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Plasma from hibernating (HIB) woodchucks (Marmota monax) or 13-lined ground squirrels (Ictidomys tridecemlineatus) suppressed 3H-thymidine uptake in mouse spleen cell cultures stimulated with Concanavalin A (ConA); plasma from non-hibernating animals were only slightly inhibitory. Maximum inhibition occurred when HIB plasma was added to the cultures prior to ConA. After HPLC size exclusion chromatography of the HIB ground squirrel plasma, a single fraction (fraction-14) demonstrated inhibitory activity. Assay of fraction-14 from 8 HIB squirrels showed inhibition ranging from 13 to 95%; inhibition was correlated to the time the squirrels were exposed to cold prior to hibernation. Western blot analysis showed the factor to be a large molecular weight protein (>300 kDa), and mass spectrometry identified sequences that were 100% homologous with alpha-2-macroglobulin from humans and other species. These findings indicate a hibernation-related protein that may be responsible for immune system down regulation.

Krüppel-like factor 4 (KLF4), a transcription factor, plays a key role in the pluripotency of stem cells. We sought to determine the function of KLF4 in T-cell development and differentiation by using T-cell-specific Klf4-knockout (KO) mice. We found that KLF4 was highly expressed in thymocytes and mature T cells and was rapidly down-regulated in mature T cells after activation. In Klf4-KO mice, we observed a modest reduction of thymocytes (27%) due to the reduced proliferation of double-negative (DN) thymocytes. We demonstrated that a direct repression of Cdkn1b by KLF4 was a cause of decreased DN proliferation. During in vitro T-cell differentiation, we observed significant reduction of IL-17-expressing CD4+ T cells (Th17; 24%) but not in other types of Th differentiation. The reduction of Th17 cells resulted in a significant attenuation of the severity (35%) of experimental autoimmune encephalomyelitis in vivo in Klf4-KO mice as compared with the Klf4 wild-type littermates. Finally, we demonstrated that KLF4 directly binds to the promoter of Il17a and positively regulates its expression. In summary, these findings identify KLF4 as a critical regulator in T-cell development and Th17 differentiation.—An, J., Golech, S., Klaewsongkram, J., Zhang, Y., Subedi, K., Huston, G. E., Wood, W. H., III, Wersto, R. P., Becker, K. G., Swain, S. L., Weng, N. Krüppel-like factor 4 (KLF4) directly regulates proliferation in thymocyte development and IL-17 expression during Th17 differentiation.

Krüppel-like factor 4 (Klf4) is a transcription factor and functions in regulating cell differentiation, cell growth, and cell cycle. Although Klf4 is expressed in lymphocytes, its function in lymphocytes is unknown. In this study, we report that the levels of Klf4 expression were low in pro-B cells and continuously increased in pre-B and in mature B cells. Upon activation, Klf4 was rapidly decreased in mature B cells after 2 h of activation. A modest decrease in numbers of pre-B cells in bone marrow and mature B cells in spleen was observed in Klf4-deficient mice. In the absence of Klf4, fewer B cells entered the S phase of the cell cycle and completed cell division in response to the engagement of BCR and/or CD40 in vitro. Furthermore, the delay in entering the cell cycle is associated with decreased expression of cyclin D2 in B cells that lack Klf4 expression. We then demonstrated that Klf4 directly bound to the promoter of cyclin D2 and regulated its expression. These findings demonstrate that Klf4 regulates B cell number and activation-induced B cell proliferation through directly acting on the promoter of cyclin D2.

The majority of highly activated CD4 T cell effectors die after antigen clearance, but a small number revert to a resting state, becoming memory cells with unique functional attributes. It is currently unclear when after antigen clearance effectors return to rest and acquire important memory properties. We follow well-defined cohorts of CD4 T cells through the effector-to-memory transition by analyzing phenotype, important functional properties, and gene expression profiles. We find that the transition from effector to memory is rapid in that effectors rested for only 3 d closely resemble canonical memory cells rested for 60 d or longer in the absence of antigen. This is true for both Th1 and Th2 lineages, and occurs whether CD4 T cell effectors rest in vivo or in vitro, suggesting a default pathway. We find that the effector–memory transition at the level of gene expression occurs in two stages: a rapid loss of expression of a myriad of effector-associated genes, and a more gradual gain of expression of a cohort of genes uniquely associated with memory cells rested for extended periods.

We have outlined the carefully orchestrated process of CD4+ T-cell differentiation from naïve to effector and from effector to memory cells with a focus on how these processes can be studied in vivo in responses to pathogen infection. We emphasize that the regulatory factors that determine the quality and quantity of the effector and memory cells generated include (i) the antigen dose during the initial T-cell interaction with antigen-presenting cells; (ii) the dose and duration of repeated interactions; and (iii) the milieu of inflammatory and growth cytokines that responding CD4+ T cells encounter. We suggest that heterogeneity in these regulatory factors leads to the generation of a spectrum of effectors with different functional attributes. Furthermore, we suggest that it is the presence of effectors at different stages along a pathway of progressive linear differentiation that leads to a related spectrum of memory cells. Our studies particularly highlight the multi-faceted roles of CD4+ effector and memory T cells in protective responses to influenza infection and support the concept that efficient priming of CD4+ T cells that react to shared influenza proteins could contribute greatly to vaccine strategies for influenza.

