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Keywords:

  • Animal models;
  • Cell differentiation;
  • Cytokines;
  • T cells;
  • Thymopoiesis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Human Immune System (HIS) mice represent a novel biotechnology platform to dissect human haematopoiesis and immune responses. However, the limited human T-cell development that is observed in HIS mice restricts its utility for these applications. Here, we address whether reduced thymopoiesis in HIS mice reflects an autonomous defect in T-cell precursors and/or a defect in the murine thymic niche. Human thymocyte precursors seed the mouse thymus and their reciprocal interactions with murine thymic epithelial cells (TECs) led to both T-cell and TEC maturation. The human thymocyte subsets observed in HIS mice demonstrated survival, proliferative and phenotypic characteristics of their normal human counterparts, suggesting that the intrinsic developmental program of human thymocytes unfolds normally in this xenograft setting. We observed that exogenous administration of human IL-15/IL-15Rα agonistic complexes induced the survival, proliferation and absolute numbers of immature human thymocyte subsets, without any obvious effect on cell-surface phenotype or TCR Vβ usage amongst the newly selected mature single-positive (SP) thymocytes. Finally, when IL-15 was administered early after stem cell transplantation, we noted accelerated thymopoiesis resulting in the more rapid appearance of peripheral naïve T cells. Our results highlight the functional capacity of murine thymic stroma cells in promoting human thymopoiesis in HIS mice but suggest that the “cross-talk” between murine thymic stroma and human haematopoietic precursors may be suboptimal. As IL-15 immunotherapy promotes early thymopoiesis, this novel approach could be used to reduce the period of T-cell immunodeficiency in the post-transplant clinical setting.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Sixty million years of evolution has generated important differences between the murine and human immune systems (HISs). As such, our knowledge of lymphocyte development, homeostasis and immune responses that is largely derived from mouse studies may not be directly applicable to man. The recent advances in the development of HIS mice provide a new model to experimentally dissect human lymphocyte biology that may eventually allow us to bridge the knowledge gap between murine and human in vivo studies of immune responses. One well-described HIS mouse model involves engraftment of newborn Balb/c Rag2−/−γc−/− mice with human fetal liver or cord blood haematopoietic stem cells (HSCs), which seed the murine BM and thymus and in turn differentiate into an almost complete spectrum of mature human myeloid and lymphoid cell subsets 1–3. Human B-cell development predominates in this model, with T cells encompassing <10% of the peripheral lymphocyte pool 1–3. The reasons for the reduced T-cell homeostasis is not known, but could involve central and/or peripheral mechanisms. Previous studies have shown that peripheral human T cells in Balb/c Rag2−/−γc−/− HIS mice exhibit an abnormally high turnover rate yet fail to accumulate 1–5. On the other hand, interaction of haematopoietic precursors with murine thymic epithelial cell (TEC) ligands or soluble factors during thymopoiesis and subsequent selection of T cells on murine major histocompatibility complexes (MHC) may generate suboptimal signals for T-cell maintenance.

T-cell development depends on instructive temporal–spatial signals provided by a resident cellular network known as thymic stroma 6, 7. TECs represent the main thymic stromal compartment in the pre-involution thymus and constitute a multifunctional platform that supports thymocyte commitment, survival, division, migration and selection. TECs are classically divided into two specialized functional subsets, cortical (cTECs) and medullary (mTECs). While cTECs are important at early stages of T-cell development and mediate positive selection, mTECs are critical at later stages governing negative selection and providing survival signals to mature single-positive (SP) thymocytes 6, 8, 9. The generation of these two functionally competent TEC compartments is a prerequisite for normal thymopoiesis. TEC maturation requires reciprocal signals from thymocytes, a symbiotic bi-directional relationship known as “thymic cross-talk” 7, 10, 11.

