CyTOF mass cytometry reveals phenotypically distinct human blood neutrophil populations differentially correlated with melanoma stage

BACKGROUND: Understanding neutrophil heterogeneity and its relationship to disease progression has become a recent focus of cancer research. Indeed, several studies have identified neutrophil subpopulations associated with protumoral or antitumoral functions. However, this work has been hindered by a lack of widely accepted markers with which to define neutrophil subpopulations. METHODS: To identify markers of neutrophil heterogeneity in cancer, we used single-cell cytometry by time-of-flight (CyTOF) coupled with high-dimensional analysis on blood samples from treatment-naïve patients with melanoma. RESULTS: Our efforts allowed us to identify seven blood neutrophil clusters, including two previously identified individual populations. Interrogation of these neutrophil subpopulations revealed a positive trend between specific clusters and disease stage. Finally, we recapitulated these seven blood neutrophil populations via flow cytometry and found that they exhibited diverse capacities for phagocytosis and reactive oxygen species production in vitro. CONCLUSIONS: Our data provide a refined consensus on neutrophil heterogeneity markers, enabling a prospective functional evaluation in patients with solid tumors.</p

Immunogenetic analysis of human follicle centre lymphomas and diffuse large cell lymphomas

This study focuses on diffuse large cell lymphomas (DLCL) and follicle center lymphomas (FCL), two entities that encompass ca 70-80% of B-cell malignancies. The aim was to characterize the VH gene usage and mutation patterns at diagnosis and to detect changes in these patterns over time. No skewing of VH gene segment usage was detected. VH genes showed a significant burden of somatic mutations, consistent with a germinal center (GC) origin. Analysis of the mutation patterns suggests antigen selection had occurred with selective pressure to maintain the framework regions of the VH genes. Detailed analysis of a large number of templates from presentation biopsies revealed that commonly DLCL are still responsive to the somatic mutation mechanisms in the GC, resulting in introclonal sequence heterogeneity. Lymphoma progression however can lead to the selection of a subclone with subsequent loss of this heterogeneity.FCL and DLCL appear to commonly express RNA transcripts for multiple Ig isotypes. The sequencing data indicate the derivation of these transcripts from different cell populations and immunocytochemistry suggests that at least some of these transcripts are translated into protein and represent istoype switch events.VH and VL sequences also provide patient specific tumour-associated antigens, which can be used as a target for immune attack. A phase I/II study is currently being undertaken based on this concept. Vaccines consisting of DNA containing VH and VL genes are being used to activate immunity against the cancer in patients with FCL. In a second part of this thesis heavy and light chain variable genes from patients with FCL were identified in preparation for a DNA vaccine study in this patient group. A pilot study of expressing the complete IgM of one of these patients in the baculovirus system was performed, as a tool for measuring anti-adiotype responses in patients with FCL before and after vaccination.</p

M1(hot)tumor-associated macrophages boost tissue-resident memory T cells infiltration and survival in human lung cancer

Background: the role of Tumour-Associated Macrophages (TAMs) in determining the outcome between the anti-tumour effects of the adaptive immune system and the tumour’s anti-immunity stratagems, is controversial. Macrophages modulate their activities and phenotypes by integration of signals in the tumour micro-environment. Depending on how macrophages are activated, they may adopt so-called M1-like, anti-tumour or M2-like, pro-tumour profiles. In many solid tumours, a dominance of M2-like macrophages is associated with poor outcomes but in some tumour types, strong M1-like profiles are linked to better outcomes. We aimed to investigate the inter-relationship of these TAM populations to establish how they modulate the efficacy of the adaptive immune system in early lung cancer.Methods: macrophages from matched lung (NTAMs) and tumour samples (TAMs) from resected lung cancers were assessed by bulk and single-cell transcriptomic analysis. Protein expression of genes characteristic of M1-like (CXCL9) or M2-like (MMP12) functions was confirmed by confocal microscopy. Immunohistochemistry related the distribution of TAM transcriptomic signatures to density of CD8+ tissue-resident memory T cells (TRM) in tumours and survival data from an independent cohort of 393 lung cancer patientsResults: TAMs have significantly different transcriptomic profiles from NTAMs with &gt;1000 differentially expressed genes. TAMs displayed a strong M2-like signature with no significant variation between patients. However, single-cell RNA-seq supported by immuno-stained cells revealed that additionally, in 25% of patients the M2-like TAMs also co-expressed a strong/hot M1-like signature (M1hot). Importantly, there was a strong association between the density of M1hot TAMs and TRM cells in tumours that was in turn linked to better survival. Our data suggests a mechanism by which M1hot TAMs may recruit TRM cells via CXCL9 expression and sustain them by making available more of the essential fatty acids on which TRM depend.Conclusions: we showed that in early lung cancer, expression of M1-like and M2-like gene signatures are not mutually exclusive since the same TAMs can simultaneously display both gene-expression profiles. The presence of M1hot TAMs was associated with a strong TRM tumour-infiltrate and better outcomes. Thus, therapeutic approaches to re-program TAMs to an M1hot phenotype are likely to augment the adaptive anti-tumour responses