subset in the thymus than the periphery implies roles it might play in intrathymic T cell development and may provide a framework upon which mechanisms of developmentally regulated IL-4 gene inducibility can be studied.
Assuntos
Antígenos CD1/metabolismo , Interleucina-4/genética , Células Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Subpopulações de Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Antígenos CD1d , Antígenos de Superfície/metabolismo , Sequência de Bases , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular , Regiões Determinantes de Complementaridade , DNA/genética , Galactosilceramidas/imunologia , Galactosilceramidas/farmacologia , Regulação da Expressão Gênica , Interleucinas/biossíntese , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Lectinas Tipo C/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Dados de Sequência Molecular , Subfamília B de Receptores Semelhantes a Lectina de Células NK , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/efeitos dos fármacosRESUMO
Dominant type-1 cytokine production is induced in a murine model of systemic histoplasmosis. We used this model to investigate whether the presence of antagonistic cytokines during T cell priming changes the polarity of T cells in response to Histoplasma infection. Before infection with Histoplasma capsulatum, mice were injected twice with goat anti-mouse IgD antiserum (GalphaMdelta), which induced expression of dominant type-2 cytokines. At days 7 and 14 after infection, the GalphaMdelta-treated mice had suppressed IFN-gamma response and a significantly greater fungal burden in their spleens and lungs. The number of IFN-gamma-producing cells as well as the level of IFN-gamma produced per cell was greatly reduced. Not only CD4+ T cells but also CD8+ T cells were affected. The number of Histoplasma-induced IFN-gamma-producing cells was partially restored in GalphaMdelta-treated IL-4-/- and IL-10-/- mice and completely restored in IL-4-/- IL-10-/- mice. Thus, the combined effect of IL-4 and IL-10 suppressed the generation of IFN-gamma-producing cells. A longitudinal study demonstrated that as IL-4 and IL-10 decreased, the number of Histoplasma-induced IFN-gamma-producing cells rapidly increased, and fungal clearance improved, demonstrating that the presence of IL-4 and IL-10 did not permanently change the polarity of T cells.