Recombinant IL-7/HGFß efficiently induces transplantable murine hematopoietic stem cells.

Difficulty obtaining sufficient hematopoietic stem cells (HSCs) directly from the donor has limited the clinical use of HSC transplantation. Numerous attempts to stimulate the ex vivo growth of purified HSCs with cytokines and growth factors generally have induced only modest increases in HSC numbers while decreasing their in vivo reconstituting ability. We previously developed a recombinant single-chain form of a naturally occurring murine hybrid cytokine of IL-7 and the ß chain of hepatocyte growth factor (rIL-7/HGFß) that stimulates the in vitro proliferation and/or differentiation of common lymphoid progenitors, pre-pro-B cells, and hematopoietic progenitor cells (day 12 spleen colony-forming units) in cultures of mouse BM. Here we used the rIL-7/HGFß in culture to induce large numbers of HSCs from multiple cell sources, including unseparated BM cells, purified HSCs, CD45- BM cells, and embryonic stem cells. In each instance, most of the HSCs were in the G0 phase of the cell cycle and exhibited reduced oxidative stress, decreased apoptosis, and increased CXCR4 expression. Furthermore, when injected i.v., these HSCs migrated to BM, self-replicated, provided radioprotection, and established long-term hematopoietic reconstitution. These properties were amplified by injection of rIL-7/HGFß directly into the BM cavity but not by treatment with rIL-7, rHGF, and/or rHGFß.

A human recombinant IL-7/HGFα hybrid cytokine enhances T-cell reconstitution in mice after syngeneic bone marrow transplantation.

BACKGROUND: T-cell regeneration after bone marrow transplantation (BMT) is often slow and incomplete. Therefore, the enhancement of T-cell reconstitution after BMT is required. We have previously described a naturally occurring hybrid cytokine consisting of interleukin (IL)-7 and the ß-chain of hepatocyte growth factor (HGF) that had lymphopoietic stimulatory activity in vitro. We have also reported that a murine recombinant (r) IL-7/HGFß protein significantly enhances T-cell regeneration in mice after BMT. METHODS: To determine whether a human form of rIL-7/HGFß has similar effects as the murine form, we have cloned and expressed the human IL-7 and HGFß genes to produce a single-chain hrIL-7/HGFß protein. We have also cloned and expressed a human form of hybrid cytokine containing IL-7 and the α-chain of HGF (HGFα) (hrIL-7/HGFα). We then determined their ability to enhance T-cell reconstitution in mice after BMT. RESULTS: We found that hrIL-7/HGFα had higher in vitro and in vivo thymocyte-stimulatory activities than did the hrIL-7/HGFß. Therefore, we focused on the functional properties of hrIL-7/HGFα and showed that administration of hrIL-7/HGFα significantly enhanced thymopoiesis in mice after syngeneic BMT by increasing the numbers of thymocytes, early thymocyte progenitors, and thymic epithelial cells. hrIL-7/HGFα cross-linked the IL-7 and HGF receptors on thymocytes, and the in vivo thymocyte-stimulatory activity was mediated by both receptors. Consequently, hrIL-7/HGFα treatment significantly increased the numbers of total and naïve T cells in the periphery. CONCLUSION: In vivo administration of hrIL-7/HGFα efficiently restores thymopoiesis and naïve T-cell reconstitution in mice after syngeneic BMT.

In vivo administration of the recombinant IL-7/hepatocyte growth factor ß hybrid cytokine efficiently restores thymopoiesis and naive T cell generation in lethally irradiated mice after syngeneic bone marrow transplantation.

