IN THIS ISSUE

The chaperone-based tumor vaccine heat shock protein 70-peptide complex-F (HSP70.PC-F), derived from a tumor:dendritic cell (DC) fusion, reverses T cell tolerance to a defined tumor Ag. The mechanisms behind its antitumor activity are poorly understood. Gong et al. (p. 3092) present evidence that HSP70.PC-F facilitates tumor immunity through its detection by the scavenger receptor expressed by endothelial cells-1 (SREC-1) and activation of TLR signaling. T cells isolated from wild-type (WT) mice immunized with HSP70.PC-F proliferated rapidly and had potent tumor killing activity, but these responses were reduced dramatically in T cells from MyD88 knockout (myd88^−/−) or TLR2/TLR4 double knockout (tlr2^−/−/tlr4^−/−) mice. Defects in T cell responses were alleviated by immunizing myd88^−/− or tlr2^−/−/tlr4^−/− mice with HSP70.PC-F-pulsed WT DCs, confirming that TLR signaling in DCs was involved in the facilitation of antitumor responses. SREC-1, an HSP70 receptor, was also involved in tumor immunity, as knockdown of SREC-1 expression in DCs blunted their ability to stimulate T cells both in vitro and in vivo. These findings indicate that HSP70.PC-F vaccination promotes antitumor responses by DCs through both SREC-1 recognition of HSP70.PC-F and TLR signaling, thus better defining the mechanisms employed by this unique vaccine.

Toll-like receptor 9 was first described as a microbial DNA sensor through its recognition of unmethylated double-stranded DNA sequences containing unique CpG motifs. Contradictory data confound evidence that TLR9 can detect mammalian DNA, despite a link between the presence of immune complexes (ICs) containing self-DNA and systemic lupus erythematosus (SLE). Yasuda et al. (p. 3109) have put forth data that TLR9-mediated dendritic cell (DC) activation by mammalian DNA incorporated into ICs required the presence of unmethylated CpG motifs. ICs formed with biotin-labeled DNA fragments containing optimal or suboptimal CpG sequences complexed with anti-biotin Abs activated TLR9 in Fms-like tyrosine kinase 3 ligand-stimulated mouse DCs (FL-DCs). DNA fragments lacking CpG motifs or methylation of suboptimal motifs prevented TLR9 stimulation by ICs. In contrast, DCs were stimulated by liposome transfection of CpG-free DNA, which may have been triggered by the delivery of a greater amount of DNA to TLR9-containing compartments. Notably, ICs with fragments from CpG-rich promoter regions of murine DNA induced TLR9-driven cytokine production by DCs, and this response was enhanced by IFN-β pretreatment. These results provide evidence that mammalian DNA with CpG sequences can stimulate TLR9 in DCs and supports data that TLR9 activation by self -DNA can contribute to SLE.

Axl is a transmembrane receptor tyrosine kinase that is part of the Tyro3, Axl and Mer (TAM) family. TAM signaling has been shown to modulate responses to TLR ligands in murine dendritic cells (DCs), and Axl expression is up-regulated in response to TLR-driven type I IFN production. Scutera et al. (p. 3004) characterized Axl expression in human DCs treated with IFN-α. Cell surface expression of Axl increased in monocyte-derived DCs differentiated with GM-CSF and IFN-α (IFN/DCs). Axl expression was also induced in IL-4 and GM-CSF-treated DCs (IL-4/DCs) with the addition of IFN-α. Axl surface expression was reduced by TLR stimulation of IFN/DCs with LPS, poly(I:C), or TLR7/8 ligand. These TLR ligands also promoted an increase in soluble Axl, which was likely caused by proteolysis and prevented by treatment with protease inhibitors. Axl stimulation by its ligand, growth arrest gene 6 (Gas6), rendered IFN/DCs resistant to subsequent LPS-induced Axl shedding and protected cells against apoptosis. Unexpectedly, Axl-expressing IFN/DCs migrated in response to Gas6, thus revealing a new role for Axl and Gas6 in human DC chemotaxis. These results shed light on the roles of Axl and Gas6 in IFN-α-treated human DCs and provide insight into potential mechanisms of autoimmune responses associated with excessive IFN-α production.

Lymphopenia induces homeostatic proliferation (HP) of T cells with a “memory-like” phenotype, but it is not known whether HP-memory T cells can function like Ag-induced “true memory” T cells. Cheung et al. (p. 3364) addressed this subject by using an adoptive transfer approach with OVA-specific CD8⁺ T cells. HP- or true-memory CD8⁺ T cells from OT-1 TCR transgenic mice proliferated and formed secondary memory populations in response to infection with OVA-expressing Listeria monocytogenes (Lm.ova) when each was transferred into C57BL/6 recipient mice. True-memory cells outcompeted HP-memory cells during Lm.ova infection following cotransfer, as true-memory cells showed greater expansion and secondary memory development compared with HP-memory cells. Lm.ova challenge revealed differences between HP- and true-memory cells with respect to chemokine expression patterns and spleen localization at later times during infection. These differences suggested that HP-memory cells formed secondary memory responses, but they could not respond to signals for tissue localization and memory formation in the same way as true-memory cells. These results indicated that lymphopenia-induced HP-memory cells did not deter proliferation and differentiation of Ag-specific true-memory cells, thus allaying some fears that lymphopenia is harmful to memory responses.

