Aberrant Lipid Metabolism in Macrophages Is Associated with Granuloma Formation in Sarcoidosis.

Rationale: Chronic sarcoidosis is a complex granulomatous disease with limited treatment options that can progress over time. Understanding the molecular pathways contributing to disease would aid in new therapeutic development. Objectives: To understand whether macrophages from patients with nonresolving chronic sarcoidosis are predisposed to macrophage aggregation and granuloma formation and whether modulation of the underlying molecular pathways influence sarcoidosis granuloma formation. Methods: Macrophages were cultivated in vitro from isolated peripheral blood CD14+ monocytes and evaluated for spontaneous aggregation. Transcriptomics analyses and phenotypic and drug inhibitory experiments were performed on these monocyte-derived macrophages. Human skin biopsies from patients with sarcoidosis and a myeloid Tsc2-specific sarcoidosis mouse model were analyzed for validatory experiments. Measurements and Main Results: Monocyte-derived macrophages from patients with chronic sarcoidosis spontaneously formed extensive granulomas in vitro compared with healthy control participants. Transcriptomic analyses separated healthy and sarcoidosis macrophages and identified an enrichment in lipid metabolic processes. In vitro patient granulomas, sarcoidosis mouse model granulomas, and those directly analyzed from lesional patient skin expressed an aberrant lipid metabolism profile and contained increased neutral lipids. Conversely, a combination of statins and cholesterol-reducing agents reduced granuloma formation both in vitro and in vivo in a sarcoidosis mouse model. Conclusions: Together, our findings show that altered lipid metabolism in sarcoidosis macrophages is associated with its predisposition to granuloma formation and suggest cholesterol-reducing therapies as a treatment option in patients.

Modular chimeric cytokine receptors with leucine zippers enhance the antitumour activity of CAR T cells via JAK/STAT signalling.

The limited availability of cytokines in solid tumours hinders maintenance of the antitumour activity of chimeric antigen receptor (CAR) T cells. Cytokine receptor signalling pathways in CAR T cells can be activated by transgenic expression or injection of cytokines in the tumour, or by engineering the activation of cognate cytokine receptors. However, these strategies are constrained by toxicity arising from the activation of bystander cells, by the suboptimal biodistribution of the cytokines and by downregulation of the cognate receptor. Here we show that replacement of the extracellular domains of heterodimeric cytokine receptors in T cells with two leucine zipper motifs provides optimal Janus kinase/signal transducer and activator of transcription signalling. Such chimeric cytokine receptors, which can be generated for common γ-chain receptors, interleukin-10 and -12 receptors, enabled T cells to survive cytokine starvation without induction of autonomous cell growth, and augmented the effector function of CAR T cells in vitro in the setting of chronic antigen exposure and in human tumour xenografts in mice. As a modular design, leucine zippers can be used to generate constitutively active cytokine receptors in effector immune cells.

Human skin-resident CD8+ T cells require RUNX2 and RUNX3 for induction of cytotoxicity and expression of the integrin CD49a.

The integrin CD49a marks highly cytotoxic epidermal-tissue-resident memory (TRM) cells, but their differentiation from circulating populations remains poorly defined. We demonstrate enrichment of RUNT family transcription-factor-binding motifs in human epidermal CD8+CD103+CD49a+ TRM cells, paralleled by high RUNX2 and RUNX3 protein expression. Sequencing of paired skin and blood samples revealed clonal overlap between epidermal CD8+CD103+CD49a+ TRM cells and circulating memory CD8+CD45RA-CD62L+ T cells. In vitro stimulation of circulating CD8+CD45RA-CD62L+ T cells with IL-15 and TGF-ß induced CD49a expression and cytotoxic transcriptional profiles in a RUNX2- and RUNX3-dependent manner. We therefore identified a reservoir of circulating cells with cytotoxic TRM potential. In melanoma patients, high RUNX2, but not RUNX3, transcription correlated with a cytotoxic CD8+CD103+CD49a+ TRM cell signature and improved patient survival. Together, our results indicate that combined RUNX2 and RUNX3 activity promotes the differentiation of cytotoxic CD8+CD103+CD49a+ TRM cells, providing immunosurveillance of infected and malignant cells.

