Stem cells are capable of both self-renewal (proliferation) and differentiation. Determining the regulatory mechanisms controlling the balance between stem cell proliferation and differentiation is not only an important biological question, but also holds the key for using stem cells as therapeutic agents. The Caenorhabditis elegans germ line has emerged as a valuable model to study the molecular mechanisms controlling stem cell behavior. In this study, we describe a large-scale RNAi screen that identified kin-10, which encodes the β subunit of protein kinase CK2, as a novel factor regulating stem cell proliferation in the C. elegans germ line. While a loss of kin-10 in an otherwise wild-type background results in a decrease in the number of proliferative cells, loss of kin-10 in sensitized genetic backgrounds results in a germline tumor. Therefore, kin-10 is not only necessary for robust proliferation, it also inhibits the proliferative fate. We found that kin-10's regulatory role in inhibiting the proliferative fate is carried out through the CK2 holoenzyme, rather than through a holoenzyme-independent function, and that it functions downstream of GLP-1/Notch signaling. We propose that a loss of kin-10 leads to a defect in CK2 phosphorylation of its downstream targets, resulting in abnormal activity of target protein(s) that are involved in the proliferative fate vs. differentiation decision. This eventually causes a shift towards the proliferative fate in the stem cell fate decision.

y Shared senior authorship.Monitoring of cytomegalovirus cell-mediated immunity is a promising tool for the refinement of preventative and therapeutic strategies posttransplantation. Typically, the interferon-g response to T cell stimulation is measured. We evaluated a broad range of cytokine and chemokines to better characterize the ex vivo hostresponse to CMV peptide stimulation. In a cohort of CMV viremic organ transplant recipients, chemokine expression-specifically CCL8 (AUC 0.849 95% CI 0.721-0.978; p ¼ 0.003) and CXCL10 (AUC 0.841, 95% CI 0.707-0.974; p ¼ 0.004)-was associated with control of viral replication. In a second cohort of transplant recipients at high-risk for CMV, the presence of a polymorphism in the CCL8 promoter conferred an increased risk of viral replication after discontinuation of antiviral prophylaxis (logrank hazard ratio 3.6; 95% CI 2.077-51.88). Using cell-sorting experiments, we determined that the primary cell type producing CCL8 in response to CMV peptide stimulation was the monocyte fraction. Finally, in vitro experiments using standard immunosuppressive agents demonstrated a dose-dependent reduction in CCL8 production. Chemokines appear to be important elements of the cellmediated response to CMV infection posttransplant, as here suggested for CCL8, and translation of this knowledge may allow for the tailoring and improvement of preventative strategies.

The mouse MHC class Ib gene H2-T11 is 95% identical at the DNA level to H2-T23, which encodes Qa-1, one of the most studied MHC class Ib molecules. H2-T11 mRNA was observed to be expressed widely in tissues of C57BL/6 mice, with highest levels in thymus. To circumvent the availability of a specific mAb, cells were transduced with cDNA encoding T11 with a substituted α3 domain. Hybrid T11D3 protein was expressed at high levels similar to control T23D3 molecules on the surface of both TAP+ and TAP− cells. Soluble T11D3 was generated by folding in vitro with Qdm, the dominant peptide presented by Qa-1. The circular dichroism spectrum of this protein was similar to that of other MHC class I molecules, and it was observed to bind labeled Qdm peptide with rapid kinetics. By contrast to the Qa-1 control, T11 tetramers did not react with cells expressing CD94/NKG2A, supporting the conclusion that T11 cannot replace Qa-1 as a ligand for NK cell inhibitory receptors. T11 also failed to substitute for Qa-1 in the presentation of insulin to a Qa-1-restricted T cell hybridoma. Despite divergent function, T11 was observed to share peptide-loading specificity with Qa-1. Direct analysis by tandem mass spectrometry of peptides eluted from T11D3 and T23D3 isolated from Hela cells demonstrated a diversity of peptides with a clear motif that was shared between the two molecules. Thus T11 is a paralog of T23 encoding an MHC class Ib molecule that shares peptide-binding specificity with Qa-1 but differs in function.

Conventional MHC class Ia-restricted CD8+ T cells play a dominant role in the host response to virus infections but recent studies indicate that T cells with specificity for nonclassical MHC class Ib molecules may also participate in host defense. To investigate the potential role of class Ib molecules in anti-viral immune responses, Kb−/−Db−/−CIITA−/− mice lacking expression of MHC class Ia and class II molecules were infected with LCMV. These animals have a large class Ib-selected CD8+ T cell population and they were observed to mediate partial (but incomplete) virus clearance during acute LCMV infection as compared to Kb−/−Db−/−®2M−/− mice that lack expression of both MHC class Ia and class Ib molecules. Infection was associated with expansion of splenic CD8+ T cells and induction of granzyme B and IFN© effector molecules in CD8+ T cells. Partial virus clearance was dependent on CD8〈®+ cells. In vitro T cell re-stimulation assays demonstrated induction of a population of ®2M-dependent, MHC class Ib-restricted CD8+ T cells with specificity for viral antigen(s) and a yet to be defined nonclassical MHC molecule(s). MHC class Ib-restricted CD8+ T cell responses were also observed after infection of Kb−/−Db−/− mice, despite the low number of CD8+ T cells in these animals. Long term infection studies demonstrated chronic infection and gradual depletion of CD8+ T cells in Kb−/−Db−/−CIITA−/− mice, demonstrating that class Ia molecules are required for viral clearance. These findings demonstrate that class Ib-restricted CD8+ T cells have the potential to participate in the host immune response to LCMV.

Conventional MHC Ia & II molecules (H-2K/D/L & I-A/E in mouse) select α/β CD8 & CD4 T cell development, respectively. Although unconventional MHC class Ib, mainly encoded in H-2 Q, T & M regions in mouse, contains more than 20 MHC Ib genes, their functions remain largely unknown. We developed a mouse model, in which 3 genes, Kb, Db & CIITA were deleted, thus called triple knock-out (TKO) mouse. In TKO, CD8 T cells are selected only by MHC Ib. Previously we have shown that α/β CD8 T cells develop well in TKO. Functionally, we observe that TKO mice mount a protective immune response against in vivo acute infections of LCMV. The protection is CD8 T cell mediated & β2m dependent. Rapid production of IFNγ and Granzyme B is detected in expended CD8 cells. Intriguingly, TKO mice do not appear to mount a memory response to a second infection. We report that splenocytes, from in vivo LCMV primed TKO, can be restimulated in vitro, by LCMV infected peritoneal macrophages or bone marrow derived dendritic cells, to produce IFNγ, but little IL-2. This MHC Ib-dependent response appears to be partially mediated by Qa-2. Therefore we have established an in vitro system to analyze MHC Ib-restricted T cell responses to LCMV & we are attempting to map the MHC Ib restriction element(s) and target epitopes. Collectively, our results suggest that MHC Ib can select functional CD8 T cells & these T cells participate in, at least under some circumstances, protective immune responses to pathogens.