Abstract: Cyclin-dependent kinase 6 (CDK6) promotes cell cycle progression and is overexpressed in human lymphoid malignancies. To determine the role of CDK6 in development and tumorigenesis, we generated and analyzed knockout mice. Cdk6-deficient mice show pronounced thymic atrophy due to reduced proliferative fractions and concomitant transitional blocks in the double-negative stages. Using the OP9-DL1 system to deliver temporally controlled Notch receptor–dependent signaling, we show that CDK6 is required for Notch-dependent survival, proliferation, and differentiation. Furthermore, CDK6-deficient mice were resistant to lymphomagenesis induced by active Akt, a downstream target of Notch signaling. These results show a critical requirement for CDK6 in Notch/Akt-dependent T-cell development and tumorigenesis and strongly support CDK6 as a specific therapeutic target in human lymphoid malignancies. [Cancer Res 2009;69(3):810–8]

Abstract: Most pathogens establish infection through mucosa, where secretory immunoglobulin A (sIgA) plays an ‘immune exclusion’ role in humoral defense. Extravasation of intravenously (i.v.) administrated therapeutic immunoglobulin G (IgG) mainly relies on convection and/or neonatal Fc receptor-mediated transcytosis from circulation into interstitial space. Active transport of interstitial IgG further across epithelium into mucosa, like sIgA, is a much desired feature for the next generation of therapeutic antibodies, especially for anti-infection purposes. For the first time, we report the engineering of an IgA mimicry of IgG, with its Fc portion in fusion with the 18-aa tail piece (tp) of sIgA and the J chain, possessing sIgA’s full binding activity towards polymeric immunoglobulin receptor that mediates mucosa transcytosis. In a diphtheria toxin receptor (DTR) knockin mouse model, i.v. injected anti-diphtheria toxin (DT) IgG(tp)J protected DTR+ cells from deletion upon DT injection. The compact design of IgG(tp)J opens new revenues for more effective therapeutic IgG mimicking some of the important biological functions of IgA.

Abstract: Expressing afucosylated human IgG1 antibodies with Chinese hamster ovary (CHO) cells deficient of α-(1,6)-fucosyltransferase (FUT8) is being more and more accepted as a routine method to enhance antibody-dependent cellular cytotoxicity (ADCC) of therapeutic antibodies, especially for anti-cancer regimens. However, in pre-clinical studies relying on disease models other than mice and primates, e.g., those underrepresented species for infectious diseases, it is less clear whether such afucosylated antibodies can demonstrate enhanced therapeutic index. This is because the orthologues of human FcγRIIIA or mouse FcγRIV from those species have not been well characterized. Methods We set up a luciferase-based ADCC assay with Jurkat reporter cells expressing FcγRIIIA/FcγRIV from human, mouse, rat, hamster, guinea pig, ferret, rabbit, cat, dog, pig and monkey, and also produced human, mouse, hamster, rabbit and pig IgG from wild type and Fut8−/− CHO cells or hybridomas. Results We confirmed that enhanced stimulation through FcγRIIIA/FcγRIV by afucosylated IgG, as compared with wild type IgG, is a cross-species phenomenon. Conclusions Thus, efficacy and toxicology studies of the next generation afucosylated therapeutic IgG and Fc fusion proteins in these underrepresented animal models should be expected to generate translatable data for treating human diseases, leading to the expanded applications of this new class of glycoengineered biologics.

Abstract: L2pB1 cells are a subpopulation of B‐1a B cells that express programmed death ligand 2 (PD‐L2) as their unique cell surface marker. In mice, about 50% of peritoneal B‐1a cells are L2pB1 cells. The remaining B‐1a cells are L2nB1 (PD‐L2 – ) B‐1a cells. L2pB1 cells differ from L2nB1 cells in their immunoglobulin repertoire, expression of interleukin 10, and their capacity to phagocytose phosphatidylcholine. The physiological roles of L2pB1 cells have not been investigated owing to the lack of an animal model that allows for specific depletion of L2pB1 cells. Here, we report a mouse model that enables specific tracking and inducible depletion of L2pB1 cells in vivo . Our data show that depletion of L2pB1 cells significantly reduces serum anti‐phosphorylcholine (PC) IgM levels and IL‐10 expression in the peritoneal cavity. This animal model provides a tool for the study of the immune regulatory functions of L2pB1 cells in health and disease.

