RESUMEN
Homeostatic programs balance immune protection and self-tolerance. Such mechanisms likely impact autoimmunity and tumor formation, respectively. How homeostasis is maintained and impacts tumor surveillance is unknown. Here, we find that different immune mononuclear phagocytes share a conserved steady-state program during differentiation and entry into healthy tissue. IFNγ is necessary and sufficient to induce this program, revealing a key instructive role. Remarkably, homeostatic and IFNγ-dependent programs enrich across primary human tumors, including melanoma, and stratify survival. Single-cell RNA sequencing (RNA-seq) reveals enrichment of homeostatic modules in monocytes and DCs from human metastatic melanoma. Suppressor-of-cytokine-2 (SOCS2) protein, a conserved program transcript, is expressed by mononuclear phagocytes infiltrating primary melanoma and is induced by IFNγ. SOCS2 limits adaptive anti-tumoral immunity and DC-based priming of T cells in vivo, indicating a critical regulatory role. These findings link immune homeostasis to key determinants of anti-tumoral immunity and escape, revealing co-opting of tissue-specific immune development in the tumor microenvironment.
Asunto(s)
Interferón gamma/inmunología , Melanoma/inmunología , Monocitos/inmunología , Metástasis de la Neoplasia/patología , Neoplasias Cutáneas/inmunología , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Microambiente Tumoral , Animales , Diferenciación Celular , Células Dendríticas/inmunología , Homeostasis , Humanos , Melanoma/genética , Melanoma/patología , Ratones , Monocitos/patología , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , TranscriptomaRESUMEN
Humoral immunity is reliant on efficient recruitment of circulating naïve B cells from blood into peripheral lymph nodes (LN) and timely transition of naive B cells to high affinity antibody (Ab)-producing cells. Current understanding of factor(s) coordinating B cell adhesion, activation and differentiation within LN, however, is incomplete. Prior studies on naïve B cells reveal remarkably strong binding to putative immunoregulator, galectin (Gal)-9, that attenuates BCR activation and signaling, implicating Gal-9 as a negative regulator in B cell biology. Here, we investigated Gal-9 localization in human tonsils and LNs and unearthed conspicuously high expression of Gal-9 on high endothelial and post-capillary venules. Adhesion analyses showed that Gal-9 can bridge human circulating and naïve B cells to vascular endothelial cells (EC), while decelerating transendothelial migration. Moreover, Gal-9 interactions with naïve B cells induced global transcription of gene families related to regulation of cell signaling and membrane/cytoskeletal dynamics. Signaling lymphocytic activation molecule F7 (SLAMF7) was among key immunoregulators elevated by Gal-9-binding, while SLAMF7's cytosolic adapter EAT-2, which is required for cell activation, was eliminated. Gal-9 also activated phosphorylation of pro-survival factor, ERK. Together, these data suggest that Gal-9 promotes B cell - EC interactions while delivering anergic signals to control B cell reactivity.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , Endotelio Vascular/metabolismo , Galectinas/metabolismo , Inmunomodulación , Transducción de Señal , Linfocitos B/citología , Biomarcadores , Adhesión Celular , Comunicación Celular/inmunología , Diferenciación Celular/inmunología , Movimiento Celular , Humanos , Inmunohistoquímica , Inmunofenotipificación , Activación de Linfocitos , Transporte de ProteínasRESUMEN
The IL-1 superfamily of cytokines and receptors has been studied extensively. However, the specific roles of IL-1 elements in host immunity to cutaneous viral infection remain elusive. In this study, we applied vaccinia virus (VACV) by scarification to IL-1R1 knockout mice (IL-1R1-/-) and found that these mice developed markedly larger lesions with higher viral genome copies in skin than did wild-type mice. The phenotype of infected IL-1R1-/- mice was similar to eczema vaccinatum, a severe side effect of VACV vaccination that may develop in humans with atopic dermatitis. Interestingly, the impaired cutaneous response of IL-1R1-/- mice did not reflect a systemic immune deficiency, because immunized IL-1R1-/- mice survived subsequent lethal VACV intranasal challenge, or defects of T cell activation or T cell homing to the site of inoculation. Histologic evaluation revealed that VACV infection and replication after scarification were limited to the epidermal layer of wild-type mice, whereas lack of IL-1R1 permitted extension of VACV infection into dermal layers of the skin. We explored the etiology of this discrepancy and determined that IL-1R1-/- mice contained significantly more macrophages and monocyte-derived dendritic cells in the dermis after VACV scarification. These cells were vulnerable to VACV infection and may augment the transmission of virus to adjacent skin, thus leading to larger skin lesions and satellite lesions in IL-1R1-/- mice. These results suggest new therapeutic strategies for treatment of eczema vaccinatum and inform assessment of risks in patients receiving IL-1 blocking Abs for treatment of chronic inflammatory disorders.
