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PI 3,4,5-trisphosphate [PI(3,4,5)P3; PIP3]-dependent Rac exchanger 1 (P-Rex1) is a Rac-specific guanine nucleotide exchange factor abundant in neutrophils and myeloid cells. As a selective catalyst for Rac2 activation, P-Rex1 serves as an important regulator of human neutrophil NADPH oxidase activity and chemotaxis in response to a variety of extracellular stimuli. The exchange activity of P-Rex1 is synergistically activated by the binding of PIP3and βγ subunits of heterotrimeric G proteins in vitro, suggesting that the association of P-Rex1 with membranes is a prerequisite for cellular activation. However, the spatial regulation of endogenous P-Rex1 has not been well defined, particularly in human neutrophils activated through G protein-coupled receptors. Upon stimulation of neutrophil chemoattractant receptors, we observed that P-Rex1 translocated from cytoplasm to the leading edge of polarized cells in a G protein βγ subunit- and PIP3-dependent manner, where it colocalized with F-actin and its substrate, Rac2. Redistribution of P-Rex1 to the leading edge was also dependent on tyrosine kinase activity and was modulated by cell adhesion. Furthermore, we observed that activation of cAMP-dependent protein kinase A (PKA), which phosphorylates and inactivates P-Rex1, inhibited its translocation. Our data indicate that endogenous P-Rex1 translocates to areas of Rac2 and cytoskeletal activation at the leading edge in response to chemoattractant stimuli in human neutrophils and that this translocation can be negatively modulated by activation of PKA and by cell adhesion.

Although class I myosins are known to play a wide range of roles, the physiological function of long-tailed class I myosins in vertebrates remains elusive. We demonstrated that one of these proteins, Myo1f, is expressed predominantly in the mammalian immune system. Cells from Myo1f-deficient mice exhibited abnormally increased adhesion and reduced motility, resulting from augmented exocytosis of β2 integrin–containing granules. Also, the cortical actin that co-localizes with Myo1f was reduced in Myo1f-deficient cells. In vivo, Myo1f-deficient mice showed increased susceptibility to infection byListeria monocytogenesand an impaired neutrophil response. Thus, Myo1f directs immune cell motility and innate host defense against infection.

Chemotaxis is a fascinating biological process, through which a cell migrates along a shallow chemoattractant gradient that is less than 5% difference between the anterior and posterior of the cell. Chemotaxis is composed of two independent, but interrelated processes--motility and directionality--both of which are regulated by extracellular stimuli and chemoattractants; small GTPases have been shown to be involved. In this chapter, the methods that are used to prepare mouse neutrophils and study their chemotactic behaviors and morphological and biochemical changes in response to chemoattractant stimulation are described.

Chemotaxis is a fascinating biological process, through which a cell migrates along a shallow chemoattractant gradient that is less than 5% difference between the anterior and posterior of the cell. Chemotaxis is composed of two independent, but interrelated processes—motility and directionality—both of which are regulated by extracellular stimuli and chemoattractants; small GTPases have been shown to be involved. In this chapter, the methods that are used to prepare mouse neutrophils and study their chemotactic behaviors and morphological and biochemical changes in response to chemoattractant stimulation are described.

Follicular helper T (Tfh) cells promote germinal center (GC) formation and subsequent B cell maturation through the secretion of cytokines such as IL-21 and IL-4. Achaete-scute complex homologue 2 (Ascl2), a transcription factor previously shown to have a role in stem cell maintenance, is highly upregulated in the Tfh-cell subset of CD4+ T cells. However, its role in Tfh-cell development and function has not been established. We generated mice with conditional deletion of Ascl2 in the CD4 T cell compartment to assess whether this protein has a functional role in Tfh cells. Tfh cells deficient in Ascl2 promote a more robust germinal center response, with larger germinal centers and an increased percentage of GC B cells after immunization. This phenotype was coupled with augmented class-switched antibody production in the Ascl2-deficient mice. The expansion of GCs and class-switched antibody production is likely a result of increased IL-4 production by Tfh cells in the Ascl2-deficient mice. These data suggest that Ascl2 may play a role in limiting the functionality of Tfh cells by preventing their secretion of IL-4 and repressing their potential as cell helpers.

Follicular helper T cells (TFH), defined by expression of the surface markers CXCR5, PD-1 and ICOS, are located in germinal centers where they provide help for B cell maturation through the secretion of IL-21. The transcriptional repressor Bcl6 is upregulated in TFH cells; however, the role that it plays in their development is unknown. We describe downregulation of P-selectin glycoprotein ligand-1 (PSGL1) as a new surface marker for TFH cells, and show that Bcl6 is required for the development of TFH cells that are PSGL1lo. Activated antigen-specific CD4 PSGL1lo T cells are enriched for expression of CXCR5 and PD-1, and highly upregulate IL-21 and Bcl6. Over-expression of Bcl6 leads to an increase in PSGL1lo TFH cells; however, it cannot rescue TFH cell development in the absence of ICOS. Consistent with others, we find B cells are required for full development of TFH cells, but not for downregulation of PSGL1, suggesting there are multiple steps in development of the subset. This data demonstrates that Bcl6, independent of ICOS signaling, is critical for the development of this lineage.