Bone marrow transplantation (BMT) is often followed by a prolonged period of T cell deficiency. Therefore, the enhancement of T cell reconstitution is an important clinical goal. We have identified a novel hybrid cytokine containing IL-7 and the ß-chain of hepatocyte growth factor (HGF) in the supernatant of cultured mouse BM stromal cells. We have cloned and expressed the IL-7/HGFß gene to produce a single-chain rIL-7/HGFß protein that stimulates the in vitro proliferation of thymocytes, early B-lineage cell, and day 12 spleen CFUs. In this study, we show that, following syngenic BMT, the in vivo administration of rIL-7/HGFß supports the rapid and complete regeneration of the thymus and efficiently reconstitutes the pool of naive T cells having a normally diverse TCR repertoire. The rIL-7/HGFß hybrid cytokine was significantly more effective quantitatively than was rIL-7 and differed qualitatively in its ability to cross-link c-Met and IL-7Rα and to stimulate the expansion of early thymocyte progenitors and thymic epithelial cells. It also supports the maturation and homeostatic expansion of peripheral T cells. Consequently, the in vivo administration of rIL-7/HGFß may offer a new approach to preventing and/or correcting post-BMT T cell immune deficiency.

In vivo antitumor activity of a recombinant IL-7/HGFbeta hybrid cytokine in mice.

The immune cytokine interleukin (IL)-7 and the ß-chain of hepatocyte growth factor (HGF) aggregate to form a naturally occurring heterodimer that stimulates the growth of common lymphoid progenitors and immature B and T lymphoid cells. We have cloned and expressed the heterodimer as a single-chain hybrid cytokine [recombinant (r) IL-7/HGFß], which stimulates short-term hematopoietic stem cells as well as lymphoid precursors. Inasmuch as IL-7 and HGF are known to have antitumor and protumor activities, respectively, we determined here whether either of these activities is exhibited by rIL-7/HGFß. We show that the in vivo administration of rIL-7/HGFß markedly inhibits the growth of newly initiated and established tumors and the formation of pulmonary metastases in murine models of colon cancer and melanoma. The antitumor effect of rIL-7/HGFß correlated with a marked increase in the number of tumor-infiltrating CD4(+) and CD8(+) T cells and activated dendritic cells. A major role for these immune cells in tumor suppression was indicated by the inability of rIL-7/HGFß to inhibit the growth of tumor cells in vitro and in congenitally athymic mice. Analysis of interferon-γ-secreting T cells showed that the immune response was tumor specific. Our findings justify further evaluation of rIL-7/HGFß as a novel experimental cancer therapy.

gp96, an endoplasmic reticulum master chaperone for integrins and Toll-like receptors, selectively regulates early T and B lymphopoiesis.

Integrins contribute to lymphopoiesis, whereas Toll-like receptors (TLRs) facilitate the myeloid replenishment during inflammation. The combined role of TLRs and integrin on hematopoiesis remains unclear. gp96 (grp94, HSP90b1) is an endoplasmic reticulum master chaperone for multiple TLRs. We report herein that gp96 is also essential for expression of 14 hematopoietic system-specific integrins. Genetic deletion of gp96 thus enables us to determine the collective roles of gp96, integrins, and TLRs in hematopoiesis. We found that gp96-null hematopoietic stem cells could support long-term myelopoiesis. B- and T-cell development, however, was severely compromised with transitional block from pro-B to pre-B cells and the inability of thymocytes to develop beyond the CD4(-)CD8(-) stage. These defects were cell-intrinsic and could be recapitulated on bone marrow stromal cell culture. Furthermore, defective lymphopoiesis correlated strongly with failure of hematopoietic progenitors to form close contact with stromal cell niche and was not the result of the defect in the assembly of antigen receptor or interleukin-7 signaling. These findings define gp96 as the only known molecular chaperone to specifically regulate T- and B-cell development.

Thymus-homing peripheral dendritic cells constitute two of the three major subsets of dendritic cells in the steady-state thymus.

