Impaired CD8 T cell memory and CD4 T cell primary responses in IL-7R alpha mutant mice.

Loss of interleukin (IL)-7 or the IL-7 receptor alpha (IL-7Ralpha, CD127) results in severe immunodeficiencies in mice and humans. To more precisely identify signals governing IL-7 function in vivo, we have disrupted the IL-7Ralpha Y449XXM motif in mice by knock-in mutagenesis (IL-7Ralpha(449F)). Thymic precursors were reduced in number in IL-7Ralpha(449F) mice, but in marked contrast to IL-7Ralpha(-/-) knockout mice, thymocytes and peripheral T cells developed normally. Strikingly, Listeria infection revealed that CD4 and CD8 T cells had different requirements for IL-7Ralpha signals. CD4 T cells failed to mount a primary response, but despite normal CD8 primary responses, maintenance of CD8 memory was impaired in IL-7Ralpha(449F) mice. Furthermore, we show that Bcl-2 is IL-7Ralpha Y449 independent and insufficient for IL-7-mediated maintenance of CD8 memory.

Haploinsufficiency identifies STAT5 as a modifier of IL-7-induced lymphomas.

The requirement for receptor components and the signalling effector, signal transducer and activator of transcription (STAT) 5A/5B, was assessed genetically in a lymphoma development model induced by interleukin-7 (IL-7). This growth factor for T- and B-cell progenitors and mature lymphocytes activates survival and proliferative pathways including Bcl-2, phosphatidylinositol-3 kinase and STAT5. Overexpression of IL-7 in vivo causes early mortality from lymphoma development. Mice overexpressing IL-7 that were heterozygous for the IL-7Ralpha subunit showed improved survival compared to wild-type mice. In addition, STAT5A/5B+/- compound heterozygous mice with one targeted allele each of STAT5A and STAT5B showed striking amelioration of IL-7-induced mortality and disease development. STAT5A/5B+/- compound heterozygous mice were otherwise normal in stem cell and lymphocyte development and cellularity. Lower STAT5 protein levels accompanied the reduction in STAT5A/5B copy number, which suggests that STAT5 haploinsufficiency is a modifier of IL-7 signal strength.

Bone marrow transplant completely rescues hematolymphoid defects in STAT5A/5B-deficient mice.

OBJECTIVE: STAT5A/5B-deficient mice are recognized to manifest defects in multiple cell types and tissues. In particular, the hematopoietic defects in these mice are widespread, affecting multiple lineages and multiple stages of development. Previous studies indicate that deficiencies intrinsic to hematopoietic cells contribute substantially to the observed defects. However, in light of the broad physiologic effects of STAT5 in the context of the organism outside the blood system, we wished to investigate the possibility of STAT5-dependent environmental influence of nonhematopoietic origin on hematopoietic development in these mice. MATERIALS AND METHODS: We transplanted wild-type bone marrow into STAT5A/5B-deficient mice to determine the effects of loss of STAT5 in nonhematopoietic tissue on hematopoietic development. RESULTS: We observed that transplantation of wild-type marrow completely corrects hematopoietic defects in STAT5A/5B-deficient recipient mice, including peripheral blood counts, bone marrow cellularity, and reductions in specific progenitor subsets. CONCLUSION: These results indicate that the important role of STAT5 in hematolymphoid development are mediated directly through effects on hematopoietic cells and not indirectly.

Transgenic bcl-2 is not sufficient to rescue all hematolymphoid defects in STAT5A/5B-deficient mice.

OBJECTIVE: Cytokines bind high-affinity receptors expressed on hematopoietic cells to initiate signaling cascades that regulate differentiation, proliferation, and survival. Previous studies have established a role for STAT5 in transducing survival signals for hematopoietic progenitor cells in response to cytokines. MATERIALS AND METHODS: To determine if constitutive expression of a member of the bcl-2 family of anti-apoptotic proteins could compensate for the loss of STAT5, we utilized combinatorial genetics to generate STAT5A/5B-deficient mice expressing a bcl-2 transgene. RESULTS: Although bcl-2 expression restored peripheral blood counts to normal in STAT5A/5B(-/-) mice, we noted a striking failure of this transgene to correct defects in hematopoietic stem and progenitor cells. CONCLUSION: These data imply important effects of STAT5 in modulating hematopoietic cells in addition to promoting survival per se.

