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1.
Cell ; 181(2): 442-459.e29, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32302573

RESUMO

Single-cell RNA sequencing (scRNA-seq) is a powerful tool for defining cellular diversity in tumors, but its application toward dissecting mechanisms underlying immune-modulating therapies is scarce. We performed scRNA-seq analyses on immune and stromal populations from colorectal cancer patients, identifying specific macrophage and conventional dendritic cell (cDC) subsets as key mediators of cellular cross-talk in the tumor microenvironment. Defining comparable myeloid populations in mouse tumors enabled characterization of their response to myeloid-targeted immunotherapy. Treatment with anti-CSF1R preferentially depleted macrophages with an inflammatory signature but spared macrophage populations that in mouse and human expresses pro-angiogenic/tumorigenic genes. Treatment with a CD40 agonist antibody preferentially activated a cDC population and increased Bhlhe40+ Th1-like cells and CD8+ memory T cells. Our comprehensive analysis of key myeloid subsets in human and mouse identifies critical cellular interactions regulating tumor immunity and defines mechanisms underlying myeloid-targeted immunotherapies currently undergoing clinical testing.


Assuntos
Neoplasias do Colo/patologia , Células Mieloides/metabolismo , Análise de Célula Única/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Sequência de Bases/genética , Linfócitos T CD8-Positivos/imunologia , China , Neoplasias do Colo/terapia , Neoplasias Colorretais/patologia , Células Dendríticas/imunologia , Feminino , Humanos , Imunoterapia , Macrófagos/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Análise de Sequência de RNA/métodos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia
2.
Nat Immunol ; 20(6): 724-735, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30936494

RESUMO

Regulatory T cells (Treg cells) maintain host self-tolerance but are a major barrier to effective cancer immunotherapy. Treg cells subvert beneficial anti-tumor immunity by modulating inhibitory receptor expression on tumor-infiltrating lymphocytes (TILs); however, the underlying mediators and mechanisms have remained elusive. Here, we found that the cytokines IL-10 and IL-35 (Ebi3-IL-12α heterodimer) were divergently expressed by Treg cell subpopulations in the tumor microenvironment (TME) and cooperatively promoted intratumoral T cell exhaustion by modulating several inhibitory receptor expression and exhaustion-associated transcriptomic signature of CD8+ TILs. While expression of BLIMP1 (encoded by Prdm1) was a common target, IL-10 and IL-35 differentially affected effector T cell versus memory T cell fates, respectively, highlighting their differential, partially overlapping but non-redundant regulation of anti-tumor immunity. Our results reveal previously unappreciated cooperative roles for Treg cell-derived IL-10 and IL-35 in promoting BLIMP1-dependent exhaustion of CD8+ TILs that limits effective anti-tumor immunity.


Assuntos
Imunidade Celular , Interleucina-10/metabolismo , Interleucinas/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Transferência Adotiva , Animais , Citocinas/genética , Citocinas/metabolismo , Perfilação da Expressão Gênica , Humanos , Melanoma Experimental , Camundongos , Neoplasias/patologia , Transdução de Sinais , Transcriptoma
3.
Immunity ; 44(2): 316-29, 2016 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-26872697

RESUMO

Regulatory T (Treg) cells pose a major barrier to effective anti-tumor immunity. Although Treg cell depletion enhances tumor rejection, the ensuing autoimmune sequelae limits its utility in the clinic and highlights the need for limiting Treg cell activity within the tumor microenvironment. Interleukin-35 (IL-35) is a Treg cell-secreted cytokine that inhibits T cell proliferation and function. Using an IL-35 reporter mouse, we observed substantial enrichment of IL-35(+) Treg cells in tumors. Neutralization with an IL-35-specific antibody or Treg cell-restricted deletion of IL-35 production limited tumor growth in multiple murine models of human cancer. Limiting intratumoral IL-35 enhanced T cell proliferation, effector function, antigen-specific responses, and long-term T cell memory. Treg cell-derived IL-35 promoted the expression of multiple inhibitory receptors (PD1, TIM3, LAG3), thereby facilitating intratumoral T cell exhaustion. These findings reveal previously unappreciated roles for IL-35 in limiting anti-tumor immunity and contributing to T cell dysfunction in the tumor microenvironment.