Many dendritic cells (DCs) in the normal mouse thymus are generated intrathymically from common T cell/DC progenitors. However, our previous work suggested that at least 50% of thymic DCs originate independently of these progenitors. We now formally demonstrate by parabiotic, adoptive transfer, and developmental studies that two of the three major subsets of thymic DCs originate extrathymically and continually migrate to the thymus, where they occupy a finite number of microenvironmental niches. The thymus-homing DCs consisted of immature plasmacytoid DCs (pDCs) and the signal regulatory protein alpha-positive (Sirpalpha(+)) CD11b(+) CD8alpha(-) subset of conventional DCs (cDCs), both of which could take up and transport circulating antigen to the thymus. The cDCs of intrathymic origin were mostly Sirpalpha(-) CD11b(-) CD8alpha(hi) cells. Upon arrival in the thymus, the migrant pDCs enlarged and up-regulated CD11c, major histocompatibility complex II (MHC II), and CD8alpha, but maintained their plasmacytoid morphology. In contrast, the migrant cDCs proliferated extensively, up-regulated CD11c, MHC II, and CD86, and expressed dendritic processes. The possible functional implications of these findings are discussed.

Cyclical mobilization and gated importation of thymocyte progenitors in the adult mouse: evidence for a thymus-bone marrow feedback loop.

It has recently been observed, as in the fetal thymus, that the importation of hematogenous thymocyte progenitors by the adult thymus is a gated phenomenon, whereby saturating numbers of progenitors periodically enter the thymus and occupy a finite number of intrathymic niches. In addition, the mobilization of thymocyte progenitors from the bone marrow appears to be a cyclical process that coincides temporally with the periods of thymic receptivity (open gate). It is proposed that these events are coordinated by a thymus-bone marrow feedback loop in which a wave of developing triple negative (CD3- CD4- CD8-) thymocytes interacts with stromal cells in the stratified regions of the thymus cortex to sequentially induce the release of diffusible cytokines that regulate the production, mobilization, and recruitment of thymocyte progenitors. The likely components of this feedback loop are described here, as are the properties of the intrathymic vascular gates and niches for thymocyte progenitors. The cyclical production and release of thymocyte progenitors from the bone marrow is placed in the context of a general phenomenon of oscillatory feedback regulation involving all lymphohemopoietic cell lineages. Lastly, the question of whether the gated (as opposed to the continuous) entry of thymocyte progenitors is essential for normal thymocytopoiesis in adult life is discussed.

A recombinant single-chain IL-7/HGFbeta hybrid cytokine induces juxtacrine interactions of the IL-7 and HGF (c-Met) receptors and stimulates the proliferation of CFU-S12, CLPs, and pre-pro-B cells.

A novel recombinant interleukin-7/hepatocyte growth factor beta-chain (IL-7/HGFbeta) hybrid cytokine was constructed as a single chain (sc) composed of IL-7 and HGFbeta connected by a flexible linker. Unlike recombinant (r) IL-7, which stimulated pro-B cells and pre-B cells only, scIL-7/HGFbeta stimulated the proliferation of pre-pro-B cells, common lymphoid progenitors (CLPs), and colony-forming unit (CFU)-S12 in cultures of IL-7-/- mouse BM cells. When injected in vivo, 3- to 4-fold more splenic B-lineage cells appeared in recipients of bone marrow (BM) cells from the scIL-7/HGFbeta-stimulated cultures than from rIL-7-stimulated cultures. Moreover, on a per-cell basis, scIL-7/HGFbeta culture-generated cells produced 16- to 20-fold more BM and splenic B-lineage cells than did normal BM cells. Antibody blocking, receptor phosphorylation, and confocal microscopy demonstrated that scIL-7/HGFbeta signals though both the IL-7 and HGF (c-Met) receptors, which form IL-7R/c-Met complexes on the surface of CLPs and pre-pro-B cells. In addition, the IL-7Ralpha chain, gammac chain, and c-Met were coisolated from purified CLPs and pre-pro-B cells on scIL-7/HGFbeta affinity gels, indicating that they are major components of the IL-7/HGFbeta receptor. Hence, the present results demonstrate that the IL-7/HGFbeta hybrid cytokine efficiently and selectively stimulates the most primitive B-lineage precursors in BM by inducing juxtacrine interactions between the IL-7 and c-Met receptors.

