CAPÍTULO I: EL PROBLEMA
7. CAPÍTULO VII: RECOMENDACIONES
The authors have no conflicts of interest to disclose.
Acknowledgements
This work was supported by a grant from the Dutch government to the Netherlands Institute for Regenerative Medicine (NIRM, grant no. FES0908) and JSH/EHA fellowship to MHB. We are indebted to Dr. Ramon Arens for critically reading the manuscript and to Jolanda de Roo for providing materials.
cells40 and PLZF1+ innate T cells41 which are both critical components of immune competence
do not develop or function properly in NSG mice. This likely relates to the fact that MHC restriction occurs mostly on mouse epithelial cells and less frequently on human APC or via T-T interactions. Thus, clinically significant differences in the TCR repertoires of T cells selected on mouse thymus versus T cells that are selected on human thymus exist. Nevertheless, in experiments where CD34+ cells were transplanted into NSG mice, a survey of the IgH locus and
TCR showed a comparable combinatorial diversity42, although the screening method employed
in this study (multiplexed PCR of V-J rearrangements) might not be sensitive enough to detect small differences in TCR repertoire between human and xenografted T cell samples. Moreover, in similar experiments using the same barcode library with mouse LSK hematopoietic stem/ progenitor cells transplanted into congenic mice, we observed a similar thymic clonal restriction (not shown), suggesting a general applicability of the observed clonal restriction for the overall T-lymphocyte pool, with a caveat on their MHC restriction and development of specialized T-cell subpopulations.
Recent work by Rodewald and co-workers proposed cell competition as a mechanism underlying selection for cellular fitness during T-cell development43. This hypothesis explains the further
clonal restriction at DN3, DP and SP stages that we observe in many mice. Apparently there is selection for progeny of certain HSCs clones while others are selected against. While this could be merely stochastic (neutral competition), the possibility that clonal fitness (e.g. metabolic activity, quiescence, other factors) plays a role is not unlikely given the existence of thymic stem cell clones with differential gene expression programs43. We therefore propose that the
progeny from different HSC clones have inherently different capacity to pass through various developmental checkpoints in the thymus. Alternatively, in the experiments were barcoded CD34+ cells were transplanted, we cannot exclude that in some cases progenitors rather than
bona fide HSCs would be the ancestors of the developing thymocytes. These progenitors are likely to have reduced fitness as well. In the experiments where phenotypically defined HSCs were transplanted, a role for such progenitors seems much less likely due to the purity of the transplanted cells. This notion is supported by the fact that in human gene therapy settings, a considerable fraction of T cells may have arisen from progenitors rather than from HSCs44.
As a consequence, immune incompetence after HSCT is not related to the transplantation of limited numbers of HSC but to intrathymic events. While transplantation of more HSC may improve hematological reconstitution, our data suggests it will do little to improve immunological reconstitution. Thymus seeding is quickly saturated when the transplanted cell dose increases, therefore, efforts aimed at improving function of the thymic epithelium45 most
likely will be most successful in improving immunological immune reconstitution after stem cell transplantation.
5
18. Dik WA, et al. (2005) New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling. J Exp Med 201:1715–23.
19. Hill MO (1973) Diversity and Evenness: A Unifying Notation and Its Consequences. Ecology 54(2): 427–432.
20. Schmidt M, et al. (2002) Polyclonal long-term repopulating stem cell clones in a primate model. Blood 100(8):2737–2743.
21. Kuramoto K, et al. (2004) The impact of low-dose busulfan on clonal dynamics in nonhuman primates. Blood 104(5):1273–1280.
22. Roeder I, et al. (2008) Characterization and quantification of clonal heterogeneity among hematopoietic stem cells: a model-based approach. Blood 112(13):4874–4883.
23. Lu R, Neff NF, Quake SR, Weissman IL (2011) Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding. Nat Biotechnol 29(10): 928–933.
24. Grosselin J, et al. (2013) Arrayed lentiviral barcoding for quantification analysis of hematopoietic dynamics: Arrayed Barcoding to Quantify Hematopoiesis. STEM CELLS 31(10):2162–2171.
25. Morrissey PJ, et al. (1991) Administration of IL-7 to mice with cyclophosphamide-induced lymphopenia accelerates lymphocyte repopulation. J Immunol Baltim Md 1950 146(5):1547–1552.