Assuntos
Anticorpos Bloqueadores/administração & dosagem , Interleucinas/metabolismo , Melanoma Experimental/imunologia , Neoplasias Cutâneas/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Memória Imunológica , Interleucinas/genética , Interleucinas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Microambiente Tumoral , Proteína do Gene 3 de Ativação de Linfócitos
5.
Vet Pathol ; 55(1): 76-97, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28494703

RESUMO

Only 2 major mast cell (MC) subtypes are commonly recognized in the mouse: the large connective tissue mast cells (CTMCs) and the mucosal mast cells (MMCs). Interepithelial mucosal inflammatory cells, most commonly identified as globule leukocytes (GLs), represent a third MC subtype in mice, which we term interepithelial mucosal mast cells (ieMMCs). This term clearly distinguishes ieMMCs from lamina proprial MMCs (lpMMCs) while clearly communicating their common MC lineage. Both lpMMCs and ieMMCs are rare in normal mouse intestinal mucosa, but increased numbers of ieMMCs are seen as part of type 2 immune responses to intestinal helminth infections and in food allergies. Interestingly, we found that increased ieMMCs were consistently associated with decreased mucosal inflammation and damage, suggesting that they might have a role in controlling helminth-induced immunopathology. We also found that ieMMC hyperplasia can develop in the absence of helminth infections, for example, in Treg-deficient mice, Arf null mice, some nude mice, and certain graft-vs-host responses. Since tuft cell hyperplasia plays a critical role in type 2 immune responses to intestinal helminths, we looked for (but did not find) any direct relationship between ieMMC and tuft cell numbers in the intestinal mucosa. Much remains to be learned about the differing functions of ieMMCs and lpMMCs in the intestinal mucosa, but an essential step in deciphering their roles in mucosal immune responses will be to apply immunohistochemistry methods to consistently and accurately identify them in tissue sections.


Assuntos
Intestinos/citologia , Leucócitos/citologia , Mastócitos/citologia , Animais , Modelos Animais de Doenças , Helmintíase Animal/imunologia , Helmintíase Animal/patologia , Mucosa Intestinal/citologia , Mucosa Intestinal/patologia , Intestinos/patologia , Leucócitos/patologia , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL
6.
Immunol Rev ; 259(1): 173-91, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24712466

RESUMO

Regulatory T cells (Tregs) prevail as a specialized cell lineage that has a central role in the dominant control of immunological tolerance and maintenance of immune homeostasis. Thymus-derived Tregs (tTregs) and their peripherally induced counterparts (pTregs) are imprinted with unique Forkhead box protein 3 (Foxp3)-dependent and independent transcriptional and epigenetic characteristics that bestows on them the ability to suppress disparate immunological and non-immunological challenges. Thus, unidirectional commitment and the predominant stability of this regulatory lineage is essential for their unwavering and robust suppressor function and has clinical implications for the use of Tregs as cellular therapy for various immune pathologies. However, recent studies have revealed considerable heterogeneity or plasticity in the Treg lineage, acquisition of alternative effector or hybrid fates, and promotion rather than suppression of inflammation in extreme contexts. In addition, the absolute stability of Tregs under all circumstances has been questioned. Since these observations challenge the safety and efficacy of human Treg therapy, the issue of Treg stability versus plasticity continues to be enthusiastically debated. In this review, we assess our current understanding of the defining features of Foxp3(+) Tregs, the intrinsic and extrinsic cues that guide development and commitment to the Treg lineage, and the phenotypic and functional heterogeneity that shapes the plasticity and stability of this critical regulatory population in inflammatory contexts.


