Insights into thymic involution in tumor-bearing mice

The thymus is a central lymphoid organ critical for the development and maintenance of an effective peripheral T-cell repertoire. Most important, it provides a specialized environment for the selection of rearranged clones that will function appropriately in the adaptive immune response. Thymic involution has been observed in several model systems; including graft-versus-host disease, aging, viral infection, and tumor development, however, the precise mechanisms involved in this phenomenon remain poorly defined. Here, we review some of our results related to the studies of the cell-mediated immunity in a mammary tumor model; more specifically, those related to the tumor-induced impaired T-cell development and thymic involution. Collectively, the understanding of the mechanisms and pathways associated with the tumor-induced thymic involution is essential for the development of innovative and safe therapies to fight against the immune suppression caused by the tumor development

Impaired thymopoiesis occurring during the thymic involution of tumor-bearing mice is associated with a down-regulation of the antiapoptotic proteins Bcl-XL and A1

The thymus is a central lymphoid organ in which T lymphocytes undergo differentiation and maturation without the need for antigenic stimulation. Apoptosis (programmed cell death), plays a critical role in shaping the T cell repertoire, deleting unproductive as well as potentially autoreactive T cells. Thymic atrophy has been observed in several model systems, including aging, graft-vs-host-disease and tumor development. However, the mechanisms involved in this phenomenon remain to be completely elucidated. We have previously shown that the progressive growth of D1-DMBA-3 mammary tumor leads to extreme thymic atrophy in the host. This thymic involution is associated with an early block in T cell maturation at the triple negative stage of differentiation. In the present study we have used our murine mammary tumor model to further analyze the specific T cell subpopulations present in the thymus of tumor-bearing animals as well as to characterize the alterations of the apoptotic process present during the impaired thymopoiesis associated to this thymic involution. Flow cytometric analysis revealed a moderate increase in the percentages of single positive CD4+ and CD8+ cells within the CD3 negative population in the thymuses of tumor-bearing mice. Moreover, we observed a prolonged increase in apoptosis among thymocytes from tumor-bearing mice compared with thymocytes from normal mice during tumor development. Lastly, we found a major decrease of Bcl-XL and A1, two crucial anti-apoptotic Bcl-2 family members that are developmentally regulated in T cells. Together, our data suggest that the severe thymic involution seen in mammary tumor bearers is due to an arrest in at least two steps of T cell differentiation and a down-regulation of important molecules that control programmed cell death

uPA and uPA-receptor are involved in cancer-associated myeloid-derived suppressor cell accumulation

Myeloid-derived suppressor cells (MDSC) have been shown to play a critical role in tumor-induced immunosuppression, in many mouse and human cancers. The aim of this study was to show that MDSC accumulation is tumor burden-dependent, and to investigate the role of the tumor-derived urokinase plasminogen activator (uPA) and its receptor (uPAR) on MDSC recruitment. Levels of MDSC were assessed in tumor-bearers, and the ability to recruit MDSC by uPA was investigated in normal, tumor-bearers, uPAR(-/-), and CD11b(-/-) mice. uPAR expression in MDSC was also explored. MDSC accumulate to dramatic levels in tumor-bearers, and tumor-derived factors such as uPA also increase to great levels in circulation. MDSC can be recruited by uPA, and uPAR but not CD11b are required for such recruitment. MDSC accumulation is tumor burden-dependent, and tumor-derived factors such as uPA and its receptor uPAR play a role in their recruitment

Modeling the CD8+ T effector to memory transition in adoptive T-cell antitumor immunotherapy