T-cell homeostasis comprises T-cell generation in the thymus, export to the periphery, maintenance of the peripheral naïve T-cell pool and regulation of activated effectors and memory T-cell compartments 12. Human T-cell development within the thymus has been extensively characterized and critical stages can be defined by sequential expression of cell-surface antigens on differentiating thymocytes. Commitment to the T-cell lineage coincides with expression of CD1a on CD7+CD3CD4CD8 cells and denotes the beginning of gene rearrangement of the TCRγ, δ and β chain loci. Subsequent CD4 up-regulation generates immature single-positive (ISP, CD3) cells that survive, proliferate and undergo further differentiation to the CD4+CD8+ DP stage upon successful TCR-β chain gene rearrangement and pairing with the pre-TCR-α (pTα) chain (β-selection) 13. At the DP stage, cell proliferation is rapid and the anti-apoptotic factor Bcl-xL is up-regulated 14, 15, allowing thymocyte survival as TCR-α chain gene rearrangements begin. CD4+CD8+ DP cells expressing a functional TCRαβ with low affinity for self-peptide–MHC complexes expressed on the cTECs are then positively selected. T cells expressing high-affinity receptors for self-peptide–MHC complexes are negatively selected, whereas those which do not interact with MHC molecules will die by neglect. Positively selected T cells down-regulate Bcl-xL, up-regulate Bcl-2 and further differentiate into either CD4+ or CD8+ SP T cells, before migrating to the periphery as naïve T cells. Peripheral T-cell homeostasis is driven by cytokines (primarily IL-7 produced by reticular cells) and MHC interactions (with a role for DCs providing MHC class II) 16–18.

As HIS mice represent a chimeric system comprising both human and mouse-derived signals, incompatibilities between human and mouse receptor–ligand interactions are possible that could limit the efficiency of the development of human lymphocytes and/or their survival, activation or tissue localization in peripheral lymphoid organs. In this study, we analyze human T-cell development within the thymus of reconstituted HIS mice, the effects of human thymopoiesis on murine TEC differentiation and the role of exogenous IL-15/IL-15Rα agonists in promoting early human thymocyte development in this humanized mouse model.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Organized thymic epithelial patterning in reconstituted HIS mice

To determine whether the correct compartmentalization and phenotypic differentiation of the murine TEC subsets was induced in reconstituted HIS mice, thymus sections were analyzed by immunohistology using antibodies specific for mTECs (MTS10), cTECs (CDR1), human T cells (CD3) and immature T cells (CD38). As reported in earlier studies 19, the block in thymopoiesis due to deficiencies in Rag and γc results in a thymic rudiment devoid of an anatomically defined medullary region with a predominance of cortical-like TECs in the remaining the thymic stroma (Fig. 1A). In contrast, the thymus of Balb/c Rag2−/−γc−/− mice reconstituted with human HSCs contains clear medullary regions harboring mTECs (MST10+CDR1) that co-localize with human CD3+ T cells. These medullary regions do not stain with CD38, which although expressed by mature T cells is expressed at higher levels by immature thymocytes (DP, iSP4) and is only detected by histology on CD3 thymocytes (Fig. 1A and C). Accordingly, the CD38+ immature thymocytes were restricted to the cortical regions co-localizing with CDR1+ cTECs. It is important to note the discrepancy in thymocyte density between the human thymus (Fig. 1B and C) and the mouse thymus in reconstituted HIS mice (Fig. 1A), with the latter being clearly less densely populated compared with that of the fetal thymus, although maintaining the same cortico-medullary partitioning. These data demonstrate a normal murine TEC compartmentalization in the thymus of reconstituted HIS mice and indicate that a productive “cross-talk” between human thymocytes and murine TECs can take place in this xenograft setting.

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Figure 1. Physiological interactions between human thymocytes and murine TECs. (A) Frozen sections of thymus from (left) Balb/c Rag2−/−γc−/− mice and (right) Balb/c Rag2−/−γc−/− reconstituted with human HSCs 12 wk earlier were stained with the indicated antibodies against mouse epithelial cells and human thymocytes. Magnification (×100). (B, C) Frozen sections of human fetal thymus (gestation age of 17 wk) were stained with the indicated antibodies against human thymocytes. Magnification (×100). Photomicrographs are representive of 3 thymi per group and 1 fetal human thymus.