Asunto(s)
Proteína Antagonista del Receptor de Interleucina 1/deficiencia , Proteína Antagonista del Receptor de Interleucina 1/inmunología , Enfermedades Cutáneas Infecciosas/inmunología , Piel/patología , Virus Vaccinia/inmunología , Vaccinia/inmunología , Administración Cutánea , Animales , Linfocitos T CD8-positivos/inmunología , Proteína Antagonista del Receptor de Interleucina 1/genética , Erupción Variceliforme de Kaposi/inmunología , Erupción Variceliforme de Kaposi/fisiopatología , Erupción Variceliforme de Kaposi/terapia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Piel/anatomía & histología , Piel/inmunología , Piel/virología , Vacunación , Virus Vaccinia/fisiología , Replicación ViralRESUMEN
Ly6G is a glycosylphosphatidylinositol (GPI)-anchored protein of unknown function that is commonly targeted to induce experimental neutrophil depletion in mice. In the present study, we found that doses of anti-Ly6G Abs too low to produce sustained neutropenia remained capable of inhibiting experimental arthritis, leaving joint tissues free of infiltrating neutrophils. Thioglycollate-stimulated peritonitis was also attenuated. No alteration in neutrophil apoptosis was observed, implicating impaired recruitment. Indeed, Ly6G ligation abrogated neutrophil migration toward LTB(4) and other chemoattractants in a transwell system. Exploring the basis for this blockade, we identified colocalization of Ly6G and ß2-integrins by confocal microscopy and confirmed close association by both coimmunoprecipitation and fluorescence lifetime imaging microscopy. Anti-Ly6G Ab impaired surface expression of ß2-integrins in LTB(4)-stimulated neutrophils and mimicked CD11a blockade in inhibiting both ICAM-1 binding and firm adhesion to activated endothelium under flow conditions. Correspondingly, migration of ß2-integrin-deficient neutrophils was no longer inhibited by anti-Ly6G. These results demonstrate that experimental targeting of Ly6G has functional effects on the neutrophil population and identify a previously unappreciated role for Ly6G as a modulator of neutrophil migration to sites of inflammation via a ß2-integrin-dependent mechanism.
