Interactions between LAMP3+ dendritic cells and T-cell subpopulations promote immune evasion in papillary thyroid carcinoma.

BACKGROUND: The incidence of papillary thyroid cancer (PTC) continues to rise all over the world, 10-15% of the patients have a poor prognosis. Although immunotherapy has been applied in clinical practice, its therapeutic efficacy remains far from satisfactory, necessitating further investigation of the mechanism of PTC immune remodeling and exploration of novel treatment targets. METHODS: This study conducted a single-cell RNA sequencing (scRNA-seq) analysis using 18 surgical tissue specimens procured from 14 patients diagnosed with adjacent tissues, non-progressive PTC or progressive PTC. Key findings were authenticated through spatial transcriptomics RNA sequencing, immunohistochemistry, multiplex immunohistochemistry, and an independent bulk RNA-seq data set containing 502 samples. RESULTS: A total of 151,238 individual cells derived from 18 adjacent tissues, non-progressive PTC and progressive PTC specimens underwent scRNA-seq analysis. We found that progressive PTC exhibits the following characteristics: a significant decrease in overall immune cells, enhanced immune evasion of tumor cells, and disrupted antigen presentation function. Moreover, we identified a subpopulation of lysosomal associated membrane protein 3 (LAMP3+) dendritic cells (DCs) exhibiting heightened infiltration in progressive PTC and associated with advanced T stage and poor prognosis of PTC. LAMP3+ DCs promote CD8+ T cells exhaustion (mediated by NECTIN2-TIGIT) and increase infiltration abundance of regulatory T cells (mediated by chemokine (C-C motif) ligand 17 (CCL17)-chemokine (C-C motif) receptor 4 (CCR4)) establishing an immune-suppressive microenvironment. Ultimately, we unveiled that progressive PTC tumor cells facilitate the retention of LAMP3+ DCs within the tumor microenvironment through NECTIN3-NECTIN2 interactions, thereby rendering tumor cells more susceptible to immune evasion. CONCLUSION: Our findings expound valuable insights into the role of the interaction between LAMP3+ DCs and T-cell subpopulations and offer new and effective ideas and strategies for immunotherapy in patients with progressive PTC.

FOXP3, CD208, and CD206 Expression in Canine Cutaneous Histiocytoma.

Canine cutaneous histiocytoma (CCH) is a noninfectious tumor that spontaneously regresses. It is suggested that this regression is due to tumor cell maturation, which is responsible for CD8 lymphocyte activation and tumor cell destruction. Nevertheless, the possible role of the immune microenvironment in tumor regression has not been investigated to date. The aim of this study was to investigate the expression of CD208 and FoxP3 as markers of dendritic cells and regulatory T lymphocytes, respectively, and tumor cell expression of CD206 as a marker of Langerhans cell activation, and relate these parameters to the different phases of CCH and to intratumoral T cell infiltration. Formalin-fixed, paraffin-embedded samples from 31 CCH were evaluated. In each case, the mitotic count and regression phase were recorded. Within the tumor, a quantitative evaluation of immunolabeled CD208+ cells, FoxP3+ cells, and CD3+ lymphocytes was performed, as well as the CD206+ tumor cell location. Intratumoral CD208+ cells correlated with CD3+ lymphocytic infiltration. The possible role of dendritic cells in tumor regression was not confirmed since CD208 seemed to be a nonspecific marker for canine dendritic cells. FoxP3+ lymphocyte density was not correlated with any parameter. Neoplastic Langerhans cells presented progressive CD206 expression, from the bottom of the tumor to the epidermis, which correlated with the tumor regression phase and with intratumoral T lymphocyte infiltration. In conclusion, we confirmed a CD206 phenotype change in tumor cells in a spatial group-related pattern, supporting the hypothesis that tumoral Langerhans cells acquire a mature phenotype with tumor regression.

Recessive missense LAMP3 variant associated with defect in lamellar body biogenesis and fatal neonatal interstitial lung disease in dogs.

