Dendritic cells (DCs) are critical for the initiation of immune responses including activation of CD8 T cells. Intracellular reactive oxygen species (ROS) levels influence DC maturation and function. ...Intracellular heme, a product of catabolism of heme-containing metalloproteins, is a key inducer of ROS. Intracellular heme levels are regulated by heme oxygenase-1 (HO-1), which catalyzes the degradation of heme. Heme oxygenase-1 has been implicated in regulating DC maturation; however, its role in other DC functions is unclear. Furthermore, the signaling pathways modulated by HO-1 in DCs are unknown. In this study, we demonstrate that inhibition of HO-1 activity in murine bone marrow-derived immature DCs (iDCs) resulted in DCs with raised intracellular ROS levels, a mature phenotype, impaired phagocytic and endocytic function, and increased capacity to stimulate antigen-specific CD8 T cells. Interestingly, our results reveal that the increased ROS levels following HO-1 inhibition did not underlie the changes in phenotype and functions observed in these iDCs. Importantly, we show that the p38 mitogen-activated protein kinase (p38 MAPK), cAMP-responsive element binding protein (CREB), and activating transcription factor 1 (ATF1) pathway is involved in the mediation of the phenotypic and functional changes arising from HO-1 inhibition. Furthermore, up-regulation of HO-1 activity rendered iDCs refractory to lipopolysaccharide-induced activation of p38 MAPK-CREB/ATF1 pathway and DC maturation. Finally, we demonstrate that treatment of iDC with the HO-1 substrate, heme, recapitulates the effects that result from HO-1 inhibition. Based on these results, we conclude that HO-1 regulates DC maturation and function by modulating the p38 MAPK-CREB/ATF1 signaling axis.
HO-1 contributes to redox homeostasis and regulation of immature dendritic cell (DC) phenotype.
HO-1 inhibition results in increased ROS, activation of p38 MAPK-CREB/ATF1 pathway, and dysregulation of DC phenotype and function.
HO-1 influences DC function through effects on p38 MAPK-CREB/ATF1 signaling pathway.
This study provides new insights into the molecular pathways influenced by HO-1 in DCs.
Here we describe the application of mass cytometry to analyze tumor-infiltrating lymphocytes in human melanoma. Mass cytometry is the coupling of flow cytometry and mass spectrometry, which allows ...for the simultaneous measurement of 40+ cell parameters on a per cell basis. Heavy metal-labeled antibodies can bind to proteins (CD markers, transcription factors, cytokines) on the cell surface and in the cytoplasm/nucleus. As labeled cells pass through the CyTOF, the instrument detects the heavy metals. Combining these signals allows description of melanoma tumor-infiltrating lymphocytes at a greater depth than alternative phenotyping strategies and enables detailed analyses of a variety of cellular parameters, including immune cell lineage, activation status, and functional polarization.
Abstract
Background
Understanding the drivers of recurrence in aggressive prostate cancer requires detailed molecular and genomic understanding in order to aid therapeutic interventions.
We provide ...here a case report of histological, transcriptional, proteomic, immunological, and genomic features in a longitudinal study of multiple biopsies from diagnosis, through treatment, and subsequent recurrence.
Case presentation
Here we present a case study of a male in 70 s with high-grade clinically-localised acinar adenocarcinoma treated with definitive hormone therapy and radiotherapy. The patient progressed rapidly with rising PSA and succumbed without metastasis 52 months after diagnosis.
We identified the expression of canonical histological markers of neuroendocrine PC (NEPC) including synaptophysin, neuron-specific enolase and thyroid transcription factor 1, as well as intact AR expression, in the recurrent disease only.
The resistant disease was also marked by an extremely low immune infiltrate, extensive genomic chromosomal aberrations, and overactivity in molecular hallmarks of NEPC disease including Aurora kinase and E2F, as well as novel alterations in the cMYB pathway. We also observed that responses to both primary treatments (high dose-rate brachytherapy and androgen deprivation therapies) were consistent with known optimal responses—ruling out treatment inefficacy as a factor in relapse.
