Engineering the human thymic microenvironment to support thymopoiesis in vivo.

A system that allows manipulation of the human thymic microenvironment is needed both to elucidate the extrinsic mechanisms that control human thymopoiesis and to develop potential cell therapies for thymic insufficiency. In this report, we developed an implantable thymic microenvironment composed of two human thymic stroma populations critical for thymopoiesis; thymic epithelial cells (TECs) and thymic mesenchyme (TM). TECs and TM from postnatal human thymi were cultured in specific conditions, allowing cell expansion and manipulation of gene expression, before reaggregation into a functional thymic unit. Human CD34+ hematopoietic stem and progenitor cells (HSPC) differentiated into T cells in the aggregates in vitro and in vivo following inguinal implantation of aggregates in immune deficient mice. Cord blood HSPC previously engrafted into murine bone marrow (BM), migrated to implants, and differentiated into human T cells with a broad T cell receptor repertoire. Furthermore, lentiviral-mediated expression of vascular endothelial growth factor in TM enhanced implant size and function and significantly increased thymocyte production. These results demonstrate an in vivo system for the generation of T cells from human HSPC and represent the first model to allow manipulation of gene expression and cell composition in the microenvironment of the human thymus.

Combined effects of interleukin-7 and stem cell factor administration on lymphopoiesis after murine bone marrow transplantation.

The decreased ability of the thymus to generate T cells after bone marrow transplantation (BMT) is a clinically significant problem. Interleukin (IL)-7 and stem cell factor (SCF) induce proliferation, differentiation, and survival of thymocytes. Although previous studies have shown that administration of recombinant human IL-7 (rhIL-7) after murine and human BMT improves thymopoiesis and immune function, whether administration of SCF exerts similar effects is unclear. To evaluate independent or combinatorial effects of IL-7 and SCF in post-BMT thymopoiesis, bone marrow (BM)-derived mesenchymal stem cells transduced ex vivo with the rhIL-7 or murine SCF (mSCF) genes were cotransplanted with T cell-depleted BM cells into lethally irradiated mice. Although rhIL-7 and mSCF each improved immune reconstitution, the combination treatment had a significantly greater effect than either cytokine alone. Moreover, the combination treatment significantly increased donor-derived common lymphoid progenitors (CLPs) in BM, suggesting that transplanted CLPs expand more rapidly in response to IL-7 and SCF and may promote immune reconstitution. Our findings demonstrate that IL-7 and SCF might be therapeutically useful for enhancing de novo T cell development. Furthermore, combination therapy may allow the administration of lower doses of IL-7, thereby decreasing the likelihood of IL-7-mediated expansion of mature T cells.

Importance of interleukin-7 in the development of experimental graft-versus-host disease.

Interleukin (IL)-7 promotes both thymopoiesis and mature T lymphocyte survival and proliferation in experimental murine models of hematopoietic stem cell (HSC) transplantation. Because HSC products for transplantation also may contain IL-7-responsive mature T lymphocytes, we examined whether IL-7 is necessary for the induction of GVHD after allogeneic bone marrow transplantation (BMT). Lethally irradiated C57BL6J (B6) and B6.IL-7(-/-) (both H2K(b)) recipient mice were co-transplanted with T cell-depleted (TCD) bone marrow cells and lymph nodes (LNs) from either congenic B6.SJL (CD45.1(+)) or allogeneic BALB/c (H2K(d)) donor mice. After transplantation, the recipient mice were subcutaneously injected with either human recombinant IL-7 or phosphate-buffered saline (PBS) for 60 days. No evidence of GVHD was detected in the congenic recipients or in the allogeneic B6/IL-7(-/-) recipients treated with PBS; in contrast, significantly increased rates of GVHD-related mortality and morbidity were found in the allogeneic B6.IL-7(-/-) recipients treated with IL-7. The proliferation and number of donor T cells were significantly lower at day 30 post-BMT in the PBS-treated B6.IL-7(-/-) recipients compared with the IL-7-treated B6.IL-7(-/-) mice. These experiments demonstrate that IL-7 is an important factor in the development of GVHD, presumably by supporting the survival, proliferation, and possibly activation of alloreactive donor-derived T cells in the recipients.