Assuntos
Linhagem da Célula/imunologia , Linfócitos T Reguladores/imunologia , Animais , Diferenciação Celular , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Tolerância Imunológica , Imunomodulação , Modelos Imunológicos , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo
7.
J Immunol ; 192(6): 2904-12, 2014 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-24532574

RESUMO

Foxp3(+) regulatory T cells (Tregs) have a well-characterized role in limiting autoimmunity and dampening deleterious immune responses. However, a potential consequence of the immunosuppressive function of Tregs can be the limitation of protective immunity to infectious pathogens. Parasitic infections are a potent stimulus for the generation of Treg responses, which may be beneficial to both the parasite and the host by promoting persistence of infection and limiting immune-mediated pathology, respectively. In this study, we explore the functional role of Tregs post-low-dose infection with the intestinal helminth parasite Trichuris muris, which yields a chronic infection because of inefficient induction of Th2 responses. Early Treg depletion postinfection resulted in expedited worm clearance, and was associated with reduced Th1-mediated inflammation of the intestinal environment. Interestingly, this protective immunity was lost, and worm burden enhanced if Tregs were depleted later once the infection was established. Early and late Treg depletion resulted in enhanced Th2 and reduced Th1 cytokine and humoral responses. Blockade of the Th2 cytokine IL-4 resulted in a moderate increase in Th1 but had no effect on worm burden. Our findings suggest that Tregs preferentially limit Th2 cell expansion, which can impact infections where clear immune polarity has not been established. Thus, the impact of Treg depletion is context and time dependent, and can be beneficial to the host in situations where Th1 responses should be limited in favor of Th2 responses.


Assuntos
Imunidade Adaptativa/imunologia , Intestinos/imunologia , Linfócitos T Reguladores/imunologia , Tricuríase/imunologia , Trichuris/imunologia , Animais , Anticorpos Anti-Helmínticos/sangue , Anticorpos Anti-Helmínticos/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Toxina Diftérica/administração & dosagem , Toxina Diftérica/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-4/imunologia , Interleucina-4/metabolismo , Intestinos/parasitologia , Intestinos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Células Th2/metabolismo , Fatores de Tempo , Tricuríase/sangue , Tricuríase/parasitologia , Trichuris/fisiologia
8.
Immunology ; 145(1): 11-23, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25262912

RESUMO

Foxp3(+) regulatory T (Treg) cells are essential to maintain immune homeostasis, yet controversy exists about the stability of this cell population. Bcl6-deficient (Bcl6(-/-) ) mice develop severe and spontaneous T helper type 2 (Th2) inflammation and Bcl6-deficient Treg cells are ineffective at controlling Th2 responses. We used a lineage tracing approach to analyse the fate of Treg cells in these mice. In the periphery of Bcl6(-/-) mice, increased numbers of Foxp3-negative 'exTreg' cells were found, particularly in the CD25(+) population. ExTreg cells from Bcl6(-/-) mice expressed increased interleukin-17 (IL-17) and extremely elevated levels of Th2 cytokines compared with wild-type exTreg cells. Although Treg cells normally express only low levels of cytokines, Treg cells from Bcl6(-/-) mice secreted higher levels of IL-4, IL-5, IL-13 and IL-17 than wild-type conventional T cells. Next, Treg-specific conditional Bcl6-deficient (Bcl6(Foxp3-/-) ) mice were analysed. Bcl6(Foxp3-/-) mice do not develop inflammatory disease, indicating a requirement for non-Treg cells for inflammation in Bcl6(-/-) mice, and have normal numbers of exTreg cells. We induced Th2-type allergic airway inflammation in Bcl6(Foxp3-/-) mice, and found that while exTreg cytokine expression was normal, Bcl6-deficient Treg cells expressed higher levels of the Th2-specific regulator Gata3 than Bcl6(+) Treg cells. Bcl6(Foxp3-/-) mice had increased numbers of Th2 cells after induction of airway inflammation and increased T cells in the bronchoalveolar lavage fluid. These data show both Treg-intrinsic and Treg-extrinsic roles for Bcl6 in controlling Treg cell stability and Th2 inflammation, and support the idea that Bcl6 expression in Treg cells is critical for controlling Th2 responses.