Conclusions
These data provide novel insights into a case of locally recurrent aggressive prostate cancer harbouring NEPC pathology, in the absence of detected metastasis.
Objective
The immune system can halt cancer progression by suppressing outgrowth of clinically occult micrometastases in a state of cancer‐immune equilibrium. Cutaneous melanoma provides a unique ...opportunity to study the immune contexture of such lesions, as miniscule skin metastases are accessible to clinical inspection and diagnostic biopsy.
Methods
Here, we analysed by multiplex immunofluorescence microscopy samples from a melanoma patient presenting with an overt and an occult in‐transit metastasis (ITM), the latter of which appeared as a small erythematous papule.
Results
Microarchitecture and immune composition in the two lesions were vastly different. CD4+ and CD8+ T cells accumulated around the margin of the overt SOX10+ Melan A+ ITM but were largely excluded from the tumor centre. By contrast, the occult micrometastasis contained only few SOX10+ Melan A− melanoma cells which were scattered within a dense infiltrate of T cells, including a prominent population of CD103+ CD8+ T cells resembling tissue‐resident memory T (TRM) cells. Notably, almost every single melanoma cell in the micrometastasis was in close proximity to these TRM‐like cells.
Conclusion
Such results support the emerging concept that CD103+ CD8+ TRM cells are key mediators of cancer surveillance and imply an important function of these cells in controlling clinically occult micrometastases in humans.
Analysing an occult in‐transit metastasis in a melanoma patient, we identify CD103+CD8+ T cells with a resident memory phenotype as the dominant T‐cell subset. Such results support the emerging concept that CD103+CD8+ tissue‐resident memory T cells are key mediators of cancer surveillance and imply an important function of these cells in controlling clinically occult micrometastases in humans.
BackgroundMucosal melanoma is a rare subtype of melanoma originating from mucosal tissues (1), metastases are very aggressive and respond poorly to therapy, including immune checkpoint inhibitors ...(ICI) such as anti-CTLA4 and anti-PD1 antibodies (2–5). CD8+ T cells constitute the most abundant immune infiltrate in metastatic melanoma, of which the Tissue Resident Memory subset (TRM) is of particular interest (6). CD8+ TRM cells express the highest levels of immune checkpoint receptors, proliferate in response to ICI and correlate with longer disease-free and overall survival (6–8). The immune landscape in mucosal melanoma remains poorly characterized. We aimed to: 1) phenotype CD8+ T cells and TRM infiltrating metastatic mucosal melanoma, 2) characterize the clonality of TRM in relation to other CD8+ T cell subsets and 3) define the capacity of CD8+ T cells and TRM to respond to melanoma cells and to in vivo and in vitro anti-PD1 treatment.MethodsWe investigated the CD8+ T and TRM cells infiltrating two temporally- and spatially-distant subcutaneous metastases, these originated from a primary vaginal mucosal melanoma. One metastasis was excised prior to anti-PD1 treatment and one was anti-PD1 refractory, having progressed on treatment. We used mass cytometry and single-cell RNA and TCR sequencing to characterise the phenotype and clonality of the T cells, multiplex immunohistochemistry to define their spatial relationship with tumour cells and other T cells, and functional assays to determine TRM response to tumour cells (figure 1).ResultsCD8+ TRM frequency increased with time and anti-PD1 treatment, forming clusters at the tumour margin. T cells in the anti-PD1 refractory lesion were more activated than T cells in the first tumour and were bound by anti-PD1 antibody in vivo. T cells could not be stimulated by anti-PD1 directly ex vivo. Both metastatic