Table 1.

Methods to examine the regulation of T cell repertoires by gut microbiota.

Methods	Advantages	Disadvantages
TCR-Tg cell transfer	- Easier identification of Ag-specific T cells by using congenic marker - Precise determination of Ag-specific T cell proliferation (CFSE or CTV labeled donor T cells) - Examination of the interaction between Ag-specific T cells and other immune cells (i.e., B cells) - Single TCR specificity	- Not reflect the physiological frequency of cognate Ag specific T cells - Intra-clonal competition distorts natural Ag-specific T cell responses against microbiota. - Limited use depending on HLA types
pMHC Tetramer staining	- Direct examination and quantification of Ag-specific T cell frequency under steady state and inflammatory settings - Examination of physiological Ag-specific T cell responses	- Relatively lower affinity between pMHC tetramer and TCR (limitation of visualization in flow cytometry) - Limited use depending on HLA types - Broad TCR specificities within pMHC tetramer+ T cells
Bulk TCR gene sequencing	- Less expensive than single cell based TCR gene sequencing - Relatively bigger sample size	- Lack of information on the TCR diversity created by TCR chain pairing* - Limited association between TCR clonality and T cell subset/function (cytokine, transcription factor expression)**
Single cell-based TCR gene sequencing	- Strong association with TCR clonality and T cell subset/function - Possible examination of the TCR diversity created by TCR chain pairing - Combination with single cell-based transcriptomics & other -omics	- Relatively expensive and limitation on sample size (typically less than 2-3 x 104) - Requirement of complex analytical methods

*Assessment of TCR chain paring is not required in case of bulk TCR gene sequencing based on TCRα or TCRβ-restricted mice., **addressed by TCR sequencing on T cells sorted based on the transcription factor or cytokine reporters.