Adoptive T-cell therapy with CD8(+) CTLs is often characterized by poor persistence of the transferred T cells and limited effector responses. Improved persistence and therapeutic efficacy have been noted when antigen-activated CD8(+) T cells express properties of memory cells. The current study was undertaken to more precisely characterize the development of memory-like CD8(+) T cells from short-term CTLs in vitro and upon transfer in vivo, including their antitumor activity. Ovalbumin (OVA)-specific OT-I CTLs acquired phenotypic and functional properties of memory cells 2 to 3 days later either by lowering the concentration of antigen to a level that does not support primary responses and providing a survival signal through transgenic Bcl-2 in vitro or simply by transferring early day 3 CTLs to antigen-free lymphoid-replete mice. In lymphoid-replete mice, established OVA-expressing E.G7 tumor was rejected by short-term CTLs that simultaneously acquired memory-like properties in secondary lymphoid tissues, where tumor antigen level remained low. Collectively, these data indicate that CTLs readily converted to memory-like cells upon lowering antigen to a concentration that selectively supports memory responses and suggest that such conversion predicts successful adoptive immunotherapy

Initial Antigen Encounter Programs CD8 +

Although much is known concerning the immunobiology of CD8+ T memory cells, the initial events favoring the generation of CD8+ T memory cells remain poorly defined. Using a culture system that yields memory-like CD8+ T cells, we show that 1 day after Ag encounter, Ag-activated T cells developed into memory-like T cells, but this optimally occurred 3 days after Ag encounter. Key phenotypic, functional, and molecular properties that typify central memory T cells were expressed within 48 h when the activated CD8+ T cells were cultured with IL-7 or IL-15 in the absence of Ag or following transfer into normal mice. These data support a model whereby Ag activation of naive CD8+ T cells not only programs effector cell expansion and contraction but the potential to develop into a memory cell which ensues in an Ag-free environment containing IL-7 or IL-15

Downregulation of interleukin-7 and hepatocyte growth factor in the thymic microenvironment is associated with thymus involution in tumor-bearing mice

During mammary tumorigenesis, there is a profound thymic involution associated with severe depletion of the most abundant subset of thymocytes, CD4(+)CD8(+) immature cells, and an early arrest in at least two steps of T cell differentiation. Thymic atrophy that is normally related with aging has been observed in other model systems, including graft-vs-host disease (GVHD) and tumor development. However, the mechanisms involved in this phenomenon remain to be elucidated. Vascular endothelial growth factor (VEGF) has been associated with thymic involution, when expressed at high levels systemically. In thymuses of D1-DMBA-3 tumor-bearing mice, this growth factor is diminished relative to the level of normal thymuses. Interestingly, the expression of hepatocyte growth factor (HGF), which has been associated with proliferation, cell survival, angiogenesis and B-cell differentiation, is profoundly down-regulated in thymuses of tumor bearers. In parallel, IL-7 and IL-15 mRNA, crucial cytokines involved in thymocytes development and cellular homeostasis, respectively, are also down-regulated in the thymuses of tumor hosts as compared to those of normal mice. Injection of HGF into mice implanted with mammary tumors resulted in normalization of thymic volume and levels of VEGF, IL-7 and IL-15. While, injections of IL-7 partially restored the thymic involution observed in the thymuses of tumor-bearing mice, injection of IL-15 did not have any significant effects. Our data suggest that the downregulation of HGF and IL-7 may play an important role in the thymic involution observed in tumor-bearing hosts

Adipocyte and leptin accumulation in tumor-induced thymic involution

Cell-mediated immunity is an important defense mechanism against pathogens and developing tumor cells. The thymus is the main lymphoid organ involved in the formation of the cell-mediated immune response by the maturation and differentiation of lymphocytes that travel from the bone marrow, through the lymphatic ducts, to become T lymphocytes. Thymic involution has been associated with aging; however, other factors such as obesity, viral infection and tumor development have been shown to increase the rate of shrinkage of this organ. The heavy infiltration of adipocyte fat cells has been reported in the involuted thymuses of aged mice. In the present study, the possible accumulation of such cells in the thymus during tumorigenesis was examined by immunohistochemistry. A significant number of adipocytes around and infiltrating the thymuses of tumor-bearing mice was observed. Leptin is a pro-inflammatory adipocytokine that enhances thymopoiesis and modulates T cell immune responses. The levels of leptin and adiponectin, another adipocytokine that has anti-inflammatory properties, were examined by western blot analysis. While no changes were observed in the amounts of adiponectin present in the thymuses of the normal and tumor-bearing mice, significantly higher levels of leptin were detected in the thymocytes of the tumor-bearing mice. This correlated with an increase in the expression of certain cytokines, such as interleukin (IL)-2, interferon (IFN)-γ and granulocyte-macrophage colony-stimulating factor (GM-CSF). The co-culture of thymocytes isolated from normal mice with ex vivo isolated adipocytes from tumor-bearing mice yielded similar results. Our findings suggest that the infiltration and accumulation of adipocytes in the thymuses of tumor-bearing mice play an important role in their altered morphology and functions