Assuntos
Asma/imunologia , Proteínas de Ligação a DNA/imunologia , Regulação da Expressão Gênica/imunologia , Linfócitos T Reguladores/imunologia , Células Th2/imunologia , Animais , Asma/genética , Asma/patologia , Citocinas/genética , Citocinas/imunologia , Proteínas de Ligação a DNA/genética , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/imunologia , Regulação da Expressão Gênica/genética , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-bcl-6 , Linfócitos T Reguladores/patologia , Células Th2/patologia
9.
J Immunol ; 191(7): 3705-11, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-23980208

RESUMO

The transcriptional repressor Bcl6 controls development of the follicular Th cell (T(FH)) lineage, but the precise mechanisms by which Bcl6 regulates this process are unclear. A model has been proposed whereby Bcl6 represses the differentiation of T cells into alternative effector lineages, thus favoring T(FH) cell differentiation. Analysis of T cell differentiation using Bcl6-deficient mice has been complicated by the strong proinflammatory phenotype of Bcl6-deficient myeloid cells. In this study, we report data from a novel mouse model where Bcl6 is conditionally deleted in T cells (Bcl6(fl/fl)Cre(CD4) mice). After immunization, programmed death -1 (PD-1)(high) T(FH) cells in Bcl6(fl/fl)Cre(CD4) mice are decreased >90% compared with control mice, and Ag-specific IgG is sharply reduced. Residual PD-1(high)CXCR5(+) T(FH) cells in Bcl6(fl/fl)Cre(CD4) mice show a significantly higher rate of apoptosis than do PD-1(high)CXCR5(+) T(FH) cells in control mice. Immunization of Bcl6(fl/fl)Cre(CD4) mice did not reveal enhanced differentiation into Th1, Th2, or Th17 lineages, although IL-10 expression by CD4 T cells was markedly elevated. Thus, T cell-extrinsic factors appear to promote the increased Th1, Th2, and Th17 responses in germline Bcl6-deficient mice. Furthermore, IL-10 may be a key target gene for Bcl6 in CD4 T cells, which enables Bcl6 to promote the T(FH) cell phenotype. Finally, our data reveal a novel mechanism for the role of Bcl6 in promoting T(FH) cell survival.


Assuntos
Proteínas de Ligação a DNA/genética , Mutação , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Animais , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Técnicas de Inativação de Genes , Ordem dos Genes , Marcação de Genes , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-bcl-6 , Linfócitos T Auxiliares-Indutores/citologia
10.
J Virol ; 87(21): 11884-93, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23986597

RESUMO

Recent evidence has identified the role of granzyme B- and perforin-expressing CD4(+) T cells with cytotoxic potential in antiviral immunity. However, the in vivo cytokine cues and downstream pathways governing the differentiation of these cells are unclear. Here, we have identified that CD4(+) T cells with cytotoxic potential are specifically induced at the site of infection during influenza virus infection. The development of CD4(+) T cells with cytotoxic potential in vivo was dependent on the cooperation of the STAT2-dependent type I interferon signaling and the interleukin-2/interleukin-2 receptor alpha pathway for the induction of the transcription factors T-bet and Blimp-1. We showed that Blimp-1 promoted the binding of T-bet to the promoters of cytolytic genes in CD4(+) T cells and was required for the cytolytic function of the in vitro- and in vivo-generated CD4(+) T cells with cytotoxic potential. Thus, our data define the molecular basis of regulation of the in vivo development of this functionally cytotoxic Th subset during acute respiratory virus infection. The potential implications for the functions of these cells are discussed.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Citocinas/imunologia , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais
11.
J Immunol ; 189(10): 4759-69, 2012 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-23053511