26. Min D, et al. (2007) Sustained thymopoiesis and improvement in functional immunity induced by exogenous KGF administration in murine models of aging. Blood 109(6):2529–2537.
27. Wils E-J, et al. (2012) Keratinocyte growth factor and stem cell factor to improve thymopoiesis after autologous CD34+ cell transplantation in rhesus macaques. Biol Blood Marrow Transplant J Am Soc Blood Marrow Transplant 18(1):55–65.
28. Fry TJ, et al. (2004) Flt3 ligand enhances thymic-dependent and thymic-independent immune reconstitution. Blood 104(9):2794–2800.
29. Welniak LA, Sun R, Murphy WJ (2002) The role of growth hormone in T-cell development and reconstitution. J Leukoc Biol 71(3):381–387.
30. van der Weerd K, et al. (2014) Thyrotropin acts as a T-cell developmental factor in mice and humans. Thyroid Off J Am Thyroid Assoc 24(6):1051–1061.
31. Velardi E, et al. (2014) Sex steroid blockade enhances thymopoiesis by modulating Notch signaling. J Exp Med 211(12):2341–2349.
32. Zakrzewski JL, et al. (2006) Adoptive transfer of T-cell precursors enhances T-cell reconstitution after allogeneic hematopoietic stem cell transplantation. Nat Med 12(9):1039–1047.
33. van Rood JJ, Roelen DL, Claas FHJ (2005) The effect of noninherited maternal antigens in allogeneic transplantation. Semin Hematol 42(2):104–111.
34. van Rood JJ, et al. (2002) Effect of tolerance to noninherited maternal antigens on the occurrence of graft-versus-host disease after bone marrow transplantation from a parent or an HLA-haploidentical sibling. Blood 99(5):1572–1577.
35. van Rood JJ, et al. (2009) Reexposure of cord blood to noninherited maternal HLA antigens improves transplant outcome in hematological malignancies. Proc Natl Acad Sci U S A 106(47):19952–19957. 36. Ishikawa F, et al. (2005) Development of functional human blood and immune systems in NOD/SCID/
IL2 receptor {gamma} chain(null) mice. Blood 106(5):1565–1573.
References
1. Szabolcs P, Cairo MS (2010) Unrelated umbilical cord blood transplantation and immune reconstitution. Semin Hematol 47(1):22–36.
2. Talvensaari K, et al. (2002) A broad T-cell repertoire diversity and an efficient thymic function indicate a favorable long-term immune reconstitution after cord blood stem cell transplantation. Blood 99(4):1458–1464.
3. Admiraal R, et al. (2015) Association between anti-thymocyte globulin exposure and CD4+ immune reconstitution in paediatric haemopoietic cell transplantation: a multicentre, retrospective pharmacodynamic cohort analysis. Lancet Haematol 2(5):e194–e203.
4. Wilkinson B, Owen JJ, Jenkinson EJ (1999) Factors regulating stem cell recruitment to the fetal thymus. J Immunol Baltim Md 1950 162(7):3873–3881.
5. Champion S, Imhof BA, Savagner P, Thiery JP (1986) The embryonic thymus produces chemotactic peptides involved in the homing of hemopoietic precursors. Cell 44(5):781–790.
6. Hao QL, et al. (2001) Identification of a novel, human multilymphoid progenitor in cord blood. Blood 97(12):3683–3690.
7. Haddad R, et al. (2004) Molecular characterization of early human T/NK and B-lymphoid progenitor cells in umbilical cord blood. Blood 104(13):3918–3926.
8. Kohn LA, et al. (2012) Lymphoid priming in human bone marrow begins before expression of CD10 with upregulation of L-selectin. Nat Immunol 13(10):963–971.
9. Weerkamp F, et al. (2006) Human thymus contains multipotent progenitors with T/B lymphoid, myeloid, and erythroid lineage potential. Blood 107(8):3131–3137.
10. Wiekmeijer A-S, et al. (2014) Sustained Engraftment of Cryopreserved Human Bone Marrow CD34(+) Cells in Young Adult NSG Mice. BioResearch Open Access 3(3):110–116.
11. Shultz LD, et al. (2005) Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. J Immunol Baltim Md 1950 174(10):6477–6489.