lesions shared common T cell clusters including TRM. Furthermore, TRM in each tumour shared T cell clones, suggesting the presence of common antigens between metastatic sites. Indeed, the two metastases had a similar mutational profile. In vitro expanded tumour infiltrating lymphocytes from both lesions recognized tumour cells from both lesions and the same neoantigen generated from a single point mutation in the gene CDKN1C. Finally, tumour cells stimulated TRM cells more robustly than other T cells subsets.Abstract 548 Figure 1Graphical depiction of the methods used to characterise T cells in mucosal metastatic melanomaConclusionsIn this patient with vaginal mucosal melanoma, subsequent melanoma metastases of clonal origin attracted CD8+ T cells of similar specificity, among which TRM cells responded more vigorously to tumour cells than other T cells subsets.AcknowledgementsThe authors would like to acknowledge imCORE La Hoffmann- Roche Ltd. for funding.Ethics ApprovalPatients diagnosed with stage 3 or 4 metastatic melanoma and undergoing clinically indicated surgery were enrolled in prospective studies approved by the Peter MacCallum Cancer Centre human ethics research committee (13/141). All experimental protocols have been approved and clinical data has been collected prospectively.ReferencesCarvajal RD, Hamid O, Ariyan C. Mucosal Melanoma. cited 2020 Apr 1; Available from: https://www.uptodate.com/contents/mucosal-melanomaDel Vecchio M, Di Guardo L, Ascierto PA, Grimaldi AM, Sileni VC, Pigozzo J, et al. Efficacy and safety of ipilimumab 3 mg/kg in patients with pretreated, metastatic, mucosal melanoma. Eur J Cancer Oxf Engl 1990; 2014 Jan;50(1):121–7.Postow MA, Luke JJ, Bluth MJ, Ramaiya N, Panageas KS, Lawrence DP, et al. Ipilimumab for patients with advanced mucosal melanoma. The Oncologist 2013 Jun;18(6):726–32.D’Angelo SP, Larkin J, Sosman JA, Lebbé C, Brady B, Neyns B, et al. Efficacy and safety of nivolumab alone or in combination with ipilimumab in patients with mucosal melanoma: a pooled analysis. J Clin Oncol Off J Am Soc Clin Oncol. 2017 Jan 10;35(2):226–35.Hamid O, Robert C, Ribas A, Hodi FS, Walpole E, Daud A, et al. Antitumour activity of pembrolizumab in advanced mucosal melanoma: a post-hoc analysis of KEYNOTE-001, 002, 006. Br J Cancer 2018;119(6):670–4.Boddupalli CS, Bar N, Kadaveru K, Krauthammer M, Pornputtapong N, Mai Z, et al. Interlesional diversity of T cell receptors in melanoma with immune checkpoints enriched in tissue-resident memory T cells. JCI Insight Internet. 2016 Dec 22 cited 2019 Apr 24;1(21). Available from: https://insight.jci.org/articles/view/88955Edwards J, Wilmott JS, Madore J, Gide TN, Quek C, Tasker A, et al. CD103+ Tumor-resident CD8+ T cells are associated with improved survival in immunotherapy-naïve melanoma patients and expand significantly during anti-PD-1 treatment. Clin Cancer Res Off J Am Assoc Cancer Res 2018 Jul 1;24(13):3036–45.Savas P, Virassamy B, Ye C, Salim A, Mintoff CP, Caramia F, et al. Single-cell profiling of breast cancer T cells reveals a tissue-resident memory subset associated with improved prognosis. Nat Med 2018 Jul;24(7):986–93.
Most knowledge on NK cells are based on studies of what are now known as conventional NK (cNK) cells in the mouse spleen or human peripheral blood. However, recent studies in mice indicate the ...presence of tissue-resident NK (trNK) cells in certain organs, such as the liver, that display different markers and transcription factor dependencies as compared to cNK cells. Here we provide evidence from cytometry by time-of-flight analysis and humanized mice indicating that human CD49e-negative NK cells are tissue-resident in the liver. Thus, these studies indicate that trNK cells are evolutionarily conserved in humans and mice, providing a foundation to explore their role in human disease.
