Next-Generation Sequencing Analysis of the Human TCRγδ plus T-Cell Repertoire Reveals Shifts in Vγ- and Vδ-Usage in Memory Populations upon Aging

Immunological aging remodels the immune system at several levels. This has been documented in particular for the T-cell receptor (TCR)alpha beta+ T-cell compartment, showing reduced naive T-cell outputs and an accumulation of terminally differentiated clonally expanding effector T-cells, leading to increased proneness to autoimmunity and cancer development at older age. Even though TCR alpha beta+ and TCR gamma delta+ T-cells follow similar paths of development involving V(D) J-recombination of TCR genes in the thymus, TCR gamma delta+ T-cells tend to be more subjected to peripheral rather than central selection. However, the impact of aging in shaping of the peripheral TRG/TRD repertoire remains largely elusive. Next-generation sequencing analysis methods were optimized based on a spike-in method using plasmid vector DNA-samples for accurate TRG/TRD receptor diversity quantification, resulting in optimally defined primer concentrations, annealing temperatures and cycle numbers. Next, TRG/TRD repertoire diversity was evaluated during TCR gamma delta+ T-cell ontogeny, showing a broad, diverse repertoire in thymic and cord blood samples with Gaussian CDR3-length distributions, in contrast to the more skewed repertoire in mature circulating TCR gamma delta+ T-cells in adult peripheral blood. During aging the naive repertoire maintained its diversity with Gaussian CDR3-length distributions, while in the central and effector memory populations a clear shift from young (V gamma 9/V delta 2 dominance) to elderly (V gamma 2/V delta 1 dominance) was observed. Together with less clear Gaussian CDR3-length distributions, this would be highly suggestive of differentially heavily selected repertoires. Despite the apparent age-related shift from V gamma 9/V delta 2 to V gamma 2/V delta 1, no clear aging effect was observed on the V delta 2 invariant T nucleotide and canonical V gamma 9-J gamma 1.2 selection determinants. A more detailed look into the healthy TRG/TRD repertoire revealed known cytomegalovirus-specific TRG/TRD clonotypes in a few donors, albeit without a significant aging-effect, while Mycobacterium tuberculosis-specific clonotypes were absent. Notably, in effector subsets of elderly individuals, we could identify reported TRG and TRD receptor chains from TCR gamma delta+ T-cell large granular lymphocyte leukemia proliferations, which typically present in the elderly population. Collectively, our results point to relatively subtle age-related changes in the human TRG/TRD repertoire, with a clear shift in V gamma/V delta usage in memory cells upon aging.