Abstract: Pre-TCR signals regulate the transition of the double-negative (DN) 3 thymocytes to the DN4, and subsequently to the double-positive (DP) stage. In this study, we show that pre-TCR signals activate Akt and that pharmacological inhibition of the PI3K/Akt pathway, or combined ablation of Akt1 and Akt2, and to a lesser extent Akt1 and Akt3, interfere with the differentiation of DN3 and the accumulation of DP thymocytes. Combined ablation of Akt1 and Akt2 inhibits the proliferation of DN4 cells, while combined ablation of all Akt isoforms also inhibits the survival of all the DN thymocytes. Finally, the combined ablation of Akt1 and Akt2 inhibits the survival of DP thymocytes. Constitutively active Lck-Akt1 transgenes had the opposite effects. We conclude that, following their activation by pre-TCR signals, Akt1, Akt2, and, to a lesser extent, Akt3 promote the transition of DN thymocytes to the DP stage, in part by enhancing the proliferation and survival of cells undergoing β-selection. Akt1 and Akt2 also contribute to the differentiation process by promoting the survival of the DP thymocytes.

Abstract: The findings in this report support a model in which Tpl2 ablation promotes intestinal inflammation in Apc min/+ mice by down-regulating the expression of the antiinflammatory cytokine IL-10 in myeloid cells in the intestinal mucosa and by interfering with the development and function of Tregs, which also inhibit inflammation. Developing polyps produce proinflammatory molecules that suppress Tregs and Tr1 cells and stimulate IL-17–producing T cells. This positive feedback loop is more robust in Apc min/+ /Tpl2 −/− mice, perhaps because the tumor burden of these mice is higher throughout life, as a result of the enhancement of tumor initiation ( Fig. P1 ). Overall, our data provide a unique genetic link between inflammation and cancer. Moreover, the animal model described here provides a valuable tool to address the pathophysiology of inflammatory bowel disease and colorectal cancer mechanistically in animals and humans. Experiments of Treg induction in culture provided evidence that the low numbers of Tregs in Tpl2 −/− mice result from a T cell-intrinsic defect in Treg generation and that only a small fraction of these Tpl2 −/− Tregs produce IL-10. Other in vivo studies reported here showed that the numbers of IL-10 + Tregs and type 1 regulatory T (Tr1) cells were lower in the intestines of Apc min/+ /Tpl2 −/− mice. The low numbers of Tregs and Tr1 cells were accompanied by high numbers of proinflammatory Foxp3 + and Foxp3 − IL-17–producing T cells. Moreover, the numbers of Tregs decreased and the numbers of IL-17–producing T cells increased with time faster in the Apc min/+ /Tpl2 −/− mice than in the Apc min/+ /Tpl2 +/+ mice. The more rapid change in the numbers of Tregs and IL-17–producing T cells in Apc min/+ /Tpl2 −/− mice parallels the increase in polyp numbers with time, which is also more rapid in these mice. This suggests that Apc min/+ /Tpl2 −/− mice may exhibit an enhanced response to proinflammatory signals triggered by the developing polyps or that Apc min/+ /Tpl2 −/− polyps may produce a more robust proinflammatory environment than Apc min/+ /Tpl2 +/+ polyps. Other cells that contribute to the abundance of IL-10 in the intestines are the Tregs, which may also produce IL-10. Our studies indeed showed that the number of Tregs is reduced in the intestines and peripheral lymphoid organs, but not in the thymus, of Tpl2 −/− mice in both the Apc +/+ and Apc min/+ genetic backgrounds. Tregs from Tpl2 −/− mice are not only low in number but express low levels of Foxp3, a transcription factor essential for Treg development and function. This suggested that the Tpl2 −/− Tregs might also be functionally defective, which was confirmed with Treg transfer experiments. These experiments showed that only the WT Tregs protect Apc min/+ /Tpl2 +/+ mice from polyposis. Interestingly, the Tpl2 +/+ Tregs failed to inhibit polyposis when transplanted into Apc min/+ /Tpl2 −/− mice, suggesting that the function of WT Tregs is intact only in the Tpl2 +/+ microenvironment. Intestinal IL-10 is produced primarily