Human brain metastatic stroma attracts breast cancer cells via chemokines CXCL16 and CXCL12.

The tumor microenvironment is composed of heterogeneous populations of cells, including cancer, immune, and stromal cells. Progression of tumor growth and initiation of metastasis is critically dependent on the reciprocal interactions between cancer cells and stroma. Through RNA-Seq and protein analyses, we found that cancer-associated fibroblasts derived from human breast cancer brain metastasis express significantly higher levels of chemokines CXCL12 and CXCL16 than fibroblasts from primary breast tumors or normal breast. To further understand the interplay between cancer cells and cancer-associated fibroblasts from each site, we developed three-dimensional organoids composed of patient-derived primary or brain metastasis cancer cells with matching cancer-associated fibroblasts. Three-dimensional CAF aggregates generated from brain metastasis promote migration of cancer cells more effectively than cancer-associated fibroblast aggregates derived from primary tumor or normal breast stromal cells. Treatment with a CXCR4 antagonist and/or CXCL16 neutralizing antibody, alone or in combination, significantly inhibited migration of cancer cells to brain metastatic cancer-associated fibroblast aggregates. These results demonstrate that human brain metastasis cancer-associated fibroblasts potently attract breast cancer cells via chemokines CXCL12 and CXCL16, and blocking CXCR6-CXCL16/CXCR4-CXCL12 receptor-ligand interactions may be an effective therapy for preventing breast cancer brain metastasis.

Antigen-specific inhibition of high-avidity T cell target lysis by low-avidity T cells via trogocytosis.

Current vaccine conditions predominantly elicit low-avidity cytotoxic T lymphocytes (CTLs), which are non-tumor-cytolytic but indistinguishable by tetramer staining or enzyme-linked immunospot from high-avidity CTLs. Using CTL clones of high or low avidity for melanoma antigens, we show that low-avidity CTLs can inhibit tumor lysis by high-avidity CTLs in an antigen-specific manner. This phenomenon operates in vivo: high-avidity CTLs control tumor growth in animals but not in combination with low-avidity CTLs specific for the same antigen. The mechanism involves stripping of specific peptide-major histocompatibility complexes (pMHCs) via trogocytosis by low-avidity melanoma-specific CTLs without degranulation, leading to insufficient levels of specific pMHC on target cell surface to trigger lysis by high-avidity CTLs. As such, peptide repertoire on the cell surface is dynamic and continually shaped by interactions with T cells. These results describe immune regulation by low-avidity T cells and have implications for vaccine design.

The role of cardiac electrophysiology in myocardial regenerative stem cell therapy.

Recent advances in stem cell biology and tissue engineering have put forth new therapeutic paradigms for treatment of myocardial disease. The aim of stem cell therapy for myocardial regeneration has been directed to induce angiogenesis for ischemic heart disease and/or introduction of new cardiomyocytes to improve the mechanical function of the failing heart. Encouraged by positive preliminary results in mouse models of myocardial infarction, clinical trials have utilized autologous skeletal myoblasts and bone-marrow-derived stem cells to treat patients in various clinical settings including acute myocardial injury, chronic angina, and heart failure. These studies have collectively shown, at best, modest improvement in cardiac function. This may be due to the fact that there is little evidence to support actual formation and/or integration of transplanted cells into the recipient myocardium. More recent and emerging data supports the finding that electrical stimulation may be an effective catalyst for sustained functional organization, integration, and maturation of transplanted cell populations into the host myocardium. A therapeutic model that utilizes electrical stimulation and/or achieves cardiac resynchronization in conjunction with stem cell transplantation may be an effective means to achieve successful myocardial regenerative therapy.

Mouse mesenchymal stem cells expressing PAX-FKHR form alveolar rhabdomyosarcomas by cooperating with secondary mutations.