Epigenetic regulation of T cell lineages in skin and blood following hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem-cell transplantation (HSCT) seeks to reconstitute the host's immune system from donor stem cells. The success of HSCT is threatened by complications including leukemia relapse or graft-versus-host-disease (GvHD). To investigate the underlying regulatory processes in central and peripheral T cell recovery, we performed sequential multi-omics analysis of T cells of the skin and blood during HSCT. We detected rapid effector T cell reconstitution, while emergence of regulatory T cells was delayed. Epigenetic and gene-regulatory programs were associated with recovering T cells and diverged greatly between skin and blood T cells. The BRG1/BRM-associated factor chromatin remodeling complex and histone deacetylases (HDACs) were epigenetic regulators involved in restoration of T cell homeostasis after transplantation. In isolated T cells of patients after HSCT, we observed class I HDAC-inhibitors to modulate their dysbalance. The present study highlights the importance of epigenetic regulation in the recovery of T cells following HSCT.

Disturbances in microbial skin recolonization and cutaneous immune response following allogeneic stem cell transfer.

The composition of the gut microbiome influences the clinical course after allogeneic hematopoietic stem cell transplantation (HSCT), but little is known about the relevance of skin microorganisms. In a single-center, observational study, we recruited a cohort of 50 patients before undergoing conditioning treatment and took both stool and skin samples up to one year after HSCT. We could confirm intestinal dysbiosis following HSCT and report that the skin microbiome is likewise perturbed in HSCT-recipients. Overall bacterial colonization of the skin was decreased after conditioning. Particularly patients that developed acute skin graft-versus-host disease (aGVHD) presented with an overabundance of Staphylococcus spp. In addition, a loss in alpha diversity was indicative of aGVHD development already before disease onset and correlated with disease severity. Further, co-localization of CD45+ leukocytes and staphylococci was observed in the skin of aGVHD patients even before disease development and paralleled with upregulated genes required for antigen-presentation in mononuclear phagocytes. Overall, our data reveal disturbances of the skin microbiome as well as cutaneous immune response in HSCT recipients with changes associated with cutaneous aGVHD.

Processing human skin samples for single-cell assays.

Characterizing resident immune cells in human skin using single-cell assays provides insight into their role in infections, inflammation, and cancer. We describe an optimized protocol to rapidly isolate viable cells from 6-mm skin punch-biopsies. We provide an example in which we coupled single-cell RNA sequencing (scRNA-seq) with single-cell T-cell receptor sequencing (scTCR-seq) of skin and blood cells to study transcriptional profiles and clonotypes of skin resident and peripheral circulating, memory, and effector T cells. This is an improved protocol based on Saluzzo et al. (2021). For complete details on the use and execution of this protocol, please refer to Saluzzo et al. (2021).

Delayed antiretroviral therapy in HIV-infected individuals leads to irreversible depletion of skin- and mucosa-resident memory T cells.

People living with HIV (PLWH) are at increased risk for developing skin and mucosal malignancies despite systemic reconstitution of CD4+ T cells upon antiretroviral therapy (ART). The underlying mechanism of chronic tissue-related immunodeficiency in HIV is unclear. We found that skin CD4+ tissue-resident memory T (Trm) cells were depleted after HIV infection and replenished only upon early ART initiation. TCR clonal analysis following early ART suggested a systemic origin for reconstituting CD4+ Trm cells. Single-cell RNA sequencing in PLWH that received late ART treatment revealed a loss of CXCR3+ Trm cells and a tolerogenic skin immune environment. Human papilloma virus-induced precancerous lesion biopsies showed reduced CXCR3+ Trm cell frequencies in the mucosa in PLWH versus HIV- individuals. These results reveal an irreversible loss of CXCR3+ Trm cells confined to skin and mucosa in PLWH who received late ART treatment, which may be a precipitating factor in the development of HPV-related cancer.

Human resident memory T cells exit the skin and mediate systemic Th2-driven inflammation.

