Endothelial immunosuppressive mystery genes in spleen for alternative immunotherapy: dissecting their features via lipid nanoparticle-based target validation

The immune modulatory role of spleen: Being the largest lymphoid organ, it is widely known that the spleen can react to blood-borne antigens by initiating an immune response. The spleen is organized in two functionally distinctive regions called the red and white pulp, separated by an interface called the marginal zone. The splenic red pulp serves primarily to filter blood and recycle iron. In contrast, the white pulp allows the generation of antigen-specific immune responses that protect the body against blood-borne infections. The marginal zone is primarily made up of innate immune cells and is distinctly present in mice compared to humans. Recognition of cellular damage by the spleen is often followed by the activation of pattern recognition receptor molecules (PRR), which in turn activates the requisite T cells indicating the role of the spleen in both early innate and subsequent adaptive immune responses. PRRs are found to be expressed selectively by various cells (monocyte, macrophage) within the spleen during an infection. Studies over the past decade have established that both helper and cytotoxic T lymphocytes segregate within the periarteriolar lymphoid sheath of the white pulp during a systemic infection. Though there exists considerable evidence on the role of the spleen during various diseases not much is known about how it reacts against cancer cells. The spleen can accumulate monocyte and granulocyte precursors leading to direct replenishment of tumor-associated macrophages and neutrophils during lung cancer development. The role of the spleen weight in predicting cellular immune response in tumor-bearing models has been established in HCC as the study described the spleen weight to be directly related to the percentages of monocytic-like myeloid-derived suppressor cells (MDSCs) and polymorphonuclear-like MDSCs in tumor-bearing mice. Also, clinical studies indicated that in agreement with preclinical studies in mice, myeloid-derived suppressor cells were found to be increased in the spleen in tumor-bearing patients, limiting the productive immune responses against tumors. Thus, accumulating evidence indicates that the precursor cells in the spleen could be used as a ‘trojan horse’ to modify the immune repertoire, which by altering their immune characteristics (eg from suppressive to stimulatory), those characteristics/actions can now reach the desired locations. The immune modulatory role of vascular endothelium: Due to their ubiquitous presence, endothelial cells (EC) act as a first-line interactor during the appearance of a foreign pathogen within the circulation. Accumulating evidence suggests that in mammals, where blood vessels have evolved into EC-lined channels, ECs can affect immune surveillance. ECs can act as conditional innate immune cells that detect inflammatory stimuli and risk factors via PRRs, as a result of which it can express MHC II molecules which present endothelial antigens to immune cells. Like any nucleated cell, ECs express basal level of MHCI but in response to stimulations such as hydrogen peroxide and IFN-γ, they upregulate the expression of MHC I and induce the expression of MHC II. ECs treated with IFN-γ effectively also induce CD4+ and CD8+ memory T cells to produce cytokines and proliferate. Studies show that lung ECs express MHCII and other molecules that enable the recruitment of T regulatory cells. Upregulation of MHCII has also been reported in various inflammatory conditions. In contrast, MHCII expression is found to be downregulated in cancerous condition. Tumor ECs (TECs) upregulate programmed death ligand 1 (PDL1), an inhibitor of T cell function. TECs downregulate the lymphocyte adhesion molecules ICAM-1 and VCAM-1, and express the death mediator FasL (killing CD8+ T effector cells). All together this hinders tumor-reactive T cell activation and infiltration into the tumor, fostering an immunosuppressive environment and tumor immune escape. In 2020, the Carmeliet Lab documented that a subset of ECs in tumor capillaries and veins express gene signatures, characteristic of immune cells (ICs), suggesting that these ECs have immunomodulatory roles. However, very few papers support this immune regulatory role of ECs in spleen. Research reveals that splenic stromal cell lines modified to develop into immature ECs enable extended proliferation of dendritic cells (DCs) when co-cultured with bone marrow and treated with immunostimulatory agonists like TNF alpha and IL-4. Although Human umbilical vein endothelial cells (HUVECs) cells in the fully differentiated form do not support DC development, HUVECs treated with TNF alpha did. Recently, another study suggested that the spleen vascular endothelial cells (SVEC) and littoral cells display distinct characteristics during myelofibrosis. Mystery genes identification using AI: The Carmeliet lab previously helped to construct the gene prioritization tool ENDEAVOUr for bulk-omics data, based on how similar a candidate gene is to the profile derived from (training) genes known to be involved in the process of interest (“guilt by association”). Using the same strategy, the host lab developed the AI-based tool “single-cell MysterYdentifier” (SCMYSTERYDENTIFIER), but now for the first time using scRNA-seq data of ECs and curated sets of training genes with known immunomodulatory function to predict immunosuppressive genes in ECs: “virgin” MYSTERY genes (class I) and CRYPTIMEC genes (class II, with a known immune function in ICs, but not/poorly studied in ECs). Validation of the targets using lipid nanoparticles (LNPs): A fast and cost-efficient strategy has already been developed by the Carmeliet lab to generate mice with EC-selective knock-out of target genes. It is based on the combined use of LNPs with improved EC-selective targeting (LNPEC) containing sgRNA in endothelial Cas9 expressing mice. One of the major advantages of this technique is that it enables one to skip the traditional ‘in-vivo after in-vitro’ and validate the targets directly in in-vivo itself. A spleen specific formulation is already available using which we can validate the immunosuppressive mystery genes identified in lung tumor models. The above literature review revealed that there is a lack of knowledge on the role of splenic ECs in general and in relation to the immunoregulatory context within the spleen during tumors. An intriguing question would be to what extent we can induce immunostimulatory or immunosuppressive roles in splenic ECs with an ultimate goal of reducing tumor growth. Overall, this warrants the need to explore further the role of splenic ECs in immune regulation during tumor. The host lab has already identified a set of immunosuppressive candidates that are novel (mystery gene) using artificial intelligence (AI) tools in lung ECs. This finding suggests the possibility and feasibility of identifying candidates with disease causing or augmenting properties in other organs during cancer as well, which I will study in spleen. Functional validation of newly identified genes can be done in vivo in murine lung tumor models using a new strategy based on lipid nanoparticle (LNP) encapsulated gene- specific sgRNA administered to transgenic mice expressing cas9 exclusively in ECs ((LNP)- sgRNA/EC cas9), that generates EC-selective knockout mice rapidly and inexpensively.