Grb2 Is Important for T Cell Development, Th Cell Differentiation, and Induction of Experimental Autoimmune Encephalomyelitis.

The small adaptor protein growth factor receptor-bound protein 2 (Grb2) modulates and integrates signals from receptors on cellular surfaces in inner signaling pathways. In murine T cells, Grb2 is crucial for amplification of TCR signaling. T cell-specific Grb2(fl/fl) Lckcre(tg) Grb2-deficient mice show reduced T cell numbers due to impaired negative and positive selection. In this study, we found that T cell numbers in Grb2(fl/fl) CD4cre(tg) mice were normal in the thymus and were only slightly affected in the periphery. Ex vivo analysis of CD4(+) Th cell populations revealed an increased amount of Th1 cells within the CD4(+) population of Grb2(fl/fl) CD4cre(tg) mice. Additionally, Grb2-deficient T cells showed a greater potential to differentiate into Th17 cells in vitro. To test whether these changes in Th cell differentiation potential rendered Grb2(fl/fl) CD4cre(tg) mice more prone to inflammatory diseases, we used the murine Th1 cell- and Th17 cell-driven model of experimental autoimmune encephalomyelitis (EAE). In contrast to our expectations, Grb2(fl/fl) CD4cre(tg) mice developed a milder form of EAE. The impaired EAE disease can be explained by the reduced proliferation rate of Grb2-deficient CD4(+) T cells upon stimulation with IL-2 or upon activation by allogeneic dendritic cells, because the activation of T cells by dendritic cells and the subsequent T cell proliferation are known to be crucial factors for the induction of EAE. In summary, Grb2-deficient T cells show defects in T cell development, increased Th1 and Th17 cell differentiation capacities, and impaired proliferation after activation by dendritic cells, which likely reduce the clinical symptoms of EAE.

NFATc1 deficiency in T cells protects mice from experimental autoimmune encephalomyelitis.

NFATc1 is a member of the nuclear factor of activated T cells (NFAT) family of transcription factors. NFAT is activated upon T-cell receptor activation followed by intracytoplasmatic calcium influx where calmodulin, a calcium sensor protein, activates the phosphatase calcineurin that dephosphorylates NFAT proteins and results in NFAT nuclear import. Here, we show the analysis of conditional NFATc1-deficient mice bearing a deletion of NFATc1 in CD4(+) and CD8(+) T cells. NFATc1-deficient CD4(+) T cells polarized under Th17 conditions express reduced levels of the Th17-associated transcription factor RORγT (where ROR is RAR-related orphan receptor) as well as the Th17-associated cytokines IL-17A, IL-17F, IL-21, and IL-10. In the murine model of experimental EAE, we found a strong reduction of the disease outcome in conditional NFATc1-deficient mice, as compared with control littermates. This was accompanied by a diminished inflammation in the brain and spinal cord and reduced IL-17A and IFN-γ expression by antigen-specific spleen, spinal cord, and brain cells. Altogether, these results reveal an important role of NFATc1 in inducing Th17-cell responses and IFN-γ, both being relevant for the EAE development.

B cell homeostasis and plasma cell homing controlled by Krüppel-like factor 2.

Krüppel-like factor 2 (KLF2) controls T lymphocyte egress from lymphoid organs by regulating sphingosin-1 phosphate receptor 1 (S1Pr1). Here we show that this is not the case for B cells. Instead, KLF2 controls homeostasis of B cells in peripheral lymphatic organs and homing of plasma cells to the bone marrow, presumably by controlling the expression of ß(7)-integrin. In mice with a B cell-specific deletion of KLF2, S1Pr1 expression on B cells was only slightly affected. Accordingly, all splenic B cell subsets including B1 cells were present, but their numbers were increased with a clear bias for marginal zone (MZ) B cells. In contrast, fewer peyers patches harboring fewer B cells were found, and fewer B1 cells in the peritoneal cavity as well as recirculating B cells in the bone marrow were detected. Upon thymus-dependent immunization, IgG titers were diminished, and antigen-specific plasma cells were absent in the bone marrow, although numbers of antigen-specific splenic plasmablasts were normal. KLF2 plays also a role in determining the identity of follicular B cells, as KLF2-deficient follicular B cells showed calcium responses similar to those of MZ B cells and failed to down-regulate MZ B cell signature genes, such as CD21 and CXCR7.

Performance and Metabolic, Inflammatory, and Oxidative Stress-Related Parameters in Early Lactating Dairy Cows with High and Low Hepatic FGF21 Expression.

Induction of FGF21 expression in the liver and a significant increase in plasma FGF21 concentration have been demonstrated in cows during early lactation, but knowledge about the function of FGF21 in dairy cows remains limited. In order to improve the understanding of the physiological role of FGF21 in dairy cows, the present study aimed to investigate differences in metabolic pathways between dairy cows with high and low hepatic expression of FGF21 at week 1 of lactation (n = 8/group) by liver transcriptomics, targeted plasma metabolomics, and analysis of inflammatory and oxidative stress-related parameters. Dry matter intake, energy balance, milk yield, and energy-corrected milk yield at days 8−14 postpartum did not differ between cows with high and low hepatic FGF21 expression. However, cows with high FGF21 expression showed an upregulation of genes involved in endoplasmic reticulum stress, inflammation, and nuclear factor E2-related factor 2 (Nrf2)-dependent cytoprotection compared to cows with low FGF21 expression at week 1 postpartum (p < 0.05). Concentrations of important antioxidants (tocopherols, ß-carotene, and glutathione) in the liver and plasma, trolox equivalent antioxidant capacity in plasma, concentrations of oxidative stress-related compounds (thiobarbituric acid-reactive substances and protein carbonyls), and levels of most acute phase proteins at week 1 postpartum did not differ between cows with high or low FGF21 expression. Moreover, among a total of >200 metabolites assayed in the plasma, concentrations of only 7 metabolites were different between cows with high or low FGF21 expression (p < 0.05). Overall, the results showed that cows with high and low FGF21 hepatic expression had only moderate differences in metabolism, but FGF21 might be important in the adaptation of dairy cows to stress conditions during early lactation.

KLF2--a negative regulator of pre-B cell clonal expansion and B cell activation.

Maturation as well as antigen-dependent activation of B cells is accompanied by alternating phases of proliferation and quiescence. We and others have previously shown that Krüppel-like factor 2 (KLF2), a regulator of T cell quiescence and migration, is upregulated in small resting precursor (pre)-B cells after assembly of the immature pre-B cell receptor (pre-BCR) and is downregulated upon antigen-induced proliferation of mature B cells. These findings suggest that KLF2, besides its function in maintaining follicular B cell identity, peripheral B cell homeostasis and homing of antigen-specific plasma cells to the bone marrow, also controls clonal expansion phases in the B cell lineage. Here, we demonstrate that enforced expression of KLF2 in primary pre-B cells results in a severe block of pre-BCR-induced proliferation, upregulation of the cell cycle inhibitors p21 and p27 and downregulation of c-myc. Furthermore, retroviral KLF2 transduction of primary B cells impairs LPS-induced activation, favors apoptosis and results in reduced abundance of factors, such as AID, IRF4 and BLIMP1, that control the antigen-dependent phase of B cell activation and plasma cell differentiation. Hence, we conclude that KLF2 is not only a key player in terminating pre-B cell clonal expansion but also a potent suppressor of B cell activation.