RESUMEN
Mucosa-associated lymphoid tissue (MALT) lymphoma is a B-cell tumour that develops over many decades in the stomachs of individuals with chronic Helicobacter pylori infection. We developed a new mouse model of human gastric MALT lymphoma in which mice with a myeloid-specific deletion of the innate immune molecule, Nlrc5, develop precursor B-cell lesions to MALT lymphoma at only 3 months post-Helicobacter infection versus 9-24 months in existing models. The gastric B-cell lesions in the Nlrc5 knockout mice had the histopathological features of the human disease, notably lymphoepithelial-like lesions, centrocyte-like cells, and were infiltrated by dendritic cells (DCs), macrophages, and T-cells (CD4+ , CD8+ and Foxp3+ ). Mouse and human gastric tissues contained immune cells expressing immune checkpoint receptor programmed death 1 (PD-1) and its ligand PD-L1, indicating an immunosuppressive tissue microenvironment. We next determined whether CD40L, overexpressed in a range of B-cell malignancies, may be a potential drug target for the treatment of gastric MALT lymphoma. Importantly, we showed that the administration of anti-CD40L antibody either coincident with or after establishment of Helicobacter infection prevented gastric B-cell lesions in mice, when compared with the control antibody treatment. Mice administered the CD40L antibody also had significantly reduced numbers of gastric DCs, CD8+ and Foxp3+ T-cells, as well as decreased gastric expression of B-cell lymphoma genes. These findings validate the potential of CD40L as a therapeutic target in the treatment of human gastric B-cell MALT lymphoma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Asunto(s)
Infecciones por Helicobacter , Helicobacter pylori , Linfoma de Células B de la Zona Marginal , Neoplasias Gástricas , Animales , Ratones , Linfocitos B , Ligando de CD40 , Factores de Transcripción Forkhead/metabolismo , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/tratamiento farmacológico , Helicobacter pylori/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Linfoma de Células B de la Zona Marginal/tratamiento farmacológico , Linfoma de Células B de la Zona Marginal/genética , Linfoma de Células B de la Zona Marginal/prevención & control , Neoplasias Gástricas/patología , Microambiente TumoralRESUMEN
BACKGROUND: Understanding immune phenotypes and human gastric disease in situ requires an approach that leverages multiplexed immunohistochemistry (mIHC) with multispectral imaging to facilitate precise image analyses. METHODS: We developed a novel 4-color mIHC assay based on tyramide signal amplification that allowed us to reliably interrogate immunologic checkpoints, including programmed death-ligand 1 (PD-L1), cytotoxic T cells (CD8+T) and regulatory T cells (Foxp3), in formalin-fixed, paraffin-embedded tissues of various human gastric diseases. By observing cell phenotypes within the disease tissue microenvironment, we were able to determine specific co-localized staining combinations and various measures of cell density. RESULTS: We found that PD-L1 was expressed in gastric ulcer and in tumor cells (TCs), as well as in tumor-infiltrating immune cells (TIICs), but not in normal gastric mucosa or other gastric intraepithelial neoplastic tissues. Furthermore, we found no significant reduction in CD8+T cells, whereas the ratio of CD8+T:Foxp3 cells and CD8+T:PD-L1 cells was suppressed in tumor tissues and elevated in adjacent normal tissues. An unsupervised hierarchical analysis also identified correlations between CD8+T and Foxp3+ tumor-infiltrating lymphocyte (TIL) densities and average PD-L1 levels. Three main groups were identified based on the results of CD8+T:PD-L1 ratios in gastric tumor tissues. Furthermore, integrating CD8+T:Foxp3 ratios, which increased the complexity for immune phenotype status, revealed 6-7 clusters that enabled the separation of gastric cancer patients at the same clinical stage into different risk-group subsets. CONCLUSIONS: Characterizing immune phenotypes in human gastric disease tissues via multiplexed immunohistochemistry may help guide PD-L1 clinical therapy. Observing unique disease tissue microenvironments can improve our understanding of immune phenotypes and cell interactions within these microenvironments, providing the ability to predict safe responses to immunotherapies.