12. Eapen M, et al. (2008) Outcomes after HLA-matched sibling transplantation or chemotherapy in children with acute lymphoblastic leukemia in a second remission after an isolated central nervous system relapse: a collaborative study of the Children’s Oncology Group and the Center for International Blood and Marrow Transplant Research. Leukemia 22(2):281–286.
13. Gerrits A, et al. (2010) Cellular barcoding tool for clonal analysis in the hematopoietic system. Blood 115(13):2610–2618.
14. Verovskaya E, et al. (2014) Asymmetry in skeletal distribution of mouse hematopoietic stem cell clones and their equilibration by mobilizing cytokines. J Exp Med 211(3):487–497.
15. Notta F, et al. (2011) Isolation of single human hematopoietic stem cells capable of long-term multilineage engraftment. Science 333(6039):218–221.
16. Benz C, et al. (2012) Hematopoietic stem cell subtypes expand differentially during development and display distinct lymphopoietic programs. Cell Stem Cell 10(3):273–283.
17. Cheung AMS, et al. (2013) Analysis of the clonal growth and differentiation dynamics of primitive barcoded human cord blood cells in NSG mice. Blood 122(18):3129–3137.
5
Supplementary Figure 1: 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. A) 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. B) 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. C) In an additional experiment, 9 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. D) 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 3 bases difference. Using this graph and an empirically determined threshold of 2 bases differences between barcodes, the reads of barcodes with <3 bases difference were combined. All barcoding data in the manuscript was processed using this procedure. E) Samples of PB CD19+ B cells were analyzed in
triplicate to determine the variation of the method between samples. 37. Weerkamp F, et al. (2005) Age-related changes in the cellular composition of the thymus in children.
J Allergy Clin Immunol 115(4):834–840.
38. Halkias J, et al. (2013) Opposing chemokine gradients control human thymocyte migration in situ. J Clin Invest 123(5):2131–2142.
39. Shultz LD, et al. (2010) Generation of functional human T-cell subsets with HLA-restricted immune responses in HLA class I expressing NOD/SCID/IL2r gamma(null) humanized mice. Proc Natl Acad Sci U S A 107(29):13022–13027.
40. Halkias J, et al. (2015) Conserved and divergent aspects of human T-cell development and migration in humanized mice. Immunol Cell Biol. doi:10.1038/icb.2015.38.
41. Lee YJ, et al. (2010) Generation of PLZF+ CD4+ T cells via MHC class II-dependent thymocyte- thymocyte interaction is a physiological process in humans. J Exp Med 207(1):237–246.
42. Marodon G, et al. (2009) High diversity of the immune repertoire in humanized NOD.SCID.gamma c-/- mice. Eur J Immunol 39(8):2136–2145.
43. Martins VC, et al. (2014) Cell competition is a tumour suppressor mechanism in the thymus. Nature 509(7501):465–470.
44. Fischer A, Hacein-Bey-Abina S, Cavazzana-Calvo M (2010) 20 years of gene therapy for SCID. Nat Immunol 11(6):457–460.
45. Tuckett AZ, et al. (2014) Image-guided intrathymic injection of multipotent stem cells supports lifelong T-cell immunity and facilitates targeted immunotherapy. Blood 123(18):2797–2805.
46. Csardi G, Nepusz T (2006) The igraph software package for complex network research. InterJournal Complex Syst:1695.
47. van Dongen JJM, et al. (2003) Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 17(12):2257–231
5
Supplementary Figure 3: 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.
Supplementary Figure 2: 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 A) of 4 mice transplanted with 150,000 CD34+ cells or B) 4 mice transplanted with 26,000 CD34+ cells. Kruskal-Wallis p-values are shown in A and B. 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.
5
Supplementary Figure 5: 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).
Supplementary Figure 6: Comparison of T cell subsets in human peripheral blood and xenografted NSG mice. Peripheral blood of NSG mice (n=8) transplanted with lentivirally-transduced umbilical cord blood
CD34+ cells at 20 weeks post transplantation and human peripheral blood control samples (n=7, female,
ages 20-52 years old) 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 *.
Supplementary Figure 4: Barcoded HSC result in multlineage engraftment in NSG mice. NSG mice were
transplanted with barcoded HSC and after 21 weeks, 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.