hrIL-7/HGFα Enhances the Number of Thymocytes After BMT
To evaluate whether hrIL-7/HGFα affects thymopoiesis in vivo after BMT, lethally irradiated C57BL/6 mice were injected intravenously (IV) with T-cell–depleted (TCD)-BM from congeneic mice (B6 Ly 5.1) and intraperitoneally with different doses of hrIL-7/HGFα (2.5, 5, 10, 15, 20, and 40 μg/injection) and equimolar doses of hrIL-7, hrHGFα, or control vehicle (phosphate-buffered saline [PBS]) at 2-day intervals between days 1 and 25. The total numbers of donor-origin thymocytes (>87%) were analyzed on day 30. As shown in Figure 2(B), roughly parallel dose-response curves were observed between hrIL-7/HGFα and hrIL-7 when the results were plotted as a function of equivalent MW (∼2.5:1). However, the amplitude of the responses differed, such that, at optimal equimolar doses (15 μg vs. 6 μg), hrIL-7/HGFα was about two times more effective at enhancing thymic cellularity than was hrIL-7. In contrast, hrHGFα alone had no effect. Analysis of thymocyte subsets showed that both hrIL-7/HGFα and hrIL-7 at optimal doses (15 and 6 μg/injection, respectively) increased all thymocyte subsets, including CD4 and CD8 double-negative (DN), double-positive, and CD4 or CD8 single-positive (SP) cells (Fig. 2C). Furthermore, the increase in thymocyte numbers in the hrIL-7/HGFα-treated BMT recipients was maintained for at least 3 months (see Figure 1, SDC 1,http://links.lww.com/TP/A471).
To determine whether both the IL-7 and HGF receptors are involved in the in vivo effects of hrIL-7/HGFα, a cohort of hrIL-7/HGFα-treated BMT recipients were injected with neutralizing anti-IL-7Rα and anti-c-Met antibodies, or isotype control antibodies. As shown in Figure 2(D), both anti-IL-7Rα or anti-c-Met antibodies used alone significantly inhibited the in vivo thymocyte-stimulatory effects of hrIL-7/HGFα, and the combination of anti-IL-7Rα and anti-c-Met antibodies was more effective than either antibody alone. The data suggest that the in vivo effects of hrIL-7/HGFα are mediated through both the IL-7 and the HGF receptors.
hrIL-7/HGFα Increases the Number of ETPs After BMT
It has been reported that only a minor lin− IL-7Rα−c-Kit+CD44+CD25− subset of DN1 thymocytes, termed ETPs, contain canonical thymocyte precursors (12–14). Therefore, we determined whether hrIL-7/ HGFα influences the numbers of ETPs after BMT. As shown in Figure 3(A), the absolute numbers of donor-origin ETPs in the hrIL-7/HGFα-treated mice was increased approximately 3-fold above normal levels and 6- to 10-fold above those in the PBS-, hrIL-7-, and hrHGFα-treated mice.
rIL-7/HGFα Increases the Number of TECs After BMT
T-cell development in the thymus is supported by the thymic microenvironment, of which TECs are the major components (15, 16). When the numbers of total TECs (CD45− EpCAM+MHC II+) were determined on day 30 after BMT, the PBS-, rIL-7-, and rHGFα-treated mice had slightly reduced numbers of TECs when compared with those in non-BMT control mice (Fig. 3B). In contrast, the administration of hrIL-7/HGFα increased the number of TECs by approximately 2-fold above normal, and both cortical (c) TECs (CD45−EpCAM+MHC II+Ly51+) and medullary (m) TECs (CD45−EpCAM+MHC II+Ly51−) were affected (Fig. 3B).
hrIL-7/HGFα Increases the Numbers of Naïve T Cells in the Periphery
Naïve CD4 SP and CD8 SP T cells, newly derived from the thymus, exhibit a CD62LhiCD44lo phenotype (17, 18). As shown in Figure 4, the numbers of total and naïve donor-origin CD4+ and CD8+ T cells were increased significantly above normal levels in the hrIL-7/HGFα-treated BMT mice (Fig. 4). Treatment with hrIL-7 alone or together with hrHGFα also increased total and naïve splenic T cell numbers, but only about half as well as did hrIL-7/HGFα.
hrIL-7/HGFα-Treated BMT Mice Have a Diverse TCR Repertoire
In these experiments, we compared the TCR Vβ usage of donor-derived peripheral T cells in the blood of hrIL-7/HGFα-treated BMT mice with that of normal control mice. As shown in Supplemental Figure 2 (see SDC 1,http://links.lww.com/TP/A471), the TCR Vβ usage was indistinguishable between the two cohorts of mice. This suggested that the precursors of these newly formed T cells had undergone normal positive and negative selection in the regenerating thymus.
