Technical Advances in Single-Cell RNA Sequencing and Applications in Normal and Malignant Hematopoiesis

Single-cell RNA sequencing (scRNA-seq) has been tremendously developed in the past decade owing to overcoming challenges associated with isolation of massive quantities of single cells. Previously, cell heterogeneity and low quantities of available biological material posed significant difficulties to scRNA-seq. Cell-to-cell variation and heterogeneity are fundamental and intrinsic characteristics of normal and malignant hematopoietic cells; this heterogeneity has often been ignored in omics studies. The application of scRNA-seq has profoundly changed our comprehension of many biological phenomena, including organ development and carcinogenesis. Hematopoiesis, is actually a maturation process for more than ten distinct blood and immune cells, and is thought to be critically involved in hematological homeostasis and in sustaining the physiological functions. However, aberrant hematopoiesis directly leads to hematological malignancy, and a deeper understanding of malignant hematopoiesis will provide deeper insights into diagnosis and prognosis for patients with hematological malignancies. Here, we aim to review the recent technical progress and future prospects for scRNA-seq, as applied in physiological and malignant hematopoiesis, in efforts to further understand the hematopoietic hierarchy and to illuminate personalized therapy and precision medicine approaches used in the clinical treatment of hematological malignancies

Cancer Immunotherapy by Blocking Immune Checkpoints on Innate Lymphocytes.

Immune checkpoints refer to a plethora of inhibitory pathways of the immune system that play a crucial role in maintaining self-tolerance and in tuning the duration and amplitude of physiological immune responses to minimize collateral tissue damages. The breakdown of this delicate balance leads to pathological conditions, including cancer. Indeed, tumor cells can develop multiple mechanisms to escape from immune system defense, including the activation of immune checkpoint pathways. The development of monoclonal antibodies, targeting inhibitory immune checkpoints, has provided an immense breakthrough in cancer therapy. Immune checkpoint inhibitors (ICI), initially developed to reverse functional exhaustion in T cells, recently emerged as important actors in natural killer (NK)-cell-based immunotherapy. Moreover, the discovery that also helper innate lymphoid cells (ILCs) express inhibitory immune checkpoints, suggests that these molecules might be targeted on ILCs, to modulate their functions in the tumor microenvironment. Recently, other strategies to achieve immune checkpoint blockade have been developed, including miRNA exploiting systems. Herein, we provide an overview of the current knowledge on inhibitory immune checkpoints on NK cells and ILCs and we discuss how to target these innate lymphocytes by ICI in both solid tumors and hematological malignancies

Innate lymphoid cell differentiation and functions in intestinal homeostasis and disease

The gastrointestinal (GI) tract, which includes the small and the large intestine, is considered the largest immunological organ that plays a pivotal role in food digestion, nutrient absorption and fuel generation. Inflammatory Bowel Disease (IBD), which is broadly divided into Crohn’s disease (CD) and Ulcerative Colitis (UC), are multifactorial chronic intestinal inflammatory conditions that affect both pediatric and adult patients. Patients with IBD have a significantly increased risk of developing colorectal cancer (CRC). The immune system is undoubtably a major factor in disease pathogenesis and understanding what goes awry to cause disease is of great importance. This thesis focuses on a particular immune cell type called innate lymphoid cells (ILCs), and follows our journey to understand their role in the intestine of pediatric and adult patients with IBD as well as in CRC. Since the discovery of ILCs is fairly recent, a lot remains unknown regarding their phenotype and function at steady and disease state, particularly in humans. In this thesis we performed immunophenotypic, functional, transcriptional as well as epigenetic assays to understand many aspects of their biology, differentiation and interactions with their adaptive counterparts, T cells. In Paper I, we described the presence of CD45RA+ ILCs with naïve features in the tonsil that are transcriptionally, epigenetically and functionally distinct from the differentiated ILC subsets. We demonstrated that (CD45RA+)CD62L− ILCs were accumulated in the inflamed gut of adult patients with IBD and compared to their tonsil counterpart, these cells showed preferential differentiation towards IL22-producing ILC3s. In Paper II, we determined the landscape of innate and adaptive lymphocytes in pediatric IBD (pIBD) through single-cell RNA sequencing. First, we demonstrated that ILCs are altered in pIBD, and in line with Paper I, we show that increased frequency of CD62L− ILCs is also a feature in pediatric IBD patients. Also, we were able to uncover shared and unique transcriptional signatures between ILCs and T cells and identify with a neighbor-based computational method the most and least inflamed cells in our dataset. In Paper III, we explored the antigen-presenting properties of circulating and tissue-resident intestinal ILCs. Specifically, we were able to demonstrate that human circulating HLADR+ ILCs were able to internalize, process and present antigen to memory CD4+ T cells, and that this process is regulated by IL-1β through NF-κΒ signaling while it is suppressed by TGF-β. Additionally, via confocal microscopy we found that intestinal HLADR+ ILCs were located in close proximity to T cells, raising the possibility that the antigen presentation by ILCs could occur in vivo. Overall, the research work included in this thesis contributes to advancing our understanding of ILCs in intestinal homeostasis and disease. The characterization of novel ILC states and functions in complex intestinal diseases such as IBD and CRC could pave the way for unraveling mechanisms that drive these diseases and potentially facilitate the development of more effective therapies for those patients

