In the present study real time polymerase chain
In the present study, real-time polymerase chain reaction also confirmed that gene expressions of RT1 class II, locus Bb (RT1-Bb) was up-regulated and Spi-B transcription factor (Spib) was downregulated at day 3 after the VMH lesions. These two genes work as one of immune responses. RT1-Bb controls cell-mediated immunity in rats . While, the protein encoded by Spib acts as a lymphoid-specific enhancer .
Acknowledgements This work was supported in part by grants-in-aid from the Suzuken Memorial Foundation (Nagoya, Japan), and Grant 26461042 from the Ministry of Education, Culture, Sports, Science and Technology in Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Introduction The production of neutralizing GYKI 52466 dihydrochloride by long-lived plasma cells and memory B cells upon antigen re-exposure underpins the protection afforded by most successful vaccines (Plotkin, 2008). These outputs from the germinal center (GC) are critically dependent on sequential CD4+ T cell help provided to B cells at multiple sites including the interfollicular zone (Kerfoot et al., 2011), T-B border (Garside et al., 1998, Okada et al., 2005), and within GCs (Allen et al., 2007, MacLennan, 1994, Victora and Nussenzweig, 2012) to drive antibody affinity maturation and memory formation (Crotty, 2011). The term follicular B helper T cells (Tfh) was originally used to describe human CD4+ T cells that express the chemokine receptor CXCR5, localize to the secondary follicle of tonsils, and provide cognate help to B cells (Breitfeld et al., 2000, Schaerli et al., 2000). The importance of Tfh cells to human health is underscored by the recurrent bacterial infections that occur when they are defective, and the autoimmune pathologies that develop when they are in excess (Tangye et al., 2013). Rapid developments in the Tfh field in recent years has been facilitated by the use of cell surface molecules, such as CXCR5, PD-1, and ICOS (Haynes et al., 2007, Rasheed et al., 2006), as surrogate markers for tracking Tfh cells in human subjects and genetic mouse models. Unfortunately, these markers of CD4+ T cell activation are not unique to Tfh cells. For example, CXCR5 is upregulated by multiple CD4+ T cell lineages upon activation in vivo (Ansel et al., 1999, Schaerli et al., 2001). Nevertheless, the recognition that the transcriptional repressor Bcl-6 is absolutely required for Tfh cell development firmly established them as a distinct CD4+ T cell lineage (Chtanova et al., 2004, Johnston et al., 2009, Nurieva et al., 2009, Yu et al., 2009). However, Bcl-6 expression is also not Tfh cell-specific as it is upregulated in all dividing CD4+ T cells during their interactions with dendritic cells (DCs) (Baumjohann et al., 2011, Kitano et al., 2011). Taken together, these uncertainties make it difficult to conclusively track the origin and fate of Tfh cells in the primary and secondary antibody response. Recently, a method for in vivo photoactivation of cells expressing PA-GFP in precise microanatomical compartments was described (Victora et al., 2010), which makes it possible to optically mark Tfh cells and track them 20 hr later (Shulman et al., 2013). Unexpectedly, it was reported that Tfh cells frequently migrated out of the follicle to invade neighboring GCs and proposed that this promoted affinity maturation by providing a diverse polyclonal source of CD4+ T cell help (Shulman et al., 2013). However, the temporospatial context of such promiscuous behavior was not defined. We have developed an alternative method for optical marking by two-photon photoconversion (TPP) of cells expressing the photoconvertible fluorescent protein Kaede (KD) (Chtanova et al., 2014). Our studies using TPP show striking differences in the migration and behavior of Tfh cells during three distinct phases: the primary response by naive CD4+ T cells; the memory phase following resolution of the GC response; and the secondary response by antigen-experienced cells. We demonstrate the migration of GC Tfh cells in the primary response was confined to the GC of origin and infrequently observed to cross into the follicular mantle (FM), a distinct region in the follicle surrounding the GC (Hardie et al., 1993). Follicular memory T cells were tracked to the outer follicle where they scanned CD169+ macrophages lining the subcapsular sinus (SCS) and became activated to divide upon antigen rechallenge. There was unrestricted movement of GC Tfh cells in the secondary response, and we show that they also enter and leave the follicle via the lymphatic flow in the SCS. Finally, we use TPP and single cell gene expression and functional analyses to show that the temporospatial cues guiding the positioning of Tfh cells during these phases of the immune response were provided in part by Epstein-Barr virus-induced G protein coupled receptor 2 (EBI2).