Asunto(s)
Antígenos Ly/metabolismo , Antígenos CD18/metabolismo , Infiltración Neutrófila , Neutrófilos/patología , Animales , Anticuerpos/farmacología , Apoptosis/efectos de los fármacos , Artritis/sangre , Artritis/patología , Artritis/prevención & control , Biomarcadores/metabolismo , Calcio/metabolismo , Movimiento Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Inflamación/patología , Articulaciones/efectos de los fármacos , Articulaciones/patología , Leucotrieno B4/farmacología , Ratones , Ratones Endogámicos C57BL , Activación Neutrófila/efectos de los fármacos , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Peritoneo/efectos de los fármacos , Peritoneo/patología , Receptores de Leucotrieno B4/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
We assessed the contribution of IL1 signaling molecules to malignant tumor growth using IL1ß-/-, IL1α-/-, and IL1R1-/- mice. Tumors grew progressively in IL1R-/- and IL1α-/- mice but were often absent in IL1ß-/- mice. This was observed whether tumors were implanted intradermally or injected intravenously and was true across multiple distinct tumor lineages. Antibodies to IL1ß prevented tumor growth in wild-type (WT) mice but not in IL1R1-/- or IL1α-/- mice. Antibodies to IL1α promoted tumor growth in IL1ß-/- mice and reversed the tumor-suppressive effect of anti-IL1ß in WT mice. Depletion of CD8+ T cells and blockade of lymphocyte mobilization abrogated the IL1ß-/- tumor suppressive effect, as did crossing IL1ß-/- mice to SCID or Rag1-/- mice. Finally, blockade of IL1ß synergized with blockade of PD-1 to inhibit tumor growth in WT mice. These results suggest that IL1ß promotes tumor growth, whereas IL1α inhibits tumor growth by enhancing T-cell-mediated antitumor immunity.
Asunto(s)
Inmunidad Adaptativa , Anticuerpos Monoclonales/farmacología , Linfocitos T CD8-positivos/inmunología , Interleucina-1alfa/inmunología , Interleucina-1beta/inmunología , Neoplasias/terapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Neoplasias/inmunología , Microambiente TumoralRESUMEN
Cancer cells often display altered cell-surface glycans compared to their nontransformed counterparts. However, functional contributions of glycans to cancer initiation and progression remain poorly understood. Here, from expression-based analyses across cancer lineages, we found that melanomas exhibit significant transcriptional changes in glycosylation-related genes. This gene signature revealed that, compared to normal melanocytes, melanomas downregulate I-branching glycosyltransferase, GCNT2, leading to a loss of cell-surface I-branched glycans. We found that GCNT2 inversely correlated with clinical progression and that loss of GCNT2 increased melanoma xenograft growth, promoted colony formation, and enhanced cell survival. Conversely, overexpression of GCNT2 decreased melanoma xenograft growth, inhibited colony formation, and increased cell death. More focused analyses revealed reduced signaling responses of two representative glycoprotein families modified by GCNT2, insulin-like growth factor receptor and integrins. Overall, these studies reveal how subtle changes in glycan structure can regulate several malignancy-associated pathways and alter melanoma signaling, growth, and survival.
Asunto(s)
Melanoma/metabolismo , Melanoma/patología , N-Acetilhexosaminiltransferasas/metabolismo , Polisacáridos/metabolismo , Animales , Línea Celular Tumoral , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Humanos , Melanoma/genética , Ratones , Ratones Noqueados , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , N-Acetilhexosaminiltransferasas/genética , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Germinal centers (GC) are microanatomical niches where B cells proliferate, undergo antibody affinity maturation, and differentiate to long-lived memory B cells and antibody-secreting plasma cells. For decades, GC B cells have been defined by their reactivity to the plant lectin peanut agglutinin (PNA), which binds serine/threonine (O-linked) glycans containing the asialylated disaccharide Gal-ß1,3-GalNAc-Ser/Thr (also called T-antigen). In T cells, acquisition of PNA binding by activated T cells and thymocytes has been linked with altered tissue homing patterns, cell signaling, and survival. Yet, in GC B cells, the glycobiological basis and significance of PNA binding remains surprisingly unresolved. Here, we investigated the basis for PNA reactivity of GC B cells. We found that GC B cell binding to PNA is associated with downregulation of the α2,3 sialyltransferase, ST3GAL1 (ST3Gal1), and overexpression of ST3Gal1 was sufficient to reverse PNA binding in B cell lines. Moreover, we found that the primary scaffold for PNA-reactive O-glycans in B cells is the B cell receptor-associated receptor-type tyrosine phosphatase CD45, suggesting a role for altered O-glycosylation in antigen receptor signaling. Consistent with similar reports in T cells, ST3Gal1 overexpression in B cells in vitro induced drastic shortening in O-glycans, which we confirmed by both antibody staining and mass spectrometric O-glycomic analysis. Unexpectedly, ST3Gal1-induced changes in O-glycan length also correlated with altered binding of two glycosylation-sensitive CD45 antibodies, RA3-6B2 (more commonly called B220) and MEM55, which (in humans) have previously been reported to favor binding to naïve/GC subsets and memory/plasmablast subsets, respectively. Analysis of primary B cell binding to B220, MEM55, and several plant lectins suggested that B cell differentiation is accompanied by significant loss of O-glycan complexity, including loss of extended Core 2 O-glycans. To our surprise, decreased O-glycan length from naïve to post-GC fates best correlated not with ST3Gal1, but rather downregulation of the Core 2 branching enzyme GCNT1. Thus, our data suggest that O-glycan remodeling is a feature of B cell differentiation, dually regulated by ST3Gal1 and GCNT1, that ultimately results in expression of distinct O-glycosylation states/CD45 glycoforms at each stage of B cell differentiation.