Rethinking immune checkpoint blockade: Beyond the T cell

The clinical success of immune checkpoint inhibitors has highlighted the central role of the immune system in cancer control. Immune checkpoint inhibitors can reinvigorate anti-cancer immunity and are now the standard of care in a number of malignancies. However, research on immune checkpoint blockade has largely been framed with the central dogma that checkpoint therapies intrinsically target the T cell, triggering the tumoricidal potential of the adaptive immune system. Although T cells undoubtedly remain a critical piece of the story, mounting evidence, reviewed herein, indicates that much of the efficacy of checkpoint therapies may be attributable to the innate immune system. Emerging research suggests that T cell-directed checkpoint antibodies such as anti-programmed cell death protein-1 (PD-1) or programmed death-ligand-1 (PD-L1) can impact innate immunity by both direct and indirect pathways, which may ultimately shape clinical efficacy. However, the mechanisms and impacts of these activities have yet to be fully elucidated, and checkpoint therapies have potentially beneficial and detrimental effects on innate antitumor immunity. Further research into the role of innate subsets during checkpoint blockade may be critical for developing combination therapies to help overcome checkpoint resistance. The potential of checkpoint therapies to amplify innate antitumor immunity represents a promising new field that can be translated into innovative immunotherapies for patients fighting refractory malignancies

Blockade of the co-inhibitory molecule PD-1 unleashes ILC2-dependent antitumor immunity in melanoma.

Group 2 innate lymphoid cells (ILC2s) are essential to maintain tissue homeostasis. In cancer, ILC2s can harbor both pro-tumorigenic and anti-tumorigenic functions, but we know little about their underlying mechanisms or whether they could be clinically relevant or targeted to improve patient outcomes. Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis. ILC2s are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor, which coordinates the recruitment and activation of eosinophils to enhance antitumor responses. Tumor-infiltrating ILC2s expressed programmed cell death protein-1, which limited their intratumoral accumulation, proliferation and antitumor effector functions. This inhibition could be overcome in vivo by combining interleukin-33-driven ILC2 activation with programmed cell death protein-1 blockade to significantly increase antitumor responses. Together, our results identified ILC2s as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for antitumor immunotherapies

Group 2 innate lymphocytes at the interface between innate and adaptive immunity.

Group 2 innate lymphoid cells (ILC2) are innate immune cells that respond rapidly to their environment through soluble inflammatory mediators and cell-to-cell interactions. As tissue-resident sentinels, ILC2 help orchestrate localized type 2 immune responses. These ILC2-driven type 2 responses are now recognized in diverse immune processes, different anatomical locations, and homeostatic or pathological settings. ILC2-derived cytokines and cell surface signaling molecules function as key regulators of innate and adaptive immunity. Conversely, ILC2 are governed by their environment. As such, ILC2 form an important nexus of the immune system and may present an attractive target for immune modulation in disease

Pleiotropic Role and Bidirectional Immunomodulation of Innate Lymphoid Cells in Cancer.

Innate lymphoid cells (ILCs) are largely tissue resident and respond rapidly toward the environmental signals from surrounding tissues and other immune cells. The pleiotropic function of ILCs in diverse contexts underpins its importance in the innate arm of immune system in human health and disease. ILCs derive from common lymphoid progenitors but lack adaptive antigen receptors and functionally act as the innate counterpart to T-cell subsets. The classification of different subtypes is based on their distinct transcription factor requirement for development as well as signature cytokines that they produce. The discovery and subsequent characterization of ILCs over the past decade have mainly focused on the regulation of inflammation, tissue remodeling, and homeostasis, whereas the understanding of the multiple roles and mechanisms of ILCs in cancer is still limited. Emerging evidence of the potent immunomodulatory properties of ILCs in early host defense signifies a major advance in the use of ILCs as promising targets in cancer immunotherapy. In this review, we will decipher the non-exclusive roles of ILCs associated with both protumor and antitumor activities. We will also dissect the heterogeneity, plasticity, genetic evidence, and dysregulation in different cancer contexts, providing a comprehensive understanding of the complexity and diversity. These will have implications for the therapeutic targeting in cancer.This work was supported by KCL Breast Cancer Now Research Unit. FF-B was funded by grant KCL-Q2-Y5 and SI was by CRUK (grant C56773/A24869)

