ABSTRACT
During an immune reaction, some antigen-experienced CD4 T cells relocate from secondary lymphoid organs (SLOs) to the bone marrow (BM) in a CD49b-dependent manner and reside and rest there as professional memory CD4 T cells. However, it remains unclear how the precursors of BM memory CD4 T cells are generated in the SLOs. While several studies have so far shown that B cell depletion reduces the persistence of memory CD4 T cells in the spleen, we here show that B cell depletion enhances the establishment of memory CD4 T cells in the BM and that B cell transfer conversely suppresses it. Interestingly, the number of antigen-experienced CD4 T cells in the BM synchronizes the number of CD49b(+)T-bet(+) antigen-experienced CD4 T cells in the spleen. CD49b(+)T-bet(+) antigen-experienced CD4 T cells preferentially localize in the red pulp area of the spleen and the BM in a T-bet-independent manner. We suggest that B cells negatively control the generation of CD49b(+)T-bet(+) precursors of resting memory CD4 T cells in the spleen and may play a role in bifurcation of activated effector and resting memory CD4 T cell lineages.
ABSTRACT
Repeatedly activated T helper 1 (Th1) cells present during chronic inflammation can efficiently adapt to the inflammatory milieu, for example, by expressing the transcription factor Twist1, which limits the immunopathology caused by Th1 cells. Here, we show that in repeatedly activated murine Th1 cells, Twist1 and T-bet induce expression of microRNA-148a (miR-148a). miR-148a regulates expression of the proapoptotic gene Bim, resulting in a decreased Bim/Bcl2 ratio. Inhibition of miR-148a by antagomirs in repeatedly activated Th1 cells increases the expression of Bim, leading to enhanced apoptosis. Knockdown of Bim expression by siRNA in miR-148a antagomir-treated cells restores viability of the Th1 cells, demonstrating that miR-148a controls survival by regulating Bim expression. Thus, Twist1 and T-bet not only control the differentiation and function of Th1 cells, but also their persistence in chronic inflammation.
Subject(s)
Apoptosis Regulatory Proteins/genetics , Apoptosis/genetics , Gene Expression Regulation , Membrane Proteins/genetics , MicroRNAs/physiology , Nuclear Proteins/metabolism , Proto-Oncogene Proteins/genetics , T-Box Domain Proteins/physiology , Th1 Cells/immunology , Twist-Related Protein 1/metabolism , Animals , Arthritis, Rheumatoid/immunology , Bcl-2-Like Protein 11 , Cell Survival/immunology , Cells, Cultured , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , MicroRNAs/antagonists & inhibitors , MicroRNAs/genetics , Nuclear Proteins/genetics , RNA Interference , RNA, Small Interfering , T-Box Domain Proteins/genetics , Twist-Related Protein 1/geneticsABSTRACT
Ceramide kinase (CERK) is essential for production of ceramide-1-phosphate (C1P), a bioactive lipid whose formation critically modulates ceramide levels. To explore how CERK is regulated, we used insect cell-expressed, recombinant hCERK and searched for post-translational modifications, using mass-spectrometry techniques. This led to identification of two phosphorylated serine residues, at positions 340 and 408. Point mutations preventing phosphorylation at either of these sites did not lead to detectable changes in subcellular localization or activity. However, preventing phosphorylation at S340 resulted in CERK instability as revealed by the behavior of the S340A mutant protein under various assay conditions in vitro. Phosphorylation of a cognate serine residue in sphingosine kinases was previously shown to be important. Therefore, phosphorylation within a conserved "regulation loop" downstream of the catalytic domain emerges as a new paradigm for regulation of kinases of the diacylglycerol kinase family. This "regulation loop" is reminiscent of the "activation loop" that controls AGC protein kinases, being a similar distance from the critical ATP binding site determinants in the primary sequence.