Distinct Activation Signals Determine whether IL-21 Induces B Cell Costimulation, Growth Arrest, or Bim-Dependent Apoptosis

IL-21 costimulates B cell proliferation and cooperatively with IL-4 promotes T cell-dependent Ab responses. Somewhat paradoxically, IL-21 also induces apoptosis of B cells. The present study was undertaken to more precisely define the expression of the IL-21R, using a novel mAb, and the circumstances by which IL-21 promotes B cell growth vs death. The IL-21R was first detected during T and B cell development, such that this receptor is expressed by all mature lymphocytes. The IL-21R was further up-regulated after B and T activation, with the highest expression by activated B cells. Functional studies demonstrated that IL-21 substantially inhibited proliferation and induced Bim-dependent apoptosis for LPS or CpG DNA-activated B cells. In contrast, IL-21 induced both costimulation and apoptosis for anti-CD40-stimulated B cells, whereas IL-21 primarily costimulated B cells activated by anti-IgM or anti-IgM plus anti-CD40. Upon blocking apoptosis using C57BL/6 Bim-deficient or Bcl-2 transgenic B cells, IL-21 readily costimulated responses to anti-CD40 while proliferation to LPS was still inhibited. Engagement of CD40 or the BCR plus CD40 prevented the inhibitory effect by IL-21 for LPS-activated B cells. Collectively, these data indicate that there are three separable outcomes for IL-21-stimulated B cells: apoptosis, growth arrest, or costimulation. We favor a model in which IL-21 promotes B cell maturation during a productive T cell-dependent B cell response, while favoring growth arrest and apoptosis for nonspecifically or inappropriately activated B cells

Abstract 792: Tumor-induced thymic atrophy: Alteration in interferons and Jak/Stats signaling pathways

Abstract The thymus is the major site of T cell differentiation and a key organ of the immune system. Most important, it provides a specialized environment for the selection of rearranged clones that will function appropriately in the adaptive immune response. Thymic involution has been observed in several model systems; including graft-vs-host disease, aging, and tumor development, however, the mechanisms involved in this phenomenon remain to be elucidated. Previous results from our laboratory have reported that the severe thymic atrophy and impaired T cell development seen in mammary tumor bearers are associated with an arrest in at least two steps of T cell differentiation, changes in the levels of crucial cytokines expressed in the thymus microenvironment, and a progressive increase in apoptosis during the tumor development mainly due to downregulation of important molecules that control programmed cell death. Cytokines regulate numerous aspects of hematopoiesis via activation of the Jak/Stat pathways. In the present study we have used our mammary tumor model to investigate whether changes in the levels of cytokines in the thymus could affect the normal expression of the aforementioned pathways. RNA and protein analysis revealed an overexpression of the different interferons, a downregulation of most of the Jak/Stat pathways, and an increased expression of several suppressors of cytokine signaling (SOCS) in the thymuses of tumor bearers. Collectively, our data suggest a mechanism by which the impaired Jak/Stat signaling pathways observed in the whole thymus of tumor-bearing mice could be contributing to the abnormal T cell development and apoptosis observed during the tumor-induced thymic atrophy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 792. doi:10.1158/1538-7445.AM2011-792</jats:p