Asunto(s)
Linfocitos B/inmunología , Diferenciación Celular/inmunología , Polisacáridos/inmunología , Transducción de Señal/inmunología , Linfocitos B/citología , Linfocitos B/metabolismo , Diferenciación Celular/genética , Línea Celular Tumoral , Células Cultivadas , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Centro Germinal/citología , Centro Germinal/inmunología , Centro Germinal/metabolismo , Glicosilación , Humanos , Lectinas/inmunología , Lectinas/metabolismo , Aglutinina de Mani/inmunología , Aglutinina de Mani/metabolismo , Polisacáridos/metabolismo , Sialiltransferasas/genética , Sialiltransferasas/inmunología , Sialiltransferasas/metabolismo , Transducción de Señal/genética , beta-Galactosida alfa-2,3-SialiltransferasaRESUMEN
One strategy adopted by vaccinia virus (VV) to evade the host immune system is to encode homologs of TNF receptors (TNFRs) that block TNF-α function. The response to VV skin infection under conditions of TNF-α deficiency, however, has not been reported. We found that TNFR1-/- mice developed larger primary lesions, numerous satellite lesions, and higher skin virus levels after VV scarification. Following their recovery, VV-scarified TNFR1-/- mice were fully protected against challenge with a lethal intranasal dose of VV, suggesting these mice had developed an effective memory immune response. A functional systemic immune response was further demonstrated by enhanced production of VV-specific IFN-γ and VV-specific CD8(+) T cells in spleens and draining lymph nodes. Interestingly, bone marrow (BM)-reconstitution studies using wild-type (WT) BM in TNFR1-/- host mice, but not TNFR1-/- BM in WT host mice, reproduced the original results seen in TNFR1-/- mice, indicating that TNFR1 deficiency in resident skin cells, rather than hematopoietic cells, accounts for the impaired cutaneous immune response. Our data suggest that lack of TNFR1 leads to a skin-specific immune deficiency, and that resident skin cells have a crucial role in mediating an optimal immune defense to VV cutaneous infection via TNF-α/TNFR1 signaling.