hrIL-7/HGFα Induces Receptor Colocalization on Thymocytes That Co-express the IL-7Rα and c-Met Receptors
We previously reported that both the IL-7 and HGF receptors from rIL-7/HGFβ-stimulated thymocytes were co-immunoprecipitated by anti-IL-7Rα antibody (7), suggesting that rIL-7/HGFβ physically cross-links c-Met and IL-7Rα on the surface of thymocytes. The results in Figure 5 show that c-Met and IL-7Rα exist as aggregates that have undergone patching and capping on dual receptor-expressing thymocytes that have been stimulated with hrIL-7/HGFα. However, receptor aggregates do not form after treatment of such cells with hrIL-7 and hrHGFα. These data also suggest that hrIL-7/HGFα cross-links the IL-7 and HGF receptors on dual-expressing thymocytes and may induce functionally unique juxtacrine signaling (6).
We have demonstrated that hrIL-7/HGFα treatment is quantitatively superior to hrIL-7 and hrHGFα in increasing the number of thymocytes and naïve T cells in lethally irradiated mice after syngeneic BMT. In addition, hrIL-7/HGFα treatment differs qualitatively from hrIL-7 and hrHGFα treatment by cross-linking the IL-7 and HGF receptors on the thymocyte subsets that co-express both receptors and by markedly increasing the numbers of TECs and ETPs. Consequently, hrIL-7/HGFα treatment significantly increased the numbers of total and naïve T cells in the spleen of BMT mice. Importantly, these donor-origin T cells (presumably recent thymic emigrants) exhibited a normal pattern of TCR Vβ usage. These results closely resemble those that we observed when BMT mice were treated with the murine form of rIL-7/HGFβ (7).
Our results also demonstrated that the hrIL-7/HGFα hybrid cytokine was more potent than hrIL-7/HGFβ in stimulating thymocyte proliferation in vitro and in vivo. This may be because of the HGFα chain binds and activates c-Met more efficiently than can the HGFβ chain (9, 10). It is also possible that the hrIL-7 component of hrIL-7/HGFα can more avidly bind to the IL-7 receptor than can its counterpart in hrIL-7/HGFβ.
In retrospect, our results are not entirely surprising, as rHGF is known to protect TECs against injury caused by irradiation or graft-versus-host disease (19) and can up-regulate the production of endogenous IL-7 in the regenerating thymus, presumably secondary to its effects on TECs (20). Yet, like Imado et al. (19), we observed that rHGFα alone did not detectably enhance thymopoiesis after syngeneic BMT. These results suggest that, despite the agonistic effects of HGFα in vivo (10, 21, 22), the physical linkage of IL-7 to HGFα in the hrIL-7/HGFα molecule is required to effectively stimulate thymopoiesis after BMT. Indeed, we have shown that hrIL-7/HGFα stimulation induces co-localization of the IL-7 and HGF receptors on co-expressing thymocytes, raising the possibility that such cross-linking may cause juxtacrine receptor interactions, downstream signal cross-talk, and unique functional readouts, as shown for common lymphocyte progenitors and prepro-B cells (see Ref. 6 and our unpublished observations). We have further confirmed the involvement of both the IL-7 and HGF receptors by partial inhibition of in vivo thymocyte-stimulatory activity with either anti-IL-7Rα or anti-c-Met antibody, and the more complete inhibition by a combination of anti-IL-7Rα and anti-c-Met antibodies.
Yet, we and others have shown that although all thymocyte subsets (including ETPs) express c-Met, only the DN and SP thymocyte subsets express the IL-7Rα-chain of the high affinity IL-7R (7, 23, 24). Therefore, it is possible that rIL-7 stimulates only those thymocytes that express the IL-7 receptor, whereas hrIL-7/HGFα stimulates all thymocytes, including those that express c-Met, but not IL-7Rα by cross-linking c-Met with the γc chain or other receptor(s), such as c-kit (25). This may also be true for TECs, although their increased numbers of TECs may be secondary to their interaction with regenerating thymocytes (26, 27). It is also possible that the increased numbers of ETPs seen in the hrIL-7/HGFα-treated thymus is caused by the increased generation or migration of thymocyte progenitors in/from the BM, rather than the direct effects of hrIL-7/HGFα on ETPs themselves.
Thus, although its precise mechanisms of action remain to be determined, our demonstration that hrIL-7/HGFα induces the rapid and complete regeneration of thymocytes and clonally diverse naïve T cells in vivo suggests that it may be useful clinically in correcting post-BMT T-cell deficiency and its complications. This is especially true, because as the hrIL-7/HGFα was generated in a mammalian expression system and seems to be glycosolated. Indeed, if hrIL-7/HGFα exhibits the same ability as mrIL-7/HGFβ to generate hematopoeitic stem cells, to enhance hematopoeitic stem-cell implantation in BM, and to support long-term hematopoietic reconstitution (L. Lai and I. Goldschneider, manuscript in preparation), it might prove to be a useful adjunct for improving the success rate of BMT itself.
