Different routes of bacterial infection induce long-lived TH1 memory cells and short-lived TH17 cells.

We used a sensitive method based on tetramers of peptide and major histocompatibility complex II (pMHCII) to determine whether CD4(+) memory T cells resemble the T helper type 1 (T(H)1) and interleukin 17 (IL-17)-producing T helper (T(H)17) subsets described in vitro. Intravenous or intranasal infection with Listeria monocytogenes induced pMHCII-specific CD4(+) naive T cells to proliferate and produce effector cells, about 10% of which resembled T(H)1 or T(H)17 cells, respectively. T(H)1 cells were also present among the memory cells that survived 3 months after infection, whereas T(H)17 cells disappeared. The short lifespan of T(H)17 cells was associated with small amounts of the antiapoptotic protein Bcl-2, the IL-15 receptor and the receptor CD27, and little homeostatic proliferation. These results suggest that T(H)1 cells induced by intravenous infection are more efficient at entering the memory pool than are T(H)17 cells induced by intranasal infection.

Flow cytometric analysis of T cell receptor signal transduction.

Intracellular assays of signal transduction in T cells stimulated by antigen in vivo have been limited by requirements for large numbers of highly purified cells to perform conventional biochemical techniques. Here, we describe a technique using flow cytometry to study signaling events in T cells after antigen recognition in the body.

Tracking epitope-specific T cells.

The tracking of antigen-specific T cells in vivo is a useful approach for the study of the adaptive immune response. This protocol describes how populations of T cells specific for a given peptide-major histocompatibility complex (pMHC) epitope can be tracked based solely on T-cell receptor (TCR) specificity as opposed to other indirect methods based on function. The methodology involves the adoptive transfer of TCR transgenic T cells with defined epitope specificity into histocompatible mice and the subsequent detection of these cells through the use of congenic or clonotypic markers. Alternatively, endogenous epitope-specific T cells can be tracked directly through the use of pMHC tetramers. Using magnetic bead-based enrichment and advanced multiparameter flow cytometry, populations as small as five epitope-specific T cells can be detected from the peripheral lymphoid organs of a mouse. The adoptive transfer procedure can be completed within 3 h, whereas analysis of epitope-specific cells from mice can be completed within 6 h.

Lipopolysaccharide enhances in vivo interleukin-2 production and proliferation by naive antigen-specific CD4 T cells via a Toll-like receptor 4-dependent mechanism.

Microbial adjuvants are essential for the development of T-cell-dependent antibody production, recall T-cell proliferation and interferon-gamma production following immunization with protein antigens. Using an adoptive transfer approach, we showed that the adjuvant lipopolysaccharide enhanced the frequency of cells producing interleukin-2, enhanced clonal expansion by antigen-specific CD4 T cells and increased CD86 and interleukin-1alpha production by antigen-presenting cells. All of these effects were dependent on Toll-like receptor-4 (TLR4) expression by cells other than the antigen-specific CD4 T cells. The ability of lipopolysaccharides to increase the number of antigen-specific CD4 T cells that survive after immunization probably explains the previous finding that antigen-specific proliferation by T cells from normal mice depends on previous exposure to antigen and adjuvant.

MHC class II deprivation impairs CD4 T cell motility and responsiveness to antigen-bearing dendritic cells in vivo.

The role continuous contact with self-peptide/MHC molecules (self ligands) in the periphery plays in the function of mature T cells remains unclear. Here, we elucidate a role for MHC class II molecules in T cell trafficking and antigen responsiveness in vivo. We find that naïve CD4 T cells deprived of MHC class II molecules demonstrate a progressive and profound defect in motility (measured by real-time two-photon imaging) and that these cells have a decreased ability to interact with limiting numbers of cognate antigen-bearing dendritic cells, but they do not demonstrate a defect in their responsiveness to direct stimulation with anti-CD3 monoclonal antibody. Using GST fusion proteins, we show that MHC class II availability promotes basal activation of Rap1 and Rac1 but does not alter the basal activity of Ras. We propose that tonic T cell receptor signaling from self-ligand stimulation is required to maintain a basal state of activation of small guanosine triphosphatases critical for normal T cell motility and that T cell motility is critical for the antigen receptivity of naïve CD4 T cells. These studies suggest a role for continuous self-ligand stimulation in the periphery for the maintenance and function of mature naïve CD4 T cells.