Efficacy of DYRK1A inhibitors in novel models of Down syndrome acute lymphoblastic leukemia.

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Single cell transcriptomics reveals cell type specific features of developmentally regulated responses to lipopolysaccharide between birth and 5 years.

Background: Human perinatal life is characterized by a period of extraordinary change during which newborns encounter abundant environmental stimuli and exposure to potential pathogens. To meet such challenges, the neonatal immune system is equipped with unique functional characteristics that adapt to changing conditions as development progresses across the early years of life, but the molecular characteristics of such adaptations remain poorly understood. The application of single cell genomics to birth cohorts provides an opportunity to investigate changes in gene expression programs elicited downstream of innate immune activation across early life at unprecedented resolution. Methods: In this study, we performed single cell RNA-sequencing of mononuclear cells collected from matched birth cord blood and 5-year peripheral blood samples following stimulation (18hrs) with two well-characterized innate stimuli; lipopolysaccharide (LPS) and Polyinosinic:polycytidylic acid (Poly(I:C)). Results: We found that the transcriptional response to LPS was constrained at birth and predominantly partitioned into classical proinflammatory gene upregulation primarily by monocytes and Interferon (IFN)-signaling gene upregulation by lymphocytes. Moreover, these responses featured substantial cell-to-cell communication which appeared markedly strengthened between birth and 5 years. In contrast, stimulation with Poly(I:C) induced a robust IFN-signalling response across all cell types identified at birth and 5 years. Analysis of gene regulatory networks revealed IRF1 and STAT1 were key drivers of the LPS-induced IFN-signaling response in lymphocytes with a potential developmental role for IRF7 regulation. Conclusion: Additionally, we observed distinct activation trajectory endpoints for monocytes derived from LPS-treated cord and 5-year blood, which was not apparent among Poly(I:C)-induced monocytes. Taken together, our findings provide new insight into the gene regulatory landscape of immune cell function between birth and 5 years and point to regulatory mechanisms relevant to future investigation of infection susceptibility in early life.

Tracking of activated cTfh cells following sequential influenza vaccinations reveals transcriptional profile of clonotypes driving a vaccine-induced immune response.

Introduction: A vaccine against influenza is available seasonally but is not 100% effective. A predictor of successful seroconversion in adults is an increase in activated circulating T follicular helper (cTfh) cells after vaccination. However, the impact of repeated annual vaccinations on long-term protection and seasonal vaccine efficacy remains unclear. Methods: In this study, we examined the T cell receptor (TCR) repertoire and transcriptional profile of vaccine-induced expanded cTfh cells in individuals who received sequential seasonal influenza vaccines. We measured the magnitude of cTfh and plasmablast cell activation from day 0 (d0) to d7 post-vaccination as an indicator of a vaccine response. To assess TCR diversity and T cell expansion we sorted activated and resting cTfh cells at d0 and d7 post-vaccination and performed TCR sequencing. We also single cell sorted activated and resting cTfh cells for TCR analysis and transcriptome sequencing. Results and discussion: The percent of activated cTfh cells significantly increased from d0 to d7 in each of the 2016-17 (p < 0.0001) and 2017-18 (p = 0.015) vaccine seasons with the magnitude of cTfh activation increase positively correlated with the frequency of circulating plasmablast cells in the 2016-17 (p = 0.0001) and 2017-18 (p = 0.003) seasons. At d7 post-vaccination, higher magnitudes of cTfh activation were associated with increased clonality of cTfh TCR repertoire. The TCRs from vaccine-expanded clonotypes were identified and tracked longitudinally with several TCRs found to be present in both years. The transcriptomic profile of these expanded cTfh cells at the single cell level demonstrated overrepresentation of transcripts of genes involved in the type-I interferon pathway, pathways involved in gene expression, and antigen presentation and recognition. These results identify the expansion and transcriptomic profile of vaccine-induced cTfh cells important for B cell help.

Tissue-resident memory T cells in the era of (Neo) adjuvant melanoma management.