Abstract
Natural killer cells are a subset of innate immune lymphocytes that can kill transformed and infected cells, and produce cytokines that modulate the host immune response. Previous studies on ...human NK cells have primarily characterized NK cells isolated from peripheral blood. However, studies in mice from our lab and others indicate an additional tissue-resident NK (trNK) population that does not re-circulate in contrast to conventional NK (cNK). When the mouse liver was excised and flushed with saline, trNK cells were detected, indicating that the trNK cells reside in the liver sinusoidal space. Recent studies to identify trNK cells in the human liver have concentrated on the differential expression of CD56-dim/bright populations of NK cells and also CD49a, a marker we previously defined for mouse trNK cells in the liver. Herein we obtained samples from liver transplantation cadaveric donors. Matching donor venous blood and serial liver saline flushes were studied. Our preliminary data identifies a subset of putative liver-trNK cells based on lack of CD49e expression which appeared in the terminal liver flush aliquot and was not present in earlier liver flushes nor peripheral blood whereas CD49a expression was not discriminatory. Mass cytometry experiments with cytometry by time-of-flight instrument and a panel of 42 antibodies corroborated these findings. Interestingly, the liver-resident CD49e− cells have elevated EOMES but lower Tbet expression which contrasts the CD49a+ trNK cell populations in human and mouse liver. Finally, the putative trNK cells express different killer immunoglobulin-like receptor (KIR) repertoire than cNK cells thus, a unique population of trNK cells that reside in the sinusoidal space of the human liver.
Lentigo maligna (LM) is a common subtype of in situ melanoma on chronically sun-exposed skin, particularly the head and neck of older patients. Although surgery is the standard treatment, there is ...associated morbidity, and options such as imiquimod cream or radiotherapy may be used if surgery is refused or inappropriate. Complete response rates following imiquimod treatment are variable in the literature. The aim of this study was to evaluate the host immune response both before and following treatment with imiquimod to better identify likely responders. Paired pre- and post-imiquimod treatment specimens were available for 27 patients. Patients were treated with imiquimod 5 days per week for 12 weeks; at 16 weeks, lesions were excised for histological assessment. Of the 27 patients, 16 were responders and 11 failed to clear the disease. PDL1 protein expression was increased, accompanied by a unique gene signature in lesions from patients that subsequently histologically cleared LM by 16 weeks. This comprised 57 upregulated immune genes in signaling networks for antigen presentation, type I interferon signaling, and T-cell activation. This may represent an early responder group to imiquimod, and this unique gene signature potentially can be used as a biomarker of LM response to imiquimod.
Abstract
Nrf2 is a redox sensitive transcription factor vital for the maintenance of cellular homeostasis. Dendritic cells (DCs) are potent APCs responsible for the initiation of an immune response. ...Our previous study highlights the role of Nrf2 in DC function (increased costimulatory receptors expression and T cell stimulatory capacity) and intracellular signaling (such as ROS, NFκB and MAPKs). The p38 MAPK pathway plays a pivotal role in the up regulation of costimulatory receptors (CD86 & MHC II). However, it is unclear how Nrf2 affects the integrity of this pathway. In the current study using bone marrow-derived immature DCs from Nrf2 deficient (KO) and wild type mice, we investigate the effect of Nrf2 on various signaling events in the p38 pathway and associated functional responses. Our results indicate that the loss of Nrf2 in DCs resulted in elevated basal p38 phosphorylation. Furthermore, an increase in basal phosphorylation of CREB and ATF-1 - downstream targets of p38, was also observed. This was coupled with increased IL-10 cytokine production at basal conditions and also upon exposure to LPS. Increased costimulatory receptor expression, antigen specific T cell activation propensity and LPS stimulated IL-10 production observed in the KO DCs can be reversed by pharmacological inhibition of p38. These results suggest that deficiency of Nrf2 in DCs leads to dysregulation of the p38 signaling pathway contributing to the altered phenotype and function of these DCs.
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