Gated importation of prothymocytes by adult mouse thymus is coordinated with their periodic mobilization from bone marrow.

The wavelike pattern of fetal T cell neogenesis is largely determined by the intermittent generation and exportation of waves of prothymocytes by the hemopoietic tissues in coordination with their gated importation by the thymus. Having previously shown that the importation of prothymocytes by the adult mouse thymus is also gated and that thymocytopoiesis proceeds in discrete (albeit overlapping) waves, we now demonstrate that prothymocytes are periodically exported in saturating numbers from the adult mouse bone marrow. Experiments in normal, radioablated, and parabiotic mice document the cyclical accumulation (3-5 wk) of prothymocytes in both the steady state and regenerating bone marrow, followed by their release into the blood approximately 1 wk before intrathymic gate opening. The results also show that circulating donor-origin thymocyte precursors can transiently ( approximately 1 wk) establish high level chimerism in the bone marrow after the mobilization of endogenous prothymocytes, presumably by occupying vacated microenvironmental niches. Hence, by analogy with the fetal state, we posit the existence of a feedback loop whereby diffusible chemokines of thymic origin regulate the production and/or release of bone marrow prothymocytes during each period of thymic receptivity. Because each resulting wave of thymocytopoiesis is accompanied by a wave of intrathymic dendritic cell formation, these coordinated events may help to optimize thymocyte selection as well as production.

Two developmentally distinct populations of dendritic cells inhabit the adult mouse thymus: demonstration by differential importation of hematogenous precursors under steady state conditions.

Although a variety of lymphoid and myeloid precursors can generate thymic dendritic cells (DCs) under defined experimental conditions, the developmental origin(s) of DCs in the steady state thymus is unknown. Having previously used selective combinations of normal, parabiotic, and radioablated mice to demonstrate that blood-borne prothymocytes are imported in a gated and competitive manner, we used a similar approach in this study to investigate the importation of the hematogenous precursors of thymic DCs. The results indicate that two developmentally distinct populations of DC precursors normally enter the adult mouse thymus. The first population is indistinguishable from prothymocytes according to the following criteria: 1) inefficient (<20%) exchange between parabiotic partners; 2) gated importation by the thymus; 3) competitive antagonism for intrathymic niches; 4) temporally linked generation of thymocytes and CD8alpha(high) DCs; and 5) absence from prothymocyte-poor blood samples. The second population differs diametrically from prothymocytes in each of these properties, and appears to enter the thymus in at least a partially differentiated state. The resulting population of DCs has a CD8alpha(-/low) phenotype, and constitutes approximately 50% of total thymic DCs. The presence of two discrete populations of DCs in the steady state thymus implies functional heterogeneity consistent with evidence implicating lymphoid DCs in the negative selection of effector thymocytes and myeloid DCs in the positive selection of regulatory thymocytes.

A central role for peripheral dendritic cells in the induction of acquired thymic tolerance.

Despite extensive negative selection in the thymus, numerous clones of self-reactive T cells are normally exported to the periphery. In most instances, autoimmunity is prevented by regulatory T (Tr) cells, many of which are also of recent thymic origin. We have demonstrated recently that natural killer (NK) Tr thymocytes (THYr) can be induced by the injection of antigen into the eye, an immunologically privileged site; and that the intravenous infusion of antigen-presenting cells (APCs) from such animals also induces NKT THYr. Furthermore, we have also observed that some of these APCs migrate to the thymus as CD11c(+) dendritic cells (DCs). Other authors have correlated the migration of DCs to the thymus with the generation of CD4(+)CD25(+) THYr. We therefore propose a novel tolerance induction pathway by which tolerogenic DCs routinely transport antigen (both self and nonself) from the periphery to the thymus, where they positively select THYr. We also propose that the ability of tolerogenic DCs to induce acquired thymic tolerance on demand might have important implications for the immunotherapy of autoimmunity and allotransplantation.