Asunto(s)
Inmunohistoquímica/métodos , Gastropatías/inmunología , Gastropatías/patología , Antígeno B7-H1/metabolismo , Linfocitos T CD8-positivos/inmunología , Factores de Transcripción Forkhead/metabolismo , Humanos , FenotipoRESUMEN
Cell division autoantigen 1 (CDA1) enhances TGF-ß signaling in renal and vascular cells, and renal expression of CDA1 is elevated in animal models of diabetes. In this study, we investigated the genetic deletion of Tspyl2, the gene encoding CDA1, in C57BL6 and ApoE knockout mice. The increased renal expression of TGF-ß1, TGF-ß type I and II receptors, and phosphorylated Smad3 associated with diabetes in wild-type mice was attenuated in diabetic CDA1 knockout mice. Notably, CDA1 deletion significantly reduced diabetes-associated renal matrix accumulation and immunohistochemical staining for collagens III and IV and attenuated glomerular and tubulointerstitial injury indices, despite the presence of persistent hyperglycemia, polyuria, renal hypertrophy, and hyperfiltration. Furthermore, CDA1 deletion reduced gene expression of TGF-ß1 receptors in the kidney, resulting in a functionally attenuated response to exogenous TGF-ß, including reduced levels of phosphorylated Smad3 and ERK1/2, in primary kidney cells from CDA1 knockout animals. Taken together, these data suggest that CDA1 deletion reduces but does not block renal TGF-ß signaling. Because direct antagonism of TGF-ß or its receptors has unwanted effects, CDA1 may be a potential therapeutic target for retarding DN and perhaps, other kidney diseases associated with TGF-ß-mediated fibrogenesis.
Asunto(s)
Autoantígenos/fisiología , Nefropatías Diabéticas/etiología , Animales , Autoantígenos/genética , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Femenino , Fibrosis , Riñón/lesiones , Riñón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Proteína smad3/fisiología , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
Cell division autoantigen 1 (CDA1) modulates cell proliferation and transforming growth factor-ß (TGF-ß) signaling in a number of cellular systems; here we found that its levels were elevated in the kidneys of two animal models of diabetic renal disease. The localization of CDA1 to tubular cells and podocytes in human kidney sections was similar to that seen in the rodent models. CDA1 small interfering RNA knockdown markedly attenuated, whereas its overexpression increased TGF-ß signaling, modulating the expression of TGF-ß, TGF-ß receptors, connective tissue growth factor, collagen types I, III, IV, and fibronectin genes in HK-2 cells. CDA1 and TGF-ß together were synergistic in stimulating TGF-ß signaling and target gene expression. CDA1 knockdown effectively blocked TGF-ß-stimulated expression of collagen genes. This was due to its ability to modulate the TGF-ß type I, but not the type II, receptor, leading to increased phosphorylation of Smad3 and extracellular signal-regulated kinase mitogen-activated protein kinase. Furthermore, the Smad3 inhibitor, SIS3, markedly attenuated the activities of CDA1 in stimulating TGF-ß signaling as well as gene expression of collagens I, III, and IV. Thus, our in vitro and in vivo findings show that CDA1 has a critical role in TGF-ß signaling in the kidney.
Asunto(s)
Autoantígenos/fisiología , Nefropatías Diabéticas/etiología , Factor de Crecimiento Transformador beta/fisiología , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Autoantígenos/genética , Secuencia de Bases , Línea Celular , Cartilla de ADN/genética , Nefropatías Diabéticas/patología , Nefropatías Diabéticas/fisiopatología , Modelos Animales de Enfermedad , Fibrosis , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Ratas , Ratas Endogámicas SHR , Transducción de SeñalRESUMEN
Current studies aiming at identifying single immune markers with prognostic value have limitations in the context of complex antitumor immunity and cancer immune evasion. Here, we show how the integration of several immune markers influences the predictions of prognosis of gastric cancer (GC) patients. We analyzed Tissue Microarray (TMA) by multiplex immunohistochemistry and measured the expression of immune checkpoint molecule PD-L1 together with antitumor CD8 T cells and immune suppressive FOXP3 Treg cells in a cohort of GC patients. Unsupervised hierarchical clustering analysis of these markers was used to define correlations between CD8 T, FOXP3 Treg and PD-L1 cell densities. We found that FOXP3 and PD-L1 densities were elevated while CD8 T cells were decreased in tumor tissues compared to their adjacent normal tissues. However, patient stratification based on each one of these markers individually did not show significant prognostic value on patient survival. Conversely, combination of the ratios of CD8/FOXP3 and CD8/PD-L1 enabled the identification of patient subgroups with different survival outcomes. As such, high densities of PD-L1 in patients with high CD8/FOXP3 and low CD8/PD-L1 ratios correlated with increased survival. Collectively, this work demonstrates the need for the integration of several immune markers to obtain more meaningful survival prognosis and patient stratification. In addition, our work provides insights into the complex tumor immune evasion and immune regulation by the tumor-infiltrating effector and suppressor cells, informing on the best use of immunotherapy options for treating patients.
