GATA transcription factor required for immunity to bacterial and fungal pathogens.

In the past decade, Caenorhabditis elegans has been used to dissect several genetic pathways involved in immunity; however, little is known about transcription factors that regulate the expression of immune effectors. C. elegans does not appear to have a functional homolog of the key immune transcription factor NF-kappaB. Here we show that that the intestinal GATA transcription factor ELT-2 is required for both immunity to Salmonella enterica and expression of a C-type lectin gene, clec-67, which is expressed in the intestinal cells and is a good marker of S. enterica infection. We also found that ELT-2 is required for immunity to Pseudomonas aeruginosa, Enterococcus faecalis, and Cryptococcus neoformans. Lack of immune inhibition by DAF-2, which negatively regulates the FOXO transcription factor DAF-16, rescues the hypersusceptibility to pathogens phenotype of elt-2(RNAi) animals. Our results indicate that ELT-2 is part of a multi-pathogen defense pathway that regulates innate immunity independently of the DAF-2/DAF-16 signaling pathway.

Peripheral "CD8 tuning" dynamically modulates the size and responsiveness of an antigen-specific T cell pool in vivo.

In this study, we suggest that CD8 levels on T cells are not static, but can change and, as a result, modulate CD8(+) T cell responses. We describe three models of CD8 modulation using novel weak-agonist (K1A) and super-agonist (C2A) altered peptide ligands of the HY smcy peptide. First, we used peripheral nonresponsive CD8(low) T cells produced after peripheral HY-D(b) MHC class I tetramer stimulation of female HY TCR transgenic and wild-type mice. Second, we used genetically lowered CD8(int) T cells from heterozygote CD8(+/0) mice. Finally, we used pre-existing nonresponsive CD8(low) T cells from male HY TCR transgenic mice. In CD8(low) and CD8(high) mice, presence of a lower level of CD8 greatly decreased the avidity of the peptide-MHC for HY TCR as reflected by avidity (K(D)) and dissociation constant (T(1/2)) measurements. All three models demonstrated that lowering CD8 levels resulted in the requirement for a higher avidity peptide-MHC interaction with the TCR to respond equivalently to unmanipulated CD8(high) T cells of the same specificity. Additionally, direct injections of wild-type HY-D(b) and C2A-D(b) tetramers into female HY TCR or female B6 mice induced a high frequency of peripheral nonresponsive CD8(low) T cells, yet C2A-D(b) was superior in inducing a primed CD8(+)CD44(+) memory population. The ability to dynamically modulate the size and responsiveness of an Ag-specific T cell pool by "CD8 tuning" of the T cell during the early phases of an immune response has important implications for the balance of responsiveness, memory, and tolerance.

Memory CD8 T cells require CD8 coreceptor engagement for calcium mobilization and proliferation, but not cytokine production.

Memory T-cell responses are faster and more robust than those of their naive counterparts. The mechanisms by which memory T cells respond better to subsequent antigenic exposure remain unresolved. A portion of the more rapid response is undoubtedly the result of the increased frequency of antigen-specific cells. In addition, there are also differences in the cells themselves with respect to their requirements for costimulation and the apparent avidity of the T cells. We used major histocompatibility complex (MHC) class I tetramers to stimulate T cells to focus on the interaction of T-cell receptor (TCR)/MHC and CD8 in the absence of other molecules that are present on cell surfaces and so contribute to the activation of T cells by undefined mechanisms. Mutated MHC class I tetramers that are unable to engage CD8 were used to investigate the role of CD8 engagement in memory cell activation. Either wild-type tetramers or tetramers carrying the mutation were used to stimulate both memory and naive TCR transgenic T cells in vitro. Surprisingly, like naive cells, memory CD8(+) T cells required CD8 engagement for calcium mobilization and optimum proliferation. In contrast, the requirements for cytokine production differed. Unlike naive cells, memory cells were able to produce cytokine in the absence of CD8 engagement. This suggests both a CD8-dependent pathway for early events and a CD8-independent pathway for cytokine production in memory cells.

Interplay between TCR affinity and necessity of coreceptor ligation: high-affinity peptide-MHC/TCR interaction overcomes lack of CD8 engagement.

CD8 engagement is believed to be a critical event in the activation of naive T cells. In this communication, we address the effects of peptide-MHC (pMHC)/TCR affinity on the necessity of CD8 engagement in T cell activation of primary naive cells. Using two peptides with different measured avidities for the same pMHC-TCR complex, we compared biochemical affinity of pMHC/TCR and the cell surface binding avidity of pMHC/TCR with and without CD8 engagement. We compared early signaling events and later functional activity of naive T cells in the same manner. Although early signaling events are altered, we find that high-affinity pMHC/TCR interactions can overcome the need for CD8 engagement for proliferation and CTL function. An integrated signal over time allows T cell activation with a high-affinity ligand in the absence of CD8 engagement.

High affinity xenoreactive TCR:MHC interaction recruits CD8 in absence of binding to MHC.

The TCR from a xenoreactive murine cytotoxic T lymphocyte clone, AHIII 12.2, recognizes murine H-2D(b) complexed with peptide p1058 (FAPGFFPYL) as well as human HLA-A2.1 complexed with human self-peptide p1049 (ALWGFFPVL). To understand more about T cell biology and cross-reactivity, the ectodomains of the AHIII 12.2 TCR have been produced in E. coli as inclusion bodies and the protein folded to its native conformation. Flow cytometric and surface plasmon resonance analyses indicate that human p1049/A2 has a significantly greater affinity for the murine AHIII 12.2 TCR than does murine p1058/D(b). Yet, T cell binding and cytolytic activity are independent of CD8 when stimulated with human p1049/A2 as demonstrated with anti-CD8 Abs that block CD8 association with MHC. Even in the absence of direct CD8 binding, stimulation of AHIII 12.2 T cells with "CD8-independent" p1049/A2 produces p56(lck) activation and calcium flux. Confocal fluorescence microscopy and fluorescence resonance energy transfer flow cytometry demonstrate CD8 is recruited to the site of TCR:peptide MHC binding. Taken together, these results indicate that there exists another mechanism for recruitment of CD8 during high affinity TCR:peptide MHC engagement.