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Human thymocyte subsets developing in the HIS mouse thymus appear phenotypically normal

We next characterized the phenotype of the developing human thymocyte populations in HIS mice. Human thymocytes can be divided into the following subsets based on cell-surface antigen expression: double negative (DN; CD3CD4CD8NKp46), immature SP CD4+ (iSP4; CD3CD4+CD8NKp46), double-positive (DP; CD3CD4+CD8+NKp46), SP CD4+ (SP4; CD3+CD4+CD8NKp46) and SP CD8+ (SP8; CD3+CD8+CD4NKp46) (Fig. 2A). These five subsets were consistently detected in HIS mouse thymi when analysed at 12-wk post-engraftment. We did not detect a clear population of CD34+ pre-T/thymic stem cells in the thymus of reconstituted HIS mice that has been previously characterized in the human thymus (Fig. 2A). We next analysed BrdU incorporation and Ki67 expression to determine the proliferative state of each thymocyte subset. As expected, cell proliferation was enhanced in cells progressing through the iSP4 to DP stages of development whereas cells that had progressed past these antigen receptor selection checkpoints (SP4 and SP8) had a lower turnover rate (Fig. 2B). As expected, anti-apoptotic Bcl-xL protein levels were also substantially up-regulated in iSP4 and DP thymocytes compared with levels observed in positively selected mature T cells (SP4 and SP8: Fig. 2C). Overall, these results suggest that appropriate pre-T-cell receptor (TCR) repertoire signalling is occurring in iSP4 thymocytes resulting in increased Bcl-xL and proliferation, whereas positive selection signals restores these properties to normal basal levels. All thymocytes were CD7+ with CD1a expression evident on DN, iSP4 and DP thymocytes but absent on SP4 and SP8 (Fig. 2D). Not surprisingly, CD45RA, CCR7, CD62L, CD44 and TCRαβ were up-regulated on mature SP4 and SP8 thymocytes, these cells also showing highest levels of CD69 expression, an antigen often associated with the recent positive selection (Fig. 2D). Overall, these results suggest that intrathymic human T-cell development in reconstituted HIS mice proceeds normally.

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Figure 2. Human thymocyte differentiation in the murine thymus of reconstituted HIS mice. 12 weeks after reconstitution with human HSCs, thymi from HIS mice were analysed. (A) Five human thymocyte subsets (DN, iSP4, DP, SP4 and SP8) were identified by flow cytometry according to the indicated cell-surface phenotype. Human post-natal thymus is shown for comparison. (B–D) HIS mice were injected intra-peritoneally with BrdU 24 h before being sacrificed. DN, iSP4, DP, SP4 and SP8 thymocytes were analysed for (B) BrdU incorporation, Ki67 expression, (C) Bcl-xL expression in spleen and (D) cell-surface phenotype by flow cytometry. Data are representative of three experiments.

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Effects of exogenous human IL-15 administration on human thymopoiesis in HIS mice

We have previously demonstrated that murine IL-15 was suboptimal in stimulating human IL-15 responsive cells and shown that in vivo administration of a potent IL-15 receptor agonist (RLI; IL-15+IL-15Rα sushi domain) results in a significant increase in IL-15 responsive human lymphocytes (NK cells and CD8+ T cells) in the periphery of HIS mice 3. While IL-15 does not appear essential for thymopoiesis in mice 20, 21, previous studies have shown that TEC subsets express IL-15 22 and could “trans-present” IL-15/IL-15Rα complexes to developing thymocytes 23, 24. Since the bioavailability of human IL-15 in HIS mice appears limited 3, we next investigated the effect of exogenous IL-15 on human thymopoiesis in vivo. HIS mice were treated with RLI on weeks 8, 9, 10 and 11 post-HSC transplant and analysed on week 12 (Fig. 3A). Surprisingly, all immature populations of human thymocytes (DN, iSP4 and DP) were significantly increased in number following this IL-15 treatment regime (Fig. 3B). BrdU uptake analysis demonstrated that this could be attributed to increased proliferation of these cells (Fig. 3C). This effect was especially apparent for DN and DP cells; iSP4 cells already have a high proliferation rate, so an effect of IL-15 on this population was more difficult to appreciate (Fig. 3C). Since previous studies 25–28 have shown that IL-15 can expand activate/memory phenotype cells in mouse (in vivo) and human (in vitro), we next analyzed the cell-surface phenotype of the mature CD3+ T cells in the thymus. Interesting, while we previously demonstrated that IL-15 enhanced CD8+ T-cell proliferation and numbers in the periphery of HIS mice, turnover of both SP4 and SP8 in the thymus were unchanged in the presence of IL-15 (Fig. 3C). Furthermore, the SP4 and SP8 that developed in the presence of RLI or from the RLI-expanded progenitors, displayed a phenotype indistinguishable from PBS-treated HIS mice (Fig. 3D). Overall, these results indicate that IL-15 can drive immature thymocyte proliferation without grossly affecting the phenotype of the resulting mature T cells.

