SUMMARY We have used Drosophila ovarian Follicle Stem Cells (FSCs) to study how stem cells are regulated by external signals and draw three main conclusions. First, the spatial definition of supportive niche positions for FSCs depends on gradients of Hh and JAK-STAT pathway ligands, which emanate from opposite, distant sites. FSC position may be further refined by a preference for low-level Wnt signaling. Second, hyperactivity of supportive signaling pathways can compensate for the absence of the otherwise essential adhesion molecule, DE-cadherin, suggesting a close regulatory connection between niche adhesion and niche signals. Third, FSC behavior is determined largely by summing the inputs of multiple signaling pathways of unequal potencies. Altogether our findings indicate that a stem cell niche need not be defined by short-range signals and invariant cell contacts; rather, for FSCs, the intersection of gradients of long-range niche signals regulates the longevity, position, number and competitive behavior of stem cells.

The ubiquitin ligase, Itch, is required to prevent autoinflammatory disease in mice and humans. Itch-deficient mice develop lethal pulmonary inflammation characterized by the production of Th2 cytokines (for example, interleukin-4 (IL-4)); however, the contribution of Itch to immune defense against respiratory pathogens has not been determined. We found that Itch-deficient mice were highly susceptible to intranasal infection with the respiratory pathogen Klebsiella pneumoniae. Infected Itch-deficient mice exhibited increased immune cell infiltration, cytokine levels and bacterial burden in the respiratory tract compared with control mice. However, numbers of resident alveolar macrophages were reduced in the lungs from Itch-deficient mice both before and after infection. High levels of Th2 cytokines in the respiratory tract correlated with deceased alveolar macrophages, and genetic ablation of IL-4 restored alveolar macrophages and host defense to K. pneumoniae in Itch-deficient mice, suggesting that loss of alveolar macrophages occurred as a consequence of Th2 inflammation. Adoptive transfer of Itch CD4 T cells into Rag mice was sufficient to drive reduction in numbers of Itch-replete alveolar macrophages. Finally, we found that Stat6 signaling downstream of the IL-4 receptor directly reduced fitness of alveolar macrophages when these cells were exposed to the Itch inflamed respiratory tract. These data suggest that Th2 inflammation directly impairs alveolar macrophage fitness in Itch mice, and elucidate a previously unappreciated link between Th2 cells, alveolar macrophages and susceptibility to bacterial infection.

Antigen encounter directs CD4+ T cells to differentiate into T helper or regulatory cells. This process focuses the immune response on the invading pathogen and limits tissue damage. Mechanisms that govern T helper cell versus T regulatory cell fate remain poorly understood. Here, we show that the E3 ubiquitin ligase Cul5 determines fate selection in CD4+ T cells by regulating IL-4 receptor signaling. Mice lacking Cul5 in T cells develop Th2 and Th9 inflammation and show pathophysiological features of atopic asthma. Following T cell activation, Cul5 forms a complex with CIS and pJak1. Cul5 deletion reduces ubiquitination and subsequent degradation of pJak1, leading to an increase in pJak1 and pSTAT6 levels and reducing the threshold of IL-4 receptor signaling. As a consequence, Cul5 deficient CD4+ T cells deviate from Treg to Th9 differentiation in low IL-4 conditions. These data support the notion that Cul5 promotes a tolerogenic T cell fate choice and reduces susceptibility to allergic asthma.

To mount an antipathogen response, CD4 T cells must undergo rapid cell proliferation; however, poorly controlled expansion can result in diseases such as autoimmunity. One important regulator of T-cell activity is the E3 ubiquitin ligase Itch. Itch deficient patients suffer from extensive autoinflammation. Similarly, Itch deficient mice exhibit inflammation characterized by high numbers of activated CD4 T cells. While the role of Itch in limiting CD4 T-cell cytokine production has been extensively studied, it is less clear whether and how Itch regulates proliferation of these cells. We determined that Itch deficient CD4 T cells are hyperproliferative in vitro and in vivo, due to increased S phase entry. Whole cell proteomics analysis of Itch deficient primary mouse CD4 T cells revealed increased abundance of the β-catenin coactivator WW domain-binding protein 2 (WBP2). Furthermore, Itch deficient cells demonstrate increased WBP2 protein stability, and Itch and WBP2 interact in CD4 T cells. Knockdown of WBP2 in CD4 T cells caused reduced proliferation. Together, our data support that Itch attenuates CD4 T cell proliferation by promoting WBP2 degradation. This study identifies novel roles for Itch and WBP2 in regulating CD4 T cell proliferation, providing insight into how Itch may prevent inflammation.