The consequence of naive CD4+ T cell activation is the differentiation and generation of effector cells. How the engagement of T cell receptors and co-stimulatory receptors leads to profound differential changes is not fully understood. To assess the transcription changes during T cell activation, we developed human T cell specific cDNA microarray gene filters and examined the gene expression profiles in human naive CD4+ T cells for 10 continuous time points during the first 24 h after anti-CD3 plus anti-CD28 (anti-CD3/CD28) stimulation. We report here a global and kinetic analysis of gene expression changes during naive CD4+ T cell activation and identify 196 genes having expression levels that significantly changed after activation. Based on the temporal change, there are 15 genes that changed between 0-1 h (early), 25 genes between 2-8 h (middle) and 156 genes between 16-24 h (late) after stimulation. Further analyses of the functions of those genes indicate their roles in maintenance of resting status, activation, adhesion/migration, cell cycle progression and cytokine production. However, a significant majority of these genes are novel to T cells and their functions in T cell activation require further study. Together, these results present a kinetic view of the gene expression changes of naive CD4+ T cells in response to T cell receptor-mediated activation for the first time, and provide a basis in understanding how the complex network of gene expression regulation is programmed during CD4+ T cell activation.

The arachidonic acid derivative, 2-arachidonoyl-glycerol (2-AG), was initially isolated from gut and brain; it is also produced and released from blood and vascular cells. Many of the 2-AG-induced cellular responses (i.e., neuromodulation, cytoprotection and vasodilation) are mediated by cannabinoid receptors CB1 and CB2. The findings presented here demonstrate the expression of CB1, CB2 and TRPV1 receptors on cerebromicrovascular endothelial cells (HBEC). The expression of TRPV1, CB1 and CB2 receptor mRNA and proteins were demonstrated by RT-PCR and polyclonal antibodies, respectively. The endocannabinoid 2-AG, and other related compounds [anandamide (ANA), methanandamide (m-ANA), N-(4-hydroxyphenyl-arachidonyl-ethanolamide) (AM404) and capsaicin] dose-dependently stimulated Ca2+ influx in HBEC. The selective TRPV1 receptor antagonist (capsazepine), CB1 receptor antagonist (SR141716A) and CB2 receptor antagonist (SR144528) inhibited these responses. The effects of capsaicin, a specific agonist for TRPV1 receptors, were inhibited by capsazepine, but only weakly by CB1 or CB2 receptor antagonists. 2-AG also induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP); this response was mediated by VR1 receptors. These studies clearly indicate that 2-AG and other related compounds may function as agonists on VR1 receptors, as well as CB1 and CB2 receptors, and implicated these factors in various HBEC functions.

The involvement of the adrenergic system derived from locus ceruleus (LC) in the regulation of blood-brain barrier has been recognized to be mediated to a large extent by the endothelium.1,6 These cells, which produce many vasoactive and inflammatory substances, have been shown to participate in the interaction between immune cells and the central nervous system (CNS). For example, the infiltration of inflammatory cells into the CNS, a critical step in the pathogenesis of many CNS diseases, is limited in normal brain. In inflammation, the endothelium has been recognized as an active participant in pathogenic mechanisms by its capacity to express adhesion molecules. These molecules facilitate binding with circulatory leukocytes, which may result in their activation and ultimate passage across the endothelium. In addition, tumor necrosis factor-α (TNFα) is one of the most important endogenous cytokines that mediates local inflammation. Its proinflammatory activities toward vascular endothelium have also been described previously.2,7 Many reports also suggest that the changes in BBB properties in neurological disorders may be associated with disturbances of adrenergic input.3,4 Thus, we examined the influence of adrenergic agents on TNFα-induced intercellular adhesion molecule-1 (ICAM-1) on human brain microvascular endothelial cells (HBEC).

Krüppel-like factor 4 (Klf4), a zinc finger-containing transcription factor of the Krüppel-like factor family, plays critical role in cell differentiation, growth, and cycle. We report here a role of Klf4 in T cell development and differentiation. The expression of Klf4 was high in thymocytes and resting mature T cells, and was down-regulated in activated mature T cells. In the absence of Klf4, modest but significant reductions of T cell numbers in thymus (~27%) and in spleen (~25%), and a similar degree reduction of spleen CD4 T cells and a less degree loss of spleen CD8 T cells were found in Klf4 deficient mice. Gene expression analysis revealed an alteration of genes involved in cell cycling and proliferation in the absence of Klf4 in thymocytes, implying their role in control of T cell number. Furthermore, we found that significant reduction of IL-17 expressing CD4 T cells (TH-17) during in vitro differentiation in mice absence of Klf4 compared to the wild-type mice. We then demonstrated that Klf4 directly binds the promoter of IL17a but not to the promoter of ROR-γt gene, suggesting that Klf4 directly regulates Il17a in TH-17 differentiation. Together, these findings demonstrate that Klf4 regulates T cell development and TH-17 differentiation.