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Figure 3. IL-15 promotes thymocyte proliferation in vivo in reconstituted HIS mice. (A) Experimental scheme. Newborn Rag2−/−γc−/− mice were irradiated with 3.3 Gy and injected intra-hepatic (i.h.) with 5×104 CD34+CD38 human fetal liver cells. At 8, 9, 10 and 11 wk of age, HIS mice were injected i.p. with 2.5 μg IL-15-IL-15Rα fusion protein (RLI) or PBS. Mice were sacrificed and analyzed at 12 wk. (B) Immature thymocyte subsets were identified as in Fig. 2A by flow cytometry and cellularity was enumerated. Values represent mean+SEM of five mice per group. **Statistically significant using Student's t-test p>0.01. (C) HIS-mice treated as in (A) injected i.p. with BrdU 24 h before being sacrificed. DN, iSP4, DP, SP4 and SP8 thymocytes were analysed for BrdU incorporation. *p<0.05 by Student's t-test. Values represent mean+SEM of five mice per group. (D) SP4 and SP8 human thymocytes (hCD45+CD3+) were analyzed by flow cytometry for surface antigens. Data are representative of three individual experiments.

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Analysis of TCRβ repertoires in IL-15 “boosted” HIS mice

The maintenance of a diverse TCR repertoire is essential for efficient immune responses against the extensive number of foreign and altered-self-antigens encountered during our lifetime. It was therefore was important to know if exogenous IL-15/IL-15Rα treatment influenced TCR selection mechanisms or biased the overall TCR repertoire in reconstituted HIS mice. We examined whether RLI administration affected the TCR repertoire by analyzing CDR3 length profiles of the β, γ, and δ TCR chains expressed by thymic T cells in control and RLI-treated HIS mice. We did not observe any striking difference in the TCR repertoire following RLI administration, suggesting that IL-15 is not preferentially selecting or propagating a subset of T-cell specificities (Fig. 4A). The overall TCR diversity in HIS thymi appears similar to what we observed amongst cord blood T cells (Supporting Information Fig. 1), although the overall diversity did appear somewhat restricted compared with that of T cells in human peripheral blood. A similar analysis performed on the γ and δ variable chains demonstrated that in vivo administration of RLI does not influence the relative γ and δ chain usage or the variability in CDR3 length for any given Vγ or Vδ chains (Fig. 4B).

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Figure 4. Diverse T-cell generation in IL-15-treated HIS mice. TCR Vβ, Vγ or Vδ repertoire in Balb/c Rag2−/−γc−/− HIS mice. Thymocytes from HIS mice treated as in Fig. 3A were isolated and TCR CDR3 immunoscope analysis was performed for different Vβ families. (A) Histograms are representative of results obtained from five mice in each group and display amino acid length of CDR3 regions (x-axis) and relative frequency (y-axis). (B) Splenocytes from HIS mice treated as in Fig. 1A or human PBMCs were isolated and TCR CDR3 immunoscope analysis was performed for different Vγ and Vδ families. Histograms are representative of results obtained from three mice in each group and display amino acid length of CDR3 regions (x-axis) and relative frequency (y-axis).