Pre-pro-B cell growth-stimulating factor (PPBSF) upregulates IL-7Ralpha chain expression and enables pro-B cells to respond to monomeric IL-7.

Although pro-B cells are well represented in IL-7 knockout (KO) mice, they express abnormally low concentrations of the interleukin-7 receptor alpha-chain (IL-7Ralpha) and do not generate pre-B cells. Here, we demonstrate that pro-B cells from IL-7 KO mice can be induced to generate pre-B cells and immature B cells by exposure to recombinant IL-7 (rIL-7) in vivo but not in vitro. Experiments in recombinant activation gene-1 (RAG-1) KO mice indicate that the in vitro unresponsiveness of IL-7(-/-) pro-B cells to rIL-7 is unrelated to the absence of a functional pre-B cell receptor (pre-BCR). Rather, it appears to be due to the suboptimal expression of the IL-7Ralpha chain. Thus, IL-7(-/-) pro-B cells readily respond to rIL-7 in vitro if IL-7Ralpha chain expression is first upregulated by exposure to IL-7 in vivo or to IL-7(+/+) bone marrow (BM) stromal cells or conditioned medium (CM) therefrom in vitro. Similar results were obtained when pro-B cells from IL-7 KO mice were cultured on IL-7(-/-) BM stromal cells in the presence of rIL-7. This suggested that the recently described pre-pro-B cell growth-stimulating factor (PPBSF), a self-assembling hybrid cytokine comprising IL-7 and the stromal cell-derived hepatocyte growth factor beta-chain (HGFbeta), is required to stimulate pro-B cells from IL-7 KO mice. This inference was verified by demonstrating that purified PPBSF upregulates IL-7Ralpha chain expression on IL-7(-/-) pro-B cells in vitro and enables them to respond to rIL-7 in a stepwise manner. We, therefore, postulate that PPBSF is the operative form of IL-7 that normally induces IL-7Ralpha(lo) pre-pro-B cells to proliferate and differentiate into IL-7Ralpha(hi) pro-B cells, which then proliferate and differentiate into pre-B cells on stimulation with monomeric IL-7.

Functional demonstration of intrathymic binding sites and microvascular gates for prothymocytes in irradiated mice.

Quantitative intrathymic (i.t.) and i.v. adoptive transfer assays for prothymocytes show strict log dose saturation kinetics, consistent with a finite number of i.t. binding sites (microenvironmental niches). This inference is supported here by demonstration of competitive antagonism obeying one-on-one receptor occupancy kinetics during the establishment of thymic chimerism in irradiated adult mice. The results of primary and secondary transfer experiments suggested that hematogenous precursors (i) enter specific i.t. niches between 4 and 24 h after injection, (ii) compete reversibly with subsequently introduced precursors, (iii) establish insurmountable competition within 5-7 days, (iv) mature through the initial stages of thymocytopoiesis preceding proliferative expansion, and (v) vacate the niches between 7 and 14 days after entry. The results also suggested that, as in non-irradiated mice, prothymocyte importation in irradiated mice is a gated phenomenon. Gate closure was indicated by the inability of i.v.-, but not i.t.-, injected bone marrow (BM) cells to induce thymic chimerism when administered 7--14 days after a primary injection and gate opening by the ability of i.v.-injected BM cells to induce thymic chimerism in competition with circulating host prothymocytes. Gate closing was log dose-responsive and could be induced in individual thymic lobes by unilateral i.t. injection, whereas gate opening, which occurs bilaterally, was not initiated until most of the niches for prothymocytes had been vacated. We therefore posit the existence of a series of associated microvascular gates and microenvironmental niches that act in concert to regulate prothymocyte importation and early thymocyte differentiation.