Shrijan Khanal

● Multiparameter Flow Cytometry Analysis: Conducted advanced multiparameter flow cytometry analysis using more than 20 colors, with data analysis performed through FlowJo and OMIQ, providing detailed insights into cellular phenotypes and immune responses.

● PBMCs Isolation: Isolated Peripheral Blood Mononuclear Cells (PBMCs) by Ficoll density gradient centrifugation from buffy coats of healthy human donors, ensuring high viability and yield for downstream applications.

● Human T cells Activation and Co-Stimulation: Optimized the activation and co-stimulation of human peripheral blood T-lymphocytes, enhancing their responsiveness for CRISPR/Cas9-based gene editing applications.

● Flow Cytometry Analysis: Performed comprehensive flow cytometry analysis to assess T cells purity, viability, and activation status, ensuring robust and reliable cell populations for experimental use.

● CRISPR/Cas9 Gene Editing: Performed CRISPR/Cas9-mediated gene editing of MIR155HG in activated human T cells, optimizing transfection protocols to maximize gene editing efficiency and consistency.

● Genomic Validation Techniques: Validated the deletion of MIR155HG using a combination of genomic PCR, qRT-PCR, and ddPCR techniques, confirming precise and effective gene knockout in human T cells.

● acute GVHD Mouse Model Scoring: Conducted clinical scoring of xenogeneic acute GVHD mouse models, monitoring disease progression and therapeutic effects with precision to evaluate treatment efficacy.

● In Vivo Xenograft-versus-Leukemia Experiments: Conducted in vivo xenograft-versus-leukemia (GVL) experiments using xenogeneic GVL mouse models. Evaluated therapeutic efficacy by monitoring tumor persistence via whole-body IVIS imaging.

● Cytotoxic T Cells Killing Assay: Designed and optimized in vitro cytotoxic T lymphocyte (CTL) activity assays, increasing assay sensitivity and reliability to better measure immune responses against target leukemic cells.

● Pancreatic Tumor Model Development: Generated and characterized murine pancreatic tumor-OVA (PK51975-OVA) cell lines in vitro and in vivo using BL6 mice, ensuring robust models for tumor immunology studies.

● Lentiviral Transduction and Flow Cytometry: Transduced PK51975 cells with lentivirus and assessed transduction efficiency via flow cytometry, followed by single-cell sorting to establish clonal cell lines.

● Tumorigenicity and T Cell Stimulation: Validated that PK51975-OVA cells effectively stimulate antigen SIINFEKL (SL8)-specific T cells and demonstrated tumorigenic potential in vivo.

● Adoptive Transfer Studies: Conducted adoptive transfer studies via retro-orbital injections of antigen-specific OT-1 T cells into PK51975-OVA tumor-bearing CD45.2 B6 mice, investigating the role of TLS in supporting stem-like T cell differentiation within tumors.

● TLS and Tumor Control: Assessed theimpact of LTBR agonism on TLS induction and its potential to improve tumor control in vivo.

● IL-21 and T Cell Differentiation: Investigated the effects of IL-21 on T cell differentiation and exhaustion, contributing to a deeper understanding of immune modulation in cancer.

● Necropsy and Tissue Collection: Performed necropsy and collected various tissues, including blood, tumor-draining lymph nodes, and tumors, ensuring high-quality samples for subsequent analyses.

● Protocol Design and Optimization: Led designing and optimization of experimental protocols, resulting in improved reproducibility and accuracy across multiple assays and experimental setups.

● Collaboration: Collaborated with cross-functional research teams, fostering a collaborative environment that drove innovative solutions and advanced research objectives.

● Data Analysis and Interpretation: Conducted in-depth analysis and interpretation of experimental data, generating meaningful insights that informed subsequent research directions and experimental designs.

● Manuscript Writing and Publication: Authored and published research manuscripts in distinguished scientific journals, contributing to the advancement of knowledge in the field of immuno-oncology.