Neonatal interstitial lung diseases due to abnormal surfactant biogenesis are rare in humans and have never been reported as a spontaneous disorder in animals. We describe here a novel lung disorder in Airedale Terrier (AT) dogs with clinical symptoms and pathology similar to the most severe neonatal forms of human surfactant deficiency. Lethal hypoxic respiratory distress and failure occurred within the first days or weeks of life in the affected puppies. Transmission electron microscopy of the affected lungs revealed maturation arrest in the formation of lamellar bodies (LBs) in the alveolar epithelial type II (AECII) cells. The secretory organelles were small and contained fewer lamellae, often in combination with small vesicles surrounded by an occasionally disrupted common limiting membrane. A combined approach of genome-wide association study and whole exome sequencing identified a recessive variant, c.1159G>A, p.(E387K), in LAMP3, a limiting membrane protein of the cytoplasmic surfactant organelles in AECII cells. The substitution resides in the LAMP domain adjacent to a conserved disulfide bond. In summary, this study describes a novel interstitial lung disease in dogs, identifies a new candidate gene for human surfactant dysfunction and brings important insights into the essential role of LAMP3 in the process of the LB formation.

Immunohistochemical study of amelogenin binding proteins in an amelogenin point mutation mouse / Estudio inmunohistoquímico de proteínas de unión de la amelogenina en un ratón con mutación puntual de amelogenina

Amelogenin is one of the enamel matrices secreted by ameloblasts. A mutation of the amelogenin gene can cause hereditary dental enamel defects known as amelogenesis imperfecta (AI). Since lysosome-associated membrane protein-1 (LAMP-1), -3 (LAMP-3), and 78kDa glucose-related protein (Grp78) were identified as binding proteins of amelogenin, several studies have suggested the involvement of these binding proteins with the cell kinetics of ameloblasts in normal or abnormal conditions. The purpose of this study is to investigate the distribution of these amelogenin binding proteins in the ameloblast cell differentiation of mice with a point mutation of the amelogenin gene (Amelx*). The incisors of Amelx* mice had a white opaque color and the tooth surface was observed to be rough under a scanning electron microscope. Among the sequential ameloblast cell differentiation in the Amelx* mice, the shape of ameloblasts at the transition stage was irregular in comparison to those in wild-type (WT) mice. Immunostaining of Grp78 revealed that the whole cytoplasm of the transition stage ameloblasts was immunopositive for Grp78 antibody, while only the distal part of cell was positive in the WT mice. Furthermore, in the Amelx* mice, the cytoplasm of the transition stage ameloblasts was immunopositive for LAMP-1 and LAMP-3. These results suggest that Amelx* may cause the abnormal distribution of amelogenin binding proteins in the cytoplasm of ameloblasts.

La amelogenina es una de las matrices de esmalte secretadas por los ameloblastos. Una mutación del gen de amelogenina puede causar defectos hereditarios del esmalte dental conocidos como amelogénesis imperfecta (AI). Dado que la proteína de membrana asociada a lisosoma-1 (LAMP-1), -3 (LAMP-3) y la proteína relacionada con la glucosa de 78 kDa (Grp78) se identificaron como proteína de unión a amelogenina, varios estudios han sugerido la participación de estas proteínas con la cinética celular de los ameloblastos en condiciones normales o anormales. El objetivo del estudio fue investigar la distribución de LAMP-1, LAM-3 y Grp78 durante la diferenciación celular de ameloblastos de ratones con una mutación puntual del gen de amelogenina (Amelx*). Los incisivos de los ratones Amelx* presentaron un color blanco opaco y se observó en microscopio electrónico de barrido que la superficie del diente era áspera. La diferenciación celular secuencial y la forma de los ameloblastos en la etapa de transición en los ratones Amelx* fue irregular en comparación con los ratones silvestres (RS). La inmunotinción de Grp78 reveló que todo el citoplasma de los ameloblastos en etapa de transición fue inmunopositivo para el anticuerpo Grp78, mientras que solo la parte distal de la célula fue positiva en los ratones RS. Además, en ratones Amelx*, el citoplasma de los ameloblastos en etapa de transición fue inmunopositivo para LAMP-1 y LAMP-3. Estos resultados sugieren que Amelx* puede causar distribución anormal de proteínas de unión a amelogenina en el citoplasma de los ameloblastos.

Inhibition of the Expression of the Small Heat Shock Protein αB-Crystallin Inhibits Exosome Secretion in Human Retinal Pigment Epithelial Cells in Culture.