RESUMO

The transcriptional repressor Bcl6 is a critical arbiter of Th cell fate, promoting the follicular Th lineage while repressing other Th cell lineages. Bcl6-deficient (Bcl6(-/-)) mice develop a spontaneous and severe Th2-type inflammatory disease, thus warranting assessment of Bcl6 in regulatory T cell (Treg) function. Bcl6(-/-) Tregs were competent at suppressing T cell proliferation in vitro and Th1-type colitogenic T cell responses in vivo. In contrast, Bcl6(-/-) Tregs strongly exacerbated lung inflammation in a model of allergic airway disease and promoted higher Th2 responses, including systemic upregulation of microRNA-21. Further, Bcl6(-/-) Tregs were selectively impaired at controlling Th2 responses, but not Th1 and Th17 responses, in mixed chimeras of Bcl6(-/-) bone marrow with Foxp3(-/-) bone marrow. Bcl6(-/-) Tregs displayed increased levels of the Th2 transcription factor Gata3 and other Th2 and Treg genes. Bcl6 potently repressed Gata3 transcriptional transactivation, providing a mechanism for the increased expression of Th2 genes by Bcl6(-/-) Tregs. Gata3 has a critical role in regulating Foxp3 expression and functional fitness of Tregs; however, the signal that regulates Gata3 and restricts its transactivation of Th2 cytokines in Tregs has remained unexplored. Our results identify Bcl6 as an essential transcription factor regulating Gata3 activity in Tregs. Thus, Bcl6 represents a crucial regulatory layer in the Treg functional program that is required for specific suppression of Gata3 and Th2 effector responses by Tregs.


Assuntos
Proteínas de Ligação a DNA/imunologia , Fator de Transcrição GATA3/imunologia , Linfócitos T Reguladores/imunologia , Células Th2/imunologia , Transcrição Gênica/imunologia , Ativação Transcricional/imunologia , Animais , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Camundongos , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/imunologia , MicroRNAs/metabolismo , Pneumonia/genética , Pneumonia/imunologia , Pneumonia/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6 , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo , Células Th1/citologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th17/citologia , Células Th17/imunologia , Células Th17/metabolismo , Células Th2/citologia , Células Th2/metabolismo , Transcrição Gênica/genética , Ativação Transcricional/genética
12.
J Immunol ; 184(8): 4123-32, 2010 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-20212093

RESUMO

The transcriptional repressor protein BCL6 regulates T cell differentiation by repressing Th2 responses and promoting follicular Th cell responses. However, little is known about the role of BCL6 in Th17 responses. We found that memory T cells from BCL6-deficient mice had increased IL-17 production. Additionally, BCL6 expression is upregulated in CD4 T cells cultured under Th17 conditions. T cells from BCL6-deficient mice showed defective Th17 differentiation and enhanced IL-4 production in vitro; however, normal Th17 differentiation was obtained with BCL6-deficient T cells under culture conditions when highly pure naive CD4 T cells were used, when IL-4 production was inhibited, or when TGF-beta levels were increased. Retrovirus-mediated expression of BCL6 in CD4 T cells repressed IL-4 and augmented basal IL-17 mRNA expression. These data support the idea that BCL6 promotes Th17 differentiation through suppression of Th2 differentiation. BCL6-deficient T cells transplanted into Rag1(-/-) mice produced wild-type levels of IL-17, indicating that, in vivo, BCL6-deficient T cells develop relatively normal Th17 responses. Macrophages from BCL6-deficient mice showed strikingly increased expression of the Th17-promoting cytokines IL-6, IL-23, and TGF-beta, and conditioned media from BCL6-deficient macrophages promoted augmented IL-17 expression by T cells. We propose that the increased Th17 activity in BCL6-deficient mice is due, in part, to BCL6-deficient macrophages promoting increased Th17 differentiation in vivo. T cells may require BCL6 for optimal Th17 differentiation; however, BCL6 function in macrophages critically regulates Th17 differentiation in vivo. We hypothesize that increased Th17 differentiation aggravates the severe Th2-type inflammatory disease in BCL6-deficient mice.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Interleucina-17/metabolismo , Macrófagos/imunologia , Proteínas Repressoras/fisiologia , Subpopulações de Linfócitos T/imunologia , Transcrição Gênica/imunologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Imunofenotipagem , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interleucina-17/biossíntese , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-bcl-6 , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Índice de Gravidade de Doença , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Regulação para Cima/genética , Regulação para Cima/imunologia
13.
J Exp Med ; 218(6)2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-33900375