Asunto(s)
Anticuerpos Antivirales/sangre , Linfocitos T CD8-positivos , Receptores Tipo I de Factores de Necrosis Tumoral/inmunología , Piel/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Virus Vaccinia/inmunología , Vaccinia/inmunología , Animales , Inmunidad Innata , Inmunoglobulina G/sangre , Interferón gamma/metabolismo , Recuento de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Tipo I de Factores de Necrosis Tumoral/deficiencia , Piel/virología , Vaccinia/patología , Carga ViralRESUMEN
Selectins are carbohydrate-binding molecules involved in constitutive lymphocyte homing and chronic and acute inflammation processes. Th1 lymphocytes participate in cell-mediated inflammatory reactions, where the selectins play a role and predominate in delayed-type hypersensitivity (DTH) reactions of the skin. Of the many candidate ligands for selectins, only P-selectin glycoprotein ligand 1 (PSGL-1), which also acts as an E-selectin ligand, has been characterized extensively at molecular, cellular, and functional levels on T cells. Here, we report that the glycosylated form of CD43 expressed in Th1 cells is a functional E-selectin-specific ligand in vitro. Furthermore, we have generated PSGL-1(-/-)/CD43(-/-) double-deficient mice (double knockout (DKO)) to demonstrate the relevance of CD43 as an E-selectin ligand in vitro and in vivo. Under flow conditions, DKO Th1 cells exhibited impaired E-selectin binding as compared with wild-type, PSGL-1(-/-), or CD43(-/-) Th1 cells. DKO mice also showed diminished ear inflammation in response to dinitrofluorobenzene-induced DTH that correlated with a reduced number of T cells in infiltrates in the challenged ear. These results demonstrate that both PSGL-1 and CD43 are major E-selectin ligands and are likely to be important during leukocyte recruitment in the development of inflammatory reactions.
Asunto(s)
Selectina E/fisiología , Hipersensibilidad Tardía/etiología , Leucosialina/fisiología , Activación de Linfocitos , Células TH1/inmunología , Animales , Glicosilación , Ligandos , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
The recruitment of memory T cells from blood into tissues is a central element of immune surveillance and adaptive immune responses and a key feature of chronic cutaneous inflammatory diseases such as psoriasis and atopic dermatitis. Human memory T cells that infiltrate skin express the carbohydrate epitope cutaneous lymphocyte-associated antigen (CLA). Expression of the CLA epitope on T cells has been described on P-selectin glycoprotein ligand-1 (PSGL-1) and associated with the acquisition of both E-selectin and P-selectin ligand functions. In this report, we show that CD43, a sialomucin expressed constitutively on T cells, can also be decorated with the CLA epitope and serve as an E-selectin ligand. CLA expressed on CD43 was found exclusively on the high-molecular-weight (125 kDa) glycoform bearing core-2-branched O-linked glycans. CLA+ CD43 purified from human T cells supported tethering and rolling in shear flow via E-selectin but did not support binding of P-selectin. The identification and characterization of CD43 as a T-cell E-selectin ligand distinct from PSGL-1 expands the role of CD43 in the regulation of T-cell trafficking and provides new targets for the modulation of immune functions in skin.
Asunto(s)
Selectina E/fisiología , Leucosialina/fisiología , Glicoproteínas de Membrana/fisiología , Monocitos/inmunología , Linfocitos T/inmunología , Anticuerpos Monoclonales , Antígenos de Diferenciación de Linfocitos T , Antígenos de Neoplasias/fisiología , Humanos , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/aislamiento & purificaciónRESUMEN
Human memory T cells associated with cutaneous inflammatory responses are characterized by their expression of cutaneous lymphocyte-associated Ag (CLA), a carbohydrate determinant differentially expressed on P-selectin glycoprotein ligand-1 (PSGL-1). Although expression of the CLA epitope on PSGL-1 (CLA(+) PSGL-1) by memory T cells is associated with acquisition of E-selectin ligand activity, it is not known whether CLA(+) PSGL-1, itself, is a ligand for E-selectin on human T cells or whether other glycoproteins, with or without CLA modification, support E-selectin-dependent rolling in shear flow. To address this issue, we developed a method for real-time analysis of functional adhesive interactions between selectin-bearing cells in shear flow with leukocyte ligands resolved by SDS-PAGE and immobilized on standard Western blots. The results of these studies provide direct evidence that CLA(+) PSGL-1 is a functional ligand for both E- and P-selectin, confirm that the P-selectin ligand activity of PSGL-1 is independent of CLA modification, and identify a distinct, non-PSGL-1 E-selectin ligand on CLA-positive human memory T cells.