Early commitment of adoptively transferred CD4+ T cells following particle-mediated DNA vaccination: implications for the study of immunomodulation.

The early responses of CD4+ T cells to particle-mediated DNA immunisation were investigated using OVA-specific TCR-transgenic CD4+ T cells. Following adoptive transfer of these cells, mice were immunised by delivery into the skin of a plasmid encoding ovalbumin. Transgenic T cells underwent a rapid and transient antigen-specific activation, followed by clonal expansion (up to approximately 6% of total lymphocytes). Immunisation with ovalbumin in CFA evoked similar responses with slightly faster kinetics. Numerous antigen-specific T cells synthesising IFN-gamma (Th1) and IL-4 (Th2) were detectable using both intracellular staining and ELISPOT assays. This study provides a quantitative analysis of both T cell proliferation and Th1/Th2 balance following particle-mediated DNA immunisation and establishes a robust and sensitive model in which to assess modulation of helper T cell responses in DNA vaccination.

Expression of L-selectin on Th1 cells is regulated by IL-12.

L-selectin has become established as a key molecule in the recirculation of naïve T cells from the blood to peripheral lymph nodes, yet little is known about its role in the migration of effector or memory cells. While differentiating naïve CD4+ T cells into Th1 and Th2 subsets in vitro, it was noted that L-selectin levels were maintained on the Th1 subset of cells. The expression of L-selectin on the Th1 cells appeared to be dependent on the presence of IL-12. Th2 cells, differentiated in the absence of IL-12, failed to maintain L-selectin expression. Coculture with IL-12, IL-18, IL-4, TNF-alpha, or IFN-alpha, -beta, or -gamma demonstrated a dependence on IL-12 alone for L-selectin expression. In addition, the inclusion of heat-killed Listeria monocytogenes in the cultures also maintained L-selectin expression on the Th1 cells. In all cultures, the maintenance of L-selectin on the T cell surface could be blocked by the inclusion of anti-IL-12 Abs. Analysis of the mRNA levels for L-selectin in T cells, differentiated in the presence or absence of IL-12, showed that the cytokine appears to exert its effect on L-selectin at the transcriptional level. Given the key role played by IL-12 in the differentiation of naïve T cells into the Th1 subset, the observation that IL-12 can also regulate L-selectin expression has implications for the migration of Th1 effector cells both through the lymphatic system and to sites of inflammation.

Differential expression of alpha3 fucosyltransferases in Th1 and Th2 cells correlates with their ability to bind P-selectin.

One of the key control points in the trafficking of the T cell effector subsets, Th1 and Th2, to sites of inflammation is their migration out of the bloodstream. The mechanism by which the cells initially adhere to the endothelium is dependent on the selectin family of adhesion molecules. Only polarised Th1 cells are capable of binding P-selectin despite both Th1 and Th2 cells expressing PSGL-1, the P-selectin ligand. This may be due to a secondary modification of PSGL-1 that is present on Th1 but not Th2 cells. One key modification of PSGL-1 is the alpha3 fucosylation of the O-glycans. To address whether the binding of Th1 and Th2 cells may be regulated by fucosylation, we have studied the expression of the alpha3 fucosyltransferases, FucT-IV and VII, using in vitro differentiated mouse T cells. Messenger RNA levels for both FucT-IV and VII were found to be higher in Th1 than Th2 cells. alpha3 fucosyltransferase enzyme activities were also elevated in Th1 cells. The increased expression of the alpha3 fucosyltransferases in Th1 cells correlated with the ability of Th1, but not Th2, cells to bind to P-selectin. Thus, the regulation of the binding of effector T cells to the endothelium, and subsequent trafficking to inflammatory sites, may be controlled by the fucosylation state of PSGL-1 mediated by the selective expression of the alpha3 fucosyltransferases.