RESUMO

Single-cell RNA sequencing is a powerful tool to examine cellular heterogeneity, novel markers and target genes, and therapeutic mechanisms in human cancers and animal models. Here, we analyzed single-cell RNA sequencing data of T cells obtained from multiple mouse tumor models by PCA-based subclustering coupled with TCR tracking using the STARTRAC algorithm. This approach revealed various differentiated T cell subsets and activation states, and a correspondence of T cell subsets between human and mouse tumors. STARTRAC analyses demonstrated peripheral T cell subsets that were developmentally connected with tumor-infiltrating CD8+ cells, CD4+ Th1 cells, and T reg cells. In addition, large amounts of paired TCRα/ß sequences enabled us to identify a specific enrichment of paired public TCR clones in tumor. Finally, we identified CCR8 as a tumor-associated T reg cell marker that could preferentially deplete tumor-associated T reg cells. We showed that CCR8-depleting antibody treatment provided therapeutic benefit in CT26 tumors and synergized with anti-PD-1 treatment in MC38 and B16F10 tumor models.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias/imunologia , Linfócitos T Reguladores/imunologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/imunologia , Células Th1/imunologia
14.
Sci Transl Med ; 13(608)2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34433637

RESUMO

Therapeutic approaches are needed to promote T cell-mediated destruction of poorly immunogenic, "cold" tumors typically associated with minimal response to immune checkpoint blockade (ICB) therapy. Bispecific T cell engager (BiTE) molecules induce redirected lysis of cancer cells by polyclonal T cells and have demonstrated promising clinical activity against solid tumors in some patients. However, little is understood about the key factors that govern clinical responses to these therapies. Using an immunocompetent mouse model expressing a humanized CD3ε chain (huCD3e mice) and BiTE molecules directed against mouse CD19, mouse CLDN18.2, or human EPCAM antigens, we investigated the pharmacokinetic and pharmacodynamic parameters and immune correlates associated with BiTE efficacy across multiple syngeneic solid-tumor models. These studies demonstrated that pretreatment tumor-associated T cell density is a critical determinant of response to BiTE therapy, identified CD8+ T cells as important targets and mediators of BiTE activity, and revealed an antagonistic role for CD4+ T cells in BiTE efficacy. We also identified therapeutic combinations, including ICB and 4-1BB agonism, that synergized with BiTE treatment in poorly T cell-infiltrated, immunotherapy-refractory tumors. In these models, BiTE efficacy was dependent on local expansion of tumor-associated CD8+ T cells, rather than their recruitment from circulation. Our findings highlight the relative contributions of baseline T cell infiltration, local T cell proliferation, and peripheral T cell trafficking for BiTE molecule-mediated efficacy, identify combination strategies capable of overcoming resistance to BiTE therapy, and have clinical relevance for the development of BiTE and other T cell engager therapies.


Assuntos
Anticorpos Biespecíficos , Neoplasias , Animais , Anticorpos Biespecíficos/uso terapêutico , Antígenos CD19 , Complexo CD3 , Linfócitos T CD8-Positivos , Claudinas , Humanos , Imunoterapia , Camundongos , Neoplasias/tratamento farmacológico
15.
J Biol Methods ; 7(1): e125, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32206674

RESUMO

Bone marrow chimeras represent a key tool employed to understand biological contributions stemming from the hematopoietic versus the stromal compartment. In most institutions, cesium irradiators are used to lethally irradiate recipient animals prior to the injection of donor bone marrow. Cesium irradiators, however, have significant liabilities-including concerns around domestic security. Recently, X-ray irradiators have been implemented as a potential alternative to cesium sources. Only a small number of publications in the literature have attempted to compare these two modalities and, in most cases, the emphasis was on irradiation of human blood productions. We were able to find only a single study that directly compared X-ray and cesium technologies in the generation of murine bone marrow chimeras, a standard laboratory practice. This study focused on chimerism in the blood of recipient animals. In the present study, we begin by comparing cesium and X-ray based sources for irradiation, then transition to using X-ray-based systems for immunology models with an emphasis on immunotherapy of cancer in immunocompetent mouse models-specifically evaluating chimerism in the blood, spleen, and tumor microenvironment. While our data demonstrate that the two platforms are functionally comparable and suggest that X-ray based technology is a suitable alternative to cesium sources. We also highlight a difference in chimerism between the peripheral (blood, spleen) and tumor compartments that is observed using both technologies. While the overall degree of chimerism in the peripheral tissues is very high, the degree of chimerism in the tumor is cell type specific with T and NK cells showing lower chimerism than other cell types.