T cell-dependent (TD) Ab responses rely upon interactions between B and Th cells and the subsequent differentiation of primary B cells with high Ag affinities into extrafollicular plasmablasts (PB). Chan et al. (p. 3139) have defined the poorly understood initial events surrounding TD B cell responses by using a transgenic mouse with a BCR specific to the experimental Ag hen egg lysozyme (HEL). HEL-specific B cells proliferated in splenic B cell follicles as early as 1–3 days after Ag exposure and migrated to germinal centers (GC) and extrafollicular PB sites by days 3 and 4. IgM⁺ extrafollicular PBs differentiated into a CXCR4^highCXCR5^lowBCR^high phenotype, whereas GC B cells developed a CXCR4^highCXCR5^highBCR^low phenotype over this time period. IgM⁺ PBs declined after day 4.5 as switched IgG⁺ PBs with high Ag affinity showed enhanced expansion and reduced apoptosis compared with IgG⁺ PBs with low or intermediate Ag affinities. Extrafollicular development of high-affinity IgG⁺ PBs was associated with MHC II expression and interactions with extrafollicular Ag-specific Th cells, but not with IL-21R expression, implicating a unique T cell help mechanism for PB development that did not involve IL-21. This study defines the earliest events associated with high affinity PB development and offers insight into the enhancement of vaccine-induced Ab responses.

The effects of TLR tolerance caused by persistent TLR stimulation are not well understood in B cells. In this issue, Poovassery et al. (p. 2974) investigated the effects of chronic TLR7 stimulation on B cell function. B cells stimulated in vitro with the TLR7 ligand R848 produced large amounts of IL-6, TNF-α, and MIP-1α, but these responses fell dramatically following R848 restimulation. R848-restimulated B cells exhibited reduced proliferation, lower IgM secretion, and diminished NF-κB and MAPK activation compared with B cells stimulated only once. Remarkably, tolerance was reversed in R848-stimulated B cells by secondary costimulation of TLR7 and BCR with R848 and anti-IgM Ab, respectively. Moreover, tolerance was prevented in R848-restimulated cells by primary costimulation with R848 and anti-IgM Ab. R848 stimulation induced tolerance to secondary TLR9 ligand (CpG) stimulation as well, but was reversed by secondary costimulation with CpG and anti-IgM Ab. TLR7 tolerance was not reversed by secondary R848/anti-IgM Ab costimulation in the presence of a PI3K inhibitor. Reduced JNK phosphorylation was observed under these conditions, indicating the involvement of PI3K signaling in tolerance reversal. These data provide a new understanding of TLR tolerance in B cells and have important implications for the use of TLR7 ligands as vaccine adjuvants.

Mast cells (MCs) are vital to innate immune responses through their ability to recruit multiple cell types to infection sites, but their influence on regulatory T cell (Treg) function is not well established. Forward et al. (p. 3014) have determined that murine bone-marrow derived mast cells (BMMCs) could suppress natural Treg through the effects of histamine. CD4⁺CD25⁺ Treg inhibited proliferation of CD4⁺CD25⁻ responder T cells (Tresp) activated by bead-conjugated anti-CD3 and anti-CD28 mAbs. Suppression of Tresp proliferation was dramatically reduced in the presence of BMMCs that were activated by the crosslinking of FcεR. Treg suppressor function was also curbed by the supernatant from activated BMMCs or histamine, a molecule released from MC granules. Conversely, Treg suppressor activity was restored in the presence of BMMC, BMMC supernatant, or histamine by cotreatment with the histamine H1 receptor antagonist loratidine. The histamine response was H1 receptor specific because famotidine, an H2 receptor antagonist, did not restore Treg function. The H1 receptor was detected on both Treg and Tresp, and histamine treatment caused a decrease in CD25 and Foxp3 protein expression in Treg. This newly-defined role of BMMCs points to a mechanism for the temporary suspension of Treg suppressor activity for the enhancement of adaptive immune responses to infection.

Hedgehog (Hh) signaling is critical to thymocyte development and also activates the Gli family of transcription factors. Hager-Theodorides et al. (p. 3023) examined the underappreciated roles of Hh signaling and the Gli3 transcription factor in the thymic stroma. Gli3 transcripts, which have not been detected in adult thymocytes, were found in the thymic stroma of both fetal and adult thymi. In addition, Gli3 expression in the stroma was linked to repression of Hh signaling. PCR array analysis revealed three novel gene targets of Gli3 in the thymic stroma: Cxcl9, retinol binding protein 1, and NO synthase 2 (Nos2). These genes appeared to be up-regulated indirectly, most probably through Gli3-mediated suppression of intermediate repressors for each gene. Double positive thymocytes from Gli3 heterozygous mice were impaired in apoptosis and negative selection in vitro compared with those from wild-type mice. These heterozygotes failed to delete autoreactive thymocytes in vivo, likely due to reduced stromal expression of the proapoptotic factor Nos2. Reduced Gli3 expression was linked to decreased TCR signal strength as well, supporting a role for Gli3 in TCR repertoire development in the stroma. These data define new roles for Gli3 in the thymic stroma, including its influence on Hh signaling and development of the TCR repertoire.

Summaries written by Christiana N. Fogg, Ph.D.