It is thought that immunity depends on naive CD4 T cells that proliferate in response to microbial antigens, differentiate into memory cells that produce anti-microbial lymphokines, and migrate to sites of infection. Here we use immunohistology to enumerate individual naive CD4 T cells, specific for a model antigen, in the whole bodies of adult mice. The cells resided exclusively in secondary lymphoid tissues, such as the spleen and lymph nodes, in mice that were not exposed to antigen. After injection of antigen alone into the blood, the T cells proliferated, migrated to the lungs, liver, gut and salivary glands, and then disappeared from these organs. If antigen was injected with the microbial product lipopolysaccharide, proliferation and migration were enhanced, and two populations of memory cells survived for months: one in the lymph nodes that produced the growth factor interleukin-2, and a larger one in the non-lymphoid tissues that produced the anti-microbial lymphokine interferon-gamma. These results show that antigen recognition in the context of infection generates memory cells that are specialized to proliferate in the secondary lymphoid tissues or to fight infection at the site of microbial entry.

Unraveling the complexity of the adaptive immune system requires the study of T cells in vivo. This protocol describes how populations of T cells specific for a given peptide: Major Histocompatibility Complex (pMHC) epitope can be identified in mice and tracked throughout the course of an immune response. The methodology involves the adoptive transfer of T-cell receptor (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 multi-parameter flow cytometry, populations as small as 5 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, while analysis of epitope-specific cells from mice can be completed within 6 h.

A sensitive peptide-major histocompatibility complex II (pMHCII) tetramer-based method was used to determine whether CD4+ memory T cells resemble the TH1 and TH-17 subsets described in vitro. Intravenous or intranasal Listeria monocytogenes infection induced pMHCII-specific CD4+ naïve T cells to proliferate and produce effector cells, about 10% of which resembled TH1 or TH-17 cells, respectively. TH1 cells were also present among the memory cells that survived three months post-infection whereas TH-17 cells disappeared. The short lifespan of TH-17 cells was associated with low amounts of Bcl-2, interleukin 15 receptor, CD27 and little homeostatic proliferation. These results suggest that TH1 cells induced by intravenous infection are more efficient at entering the memory pool than TH-17 cells induced by intranasal infection.

Naive T cells undergo homeostatic proliferation in lymphopenic mice, a process that involves TCR recognition of specific self peptide/MHC complexes. Since costimulation signals regulate the T cell response to foreign Ags, we asked whether they also regulate homeostatic expansion. We report in this study that homeostatic expansion of CD4 and CD8 T cells occurs independently of costimulation signals mediated through CD28/B7, CD40L/CD40, or 4-1BB/4-1BBL interactions. Using DO11.10 TCR transgenic T cells, we confirmed that CD28 expression was dispensable for homeostatic expansion, and showed that the presence of endogenous CD4+CD25+ regulatory cells did not detectably influence homeostatic expansion. The implications of these findings with respect to regulation of T cell homeostasis and autoimmunity are discussed.

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.GTPases ͉ MHC class II deficiency ͉ self ligand