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An in-depth analysis of γ/δ TCR+ T-cell development in HIS mice has not been hitherto reported. We observed a clear population of Vγ9+Vδ2+ T cells in the thymus and LN of HIS mice and these γ/δ T cells were largely restricted to the CD161+CD3+ T-cell subset (Fig. 5A). In the thymus, we also detected a clear Vγ9Vδ2+ and a minor Vγ9+Vδ2 T-cell population (Fig. 5A). The fraction and number of γ/δ T cells also increased following RLI treatment (Fig. 5B and C). Using CDR3 immunoscope analysis of γ and δ variable chains, we confirmed that most human γ/δ T cells found in the periphery of HIS mice use Vδ2 and this preferential usage was unaffected by RLI treatment (Fig. 5D). Two major populations exist, Vγ8+ and Vγ9+ T cells which each represent between 40 and 60% of total γ/δ T cells and which pair almost exclusively with Vδ2 (Fig. 5D). In vivo administration of RLI did not influence the relative γ and δ chain usage or the variability in CDR3 length for any given Vγ or Vδ chains (Fig. 5D).

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Figure 5. Analysis of γδ TCR+ T-cell generation in HIS mice. (A) Human γδ TCR+ T cells from thymus and LN of HIS mice were analyzed by flow cytometry. Human T cells were separated into two subsets based on CD161 expression (CD3+CD161+ or CD3+CD161) and the expression of Vγ9, Vδ2, Vδ1 and CD8. (B) Total human γδ TCR+ T cells from thymus and LN from HIS mice treated as in Fig. 3A were analyzed by flow cytometry using a Pan γδ TCR antibody amongst total human T cells (hCD45+CD3+) in these organs. Flow cytometric plots are representative of (A) 4 and (B) 5 mice in each group. (C) Summary scatter plots of human γδ TCR+ T cells. (D) The relative proportion of thymocytes expressing the indicated γ or δ TCR was determined by quantitative PCR from mice treated as in Fig. 3A. Percentages represent the frequency of T cells containing the indicated Vγ or Vδ PCR products out of the total Vγ or Vδ PCR products. Histograms represent the mean percentage+SEM of three mice in each group.

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Trans-presented human IL-15 accelerates T-cell development in HIS mice

Since RLI was efficient in increasing human T-cell numbers and displayed a clear effect in the thymus, we next determined whether RLI immunotherapy could promote early thymopoeisis in HIS mice (before mature T cells are generated), and thereby accelerate production of mature T cells. We analyzed the thymus and spleen of HIS mice at various ages and found that between 5 and 6 wk after HSC engraftment represented the ideal window to commence RLI immunotherapy as the thymus was seeded with immature thymocytes, whereas mature T cells were lacking from both the thymus and spleen (Fig. 6A). These HIS mice were then treated with a more intense RLI treatment regime (2.5 μg of RLI on days 0, 3, 6 and 9) and then sacrificed on day 12. RLI immunotherapy resulted in significant increase in human T cells in all organs analyzed, promoting both SP4 and SP8 development in the thymus and resulting in a significantly increased pool of naïve mature T cells (hCD45+CD45RA+CD3+; Fig. 6B). Furthermore, this effect could be monitored over time in the blood of HIS mice receiving RLI where we detected almost a 10-fold increase in human T cells in the blood after the second injection (day 4) and increasing up to 20-fold more T cells at the end of the treatment regime (Fig. 6C).

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Figure 6. RLI accelerates thymopoeisis and the appearance of mature peripheral T cells in the early post-HSC graft period. (A) Thymus and spleen from HIS mice (5–6 wk post-HSC engraftment) were analyzed by flow cytometry for human T-cell reconstitution by hCD45, CD56, CD3, CD4, CD8 and CD45RA. (B) HIS mice (5–6 wk post-HSC engraftment) were treated every three days with 2.5 μg IL-15/IL-15Rα fusion protein (RLI) or PBS and sacrificed 12 days after the first injection. Human (hCD45+) lymphocytes from thymus, spleen and BM were analyzed for mature T cells (CD3 and CD45RA) and double-positive thymocytes (CD4 and CD8) by flow cytometry. (C) HIS mice treated as in Figure 6B were bled on days 0, 4, 7 and 12 after the first injection and the total number of human T cells (hCD45+CD3+) per mL of blood was enumerated by flow cytometry. Data represent the fold increase in T-cell number in mice treated with IL-15/IL-15Rα compared with PBS at each time point. Data are mean percentage±SEM of three mice in one experiment.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

