Identification of T Cell Receptor Repertoire Signatures in Age, Autoimmune Conditions and Cancer using Next-Generation Sequencing

Abstract

The immune system is an interactive network composed of cells, tissues and soluble factors which has evolved to protect the host from invading pathogens. T cells are specialized cells of the adaptive arm of the immune system and recognize antigens via their T cell receptor (TCR). The TCR is encoded by the random and irreversible recombination of V(D)J gene segments, a process resulting in a highly diverse repertoire of TCRs. The large diversity of the repertoire offers recognition of, and therefore immunological protection against, a wide range of antigens for the host. The clonal composition of the immune repertoire allows insights into the current state as well as the antigen exposure history of an individual and can be determined by next-generation sequencing (NGS). Increasing age and diseases like cancer can compromise this fine-tuned network thus leading to chronic inflammation, autoimmunity or insufficient responses towards harmful antigens. The work presented here discusses age-, cancer and autoimmunity-associated alterations of the TCR repertoire and summarizes fundamental aspects of T cell immunity as well as applications and challenges of adaptive immune receptor repertoire sequencing (AIRR seq). In the first publication, which arose from this thesis, we profiled more than 300 peripheral blood TCR repertoires from healthy individuals as well as patients with cancer of all age groups. These analyses revealed a continuous age-dependent decline of T cell diversity which was accompanied by a decrease in T cell richness beginning at the fourth decade of life. Moreover, we observed that patients with cancer who received chemotherapy reestablished their pre-treatment T cell diversity. Interestingly, the regeneration of the repertoire complexity after chemotherapy was best explained by rebound thymic activity rather than through recovery of T cell counts by peripheral clonal expansion only. Taken together our data indicate that TCR metrics deteriorate gradually with increasing age, but age-specific repertoire metrics are restored after chemotherapy even in elderly patients with cancer. In the second publication, we profiled the TCR repertoires of patients with autoimmune cytopenias (AIC) to understand if these patients present disease-specific immunological signatures that could reveal pathophysiological clues and eventually be exploited as bloodbased biomarker. We analyzed 25 newly diagnosed patients with primary or secondary (lymphoma-associated) AIC as well as three reference cohorts composed of age- and sexmatched healthy controls, patients with active autoimmune hepatitis (AIH; another primary autoimmune disease) as well as patients with chronic lymphocytic leukemia (CLL, without autoimmune complication). Global TCR repertoire metrics like diversity and clonality as well as VJ gene usage distribution showed uniform characteristics for patients with lymphoma, but no AIC-specific signature. However, clustering of T cells with overlapping antigen specificity using the GLIPH algorithm (grouping lymphocyte interaction by paratope hotspots) revealed a considerable lack of T cell clusters in patients with primary autoimmune disease (AIC as well as AIH) as compared to healthy donors. The functionality of these clusters will have to be elucidated in future studies. Nevertheless, the signature of T cell cluster loss may represent a readily accessible biomarker for autoimmune conditions. Taken together, AIRR seq has deepened our understanding of the adaptive immune response and has helped thus far to answer pressing questions in fields of immunodiagnostics, vaccines, cancer immunotherapy, and antibody engineering. As technical and computational challenges are being solved, this technology will come to realize its true potential.