Emigration of tissue-resident memory T cells (TRMs) was recently introduced in mouse models and may drive systemic inflammation. Skin TRMs of patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) can coexist beside donor T cells, offering a unique human model system to study T cell migration. By genotyping, mathematical modeling, single-cell transcriptomics, and functional analysis of patient blood and skin T cells, we detected a small consistent population of circulating skin-derived T cells with a TRM phenotype (cTRMs) in the blood and unveil their skin origin and striking resemblance to skin TRMs. Blood from patients with active graft-versus-host disease (GVHD) contains elevated numbers of host cTRMs producing pro-inflammatory Th2/Th17 cytokines and mediating keratinocyte damage. Expression of gut-homing receptors and the occurrence of cTRMs in gastrointestinal GVHD lesions emphasize their potential to reseed and propagate inflammation in distant organs. Collectively, we describe a distinct circulating T cell population mirroring skin inflammation, which could serve as a biomarker or therapeutic target in GVHD.

The transcription factor Bcl11b promotes both canonical and adaptive NK cell differentiation.

Epigenetic landscapes can provide insight into regulation of gene expression and cellular diversity. Here, we examined the transcriptional and epigenetic profiles of seven human blood natural killer (NK) cell populations, including adaptive NK cells. The BCL11B gene, encoding a transcription factor (TF) essential for T cell development and function, was the most extensively regulated, with expression increasing throughout NK cell differentiation. Several Bcl11b-regulated genes associated with T cell signaling were specifically expressed in adaptive NK cell subsets. Regulatory networks revealed reciprocal regulation at distinct stages of NK cell differentiation, with Bcl11b repressing RUNX2 and ZBTB16 in canonical and adaptive NK cells, respectively. A critical role for Bcl11b in driving NK cell differentiation was corroborated in BCL11B-mutated patients and by ectopic Bcl11b expression. Moreover, Bcl11b was required for adaptive NK cell responses in a murine cytomegalovirus model, supporting expansion of these cells. Together, we define the TF regulatory circuitry of human NK cells and uncover a critical role for Bcl11b in promoting NK cell differentiation and function.

Long-term skin-resident memory T cells proliferate in situ and are involved in human graft-versus-host disease.

The skin contains a population of tissue-resident memory T cells (Trm) that is thought to contribute to local tissue homeostasis and protection against environmental injuries. Although information about the regulation, survival program, and pathophysiological roles of Trm has been obtained from murine studies, little is known about the biology of human cutaneous Trm Here, we showed that host-derived CD69+ αß memory T cell clones in the epidermis and dermis remain stable and functionally competent for at least 10 years in patients with allogeneic hematopoietic stem cell transplantation. Single-cell RNA sequencing revealed low expression of genes encoding tissue egress molecules by long-term persisting Trm in the skin, whereas tissue retention molecules and stem cell markers were displayed by Trm The transcription factor RUNX3 and the surface molecule galectin-3 were preferentially expressed by host T cells at the RNA and protein levels, suggesting two new markers for human skin Trm Furthermore, skin lesions from patients developing graft-versus-host disease (GVHD) showed a large number of cytokine-producing host-derived Trm, suggesting a contribution of these cells to the pathogenesis of GVHD. Together, our studies highlighted the relationship between the local human skin environment and long-term persisting Trm, which differs from murine skin. Our results also indicated that local tissue inflammation occurs through host-derived Trm after allogeneic hematopoietic stem cell transplantation.

A discrete subset of epigenetically primed human NK cells mediates antigen-specific immune responses.