HIS mouse models represent an important new model to study human lymphopoiesis, lymphoid homeostasis, immune responses and immune-related disease pathologies 29. Nevertheless, this promising biotechnology platform has yet to be fully exploited due to certain limitations, including suboptimal human T-cell reconstitution and reduced capacity to elicit antigen-specific antibody and cellular human immune responses following vaccination 29–32. While the latter limitations may deter the pharmaceutical industry from using this technology for pre-clinical drug/vaccine screening, it remains clear that HIS mice offer an unprecedented opportunity to experimentally dissect human haematopoiesis, studies that have been previously restricted to in vitro systems. One example is the recent elucidation of a linear development of human NK-cell subsets in vivo and their dependence on trans-presented IL-15 3. Here, we use HIS mice to demonstrate that human immature thymocytes are highly responsive to trans-presented IL-15 in vivo. Exogenous IL-15 accelerated thymopoiesis in HIS mice resulting in the earlier appearance of naïve T cells in the periphery. This finding would have not been possible using in vitro studies and is not predicted from murine studies, highlighting the importance of this humanized mouse model to advance our knowledge in biology of human haematopoiesis.

Since human lymphocytes are poorly triggered by murine IL-15 3, we hypothesized that a similar mechanism may explain the weak overall thymopoiesis that is observed in HIS mice. Several signals have been implicated in controlling thymopoiesis including those emanating from the TCR following interactions with self-peptide+MHC (pMHC) and those induced by growth factors including cytokines 12. Mice lacking the common cytokine receptor gamma chain (γc) family of cytokines (which comprise IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21) have hypoplastic thymi and a reduced number of T cells, although the T cells generated were able to respond to a range of mitogens 33, 34. Humans possessing mutations in genes encoding the γc, Jak3 (both critical for signal transduction following binding γc cytokines) or the α chain of the IL-7 receptor (IL-7Rα), also display a severe block in T-cell development and resulting severe combined immunodeficiency 35. While abundant data show that IL-7 is the principle γc cytokine behind the thymic defect in γc-null mice, other γc cytokines may function in concert with IL-7 for normal thymopoeisis 36–38. Indeed, mice deficient for signalling components of IL-7 and IL-15 (IL-7Rα−/−IL-2Rβ−/− display a thymopoiesis block more closely resembling that of γc−/− mice than mice deficient for signalling components of IL-7 and IL-2 (IL-7Rα−/−IL-2Rα−/−) or IL-7 and IL-4 (IL-7Rα−/−IL-4Rα−/−) 39. That fact that mice deficient for IL-15 or IL-15Rα display no obvious abnormalities in thymopoiesis 20, 21 suggests a minor role for IL-15 in this species that is only evident when thymopoiesis is already impaired (such as in the absence of IL-7).

The bioactive form of IL-15 is complexed with the IL-15Rα chain. Thus, cells expressing IL-15 such as monocytes, DCs and parenchymal cells must also co-express the IL-15Rα in order to “trans-present” IL-15 to IL-15-responsive cells. Evidence suggests that trans-presentation of IL-15 by parenchymal cells is crucial in the development of IL-15 responsive cells such as invariant NKT cells in the thymus 23, 24. One could envisage the IL-15Rα+ thymic stroma presenting IL-15 and working in synergy with locally expressed IL-7 22 to promote immature thymocyte development and perhaps invariant NKT-cell development.

Our TCR Vβ immunoscope data indicated that IL-15 promoted the generation of a pool of T cells with diverse TCR reactivity. While Vβb appears less commonly used and occasionally absent following IL-15 treatment, this is not a consistent finding and we have previously reported diverse usage of Vβb amongst peripheral T cells in HIS mice 40. A likely reason for the rare usage of Vβb is that the primers for BV13a amplify the genes 13S1, S2, S3, S4, S6, S7, S8 et S9 whereas the primers BV13b uniquely amplify the gene 13S5.