Tissue-resident memory T (TRM) cells have emerged as key players in the immune control of melanoma. These specialized cells are identified by expression of tissue retention markers such as CD69, CD103 and CD49a with downregulation of egress molecules such as Sphingosine-1-Phosphate Receptor-1 (S1PR1) and the lymphoid homing receptor, CD62L. TRM have been shown to be integral in controlling infections such as herpes simplex virus (HSV), lymphocytic choriomeningitis virus (LCMV) and influenza. More recently, robust pre-clinical models have also demonstrated TRM are able to maintain melanoma in a dormant state without progression to macroscopic disease reminiscent of their ability to control viral infections. The discovery of the role these cells play in anti-melanoma immunity has coincided with the advent of immune checkpoint inhibitor (ICI) therapy which has revolutionized the treatment of cancers. ICIs that target programmed death protein-1 (PD-1) and cytotoxic T lymphocyte antigen-4 (CTLA-4) have led to substantial improvements in outcomes for patients with metastatic melanoma and have been rapidly employed to reduce recurrences in the resected stage III setting. While ICIs mediate anti-tumor activity via CD8+ T cells, the specific subsets that facilitate this response is unclear. TRM invariably exhibit high expression of immune checkpoints such as PD-1, CTLA-4 and lymphocyte activating gene-3 (LAG-3) which strongly implicates this CD8+ T cell subset as a crucial mediator of ICI activity. In this review, we present pre-clinical and translational studies that highlight the critical role of TRM in both immune control of primary melanoma and as a key CD8+ T cell subset that mediates anti-tumor activity of ICIs for the treatment of melanoma.

Directing the Future Breakthroughs in Immunotherapy: The Importance of a Holistic Approach to the Tumour Microenvironment.

Immunotherapy has revolutionised the treatment of cancers by exploiting the immune system to eliminate tumour cells. Despite the impressive response in a proportion of patients, clinical benefit has been limited thus far. A significant focus to date has been the identification of specific markers associated with response to immunotherapy. Unfortunately, the heterogeneity between patients and cancer types means identifying markers of response to therapy is inherently complex. There is a growing appreciation for the role of the tumour microenvironment (TME) in directing response to immunotherapy. The TME is highly heterogeneous and contains immune, stromal, vascular and tumour cells that all communicate and interact with one another to form solid tumours. This review analyses major cell populations present within the TME with a focus on their diverse and often contradictory roles in cancer and how this informs our understanding of immunotherapy. Furthermore, we discuss the role of integrated omics in providing a comprehensive view of the TME and demonstrate the potential of leveraging multi-omics to decipher the underlying mechanisms of anti-tumour immunity for the development of novel immunotherapeutic strategies.

Transcriptional profiling of circulating mononuclear cells from patients with chronic obstructive pulmonary disease receiving mesenchymal stromal cell infusions.

Chronic obstructive pulmonary disease (COPD) is an inflammatory airways disease with limited therapeutic options. We have previously shown that mesenchymal stromal cell (MSC) infusions are well tolerated in patients with COPD and reduce circulatory biomarkers associated with systemic inflammation and oxidative stress. This study aimed to delineate the underlying mechanisms further by characterizing the transcriptional networks in these patients and to explore the role of MSC-derived paracrine factors in regulating these pathways. Allogeneic, bone marrow-derived MSCs were systemically administered into patients with stable COPD (n = 9). Gene expression profiles from peripheral blood mononuclear cells (PBMCs) were analyzed across the first week after infusion. Paracrine mechanisms associated with these transcriptional changes were explored further by culturing patient PBMCs with MSC-conditioned medium (MSC-CM) or post-MSC infusion (PI) plasma to measure the regulatory effects of soluble factors that may be derived from MSCs. MSC-CM and PI-plasma were characterized further to identify potential immunoregulatory candidates. MSC infusion elicited a strong but transient transcriptional response in patient PBMCs that was sustained up to 7 days. MSC infusion strongly downregulated transcriptional pathways related to interleukin (IL)-8 and IL-1ß, which were also significantly inhibited in vitro following co-culture of PBMCs with MSC-CM and PI-plasma. MSC-derived soluble tumor necrosis factor receptor-1, transforming growth factor-ß1, and extracellular vesicle-associated microRNAs were identified as potential mechanisms promoting these changes, but depletion of these individual candidates revealed inconsistent results. MSC-derived paracrine factors modulate important inflammatory pathways that are relevant to COPD pathogenesis. These data strengthen the hypothesis that therapies using MSCs and their secreted products may be beneficial to patients with COPD.