RESUMEN
Diabetes is a negative risk factor for aortic aneurysm, but the underlying explanation for this phenomenon is unknown. We have previously demonstrated that cell division autoantigen 1 (CDA1), which enhances transforming growth factor-ß signaling, is upregulated in diabetes. We hypothesized that CDA1 plays a key role in conferring the protective effect of diabetes against aortic aneurysms. Male wild-type, CDA1 knockout (KO), apolipoprotein E (ApoE) KO, and CDA1/ApoE double-KO (dKO) mice were rendered diabetic. Whereas aneurysms were not observed in diabetic ApoE KO and wild-type mice, 40% of diabetic dKO mice developed aortic aneurysms. These aneurysms were associated with attenuated aortic transforming growth factor-ß signaling, reduced expression of various collagens, and increased aortic macrophage infiltration and matrix metalloproteinase 12 expression. In the well-characterized model of angiotensin II-induced aneurysm formation, concomitant diabetes reduced fatal aortic rupture and attenuated suprarenal aortic expansion, changes not seen in dKO mice. Furthermore, aortic CDA1 expression was downregulated â¼70% within biopsies from human abdominal aortic aneurysms. The identification that diabetes is associated with upregulation of vascular CDA1 and that CDA1 deletion in diabetic mice promotes aneurysm formation provides evidence that CDA1 plays a role in diabetes to reduce susceptibility to aneurysm formation.
Asunto(s)
Aneurisma de la Aorta Abdominal/genética , Autoantígenos/genética , Diabetes Mellitus Experimental/metabolismo , Adulto , Anciano , Angiotensina II/farmacología , Animales , Aneurisma de la Aorta/inducido químicamente , Aneurisma de la Aorta/genética , Aneurisma de la Aorta/inmunología , Aneurisma de la Aorta/metabolismo , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/inmunología , Aneurisma de la Aorta Abdominal/metabolismo , Rotura de la Aorta , Autoantígenos/metabolismo , Colágeno/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Macrófagos/inmunología , Masculino , Metaloproteinasa 12 de la Matriz/metabolismo , Ratones , Ratones Noqueados , Ratones Noqueados para ApoE , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Transducción de Señal , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Vasoconstrictores/farmacologíaRESUMEN
Cell Division Autoantigen 1 (CDA1) was discovered following screening a human expression library with serum from a patient with Discoid Lupus Erythematosus. CDA1, encoded by TSPYL2 on the X chromosome, shares anti-proliferative and profibrotic properties with TGF-b. It inhibits cell growth through p53, pERK1/2 and p21mediated pathways and is implicated in tumorigenesis and the DNA damage response. Its pro-fibrotic property is mediated through cross-talk with TGF-b that results in upregulation of extracellular matrix proteins. The latter properties have identified a key role for CDA1 in diabetes associated atherosclerosis. These dual properties place CDA1 as an attractive molecular target for treating tumors and vascular fibrosis including atherosclerosis and other vascular disorders associated with enhanced TGF-ß action and tissue scarring.
RESUMEN
We previously reported that overexpression of cell division autoantigen 1 (CDA1) in HeLa cells arrests cell growth and inhibits DNA synthesis at S-phase. Here we show that CDA1-induced arrest of cell growth is accompanied by increases in protein and mRNA levels of the cyclin-dependent kinase (Cdk) inhibitor protein, p21(Waf1/Cip1) (p21). Both p21 induction and cell growth arrest are reversed when CDA1 expression is inhibited. CDA1 also increases p53 protein, but not its mRNA, in a time- and dose-dependent manner. MDM2, a ubiquitin ligase regulating p53 degradation, is inactivated by CDA1, suggesting that p53 protein accumulation is due to decreased protein degradation. Knockdown of p53, using siRNA targeting two sites of p53 mRNA, abrogates transcriptional induction of p21 by CDA1. Deletion of the p53 responsive element in the distal region of p21 promoter attenuates promoter activity in response to CDA1. DNA damage caused by camptothecin treatment increases mRNA and protein levels of CDA1, accompanied by induction of p53. The DNA damage-induced p53 induction is markedly attenuated by CDA1 knockdown. CDA1 induces phosphorylation of ERK1/2(p44/42), an activity blocked by PD98059 and U0126, inhibitors of the upstream kinase MEK1/2. The MEK inhibitors also block induction of p21 mRNA and abrogate p21 promoter activity stimulated by CDA1. Cell cycle kinases, Cdk1, -2, -4, and -6 are inhibited by CDA1 overexpression. We conclude that CDA1 induces p53- and MEK/ERK1/2 MAPK-dependent expression of p21 by acting through the p53 responsive element in the p21 promoter and that this contributes to its antiproliferative activity.