In the thymus, essentially all steps of T lymphocyte development require interactions between thymocytes with resident thymic stromal components, which are mainly composed of TECs in the pre-involution thymus. TECs form a three-dimensional multifunctional supportive platform for T-cell production. Our data showed that murine TECs are able to sustain human thymopoiesis, indicating a certain degree of cross-reactivity between murine thymopoietic factors and human thymocytes. Nevertheless, exogenous administration of hIL-7 5 or hIL-15 (this report) can independently improve human thymopoiesis indicating human cytokines are more efficient. Multiple factors are important for the commitment (DLL4), survival and division (IL-7, IL-15, stem cell factor), migration (CXCL12 and CCL25) and selection (MHC-peptide) of developing thymocytes 7. Whether substitution (replacement) of the corresponding human factors will improve human thymopoiesis in HIS mice will require further study.

Strikingly, thymocytes and TECs have a symbiotic relationship since TEC maturation requires reciprocal signals from thymocytes, the so-called thymic “cross-talk” 7. TEC patterning is compromised by mutations that affect early thymopoiesis (Fig. 1 and 41, 42). Our findings showed that transfer of human HSCs into Rag2−/−γc−/− mice restores cortical and medullary thymic epithelial compartments, to a similar extent to that observed upon reconstitution with murine HSC precursors 43. Hence, a sufficient degree of cross-reactivity appears to exist between signals emanating from human thymocytes that provide maturation cues for the segregation and patterning of cortical and medullary TEC niches. That said, several ligand-receptor interactions taking place between species in the HIS thymus are more than likely sub-optimal, thus we cannot rule out the possibility that the clear effect of IL-15 on developing human thymocytes may be exaggerated or enhanced due to the poor reactivity to other murine-derived stimuli that drive thymopoiesis under normal circumstances.

Finally, our results indicate that modulation of IL-15/IL-15Rα availability may impact on human T-cell reconstitution following HSC transplantation. The accelerated emergence of peripheral T cells in RLI-treated HIS mice could shorten the window of relative T-cell immunodeficiency following therapeutic BM transplantation. By extension, the ability of RLI to promote human thymopoiesis and improve T-cell homeostasis offers the means to boost cellular immunity following radiotherapy, chemotherapy and/or immunotherapy of cancer and other devastating diseases.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Mice

Rag2−/−γc−/− mice on the Balb/c background have been previously described 3, 40. Mice were maintained in isolators with autoclaved food and water. Mice with a HIS were generated as previously described 1–3, 40. Briefly, newborn (3–5-day-old) mice received sub-lethal (3.3 Gy) total body irradiation from a Cs source, and were injected intra-hepatic (i.h.) with 5×104 sorted CD34+CD38 human fetal liver cells. Protocols for generating HIS mice were approved by Institut Pasteur committees and followed guidelines of the French Ministry of Agriculture.

In vivo assays and treatments

HIS mice were injected intra-peritoneally with 100 μL of 10 mg/mL of BrdU (BD Bioscience, San Jose, CA, USA) 12 and 24 h prior to analysis and treated i.p. with 100 μL of 2.5 μg RLI or PBS alone commencing either 5–6 or 8 wk after reconstitution.

Flow cytometry analysis for cell-surface and intracellular markers

The following anti-human mAb were used to stain cell suspension for FACS analysis: CD3 (SK7), CD4 (SK3), CD34 (581), Vδ2 (B6), CD8 (SK1), CD19 (HIB19), NKp46 (9E2), CD38 (HB7), CD16 (3G8), CD45RO (UCLH1), KIR2DL2/L3 (DX27), KIR3DL1 (DX9), CD122 (Mik-β3), TCR-αβ (T10B9.1A-31), CD127 (hIL-7R-M21), CD45 (2D1), CD69 (L78), BrdU (B44) from BD Bioscience, CD28 (CD28.2), CD27 (O323), CD62L (DREG-56) from Biolegend (San Diego, CA, USA) and CD25 (BC96) and CD45RA (HI100) from eBioscience, KIR2DS4 (FES172), KIR2DL1/DS1 (EB6B), KIR2DL2/L3/DS2 (GL183), KIR3DL1/DS1 (Z27.3.7), Vγ9 (IMMU 360), Bcl-xL (7B2.5) from Beckman Coulter (Fullerton, CA, USA) and CD25 (BC96), γδ TCR (B1.1), CCR7 (3D12) and CD45RA (HI100) from eBioscience (San Diego, CA, USA). CRD1 and MTS10 were kind gifts from Richard Boyd (Monash University, Melbourne, Australia). For BrdU detection, cells were incubated for 1 h at 37°C with 30 μg DNAse from BrdU flow kits (BD Bioscience). All washings and reagent dilutions were done with PBS containing 2% fetal calf serum (FCS). All acquisitions were performed using LSRII, Canto 1 or Canto 2 cytometers, cell sorting was perform using FACS ARIA, all machines were interfaced to the FACS-Diva software (BD Bioscience). Intracellular staining was performed using BD Perm/Wash and BD Cytofix/Cytoperm reagents from BD Bioscience according to the manufacturer's instruction.