MATERIALS AND METHODS
Four- to 8-week-old C57BL/6 (B6) and B6 Ly5.1 mice were purchased from the National Cancer Institute (Frederick, MD). Mice were housed, treated, and handled in accordance with the guidelines set forth by the University of Connecticut Health Center Animal Care Committee.
Construction, Expression, and Purification of Human Single-Chain IL-7/HGFα Hybrid Cytokine
The human IL-7/HGFα gene was constructed by adapting the protocol that we previously used to construct the murine IL-7/HGFβ gene (6). Briefly, human IL-7 cDNA was amplified with primers A and B (see Table, SDC 1,http://links.lww.com/TP/A471) and human cDNA for the NK1 splice variant of HGFα with primer C and D. The polymerase chain reaction (PCR) products of IL-7 and NK1 were combined and subjected to an additional round of PCR with primers A and D. Because primers B and C contained the linker sequence encoding (Gly4Ser)2, the IL-7/HGFα gene was constructed after the overlap extension PCR. The IL-7/HGFα gene was then cloned into a pOptiVEC mammalian expression vector (Invitrogen, Carlsbad, CA) and transfected into Chinese hamster ovary cell-derived DG44 cells (Invitrogen). The hrIL-7/HGFα protein was then purified from the supernatant, as we described previously (6). Briefly, the supernatant was concentrated by a prep/scale–tangential flow filter cartridge with 10 kDa MW cutoff (Millipore, Bedford, MA) and diafiltered into washing buffer. The sample was then applied to serially linked columns of DEAE and CM sepharose (GE Health Care Biosciences, Piscataway, NJ). After washing, the linked columns were separated, and protein was eluted from the CM column in the washing buffer containing NaCl gradient. The purified protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting using antibodies against both human IL-7 and HGF, and in vitro thymocyte-stimulatory activity (6). For controls, we also cloned and expressed human IL-7 (using primers A and E) and HGFα (NK1) (using primers F and G) genes individually and purified hrIL-7 and hrHGFα proteins from the expression system, respectively.
BM was obtained from mouse femurs and tibias and T-cell depletion by immunomagnetic removal of the CD4+ CD8+ Thy-1.2+ cells as described (28). Recipients (B6 mice) received 1000 cGy total body irradiation and were injected IV 2 to 4 hours later with 2×106 TCD-BM cells from congeneic (B6 Ly5.1) mice. Groups of mice were then injected intraperitoneally with hrIL-7/HGFβ, hrIL-7/HGFα, hrIL-7, and hrHGFα or PBS at 2-day intervals between days 1 and 25 after BMT. Some hrHGFα-treated BMT mice were also injected IV with 50 μg anti-c-Met antibody (R&D Systems, Minneapolis, MN) and 450 μg anti-IL-7Rα antibody (A7R34), or isotype control on the day of BMT and once per week thereafter. The A7R34 hybridoma (anti-IL-7Rα) was generated by S. Nishikawa (see Ref. 29, Kumamoto University School of Medicine, Kumamoto, Japan); We obtained the hybridoma from Dr. I. Weissman (Stanford University School of Medicine) and purified the antibody as described (30).
TECs were isolated as per Gray et al. (31). Single-cell suspensions of thymocytes and spleen cells were stained with one or more of the following fluorochrome-conjugated antibodies as described (6): CD4, CD8, CD25, CD44, CD62L, CD117, CD127, BP-1, EpCAM, CD45, I-Ab, CD45.1, and a TCR Vβ antibody panel. ETPs were identified as lin− IL-7Rα− (CD127) c-kit+ (CD117) CD44+ CD25− thymus cells. A cocktail of antibodies against TER-119, B220, CD19, IgM, Gr-1, CD11b, CD11c, NK1.1, TCRβ, CD3e, and CD8α was used to distinguish the lin− cells. The samples were analyzed on a FACSCalibur flow cytometer (Becton and Dickinson, San Jose, CA) with CellQuest acquisition software. Data analysis was done using FlowJo software (Ashland, OR).
Purified CD4− CD8− DN thymocytes were washed, and stimulated with either hrIL-7/HGFα (60 ng/mL) or the combination of hrIL-7 (24 ng/mL) and hrHGFβ (36 ng/mL) for 2 hr in serum-free medium. The cells were prepared by cytospin, fixed by 4% paraformaldehyde, and stained with fluorescein isothiocyanate -labeled rat anti-IL-7Rα antibody and purified goat anti-c-Met antibody developed with Alexa fluor-594-labeled anti-goat IgG as we described previously (6). The cells were then observed under a Zeiss LSM510 Meta laser scanning confocal microscope (Carl Zeiss, Thornwood, NY).
Statistical Analysis of Data
The values of P were based on two-sided Student's t test. A confidence level above 95% (P<0.05) was determined as significant.
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Bone marrow transplantation; T-cell regeneration; Thymus; Cytokine
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