16.
Cell Rep ; 30(4): 1039-1051.e5, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31995748

RESUMO

Interleukin-35 (IL-35) is an immunosuppressive cytokine composed of Epstein-Barr-virus-induced protein 3 (Ebi3) and IL-12α chain (p35) subunits, yet the forms that IL-35 assume and its role in peripheral tolerance remain elusive. We induce CBA-specific, IL-35-producing T regulatory (Treg) cells in TregEbi3WT C57BL/6 reporter mice and identify IL-35 producers by expression of Ebi3TdTom gene reporter plus Ebi3 and p35 proteins. Curiously, both subunits of IL-35 are displayed on the surface of tolerogen-specific Foxp3+ and Foxp3neg (iTr35) T cells. Furthermore, IL-35 producers, although rare, secrete Ebi3 and p35 on extracellular vesicles (EVs) targeting a 25- to 100-fold higher number of T and B lymphocytes, causing them to acquire surface IL-35. This surface IL-35 is absent when EV production is inhibited or if Ebi3 is genetically deleted in Treg cells. The unique ability of EVs to coat bystander lymphocytes with IL-35, promoting exhaustion in, and secondary suppression by, non-Treg cells identifies a novel mechanism of infectious tolerance.


Assuntos
Vesículas Extracelulares/metabolismo , Tolerância Imunológica , Subunidade p35 da Interleucina-12/metabolismo , Interleucinas/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Receptores de Citocinas/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Técnicas de Cocultura , Vesículas Extracelulares/imunologia , Vesículas Extracelulares/ultraestrutura , Feminino , Fatores de Transcrição Forkhead/metabolismo , Técnicas de Inativação de Genes , Transplante de Coração , Terapia de Imunossupressão , Interleucinas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Linfócitos T Reguladores/metabolismo
17.
Int J Cancer ; 125(4): 977-81, 2009 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-19405121

RESUMO

The BCL6 transcriptional repressor protein has been shown to promote B-cell lymphoma in transgenic mouse models. The mechanism by which BCL6 transforms primary B cells is unclear, although repression of the p53 tumor suppressor is thought to play a role. Here, we showed that BCL6 has critical oncogene functions that are independent of p53 repression. We found that BCL6 cooperates with constitutive CD40 signaling to rapidly transform p53-deficient primary mouse B cells in vitro. Constitutive CD40 signaling alone does not transform p53-deficient B cells, indicating that BCL6 acts specifically as an immortalizing oncogene in this system. The BCL6 transformed B cells are polyclonal and form polyclonal tumors. At the initiation of the cultures, BCL6 does not significantly alter cell cycle progression, but it does promote increased cell survival. Early cultures of BCL6-expressing B cells exhibited marked repression of ATR and p27kip1 but not other BCL6 target genes, suggesting that the ATR and p27kip1 genes have key early roles in mediating BCL6 transformation function. BCL6-transformed cell lines exhibited further decreases of ATR and p27kip1 expression plus strong decreases in Blimp1 and PDCD2 expression. Our study provides important clues about the critical target genes used by BCL6 to transform primary B cells and indicates that the CD40 signaling pathway can collaborate with BCL6 in the transformation of primary B cells. Thus, our study demonstrates a rapid in vitro system to analyze the transformation function of BCL6.


Assuntos
Linfócitos B/metabolismo , Antígenos CD40/metabolismo , Transformação Celular Neoplásica/metabolismo , Proteínas de Ligação a DNA/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Ligante de CD40/metabolismo , Células Cultivadas , Linfoma de Células B/patologia , Camundongos , Proteínas Proto-Oncogênicas c-bcl-6
18.
J Interferon Cytokine Res ; 35(7): 499-512, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25919641