Cell preparation

Human fetal liver and thymus was obtained from elective abortions, with gestational age ranging from 14 to 20 wk. Post-natal thymus was obtained from children (<3 years of age) undergoing heart surgery at the AMC. Experiments using human material were approved by the Medical and Ethical Committees at the Institut Pasteur and AMC-UvA and performed in full compliance with French law. Single-cell suspensions of fetal material was achieved by mechanical disruption using a Stomacher® Biomaster lab system (Seward, Hadleigh, UK). Magnetic enrichment of CD34+ cells (>98% pure) was performed by using the CD34 Progenitor Cell Isolation Kit (Miltenyi Biotech, Auburn, CA, USA), after preparation of single-cell suspension and isolation of mononuclear cells by density gradient centrifugation over Ficoll-Hypaque (Nycomed Pharma, Roskilde, Denmark). Cell suspensions were prepared in RPMI medium with 2% FCS. Single-cell suspensions of murine organs were prepared as previously described 44.

TCR Vβγδ and CDR3 immunoscope analysis and immunohistology

Nearly, 12 wk after CD34+CD38 HSC engraftment, HIS mice were killed and single-cell suspensions of thymocytes were prepared. Red cells lysis was performed in 1 mL of red cell lysis buffer (Sigma) for 10 min. Thymocytes were washed, resuspended in 600 μL of RLT lysis buffer (Qiagen) and homogenized by passing through a 21-gauge needle several times using Rnase-free syringes. RNA was prepared using RNeasy mini kits (Qiagen) according to the manufacturers's instructions. TCR Vβ immunoscope was performed as previously described 45. Briefly, cDNA was prepared and real-time PCR performed by combining primers for the different Vβγδ chains (Vβ1-24, Vγ2-9 and Vδ1-8). Fluorescent products were separated on ABI-Prism 3730 DNA analyzer to determine CDR3 lengths. Analysis of five individual HIS-mice from each group containing greater than 90% human chimerism in the thymus was performed. Immunohistological analysis was performed as previously described 22, 46.

Statistical analysis

Statistical analyses were performed using the GraphPad Prism (GraphPad Software, San Diego, CA, USA). All data were subjected to two-tailed unpaired Student' t-test analysis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

This work is supported by grants from National Health and Medical Research Council of Australia (N. D. H.), The Menzies Foundation (N. D. H.), The International Human Frontiers Science Program Organization (N. D. H.), Institut Pasteur (J. P. D.), INSERM (J. P. D.), Fondation pour la Recherche Médicale (J. P. D.), InCa (J. P. D.), College de France (Prof. Philippe Kourilsky), Fundação para a Ciência e a Tecnologia (N. L. A.) and a Grand Challenges in Global Health grant from the Bill & Melinda Gates Foundation (J. P. D.). We acknowledge the Bloemenhove Clinic (Heemstede, The Netherlands) for providing fetal tissues. We would like to thank Allison Bordack, Claire Leceste, Stephan Poew, Joran Volmer and Jenny Meerding for technical assistance.

Authorship. N. D. H designed and performed experiments and wrote the paper. N. L, A. L, K. W, H. S.-M., H. S and N. L. A performed experiments and supplied materials and facilities. A. P and Y. J characterized and supplied reagents. J. P. D designed experiments and wrote the paper.

Conflict of interest: The authors declare no financial or commercial conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

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