Loss of tolerance and autoimmunity affecting multiple organs in STAT5A/5B-deficient mice.

STAT5 has previously been reported to be dispensable for the maintenance of tolerance in vivo. However, in examining hemopoiesis in mice lacking both isoforms of STAT5, STAT5A, and STAT5B, we noted that a subset of these mice demonstrated dramatic alterations in several bone marrow progenitor populations concomitant with lymphocytic infiltration of the bone marrow. In addition, cellular infiltration affecting the colon, liver, and kidney was observed in these mice. Survival analysis revealed that STAT5A/5B(-/-) mice exhibited early death. The increased mortality and the pathology affecting multiple organs observed in these mice were abrogated on the recombination-activating gene 1(-/-) background. In light of the similarities between STAT5A/5B-deficient mice and mice unable to signal through the IL-2R, we hypothesized that the tolerizing role of STAT5A/5B was triggered via activation of the IL-2R. In agreement with this, we found that IL-2Rbeta chain-deficient mice exhibited similar hemopoietic abnormalities. Because IL-2 signaling is thought to contribute to tolerance through maintenance of a CD4(+)CD25(+) regulatory T cell population, we examined these cells and observed a numerical reduction in STAT5A/5B(-/-) mice along with a higher rate of apoptosis. These data provide strong evidence for a requirement for STAT5 in the maintenance of tolerance in vivo.

STAT5 promotes multilineage hematolymphoid development in vivo through effects on early hematopoietic progenitor cells.

The transcription factor signal transducers and activators of transcription 5 (STAT5) is activated by numerous cytokines that orchestrate blood cell development. Multilineage peripheral blood cytopenias were observed in adult mice lacking both isoforms of STAT5 (STAT5A and STAT5B) as well as accelerated rates of apoptosis in the bone marrow. Although the hematopoietic stem cell (HSC) population was preserved in a number of these mice, the post-HSC progenitor populations were diminished and a marked reduction in functional progenitors (spleen colony-forming units) was detected. Competitive bone marrow transplantation studies in vivo revealed a profound impairment of repopulation potential of STAT5-null HSCs, leading to complete lack of contribution to the myeloid, erythroid, and lymphoid lineages. These abnormalities were associated with heightened proliferation activity in the HSC fraction, suggesting the action of homeostatic mechanisms to maintain sufficient levels of diverse blood cell types for viability. Thus, STAT5 normally sustains the robust hematopoietic reserve that contributes to host viability through crucial survival effects on early progenitor cells.

Differential induction of cellular detachment by envelope glycoproteins of Marburg and Ebola (Zaire) viruses.

Human infection by Marburg (MBG) or Ebola (EBO) virus is associated with fatal haemorrhagic fevers. While these filoviruses may both incite disease as a result of explosive virus replication, we hypothesized that expression of individual viral gene products, such as the envelope glycoprotein (GP), may directly alter target cells and contribute to pathogenesis. We found that expression of EBO GP in 293T cells caused significant levels of cellular detachment in the absence of cell death or virus replication. This detachment was induced most potently by membrane-bound EBO GP, rather than the shed glycoprotein products (sGP or GP1), and was largely attributable to a domain within the extracellular region of GP2. Furthermore, detachment was blocked by the Ser/Thr kinase inhibitor 2-aminopurine, suggesting the importance of a phosphorylation-dependent signalling cascade in inducing detachment. Since MBG GP did not induce similar cellular detachment, MBG and EBO GP interact with target cells by distinct processes to elicit cellular dysregulation.