The Role of IL-4 Signaling in CD8+ Innate-Like Lymphocyte Development.

Abstract 2161 The innate and adaptive arms of the immune system collaborate to protect the host against invading pathogens and perform immune surveillance against malignant transformation. As key effectors of the adaptive immune system, conventional T cells develop in the thymus and exit to the periphery as naïve cells, requiring antigenic stimulation and subsequent differentiation to gain effector functions, such as cytokine secretion or cytolytic activity. In contrast to conventional T cells, non-conventional T lymphocytes possess characteristics of innate immune cells, such as expression of surface markers associated with activation/memory and acquisition of effector function during thymic development, and thus are termed innate-like lymphocytes (ILLs). Recently, an expanded population of CD8+ ILLs was identified in mice with a mutation in the T cell receptor signaling protein SLP-76 (SLP-76 Y145F mice). These CD8+ ILLs are characterized by expression of activation/memory markers CD44 and CD122, the expression of the T-box transcription factor Eomesodermin (Eomes) and rapid production of IFN-γ after ex vivo stimulation. The development of these CD8+ ILLs occurs in a cell-extrinsic manner and requires IL-4. We demonstrate that IL-4 is sufficient to upregulate Eomes expression in wild-type CD8 single-positive (SP) thymocytes in short-term in vitro culture and potentiate CD8+ ILL development in fetal thymic organ culture. Using phospho-flow cytometry, we find that CD8+ ILLs from SLP-76 Y145F mice have increased STAT6 and Akt activation vs. CD8+ non-ILLs. In CD8SP thymocytes deficient in STAT6, Eomes expression is not upregulated in response to IL-4. In addition, we demonstrate that pharmacologic inhibition of Akt in SLP-76 Y145F fetal thymic organ culture blocks CD8+ ILL development and also prevents IL-4 induced Eomes upregulation in WT CD8SP thymocytes. Importantly, we have identified CD8+ ILLs in human fetal thymocytes and umbilical cord blood and found that IL-4 is sufficient to up-regulate Eomes expression in these cells. Taken together, our data suggest that IL-4 signaling via STAT6 and Akt pathways is required for IL-4 induction of Eomes expression and CD8+ ILL development. Understanding signal transduction pathways required for CD8+ ILL development will provide insight into the development of this unique lymphocyte subset that sits at the interface between innate and adaptive immunity and has been identified in human umbilical cord blood. Disclosures: No relevant conflicts of interest to declare.

R5 Human Immunodeficiency Virus Type 1 Infection of Fetal Thymic Organ Culture Induces Cytokine and CCR5 Expression

ABSTRACT Late-stage CCR5 tropic human immunodeficiency virus type 1 (HIV-1) isolates (R5 HIV-1) can deplete nearly all CD4+ thymocytes from human thymus/liver grafts, despite the fact that fewer than 5% of these cells express CCR5. To resolve this paradox, we studied the replication and cytopathic effects (CPE) of late-stage R5 HIV-1 biological clones from two progressors and two long-term nonprogressors (LTNP) in fetal thymic organ culture (FTOC) with and without added cytokines. We found that R5 HIV-1 clones from progressors but not LTNP were cytopathic in untreated FTOC. Moreover, R5 HIV-1 clones from progressors replicated to higher levels than LTNP-derived R5 HIV-1 clones in this system. In contrast, when FTOC was maintained in the presence of interleukin 2 (IL-2), IL-4, and IL-7, both progressor and LTNP clones exhibited similar replication and CPE, which were equal to or greater than the levels achieved by progressor-derived R5 HIV-1 clones in untreated FTOC. This finding was likely due to IL-2-induced CCR5 expression on CD4+ thymocytes in FTOC. R5 HIV-1 clones showed greater pathogenesis for CCR5+ cells but also showed evidence of CPE on CCR5− cells. Furthermore, infection of FTOC by R5 HIV-1 induced IL-10 and transforming growth factor β (TGF-β) expression. Both IL-10 and TGF-β in turn induced CCR5 expression in FTOC. Induction of CCR5 expression via cytokine induction by R5 HIV-1 infection of CCR5+ thymocytes likely permitted further viral replication in newly CCR5+ thymocytes. CCR5 expression, therefore, is a key determinant of pathogenesis of R5 HIV-1 in FTOC.

VEGF inhibits T-cell development and may contribute to tumor-induced immune suppression

T-cell defects and premature thymic atrophy occur in cancer patients and tumor-bearing animals. We demonstrate that exposure of mice to recombinant vascular endothelial growth factor (VEGF) at concentrations similar to those observed in advanced stage cancer patients reproduces this profound thymic atrophy and is highlighted by a dramatic reduction in CD4+/CD8+ thymocytes. We find that VEGF does not induce thymocyte apoptosis, but instead rapidly decreases the number of the earliest observable progenitors in the thymus. VEGF does not inhibit thymocyte development in fetal thymic organ culture, further suggesting a prethymic effect. We also demonstrate that bone marrow progenitors from animals infused with recombinant VEGF and transferred to irradiated untreated animals recolonize the thymus more efficiently than progenitors from control animals. This suggests that VEGF exposure is associated with an increased population of thymus-committed progenitors in the bone marrow. We hypothesize that pathophysiologically relevant concentrations of VEGF may block the differentiation and/or emigration of these progenitors resulting in the observed thymic atrophy. Removal of VEGF via cessation of infusion or adoptive transfer of progenitors to a congenic host induces a preferential commitment of lymphoid progenitors to the T lineage and results in a restoration of the normal composition and cellularity of the thymus. These data demonstrate that at pathophysiologic concentrations, VEGF interferes with the development of T cells from early hematopoetic progenitor cells and this may contribute to tumor-associated immune deficiencies.