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 kera-tinocyte 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 mu-rine 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. (Blood. 2002;99:4592-4600)

Interleukin-7 (IL-7) is the major thymopoietic cytokine. Injections of IL-7 after murine bone marrow transplantation (BMT) correct defects in thymic differentiation, including thymic hypocellularity, abnormal differentiation of CD3 ؊ CD4 ؊ CD8 ؊ (triple-negative [TN]) thymocytes into CD4 ؉ CD8 ؉ (double-positive [DP]) cells, and antigen-specific mature T-lymphocyte proliferation. To determine whether IL-7 production is decreased in BMT recipients, BMT was performed with congenic murine donor-recipient strains and escalating doses of pre-BMT conditioning. Increasing doses of radiation resulted in decreased thymic cellularity and maturation from the TN to the DP stage. Quantitative reverse transcription-polymerase chain reaction analyses demonstrated that intrathymic production of IL-7 was significantly decreased in irradiated mice than in nonirradiated controls. Decline in IL-7 transcript levels was correlated with the dose of radiation administered. Analyses of the numbers of CD45 ؊ major histocompatibility complex class II ؉ thymic stromal cells suggested that the mechanism for the decreased IL-7 production was loss of IL-7-producing thymic stromal cells. Experiments indicated that pre-BMT conditioning with radiation led to decreased stromal production of IL-7 and consequent blocks in the maturation of thymocytes. They provided a mechanism for both the abnormal thymopoiesis observed after BMT and the previously observed beneficial effects of IL-7 administration in murine models. Impaired production of IL-7 by thymic stroma may be a general model for the clinically observed adverse effects of cytotoxic therapy on thymopoiesis. IntroductionThe immune deficiency observed after bone marrow transplantation (BMT) is a major cause of morbidity and mortality in patients who undergo transplantation and results in prolonged susceptibility to infection. 1,2 Some of the immunologic defects observed after BMT have included abnormalities of thymopoiesis, activation of T lymphocytes, and antibody production. [3][4][5][6] The thymus has been demonstrated to be a target of graft-versus-host disease (GVHD), and GVHD is associated with decreased thymopoietic capacity after BMT, resulting in decreased thymic output. 7,8 However, abnormal numbers of circulating T lymphocytes have been observed in patients without GVHD, suggesting that other mechanisms besides GVHD suppress the production of new T lymphocytes. 5 Thymopoietic defects may be due to the effects of radiation or chemotherapy on the thymic microenvironment. In addition, these effects may be age related. Analyses of patients undergoing either high-dose chemotherapy or BMT have shown an age-related decline in the production of new T lymphocytes. 9-11 Abnormal numbers of T lymphocytes are especially evident in adult recipients of T-cell-depleted, matched, unrelated donor transplants, suggesting that the combined effects of age, alloreactivity, and high-dose cytotoxic therapy result in clinically significant defects in thymopoiesis. 11 We have been studying the thymopoietic ...

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-FKHRexpressing 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. [Cancer Res 2008;68(16):6587-97]

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, prior to re-aggregation 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, 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.

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.

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 ␣ (IL- 7R␣ IntroductionGraft-versus-host disease (GVHD) continues to be a limiting factor in the use of clinical hematopoietic stem-cell transplantation (HSCT). GVHD occurs when donor T cells recognize host antigenic disparities expressed on antigen-presenting cells (APCs), resulting in activation of alloreactive T cells and destruction of host tissues. Patients with GVHD develop a wide range of symptoms, including skin rash, diarrhea, liver disease, erythema, and weight loss, which eventually result in death. [1][2][3][4][5][6][7] Immunosuppressive drug treatment or mature T-cell-depleted bone marrow transplantation (TCD BMT) have been used as effective strategies to prevent GVHD. 8,9 However, these strategies can also lead to engraftment failure, a prolonged state of immunodeficiency, and various types of opportunistic infections. Therefore, developing a therapeutic strategy to suppress GVHD without compromising the immune system will be ideal for allogeneic BMT recipients.IL-7 and Kit ligand (KL; stem-cell factor [SCF]) are the major lymphopoietic cytokines produced in the thymus and BM compartment. 10-13 IL-7 induces proliferation, differentiation, and survival of immature T lymphocytes. During normal T-cell development in the thymus, IL-7 produced by thymic epithelial cells (TECs) binds to the cognate IL-7 receptor (IL-7R). The IL-7R is composed of IL-7R␣ and common ␥ subunits and expressed on the surface of immature T-lymphoid progenitor cells. Mutations of the IL-7, IL-7R␣, and ␥ c genes result in defective thymopoiesis and impaired ability to produce T lymphocytes. 14-18 Previously we and others have shown that administration of recombinant human IL-7 following histocompatible BMT in murine recipients corrects thymopoietic defects and enhances immune reconstitution, further suggesting the importance of IL-7 in the development of T lymphocytes. 19 Besides its thymopoietic effects, IL-7 also promotes expansion and survival of mature naive and memory CD4 ϩ and CD8 ϩ T cells. Recent studies have shown that IL-7-IL-7R interactions in concert with low-affinity interactions between T-cell receptors (TCRs) and self-peptide ligands bound to major histocompatibility complex (MHC) allow proliferation of mature T cells in the periphery. [20][21][22][23][24][25][26] In addition, IL-7 enhances the survival of alloreactive donor T cells in allogeneic BMT recipients and plays a crucial role in the development and exacerbation of GVHD. [27][28][29][30][31] Based on the effects of IL-7 on mature T cells, we investigated whether GVHD could be prevented by a blockade of IL-7R␣ with an anti-IL-7R␣ monoclonal antibody. Similar to earlier experimental results ...

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.

Summary 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 (pMHC) 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.