Exosomes carry cell type-specific molecular cargo to extracellular destinations and therefore act as lateral vectors of intercellular communication and transfer of genetic information from one cell to the other. We have shown previously that the small heat shock protein αB-crystallin (αB) is exported out of the adult human retinal pigment epithelial cells (ARPE19) packaged in exosomes. Here, we demonstrate that inhibition of the expression of αB via shRNA inhibits exosome secretion from ARPE19 cells indicating that exosomal cargo may have a role in exosome biogenesis (synthesis and/or secretion). Sucrose density gradient fractionation of the culture medium and cellular extracts suggests continued synthesis of exosomes but an inhibition of exosome secretion. In cells where αB expression was inhibited, the distribution of CD63 (LAMP3), an exosome marker, is markedly altered from the normal dispersed pattern to a stacked perinuclear presence. Interestingly, the total anti-CD63(LAMP3) immunofluorescence in the native and αB-inhibited cells remains unchanged suggesting continued exosome synthesis under conditions of impaired exosome secretion. Importantly, inhibition of the expression of αB results in a phenotype of the RPE cell that contains an increased number of vacuoles and enlarged (fused) vesicles that show increased presence of CD63(LAMP3) and LAMP1 indicating enhancement of the endolysosomal compartment. This is further corroborated by increased Rab7 labeling of this compartment (RabGTPase 7 is known to be associated with late endosome maturation). These data collectively point to a regulatory role for αB in exosome biogenesis possibly via its involvement at a branch point in the endocytic pathway that facilitates secretion of exosomes.

A phase IB study on intravenous synthetic mRNA electroporated dendritic cell immunotherapy in pretreated advanced melanoma patients.

BACKGROUND: Autologous monocyte-derived dendritic cells (DCs) electroporated with synthetic messenger RNA (mRNA) encoding a CD40 ligand, a constitutively active Toll-like receptor 4 and CD70, together with mRNA encoding fusion proteins of a human leukocyte antigen (HLA)-class II targeting signal (DC-LAMP) and a melanoma-associated antigen (MAA); either MAGE-A3, MAGE-C2, tyrosinase or gp100) (TriMixDC-MEL) are superiorly immunogenic. PATIENTS AND METHODS: In this phase IB clinical trial, 24 million viable DCs were administered by four biweekly combined intradermal (id) and intravenous (iv) administrations, and a fifth administration on week 16. The number of iv-administered DCs was escalated in four sequentially treated cohorts. Immune responses were assessed by analysis of antigen specificity of blood-derived T-cells and skin infiltrating lymphocytes (SKILs). RESULTS: Fifteen patients with pretreated advanced melanoma tolerated administration of TriMixDC-MEL well. Two patients achieved a complete response and two patients a partial response. All objective responders are progression-free after a follow-up of, respectively, 24+, 28+, 33+, and 34+ months. Post-therapy antigen-specific SKILs were documented in 6 of 12 patients, and antigen-specific CD8(+) T-cells were detected in the blood of 4 of 5 patients. CONCLUSIONS: Cellular immunotherapy with TriMixDC-MEL is safe and immunogenic. Antitumor activity with durable disease control is observed across the investigated iv-dose levels. CLINICALTRIALSGOV IDENTIFIER: NCT01066390.

DC-LAMP+ dendritic cells are recruited to gastric lymphoid follicles in Helicobacter pylori-infected individuals.

Infection with Helicobacter pylori is associated with development of ulcer disease and gastrointestinal adenocarcinoma. The infection leads to a large infiltration of immune cells and the formation of organized lymphoid follicles in the human gastric mucosa. Still, the immune system fails to eradicate the bacteria, and the substantial regulatory T cell (Treg) response elicited is probably a major factor permitting bacterial persistence. Dendritic cells (DCs) are professional antigen-presenting cells that can activate naive T cells, and maturation of DCs is crucial for the initiation of primary immune responses. The aim of this study was to investigate the presence and localization of mature human DCs in H. pylori-infected gastric mucosa. Gastric antral biopsy specimens were collected from patients with H. pylori-associated gastritis and healthy volunteers, and antrum tissue was collected from patients undergoing gastric resection. Immunohistochemistry and flow cytometry showed that DCs expressing the maturation marker dendritic cell lysosome-associated membrane glycoprotein (DC-LAMP; CD208) are enriched in the H. pylori-infected gastric mucosa and that these DCs are specifically localized within or close to lymphoid follicles. Gastric DC-LAMP-positive (DC-LAMP(+)) DCs express CD11c and high levels of HLA-DR but little CD80, CD83, and CD86. Furthermore, immunofluorescence analyses demonstrated that DC-LAMP(+) DCs are in the same location as FoxP3-positive putative Tregs in the follicles. In conclusion, we show that DC-LAMP(+) DCs with low costimulatory capacity accumulate in the lymphoid follicles in human H. pylori-infected gastric tissue, and our results suggest that Treg-DC interactions may promote chronic infection by rendering gastric DCs tolerogenic.

