T cell receptor next-generation sequencing reveals cancer-associated repertoire metrics and reconstitution after chemotherapy in patients with hematological and solid tumors.

The dynamics of immunoaging and the onset of immunoparesis in healthy individuals and cancer patients has been controversially discussed. Moreover, the role of chemotherapy on T cell regeneration needs further elucidation in light of novel immunotherapies that have become standard of care for many elderly cancer patients. We used next-generation immunosequencing to study T cell receptor (TCR) repertoire metrics on 346 blood samples from healthy individuals and cancer patients producing a dataset with around 8.8 million TCR reads. This analysis showed that decline of T cell diversity and increase in T cell clonality is a continuous process beginning in healthy individuals over 40 years of age. Untreated patients with both hematological and solid tumors showed blood TCR repertoires with significantly lower diversity and higher clonality as compared to healthy individuals across all decades. Loss in T cell diversity was essentially driven by a loss in richness in aging healthy individuals, while in cancer patients a loss in repertoire evenness was an additional contributing factor. Interestingly, chemotherapy did not impair the regeneration of blood TCR repertoire diversity to pre-treatment age-specific levels. Surprisingly, even patients over the age of 70 years receiving highly T cell toxic therapies reestablished their pre-treatment T cell diversity suggesting rebound thymic activity rather than recovery of T cell counts by peripheral expansion only. Taken together, these data suggest that human TCR repertoire metrics gradually deteriorate in the aging individual, but age-specific TCR metrics are restored after T cell depleting therapy even in elderly cancer patients.

Deep sequencing of bone marrow microenvironments of patients with del(5q) myelodysplastic syndrome reveals imprints of antigenic selection as well as generation of novel T-cell clusters as a response pattern to lenalidomide.

In myelodysplastic syndromes with a partial deletion of the long arm of chromosome 5, del(5q), lenalidomide is believed to reverse anergic T-cell immunity in the bone marrow resulting in suppression of the del(5q) clone. In this study we used next-generation sequencing of immunoglobulin heavy chain (IGH) and T-cell receptor beta (TRB) rearrangements in bone marrow-residing and peripheral blood-circulating lymphocytes of patients with del(5q) myelodysplastic syndromes to assess the immune architecture and track adaptive immune responses during treatment with lenalidomide. The baseline bone marrow B-cell space in patients was comparable to that of age-matched healthy controls in terms of gene usage and IGH clonality, but showed a higher percentage of hypermutated IGH sequences, indicating an expanded number of antigen-experienced B lineage cells. Bone marrow B lineage clonality decreased significantly and hypermutated IGH clones normalized upon lenalidomide treatment, well in line with the proliferative effect on healthy antigen-inexperienced B-cell precursors previously described for this drug. The T-cell space in bone marrow of patients with del(5q) myelodysplastic syndromes showed higher TRB clonality compared to that of healthy controls. Upon lenalidomide treatment, myelodysplastic syndrome-specific T-cell clusters with low to medium spontaneous generation probabilities emerged; these clusters were shared across patients, indicating a common antigen-driven T-cell response pattern. Hence, we observed B lineage diversification and generation of new, antigen-dependent T-cell clusters, compatible with a model of adaptive immunity induced against the del(5q) clone by lenalidomide. Overall, this supports the concept that lenalidomide not only alters the functional T-cell state, but also the composition of the T- and B-cell repertoires in del(5q) myelodysplastic syndromes.

T-cell diversification reflects antigen selection in the blood of patients on immune checkpoint inhibition and may be exploited as liquid biopsy biomarker.

Cancer immunotherapy with antibodies targeting immune checkpoints, such as programmed cell death protein 1 (PD-1), shows encouraging results, but reliable biomarkers predicting response to this costly and potentially toxic treatment approach are still lacking. To explore an immune signature predictive for response, we performed liquid biopsy immunoprofiling in 18 cancer patients undergoing PD-1 inhibition before and shortly after initiation of treatment by multicolor flow cytometry and next-generation T- and B-cell immunosequencing (TCRß/IGH). Findings were correlated with clinical outcomes. We found almost complete saturation of surface PD-1 on all T-cell subsets after the first dose of the antibody. Both T- and B-cell compartments quantitatively expanded during treatment. These expansions were mainly driven by an increase in the activated T-cell compartments, as well as of naïve B- and plasma cells. Deep immunosequencing revealed a clear diversification pattern of the clonal T-cell space indicative of antigenic selection in 47% of patients, while the remaining patients showed stable repertoires. 43% of the patients with a diversification pattern showed disease control in response to the PD-1 inhibitor. No disease stabilizations were observed without clonal T-cell space diversification. Our data show for the first time a clear impact of PD-1 targeting not only on circulating T-cells, but also on B-lineage cells, shedding light on the complexity of the anti-tumor immune response. Liquid biopsy T-cell next-generation immunosequencing should be prospectively evaluated as part of a composite response prediction biomarker panel in the context of clinical studies.