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. 2015 Nov 3;112(44):E6020-7.
doi: 10.1073/pnas.1519118112. Epub 2015 Oct 19.

Development of a Diverse Human T-cell Repertoire Despite Stringent Restriction of Hematopoietic Clonality in the Thymus

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Free PMC article

Development of a Diverse Human T-cell Repertoire Despite Stringent Restriction of Hematopoietic Clonality in the Thymus

Martijn H Brugman et al. Proc Natl Acad Sci U S A. .
Free PMC article

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.

Keywords: T lymphocyte; T-cell development; T-cell receptor; hematopoietic stem cell; thymus.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Barcoding human HSC and xenotransplantation. (A) Human HSC (CD34+CD38CD45RACD90+CD49f+) or CD34+ cells were isolated and transduced with the PTGZ barcode library carrying 21 variable bases in the barcode with a total complexity of 485 different barcodes. The transduced cells were transplanted in to 5- to 6-wk-old sublethally irradiated NSG mice. The mice were bled monthly and myeloid cells, T cells, and B cells were isolated from PB. At 16 or 21 wk, the experiment was terminated and, in addition to PB, cells isolated from thymus, spleen, and BM were sorted as indicated and the barcode content of the samples was analyzed. From the thymus, DN, DP, and SP cells were isolated using the indicated markers. Chimerism in the PB of nine mice transplanted with 1,000 purified human HSCs as determined by human CD45 expression (B) and GFP marking of human cells (C). Development of CD3+ T cells (D), CD13+/CD33+ myeloid cells (E), and CD19+ B cells (F) within the human CD45+ population were followed in time after transplantation of the transduced cells.
Fig. S1.
Fig. S1.
Spike-in experiments to calibrate the DNA barcoding method. A plasmid sample was spiked in at equimolar ratio and diluted into a bulk barcoded sample. The bargraph shows the measured fraction of deep-sequencing reads in each sample, with the lowest blue bar in the graph indicating the spiked-in plasmid (A). Triplicate analysis of the fraction of reads made up by the spiked-in barcode shows the least-squares fitted line and the Pearson correlation coefficient. (B). In an additional experiment, nine repeated measures of the same sample were performed and the coefficient of variation was determined and plotted against the mean fraction of reads, showing that the precision of quantification increases with the amount of reads (C). A higher number of barcodes than the library complexity was retrieved from the analysis of these samples. To correct for these sequencing errors, the number of dissimilar bases between all barcodes was determine and a directed acyclic graph was built for all barcodes that had less than three bases difference. Using this graph and an empirically determined threshold of two bases differences between barcodes, the reads of barcodes with <three bases difference were combined. All barcoding data in the report was processed using this procedure (D). Samples of PB CD19+ B cells were analyzed in triplicate to determine the variation of the method between samples (E). Fraction of total reads is indicated on the y axis.
Fig. 2.
Fig. 2.
Clonal contribution to HSC and adaptive immune cells. Multilineage engraftment is demonstrated by contribution of barcoded clones to HSC, splenic CD3+ T cells and CD19+ B cells in nine NSG mice that were transplanted with purified barcoded human HSCs. The barcode content of these compartments was determined by comparing the normalized contribution of clones. The relative contributions to the total human HSC (BM) and T- and B-lymphoid lineages (spleen) in each mouse is shown as a ternary plot. Each point represents the contribution of a clone to HSC, CD19+ B-cell, and CD3+ T-cell lineages. The extent of contribution is depicted by the location of the point along the three axes, with clones contributing equally to all lineages being closer to the center of the triangle. In most mice, but especially in mouse 8 and mouse 9, true multilineage contribution of HSC clones can be observed. The numbers at the points of the triangle indicate the number of detected clones for which contribution to only one lineage was detected.
Fig. S2.
Fig. S2.
Barcoded HSC result in multlineage engraftment in NSG mice. NSG mice were transplanted with barcoded HSC and after 21 wk, barcode content in CD19+ B cells, CD3+ T cells, and CD13+/CD33+ myeloid cells was determined. Ternary plots show the contribution of a clone to each lineage. Multilineage contribution is defined as contribution of a clone to each of these three lineages. The total number of clones retrieved from these spleen samples is indicated, as is the number of these clones that contribute to all three lineages.
Fig. 3.
Fig. 3.
Clonal restriction during T-cell development. Thymus was homogenized and cells were sorted into their developmental stages (DN1/2, DN3, DP, SP) (A). The number of clones contributing >1% of the sample in nine mice transplanted with barcoded HSCs or CD34+ cells were counted and the Shannon diversity index calculated. The normalized Shannon diversity was compared between HSC in the BM and the developmental stages in the thymus. Mean normalized Shannon diversity indices for nine mice transplanted with 1,000 HSC are shown with error bars indicating SEM (B). Wilcoxon P values are shown in B. (C) Clonal repertoire, as determined by barcode analysis of sorted population in the thymus in stacked area graphs (as in A), with colors identifying the individual clones. Note that the colors are chosen for the purpose of display, which means that the same color depicts different clones in different mice.
Fig. S3.
Fig. S3.
Clonal restriction during T-cell development after CD34+ cell transplantation. Similar to the data in Fig. 2, the normalized Shannon diversity was compared between the developmental stages in the thymus of four mice transplanted with 150,000 CD34+ cells (A) or four mice transplanted with 26,000 CD34+ cells (B). Kruskal–Wallis P values are shown in A and B. (C and D) The clonal contribution in the thymic subsets of mice transplanted with 150,000 or 26,000 barcoded human CD34+ cells is shown in stacked area graphs using colors to mark the individual barcoded clones in mice receiving a high (150,000 cells, C) or low (26,000 cells, D) dose of barcoded CD34+ cells.
Fig. 4.
Fig. 4.
Separation of hematological and immunological clonality. TRB repertoires in DP and SP thymic subsets, and sorted CD3 splenocytes of mice transplanted with human HSC were determined. Electropherograms of two collections of primers amplifying the TRB locus (green and blue) show the TRB diversity for a representative mouse. The positive control shows peripheral blood mononuclear cells (PBMC) of a healthy individual.
Fig. S4.
Fig. S4.
Hematological and immunological clonality in splenocytes. Rearrangements of TRD, TRG, and TRB of sorted transduced human splenic CD3 cells are shown using two collections of primers to amplifying the respective loci. PBMC of a healthy individual was used a positive control and clonal rearrangements in Nalm-16 (TRD), MOLT3 (TRG), ALL-1 (TRB-A), and Jurkat (TRB-C) are shown for comparison.
Fig. S5.
Fig. S5.
GFP marking in the thymus. The percentage of GFP marking in the different thymocyte subsets after transplantation of barcoded HSC is displayed as mean + SEM (n = 9).
Fig. S6.
Fig. S6.
Comparison of T-cell subsets in human PB and xenografted NSG mice. PB of NSG mice (n = 8) transplanted with lentivirally transduced UCB CD34+ cells at 20 wk posttransplantation and human PB control samples (n = 7, female, ages 20–52 y) were analyzed for the expression of CD45RO and CD45RA in the CD4+ and CD8+ T cells by flow cytometry. Wilcoxon P values smaller than 0.05 are indicated with an asterisk.

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