Distribution of regulatory T cells and interaction with dendritic cells in the synovium of rheumatoid arthritis.

OBJECTIVES: The aim of this study was to investigate the tissue distribution of regulatory T cells (Treg cells) and their interaction with dendritic cells (DCs) in synovium from patients with rheumatoid arthritis (RA) or osteoarthritis (OA). METHODS: Immunohistochemical staining was used to investigate the distribution of Treg cells and the interaction between Treg cells and DCs in RA (n = 30) and OA synovium (n = 8). mRNA levels were measured by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Large numbers of Treg cells were observed in lymphoid aggregates and perivenular infiltration areas in the RA synovium. Specific cellular markers for Treg cells (Foxp3, CD39, LAG-3, and Nrp-1) were found in lymphoid aggregates, perivenular infiltration, and scattered in lining layer areas. As molecular markers for DCs, DC-LAMP, DEC-205, CD80/86, and CD83 were also detected in the lymphoid aggregates and perivenular infiltration areas in RA. Furthermore, the co-localization of Treg cells and DCs was confined mainly in the lymphoid aggregation areas. The number of DCs increased significantly more than the number of Treg cells with inflammatory progression in RA. mRNA expression of the cellular markers for Treg cells (Foxp3, LAG-3, and Nrp-1) and the molecular markers for DCs (DC-LAMP and DEC-205) was increased in RA compared with OA synovium. CONCLUSIONS: Our results indicate that DCs play a dominant role in regulating the activation and progression of immune responses in RA, even though the number of Treg cells was upregulated at the same time. This suggests that Treg cells do not function normally to suppress the maturation of DCs in the RA synovium.

Crystal structure of the conserved domain of the DC lysosomal associated membrane protein: implications for the lysosomal glycocalyx.

BACKGROUND: The family of lysosome-associated membrane proteins (LAMP) comprises the multifunctional, ubiquitous LAMP-1 and LAMP-2, and the cell type-specific proteins DC-LAMP (LAMP-3), BAD-LAMP (UNC-46, C20orf103) and macrosialin (CD68). LAMPs have been implicated in a multitude of cellular processes, including phagocytosis, autophagy, lipid transport and aging. LAMP-2 isoform A acts as a receptor in chaperone-mediated autophagy. LAMP-2 deficiency causes the fatal Danon disease. The abundant proteins LAMP-1 and LAMP-2 are major constituents of the glycoconjugate coat present on the inside of the lysosomal membrane, the 'lysosomal glycocalyx'. The LAMP family is characterized by a conserved domain of 150 to 200 amino acids with two disulfide bonds. RESULTS: The crystal structure of the conserved domain of human DC-LAMP was solved. It is the first high-resolution structure of a heavily glycosylated lysosomal membrane protein. The structure represents a novel ß-prism fold formed by two ß-sheets bent by ß-bulges and connected by a disulfide bond. Flexible loops and a hydrophobic pocket represent possible sites of molecular interaction. Computational models of the glycosylated luminal regions of LAMP-1 and LAMP-2 indicate that the proteins adopt a compact conformation in close proximity to the lysosomal membrane. The models correspond to the thickness of the lysosomal glycoprotein coat of only 5 to 12 nm, according to electron microscopy. CONCLUSION: The conserved luminal domain of lysosome-associated membrane proteins forms a previously unknown ß-prism fold. Insights into the structure of the lysosomal glycoprotein coat were obtained by computational models of the LAMP-1 and LAMP-2 luminal regions.

Distribution and maturation of skin dendritic cell subsets in two forms of cutaneous T-cell lymphoma: mycosis fungoides and Sézary syndrome.

Dendritic cells (DCs) critically regulate immune responses and the "immune-surveillance" of tumours. This study retrospectively analysed the distribution and maturation status of DC-subsets in T-cell lymphoma of the skin. Mycosis fungoides and Sézary syndrome (n = 25) were investigated immunohistochemically for DC subsets, based on C-type lectin receptor expression: Langerhans' cells (langerin/CD207+, DEC-205/CD205+), dermal DCs (DC-SIGN/CD209+, CD205+) and plasmacytoid DC (BDCA-2/CD303+). Maturation status was assessed by double-labelling for CD83 and CD208/DC-LAMP. DCs were interspersed between the neoplastic infiltrate, and a marked increase in numbers of all three subsets was noted, DC-SIGN+ dermal DCs constituting the majority. Substantial numbers of plasmacytoid DCs were consistently observed. Most DCs in epidermis and dermis were phenotypically immature. Amongst the relatively few mature DCs in the dermis, langerin+ cells predominated. There was a positive correlation between the histological intensity of the tumour infiltrate and DC numbers. It is possible that mature DCs reflect ongoing anti-tumour immune responses, and immature DCs the induction of tumour tolerance.