In silico Analyses of Immune System Protein Interactome Network, Single-Cell RNA Sequencing of Human Tissues, and Artificial Neural Networks Reveal Potential Therapeutic Targets for Drug Repurposing Against COVID-19

Background: There is pressing urgency to identify therapeutic targets and drugs that allow treating COVID-19 patients effectively.Methods: We performed in silico analyses of immune system protein interactome network, single-cell RNA sequencing of human tissues, and artificial neural networks to reveal potential therapeutic targets for drug repurposing against COVID-19.Results: We screened 1,584 high-confidence immune system proteins in ACE2 and TMPRSS2 co-expressing cells, finding 25 potential therapeutic targets significantly overexpressed in nasal goblet secretory cells, lung type II pneumocytes, and ileal absorptive enterocytes of patients with several immunopathologies. Then, we performed fully connected deep neural networks to find the best multitask classification model to predict the activity of 10,672 drugs, obtaining several approved drugs, compounds under investigation, and experimental compounds with the highest area under the receiver operating characteristics.Conclusion: After being effectively analyzed in clinical trials, these drugs can be considered for treatment of severe COVID-19 patients. Scripts can be downloaded at

IMMUNOSUPPRESSIVE MECHANISMS IN THE TUMOR MICROENVIRONMENT MEDIATING RESISTANCE TO IMMUNOTHERAPY

Breast cancer is the leading site of new cancer cases in women and the second leading cause of cancer related deaths. Improvements in detection and treatment in the past three decades has led to a significant decline in breast cancer deaths, yet just this year more than 42,000 people are expected to die from breast cancer. Immunotherapy, boosting the anti-tumor immune response, is a valuable advancement for the field of cancer therapy. Immune checkpoint therapy focuses on blocking inhibitory receptors expressed on activated immune cells. Two prominent checkpoint inhibitory receptors are cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and programmed cell death protein 1 (PD-1). Unfortunately, most triple negative breast cancer patients do not benefit from immune checkpoint inhibition despite immune infiltration into the tumors. In the work presented here, we aim to find ways to improve immunotherapy through projects studying the effectiveness of immune checkpoint blockade in cancer. First, we evaluated several models of breast cancer and found the heavily immune infiltrated claudin-low subtype was not able to respond to immunotherapy due to the suppressive tumor microenvironment. This subtype of breast cancer responded to checkpoint inhibition only in the context of specific and complete regulatory T cell (Treg) depletion. Second, we studied the role of PD-1 blockade on Tregs in a model of claudin-low breast cancer. We found that PD-1 blockade increased proliferation and survival of Tregs in the tumor microenvironment, leading to increased immunosuppression. Third, we observed the role of PD-1 expression on NK cells in cancer and chronic viral infection. We saw that NK cells may not be a beneficial target for immunotherapy due to the inconsistency of PD-1 expression. Together, this work provides insight into potential mechanisms involved in the poor response to immune checkpoint therapy in some cancers. Although tumors may be heavily immune infiltrated, this does not predict response to immunotherapy, and a more thorough analysis of the tumor microenvironment should be done such as determining which subsets of immune cells in the tumor expressing PD-1 can potentially be affected by checkpoint blockade.Doctor of Philosoph

Immunol Rev

For years, we have taken a reductionist approach to understanding gene regulation through the study of one gene in one cell at a time. While this approach has been fruitful it is laborious and fails to provide a global picture of what is occurring in complex situations involving tightly coordinated immune responses. The emergence of whole-genome techniques provides a system-level view of a response and can provide a plethora of information on events occurring in a cell from gene expression changes to splicing changes and chemical modifications. As with any technology, this often results in more questions than answers, but this wealth of knowledge is providing us with an unprecedented view of what occurs inside our cells during an immune response. In this review, we will discuss the current RNA-sequencing technologies and what they are helping us learn about the innate immune system.U01 IP000017/IP/NCIRD CDC HHSUnited States/R35 GM137801/GM/NIGMS NIH HHSUnited States/R01 AI148413/AI/NIAID NIH HHSUnited States