Fig. 3.

Immune repertoire diversity and validation of a TCR-pHLA association. A TCR diversity is generated by genetic recombination at the TCR locus. Within the thymus, rearrangement of V (variability), D (determining), J (joining) and C (constant) genes of the TCRB is followed by TCRA locus recombination. B Thymic selection of the recombined TCRαβ pair is based on their relative affinity for HLA molecules: a lower threshold to recognise an HLA allele (positive selection) and an upper threshold to avoid self-reactivity and autoimmunity against self-antigens (negative selection). The naïve T cell repertoire generated is further selected upon peripheral antigen encounters in the circulation and tissues, resulting in clonal proliferation and generation of adaptive memory. C The combinatorial potential of the TCR repertoire is required to cover the antigenic peptidome. However, the observed diversity is considerably lower than the theoretical diversity due to constraints such as possible and productive recombination, α-β pairing and HLA selection, in addition to sampling issues (much of the repertoire is represented by unique TCR sequences). D Immune repertoire studies address this diversity thanks to advances in next-generation sequencing (NGS) technologies. Either using DNA or RNA as starting material, TCR sequences can be enriched and prepared for sequencing using different methods, such as multiplex PCR amplification, target enrichment or 5′RACE (5′-rapid amplification of cDNA ends) and nested PCR amplification (the latter only for RNA input). Single-cell RNA sequencing (scRNA seq.) can also be used to study the immune repertoire of T cells clones. Whilst it has reduced capacity to capture the diversity due to limited cell input (max 10.000 cells), it can provide the TCRαβ pair chain sequences involved in the TCR-pHLA interaction (and transcriptome of each cells), being more suitable for the next steps of the validation of a TCR-pHLA interaction. According to pre-existing information related to the condition, (1) altered peptidome observed with, i.e. elution studies, or (2) altered immunome observed with repertoire studies, follow-up studies will seek to find the cognate TCR (TCR screening studies) or peptide (Antigen screening studies), respectively