Development of a diverse human T-cell repertoire despite stringent restriction of hematopoietic clonality in the thymus

Abstract

The fate and numbers of hematopoietic stem cells (HSC) and their progeny that seed the thymus constitute a fundamental question with important clinical implications. HSC transplantation is often complicated by limited T-cell reconstitution, especially when HSC from umbilical cord blood are used. Attempts to improve immune reconstitution have until now been unsuccessful, underscoring the need for better insight into thymic reconstitution. Here we made use of the NOD-SCID-IL-2Rγ−/− xenograft model and lentiviral cellular barcoding of human HSCs to study T-cell development in the thymus at a clonal level. Barcoded HSCs showed robust (>80% human chimerism) and reproducible myeloid and lymphoid engraftment, with T cells arising 12 wk after transplantation. A very limited number of HSC clones (<10) repopulated the xenografted thymus, with further restriction of the number of clones during subsequent development. Nevertheless, T-cell receptor rearrangements were polyclonal and showed a diverse repertoire, demonstrating that a multitude of T-lymphocyte clones can develop from a single HSC clone. Our data imply that intrathymic clonal fitness is important during T-cell development. As a consequence, immune incompetence after HSC transplantation is not related to the transplantation of limited numbers of HSC but to intrathymic events.H ematopoietic stem cell transplantation (HSCT) has become common clinical practice in the treatment of leukemia, lymphoma, and certain inherited immune and metabolic disorders. After transplantation there is an immediate need for de novo production of granulocytes, erythrocytes, and platelets, referred to as "hematopoietic reconstitution," later followed by recovery of lymphocyte numbers, termed "immune reconstitution." Although often successful, HSCT is associated with a number of complications arising from poor immune reconstitution, which presents one of the most important causes of morbidity after HSCT (1, 2). Poor myeloid reconstitution is directly linked to low numbers of HSCs in the transplant, but the reasons for poor immune and, in particular, T-lymphocyte reconstitution are much less clear.A study on the application of antithymocyte globulin in cord blood transplantation showed that antithymocyte globulin administration in pretransplantation conditioning results in decreased T-cell reconstitution and survival (3), indicating the need for a better understanding of de novo T-cell development after HSCT. As T cells develop in the thymus, in contrast to all other blood lineages that develop in the bone marrow (BM), HSCderived progenitors need to seed the thymus. Earlier work in mice has indicated that relatively few progenitors seed the thymus (4, 5), yet their numbers and the subsequent fate of the progeny derived from an HSC clone has remained elusive. The nature of the thymusseeding cell has been subject of much debate, certainly in humans. CD34+ cells (6, 7), CD34 (8), and others have been proposed as thymus-seeding cells in the human situation. In contrast to mice, the earliest hu...

The functional relationship between hematopoietic stem cells and developing T lymphocytes

“…A very limited number of HSC clones 0) repopulated the thymus, as observed by a roughly 10-fold lower number of retrieved barcodes by deep sequencing in DN thymocytes compared to transplanted HSCs in the BM. 68 This clonal restriction was observed when using either total CD34 + cells and highly purified, phenotypically defined human HSCs. Intriguingly, we observed further restriction of the number of clones during subsequent development, with another clonal restriction point at the DN3-DP transition.…”

“…Intriguingly, we observed further restriction of the number of clones during subsequent development, with another clonal restriction point at the DN3-DP transition. 68 This indicates selection of progeny of HSC clones at several key developmental checkpoints in the thymus. A recent work by Rodewald and coworkers proposed cell competition as a mechanism underlying selection for cellular fitness during T cell development.…”

“…68 Therefore, clonality in immunological terms is fully diverse, while hematological clonality is very limited, showing that these clonal systems are independently regulated. As a consequence, immune incompetence after HSC transplantation is not related to the transplantation of limited numbers of HSCs but to intrathymic events.…”

“…Despite the severe hematological clonal restriction, TCR rearrangements were polyclonal and showed a diverse repertoire, demonstrating that a multitude of T lymphocyte clones can develop from a single HSC clone. 68 Therefore, clonality in immunological terms is fully diverse, while hematological clonality is very limited, showing that these clonal systems are independently regulated. As a consequence, immune incompetence after HSC transplantation is not related to the transplantation of limited numbers of HSCs but to intrathymic events.…”

A Lentiviral Fluorescent Genetic Barcoding System for Flow Cytometry-Based Multiplex Tracking

“…While gammaretroviral or lentiviral delivery of readily detectable reporters has initially proven valuable for enrichment and tracking of transduced populations, viral integration site analysis and retroviral barcoding have further improved clonal resolutions. [1][2][3][4][5][6][7] The power of integration site analysis and DNA barcoding lies in parallel cell fate tracking, which opens up numerous experimental opportunities including in vivo applications in a reduced number of mice enabling decreased variability and improved efficiencies. 8 Application of such tracking and fate mapping approaches have for example been extensively and elegantly exploited in the context of studies in the hematopoietic system both in vivo and in vitro.…”