Fine-Tuning the Tumour Microenvironment: Current Perspectives on the Mechanisms of Tumour Immunosuppression.

Immunotherapy has revolutionised the treatment of cancers by harnessing the power of the immune system to eradicate malignant tissue. However, it is well recognised that some cancers are highly resistant to these therapies, which is in part attributed to the immunosuppressive landscape of the tumour microenvironment (TME). The contexture of the TME is highly heterogeneous and contains a complex architecture of immune, stromal, vascular and tumour cells in addition to acellular components such as the extracellular matrix. While understanding the dynamics of the TME has been instrumental in predicting durable responses to immunotherapy and developing new treatment strategies, recent evidence challenges the fundamental paradigms of how tumours can effectively subvert immunosurveillance. Here, we discuss the various immunosuppressive features of the TME and how fine-tuning these mechanisms, rather than ablating them completely, may result in a more comprehensive and balanced anti-tumour response.

A simplified protocol for profiling heparin-contaminated circulating miRNAs: by microfluidic array.

Peripheral blood is a valuable, non-invasive source of biomarkers which include circulating miRNAs. Using microfluidic array-based techniques, miRNAs can be successfully measured in small amounts of blood plasma (< 0.5 mL) using cDNA pre-amplification. However, the use of heparin-based anticoagulants for blood collection hinders the detection of circulating miRNAs due to its inhibitory effect on PCR components. Although pre-treatment with heparinase have been shown to overcome heparin contamination in blood, its effect has not been described in array-based analyses or more sensitive applications with smaller sample volumes (i.e. 200 µL plasma) requiring pre-amplification. We show that the treatment of miRNA extracted from heparinised plasma with an optimised concentration of Bacteroides heparinase I prior to cDNA pre-amplification dramatically improves the number of detectable miRNA from 2 to 67 targets on the TaqMan® Array Human MicroRNA Cards. Furthermore, the titrated amount of heparinase (3 U) gave the best miRNA detection compared to those used in previous studies (6-24 U). This study provides novel data which demonstrates that heparinase treatment is compatible with protocols that involve pre-amplification of cDNA and microfluidic array-based techniques. This an improved methodology that permits miRNA-based biomarker analysis from small volume of heparinised plasma.

Control of early HIV-1 infection associates with plasmacytoid dendritic cell-reactive opsonophagocytic IgG antibodies to HIV-1 p24.

OBJECTIVES: We have previously demonstrated that HIV-1 p24-specific plasmacytoid dendritic cell-reactive opsonophagocytic antibody (PROAb) responses associate with control of chronic HIV infection. Here, we examined whether HIV-1 p24-specific PROAbs associate with control of early HIV infection and their relationship with HIV-1 p24-specific IgG subclasses. METHODS: Plasma collected at 8 and 52 weeks following primary HIV-1 infection was obtained from antiretroviral therapy-naïve patients who were classified as 'good' (plasma HIV-1 RNA < 5000 copies/ml; n = 17) or 'poor' (HIV-1 RNA > 50 000 copies/ml; n = 15) controllers at week 52. HIV-1 p24-specific PROAb responses were assayed using a plasmacytoid dendritic cell line (Gen2.2), and HIV-1 p24-specific IgG3, IgG1 and IgG2 levels were assayed by ELISA. RESULTS: HIV-1 p24-specific PROAb responses increased in 'good controllers' (P = 0.01) but remained unchanged in 'poor controllers' between weeks 8 and 52. Of the HIV-1 p24-specific IgG subclasses measured, only IgG1 increased over this time period in 'good controllers' alone (P = 0.003), which correlated with the increase in HIV-1 p24-specific PROAb responses (r = 0.83, P < 0.0001). Depletion of IgG1 from IgG preparations of 'good controllers' resulted in the inhibition of HIV-1 p24-specific PROAb responses. In the total patient cohort, plasma HIV-1 RNA levels at week 52 correlated inversely with changes in HIV-1 p24-specific PROAb responses (r = -0.37, P = 0.04) and IgG1 (r = -0.51, P = 0.003) levels between weeks 8 and 52. CONCLUSION: Control of early HIV-1 infection was associated with an increase in HIV-1 p24-specific PROAb responses, which was mediated by HIV-1 p24-specific IgG1 antibodies. These findings provide further evidence that antibodies to HIV core proteins may contribute to control of HIV-1 infection.