This dissertation focuses on the development and application of TetTCRII-SeqHD, an innovative high-dimensional, high-throughput sequencing technology designed to study antigen-specific CD4+ T cells. These cells are central to orchestrating immune responses but remain underexplored in an unbiased way in both peripheral blood mononuclear cells (PBMCs) and tissue-specific contexts due to technical limitations. TetTCRII-SeqHD addresses these challenges by integrating peptide-major histocompatibility complex (pMHC) class II tetramers with DNA barcoding, enabling the simultaneous analysis of T cell receptor (TCR) repertoires, phenotypic characteristics, transcriptional profiles, and antigen specificities. This dissertation demonstrates the technical advancements of TetTCRII-SeqHD, including the use of affinity-matured pMHC class II tetramers, which significantly improve the detection of low-avidity CD4+ T cells. This platform was applied to profile immune responses to SARS-CoV-2 and influenza across tissues, revealing diverse phenotypic profiles and clonal expansions of antigen-specific CD4+ T cells. These findings provide critical insights into the heterogeneity of tissue-specific immunity and its impact on CD4+ T cell differentiation states and functional adaptations. By providing a comprehensive approach to analyzing CD4+ T cells, TetTCRII-SeqHD addresses major gaps in immune cell profiling. This work establishes the platform as a transformative tool for immunological research, with broad implications for understanding immune responses, advancing vaccine development, and improving therapeutic strategies for infectious diseases.