by macrophages. This observation, combined with the known defect of Tpl2 −/− macrophages in the induction of IL-10 by signals initiated by receptors triggered by bacterial products ( 4 ), suggested that the low levels of IL-10 in the intestines of Tpl2 −/− mice might be attributable to a macrophage signaling defect. Further studies showed that Tpl2 ablation in macrophages inhibits the activation of several upstream regulators of IL-10, including mTOR and Stat3 ( 5 ). Divergence in the composition of the intestinal inflammatory infiltrates of the Apc min/+ /Tpl2 −/− mice becomes apparent with the initiation of polyposis. Because polyps may stimulate inflammation ( 3 ), this observation challenged the hypothesis that the enhancement of polyposis in Apc min/+ /Tpl2 −/− mice may be secondary to the enhancement of inflammation. ELISAs addressing the expression of cytokines in the intestinal mucosa of these mice showed that the levels of intestinal IL-10 were lower in Tpl2 −/− mice than in Tpl2 +/+ mice, independent of adenomatous polyposis coli ( APC ) or polyp status. These data identified IL-10 as one of the factors that may be responsible for the initial divergence in inflammation and tumorigenesis between Apc min/+ /Tpl2 +/+ and Apc min/+ /Tpl2 −/− mice. The enhancement of tumorigenesis in Tpl2 −/− mice carrying the Apc min mutation could be the result of an intrinsic defect in the intestinal epithelia. Alternatively, it may be attributable to tumor-promoting changes in the intestinal stroma (i.e., the tissue supporting the intestinal epithelia). Bone marrow transplantation experiments showed that this enhancement is driven, at least in part, by hematopoietic cells. These results, combined with the known protumorigenic effects of inflammation, also suggested that Tpl2 ablation may promote tumorigenesis by promoting inflammation. Our analysis of the intestinal inflammatory infiltrates by flow cytometry showed that the infiltrates isolated from Apc min/+ /Tpl2 −/− mice contained a higher percentage of macrophages and tumor-associated myeloid-derived suppressor cells, and that the percentages of these cells increase with time. More important, the levels of the proinflammatory mediators, TNF-α, and IL-6 were higher in extracts of the intestinal mucosa of the Apc min/+ /Tpl2 −/− mice, and polyps from the same mice expressed higher levels of the enzyme COX-2, suggesting that Tpl2 ablation promotes the establishment of a proinflammatory environment in the intestinal mucosa. The Tpl2 gene encodes an enzyme that phosphorylates proteins on serine and threonine amino acid residues (a serine-threonine protein kinase) and is activated in retrovirus-induced tumors in animals via the integration of a DNA copy of the retroviral RNA genome within the gene. The Tpl2 kinase is activated enzymatically by signals originating in receptors that recognize bacterial products and various regulators of the immune system. Tpl2 activated by such signals plays an important role in the regulation of innate and adaptive immunity and inflammation (refs. 1 and 2 and references therein). Here, we show that mice with an inactivating mutation in the Apc gene and a second inactivating mutation in Tpl2 ( Apc min/+ /Tpl2 −/− mice) exhibit a fivefold increase in the number of intestinal polyps by comparison with the mice that carry only the Apc mutation. The increase in polyp numbers was partly hematopoietic cell-driven. The ablation of Tpl2 promoted intestinal inflammation by interfering with the secretion of the antiinflammatory molecule IL-10 in the intestinal mucosa and with the generation and function of regulatory T cells (Tregs), which also inhibit inflammation and tumorigenesis. Inflammation and polyp numbers increased with time, but the increase was faster in Apc min/+ /Tpl2 −/− mice, suggesting that the positive feedback initiated by polyp-induced inflammatory signals is more robust in these mice. Given the importance of Apc mutations in human colorectal cancer, these findings provide unique insights into the role of inflammation in this disease and into the molecular mechanisms that may influence the development of inflammation.