RESUMO

Counter-regulation afforded by specialized regulatory cell populations and immunosuppressive cytokines is critical for balancing immune outcome. The inhibitory potential of the established suppressive cytokines, IL-10 and TGFß, has been well elucidated in diverse inflammatory scenarios in conjunction with their key roles in Treg development and function. Despite the early predictions for an immunomodulatory role for the Ebi3/p35 heterodimer in placental trophoblasts, IL-35 biology remained elusive until 2007 when it was established as a Treg-restricted inhibitory cytokine. Since then, Treg-derived IL-35 has been shown to exhibit its suppressive activities in a range of autoimmune diseases and cancer models. Recent studies are beginning to explore other cellular sources of IL-35, such as Bregs and CD8(+) Tregs. Despite these new cellular sources and targets, the mode of IL-35 suppression remains restricted to inhibition of proliferation and induction of an IL-35-producing induced regulatory T cell population referred to as iTr35. In this review, we explore the early beginnings, status quo, and future prospects of IL-35 biology. The unparalleled opportunity of targeting multiple immunosuppressive populations (Tregs, Bregs, CD8(+) Tregs) through IL-35 is highly exciting and offers tremendous promise from a translational standpoint, particularly for cancer immunotherapies.


Assuntos
Interleucinas/metabolismo , Animais , Linfócitos B/imunologia , Humanos , Imunidade , Imunomodulação , Transdução de Sinais , Linfócitos T Reguladores/imunologia
19.
Microrna ; 4(1): 36-40, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25707810

RESUMO

MicroRNAs (miRs) have emerged as useful biomarkers for different disease states, including allergic inflammatory diseases such as asthma and eosinophilic esophagitis (EoE). Serum miRs are a possible non-invasive method for diagnosis of such diseases. We focused on microRNA-21 (miR-21) levels in serum, in order to assess the feasibility of using this gene as a non-invasive biomarker for these diseases in the clinic, as well as to better understand the expression pattern of miR-21 in allergic inflammation. We used quantitative PCR (QPCR) to assay miR-21 and other control miRs in esophageal biopsies from EoE patients and serum samples from EoE and asthma patients. Serum levels of miR-21 were significantly elevated in patients with asthma, whereas serum miR-21 levels were not associated with the presence of allergen-specific IgE (i.e. atopy). Esophageal biopsies showed a large elevation of miR-21 in EoE and an increase in miR-21 in EoE serum. Control U6 miR did not vary between asthma and control patients, however EoE serum had significantly decreased U6 microRNA compared to controls. The decreased U6 in EoE sera did not completely account for the relative increase in miR-21 in the sera of EoE patients. We report for the first time that miR-21 is elevated in the sera of both asthma and EoE patients. We find no relation between serum miR-21 levels and atopy. Our results thus suggest miR-21 is a novel biomarker for human allergic inflammatory diseases.


Assuntos
Asma/sangue , Esofagite Eosinofílica/sangue , MicroRNAs/sangue , Biomarcadores/sangue , Criança , Pré-Escolar , Humanos , Imunoglobulina E/sangue
20.
Mol Immunol ; 54(3-4): 435-42, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23416424

RESUMO

The transcriptional repressor Bcl6 is a critical regulator of T helper cell fate, and inhibits Th2-type inflammation. We have found that microRNA-21 (miR-21) is a novel target gene for Bcl6 in Treg cells. Bcl6 represses and Stat3 activates miR-21 transcription through a Stat3 binding element in the promoter, indicating opposing regulation of miR-21 by the two transcription factors via the same DNA site. Ectopic expression of miR-21 promoted Th2 differentiation in non-polarized T cells. The pro-Th2 activity of miR-21 was associated with increased Gata3 expression and decreased expression of the miR-21 target gene Sprouty1. Increased miR-21 promoted Th2 and Treg gene expression in wild-type Tregs. MiR-21 could thus help promote the Th2 bias of Bcl6-deficient conventional T cells and Treg cells. MiR21 expression is increased in Th2-type inflammation, and our results define miR-21 as a critical target of Bcl6, thus providing a new link between Bcl6 and Th2 inflammation. Finally, our results reveal a novel T cell autonomous role for miR-21 in promoting Th2 differentiation.


Assuntos
Proteínas de Ligação a DNA/genética , MicroRNAs/genética , Células Th2/fisiologia , Animais , Sítios de Ligação , Diferenciação Celular/genética , Proteínas de Ligação a DNA/metabolismo , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-6 , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Células Th2/citologia , Células Th2/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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