Arterial macrophages have different developmental origins, but the association of macrophage ontogeny with their phenotypes and functions in adulthood is still unclear. Here, we combine macrophage fate-mapping analysis with single-cell RNA sequencing to establish their cellular identity during homeostasis, and in response to angiotensin-II (AngII)-induced arterial inflammation. Yolk sac erythro-myeloid progenitors (EMP) contribute substantially to adventitial macrophages and give rise to a defined cluster of resident immune cells with homeostatic functions that is stable in adult mice, but declines in numbers during ageing and is not replenished by bone marrow (BM)-derived macrophages. In response to AngII inflammation, increase in adventitial macrophages is driven by recruitment of BM monocytes, while EMP-derived macrophages proliferate locally and provide a distinct transcriptional response that is linked to tissue regeneration. Our findings thus contribute to the understanding of macrophage heterogeneity, and associate macrophage ontogeny with distinct functions in health and disease.

Immune responses against the mutated region of cytoplasmatic NPM1 might contribute to the favorable clinical outcome of AML patients with NPM1 mutations (NPM1 mut )Immune responses directed against epitopes derived from the mutated region of nucleophosmin 1 (NPM1) by NPM1 mut -specific CD8(1) cytotoxic T cells (CTLs) might be involved in the rejection of NPM1 mut myeloid leukemic blasts. NPM1 mutations are one of the most frequent molecular alterations in acute myeloid leukemia (AML) and are an important prognostic marker.1 The mutations cause an abnormal shift of the NPM1 protein from the nucleus to the cytoplasm, described by Falini et al.2 AML patients with NPM1 mut , but without FLT3 internal tandem duplication (ITD) mutation, show improved overall survival.3 NPM1 mut /FLT3-ITD-negative patients do not seem to benefit from allogeneic stem cell transplantation in first-line treatment; however, this issue is still under evaluation, further clinical trials are ongoing, and also minimal residual disease (MRD) has to be considered in treatment decision. 3,4 The functional role of NPM1 mut for the improved clinical outcome is still under evaluation. Immune responses to NPM1 mut may contribute to favorable prognosis of this AML subtype. Recently, we described specific T-cell responses of CD4 1 and CD8 1 T cells against epitopes derived from mutated regions of NPM1. 5 Two NPM1 mut -derived peptides, called #1 and #3, induced specific T-cell responses in patients with NPM1 mut (33% and 44%, respectively). NPM1 mut AML patients showed a significantly higher frequency of CTL responses against peptide #3 compared with healthy volunteers (P 5 .046).5 Several leukemia-associated antigens (LAAs) have been defined, most importantly RHAMM, Proteinase 3, and Wilms' tumor antigen 1 (WT-1). These antigens were tested in clinical peptide vaccination trials. 6 Immunologic and clinical responses were detected in patients with different hematologic malignancies. 7,8 Berneman et al 9 discussed NPM1 mut as a further important LAA to attack AML and leukemic stem cells by autologous T cells.In this work, we performed survival analysis of 25 NPM1 mut patients ( Figure 1A), analyzed by enzyme-linked immunospot comparing cases with or without specific T-cell responses. Our data suggest a better overall survival of patients with specific CTL responses against peptide #1 or #3 (P 5 .004; Figure 1B).Immune responses seem to differ in dependence on the epitopes (P = .026; Figure 1C), although this finding has to be interpreted with caution due to the low number of patients. The survival rate of all NPM1 mut patients is lower due to other molecular alterations (like FLT3-ITD in 11 of 25 NPM1 mut patients) and the inclusion of elderly patients (7 of 25 were older than 60 years of age). Due to its exquisite specificity in leukemia, NPM1 mut might constitute an ideal target structure for individualized immunotherapeutic approaches. Analysis with material from larger controlled clinical trials has to be performed. Nevertheless, these data suggest...

The solute carrier (SLC) superfamily represents the biggest family of transporters with important roles in health and disease. Despite being attractive and druggable targets, the majority of SLCs remains understudied. One major hurdle in research on SLCs is the lack of tools, such as cell-based assays to investigate their biological role and for drug discovery. Another challenge is the disperse and anecdotal information on assay strategies that are suitable for SLCs. This review provides a comprehensive overview of state-of-the-art cellular assay technologies for SLC research and discusses relevant SLC characteristics enabling the choice of an optimal assay technology. The Innovative Medicines Initiative consortium RESOLUTE intends to accelerate research on SLCs by providing the scientific community with high-quality reagents, assay technologies and data sets, and to ultimately unlock SLCs for drug discovery.

No abstract

Purpose: In acute myeloid leukemia (AML) without retinoic acid receptor (RAR) rearrangement, the effect of all-trans-retinoic acid (ATRA) is still poorly understood despite an association of NPM1 mutation and ATRA response. Recently, preferentially expressed antigen in melanoma (PRAME) has been shown to be a dominant repressor of RAR signaling.Experimental Design: Thus, we further investigated ATRA response mechanisms, especially the impact of PRAME expression on ATRA responsiveness. We profiled gene expression in diagnostic samples derived from our AML HD98B trial, in which ATRA was administered in addition to intensive chemotherapy.Results: Our data revealed a PRAME expression-associated gene pattern to be significantly enriched for genes involved in the retinoic acid metabolic process. In leukemia cell line models, we could show that retinoic acid-regulated cell proliferation and differentiation are impacted by PRAME expression. In patients with primary AML, repressor activity of high-PRAME levels might be overcome by the addition of ATRA as indicated by better outcome in 2 independent studies (P ¼ 0.029).Conclusions: PRAME seems to impair differentiation and to increase proliferation likely via blocking RAR signaling, which might be reversed by ATRA. PRAME therefore represents a promising target for both ATRA treatment and possibly future immunotherapeutic approaches in AML.

Leukemic stem cells (LSC) might be the source for leukemic disease self-renewal and account for disease relapse after treatment, which makes them a critical target for further therapeutic options. We investigated the role of cytotoxic T-lymphocytes (CTL) counteracting and recognizing LSC. Leukemia-associated antigens (LAA) represent immunogenic structures to target LSC. We enriched the LSC-containing fraction of 20 AML patients and hematopoietic stem cells (HSC) of healthy volunteers. Using microarray analysis and qRT-PCR we detected high expression of several LAA in AML cells but also in LSC. PRAME (p 5 0.0085), RHAMM (p 5 0.03), WT1 (p 5 0.04) and Proteinase 3 (p 5 0.04) showed significant differential expression in LSC compared with HSC. PRAME, RHAMM and WT1 are furthermore also lower expressed on leukemic bulk. In contrast, Proteinase 3 indicates a higher expression on leukemic bulk than on LSC. In colony forming unit (CFU) immunoassays, T cells stimulated against various LAA indicated a significant inhibition of CFUs in AML patient samples. The LAA PRAME, RHAMM and WT1 showed highest immunogenic responses with a range up to 58-83%. In a proof of principle xenotransplant mouse model, PRAME-stimulated CTL targeted AML stem cells, reflected by a delayed engraftment of leukemia (p 5 0.0159). Taken together, we demonstrated the expression of several LAA in LSC. LAA-specific T cells are able to hamper LSC in immunoassays and in a mouse model, which suggests that immunotherapeutic approaches have the potential to target malignant stem cells.

No abstract

No abstract