Adaptive features of natural killer (NK) cells have been reported in various species with different underlying mechanisms. It is unclear, however, which NK cell populations are capable of mounting antigen-specific recall responses and how such functions are regulated at the molecular level. Here, we identify and characterize a discrete population of CD49a+CD16- NK cells in the human liver that displays increased epigenetic potential to elicit memory responses and has the functional properties to exert antigen-specific immunity in the skin as an effector site. Integrated chromatin-based epigenetic and transcriptomic profiling revealed unique characteristics of hepatic CD49a+CD16- NK cells when compared with conventional CD49a-CD16+ NK cells, thereby defining active genomic regions and molecules underpinning distinct NK cell reactivity. In contrast to conventional NK cells, our results suggest that adaptive CD49a+CD16- NK cells are able to bypass the KIR receptor-ligand system upon antigen-specific stimulation. Furthermore, these cells were highly migratory toward chemokine gradients expressed in epicutaneous patch test lesions as an effector site of adaptive immune responses in the skin. These results define pathways operative in human antigen-specific adaptive NK cells and provide a roadmap for harnessing this NK cell subset for specific therapeutic or prophylactic vaccine strategies.

Anti-Apoptotic Molecule BCL2 Is a Therapeutic Target in Steroid-Refractory Graft-Versus-Host Disease.

Graft-versus-host disease (GVHD) is the leading cause of mortality after hematopoietic stem cell transplantation and primarily affects barrier organs such as the skin. One-third of cases are refractory to steroid treatment resulting in poor outcomes and the need for novel therapies. Longitudinal analysis of T-cell transcriptomes in patients before the appearance of GVHD symptoms revealed the upregulation of anti-apoptotic regulator B-cell lymphoma 2 (BCL2) at GVHD initiation. To determine the potential of BCL2 inhibition in active GVHD, we analyzed tissues of 88 patients with acute or chronic GVHD. BCL2 RNA was elevated in multiple organs affected by GVHD and expression correlated with transplant-related mortality and steroid-refractory GVHD. BCL2-expressing lymphocytes were present in skin lesions and peripheral blood of patients with acute and chronic GVHD. Inhibition of BCL2 increased the CD4 to CD8 ratio in allogeneic T cells in vitro and induced apoptosis of T cells from patients with steroid-pretreated chronic GVHD ex vivo. In addition, the higher ratio of regulatory to nonregulatory T cells upon blockage of BCL2 could add to the anti-inflammatory effect of BCL2 blockage. Collectively, our results highlight BCL2 as an important factor for GVHD development and introduce BCL2 inhibition as previously unreported and urgently needed targeted therapy in the treatment of steroid-refractory GVHD.

ClinQC: a tool for quality control and cleaning of Sanger and NGS data in clinical research.

BACKGROUND: Traditional Sanger sequencing has been used as a gold standard method for genetic testing in clinic to perform single gene test, which has been a cumbersome and expensive method to test several genes in heterogeneous disease such as cancer. With the advent of Next Generation Sequencing technologies, which produce data on unprecedented speed in a cost effective manner have overcome the limitation of Sanger sequencing. Therefore, for the efficient and affordable genetic testing, Next Generation Sequencing has been used as a complementary method with Sanger sequencing for disease causing mutation identification and confirmation in clinical research. However, in order to identify the potential disease causing mutations with great sensitivity and specificity it is essential to ensure high quality sequencing data. Therefore, integrated software tools are lacking which can analyze Sanger and NGS data together and eliminate platform specific sequencing errors, low quality reads and support the analysis of several sample/patients data set in a single run. RESULTS: We have developed ClinQC, a flexible and user-friendly pipeline for format conversion, quality control, trimming and filtering of raw sequencing data generated from Sanger sequencing and three NGS sequencing platforms including Illumina, 454 and Ion Torrent. First, ClinQC convert input read files from their native formats to a common FASTQ format and remove adapters, and PCR primers. Next, it split bar-coded samples, filter duplicates, contamination and low quality sequences and generates a QC report. ClinQC output high quality reads in FASTQ format with Sanger quality encoding, which can be directly used in down-stream analysis. It can analyze hundreds of sample/patients data in a single run and generate unified output files for both Sanger and NGS sequencing data. Our tool is expected to be very useful for quality control and format conversion of Sanger and NGS data to facilitate improved downstream analysis and mutation screening. CONCLUSIONS: ClinQC is a powerful and easy to handle pipeline for quality control and trimming in clinical research. ClinQC is written in Python with multiprocessing capability, run on all major operating systems and is available at https://sourceforge.net/projects/clinqc.