Alveolar rhabdomyosarcomas (ARMS) are highly malignant soft-tissue sarcomas that arise in children, adolescents, and young adults. Although formation and expression of the PAX-FKHR fusion genes is thought to be the initiating event in this cancer, the role of PAX-FKHR in the neoplastic process remains largely unknown in a progenitor cell that is undefined. We hypothesize that PAX-FKHR determine the ARMS progenitor to the skeletal muscle lineage, which when coupled to the inactivation and/or activation of critical cell signaling pathways leads to the formation of ARMS. Because a number of studies have proposed that mesenchymal stem cells (MSC) are the progenitor for several of the sarcomas, we tested this hypothesis in MSCs. We show that PAX-FKHR induce skeletal myogenesis in MSCs by transactivating MyoD and myogenin. Despite exhibiting enhanced growth in vitro, the PAX-FKHR-expressing populations do not form colonies in soft agar or tumors in mice. Expression of dominant-negative p53, or the SV40 early region, elicits tumor formation in some of the PAX-FKHR-expressing populations. Additional activation of the Ras signaling pathway leads to highly malignant tumor formation for all of the populations. The PAX-FKHR-expressing tumors were shown to have histologic, immunohistochemical, and gene expression profiles similar to human ARMS. Our results show the critical role played by PAX-FKHR in determining the molecular, myogenic, and histologic phenotype of ARMS. More importantly, we identify MSCs as a progenitor that can give rise to ARMS.

Prevention of graft-versus-host disease by anti IL-7Ralpha antibody.

Graft-versus-host disease (GVHD) continues to be a serious complication that limits the success of allogeneic bone marrow transplantation (BMT). Using IL-7-deficient murine models, we have previously shown that IL-7 is necessary for the pathogenesis of GVHD. In the present study, we determined whether GVHD could be prevented by antibody-mediated blockade of IL-7 receptor alpha (IL-7Ralpha) signaling. C57/BL6 (H2K(b)) recipient mice were lethally irradiated and underwent cotransplantation with T-cell-depleted (TCD) BM and lymph node (LN) cells from allogeneic BALB/c (H2K(d)) donor mice. Following transplantation, the allogeneic BMT recipients were injected weekly with either anti-IL-7Ralpha antibody (100 mug per mouse per week) or PBS for 4 weeks. Anti-IL-7Ralpha antibody treatment significantly decreased GVHD-related morbidity and mortality compared with placebo (30% to 80%). IL-7Ralpha blockade resulted in the reduction of donor CD4(+) or CD8(+) T cells in the periphery by day 30 after transplantation. Paradoxically, the inhibition of GVHD by anti-IL-7Ralpha antibody treatment resulted in improved long-term thymic and immune function. Blockade of IL-7R by anti-IL-7Ralpha antibody resulted in elimination of alloreactive T cells, prevention of GVHD, and improvement of donor T-cell reconstitution.

Protection from thymic epithelial cell injury by keratinocyte growth factor: a new approach to improve thymic and peripheral T-cell reconstitution after bone marrow transplantation.

Decreased thymopoietic capacity contributes to the severe and clinically significant immune deficiency seen after bone marrow transplantation (BMT). One mechanism for thymopoietic failure is damage to the interleukin 7 (IL-7)-producing thymic epithelial cells (TECs) by irradiation and chemotherapy, which can be partially treated by IL-7 administration. Pretreatment of BMT recipients with keratinocyte growth factor (KGF, or Fgf7), an epithelial cell-specific growth factor, protects mucosal, cutaneous, and pulmonary epithelial cells from cytotoxic therapy-induced damage in experimental murine models. Like other epithelial cells, TECs specifically express KGF receptors. Because KGF specifically protects KGF receptor-bearing epithelial cells and post-BMT immune deficiency is caused by loss of TECs, we hypothesized that KGF pretreatment would improve post-BMT thymic function. To test the hypothesis, BMT recipient mice were given KGF or placebo prior to congenic or allogeneic BMT. Administration of KGF before murine BMT significantly increased the capacity of the thymus to generate donor-derived thymocytes. KGF pretreatment also normalized the proportion of thymic subpopulations, increased the number of naive T cells in the periphery, and improved the response to neoantigen immunization. KGF treatment caused increased production of intrathymic IL-7, and the thymopoietic effects of KGF required an intact IL-7 signaling pathway. These results demonstrate that KGF may have immunomodulatory effects by a unique mechanism of protection of TECs. Furthermore, thymic injury and prolonged posttransplantation immune deficiency in BMT recipients can be prevented by KGF administration.