Characterisation and expression analysis of the rainbow trout (Oncorhynchus mykiss) homologue of the human dendritic cell marker CD208/lysosomal associated membrane protein 3.

LAMP3/CD208 is a member of the lysosomal-associated membrane protein family and is used as a putative marker for mature dendritic cells (DCs) in humans since it is upregulated upon DC activation and maturation. This paper reports the cloning and sequencing of LAMP3 in rainbow trout. The predicted trout LAMP3 shares the characteristic features of LAMP family members such as a C-terminal lysosomal sorting motif (G-Y-D-R-I) in the short C-terminal cytoplasmic tail, typical for lysosomal targeting, four potential N-linked glycosylation sites (NXS/T), four conserved cysteines in the membrane-proximal domain and the luminal domain divided by a serine/proline-rich region. Expression studies revealed that trout LAMP3, like chicken LAMP3, was constitutively expressed in a wide range of lymphoid tissues, at highest levels in the head kidney, liver and spleen, respectively. LAMP3 was also constitutively expressed in trout head kidney macrophages and RTS11 cells and the expression was shown to be induced in vivo after infection with viral and bacterial pathogens and in cultured macrophages after modulation with microbial mimics (LPS and PolyIC). Thus, it is clear that if LAMP3 is expressed by trout DCs it is not exclusively expressed by them. However, the marker is valuable to further study antigen presentation in fish and to complement already known DC markers.

Increased influx of myeloid dendritic cells during acute rejection is associated with interstitial fibrosis and tubular atrophy and predicts poor outcome.

Dendritic cells are key players in renal allograft rejection and have been identified as an intrinsic part of the kidney. Here we quantified and phenotyped the dendritic cell populations in well-defined biopsies of 102 patients with acute renal allograft rejection in comparison with 78 available pretransplant biopsies. There was a strong increase in BDCA-1(+) and DC-SIGN(+) myeloid, BDCA-2(+) plasmacytoid, and DC-LAMP(+) mature dendritic cells in rejection biopsies compared with the corresponding pretransplant tissue. Mature dendritic cells were mostly found in clusters of lymphoid infiltrate and showed a strong correlation with the Banff infiltrate score. The presence of both myeloid and plasmacytoid dendritic cell subsets in the kidney during acute rejection correlated with interstitial fibrosis and tubular atrophy. Importantly, the myeloid dendritic cell density at the time of acute rejection was an independent risk factor for loss of renal function after the first year. Thus, acute renal allograft rejection is characterized by an influx of myeloid and plasmacytoid dendritic cells, strongly associated with local damage in the graft. Hence, the density of myeloid dendritic cells during acute rejection could be an important risk factor for the long-term development of chronic changes and loss of graft function.

Lysosome-associated membrane glycoprotein 3 is involved in influenza A virus replication in human lung epithelial (A549) cells.

BACKGROUND: Influenza A virus mutates rapidly, rendering antiviral therapies and vaccines directed against virus-encoded targets ineffective. Knowledge of the host factors and molecular pathways exploited by influenza virus will provide further targets for novel antiviral strategies. However, the critical host factors involved in influenza virus infection have not been fully defined. RESULTS: We demonstrated that LAMP3, a member of lysosome-associated membrane glycoprotein (LAMP) family, was significantly induced in human lung epithelial (A549) cells upon influenza A virus infection. Knockdown of LAMP3 expression by RNA interference attenuated production of viral nucleoprotein (NP) as well as virus titers. Confocal microscopy results demonstrated that viral NP is colocalized within LAMP3 positive vesicles at early stages of virus infection. Furthermore, knockdown of LAMP3 expression led to a reduction in nuclear accumulation of viral NP and impeded virus replication. CONCLUSIONS: LAMP3 is an influenza A virus inducible gene, and plays an important role in viral post-entry steps. Our observations may provide insights into the mechanism of influenza virus replication and potential targets for novel anti-influenza therapeutics.