Tumor-induced T cell tolerance is mediated by bone marrow-derived dendritic cells (DCs) and macrophages. Although immature myeloid cells (ImCs), another group of APCs, accumulate in tumor-bearing mice and suppress T cells in vitro, their role in tumor suppression in vivo is unknown. Kusmartsev et al. (p. 4583 ) adoptively transferred T cells transgenic for a specific OVA peptide into naive mice. Mice were immunized 2–3 days later with OVA peptide with or without ImCs isolated from spleens of tumor-bearing mice. Lymph node (LN) cells isolated after 10 days from animals that received ImCs did not respond to stimulation with OVA peptide compared with LN cells from control animals. ImCs, but not DCs, pulsed in vitro with OVA peptide before transfer into immunized mice reduced the in vitro LN cell response, whereas ImCs pulsed with control peptide did not. Suppression by ImCs was MHC class I restricted. Neither ImCs from tumor-free mice nor LN cells from immunized mice injected with OVA-pulsed CD11c+ DCs from tumor-bearing mice were able to suppress T cells in vitro. LPS-activated, peptide-loaded DCs from control mice transferred into mice immunized with the same peptide plus ImCs reversed the T cell tolerance. Dye-labeled ImCs from tumor-bearing mice injected into syngeneic tumor-bearing mice accumulated in tumor tissues and picked up soluble FITC-OVA injected i.p. Spleen and LN ImCs from mice injected with tumor cells transgenic for OVA expressed an OVA-derived epitope and abrogated the OVA peptide-specific CD8+ T cell response of ImCs from mice bearing the same tumor. Numbers of ImCs directly correlated with tumor size. The authors conclude that ImCs acquire tumor Ags and mediate Ag-specific CD8+ T cell tolerance in mice.

Transcription factor, T-bet, regulates IFN-γ production in a variety of cell types to overcome microbial infections. However, the role of T-bet in resistance to intracellular infections is not clear. Ravindran et al. (p. 4603 ) found that all T-bet−/− mice died after i.v. injection with attenuated Salmonella typhimurium, whereas wild-type mice exhibited only a transient infection. Viable bacteria increased markedly in spleens of T-bet−/− mice but declined in wild-type spleens at 3 wk postinfection. Only wild-type mice had elevated levels of Salmonella-specific IgG2a Ab. Wild-type splenic CD4+ T cells produced a low level of IFN-γ spontaneously in vitro and a higher level after i.v. injection with heat-killed S. typhimurium; few IFN-γ-producing cells were detected in spleens of infected T-bet−/− mice. Adoptively transferred TCR transgenic Salmonella-specific T cells had identical trafficking patterns and proliferation profiles in wild-type and T-bet−/− recipients injected with heat-killed bacteria, and both recipient strains had similar numbers of splenic IFN-γ-producing CD4+ T cells after in vivo challenge with flagellin peptide. In contrast, T-bet−/− cells and wild-type cells exhibited similar proliferative activities after injection into wild-type mice, but only wild-type cells produced IFN-γ after immunization of recipients with heat-killed bacteria and challenge with flagellin peptide. Injected T-bet−/− cells had higher levels of IL-2 production. The data demonstrate T-bet is required to increase IFN-γ production and suppress IL-2 production in CD4+ T cells in S. typhimurium-infected mice.

Neddylation of proteins in plants occurs through a series of reactions by enzymes related to those involved in ubiquitination. The ubiquitin homologue NEDD8 is conjugated only to the Cullin subunit (Cul-1) of a specific ubiquitin ligase complex. Neddylation has not been described in mammals. Collier-Hyams et al. (p. 4194 ) detected a 90-kDa protein that reacted with anti-Cul-1 and anti-NEDD8 Abs on immunoblots of lysates of human epithelial cells; the protein was lost in lysates from cells colonized in culture with some strains of nonpathogenic bacteria. Similar results were seen for Cul-1 expressed from a transfected plasmid. TNF-α stimulation of colonized cells showed phosphorylation but no ubiquitination of IκB-α, in contrast to the rapid IκB-α degradation seen in the absence of bacterial colonization. Overexpression of a dominant negative mutant of the NEDD8 transferase/ligase enzyme in epithelial cells blocked ubiquitination of overexpressed IκB-α and repressed TNF-α activation of a transfected TNF-α-induced, NF-κB-dependent reporter plasmid. The proportion of endogenous neddylated Cul-1 was reduced by introduction of NEDD8 transferase/ligase enzyme small interfering RNA (siRNA), by introduction of NEDD8 siRNA, or by cotransfection of the TNF-α-induced, NF-κB-dependent reporter plasmid plus the NEDD8 transferase/ligase siRNA. The data suggest that enteric bacteria reduce colonic inflammation by inhibiting NF-κB activation through down-regulation of Cullin neddylation.

Migration of epidermal keratinocytes is required to heal skin wounds. Although Abs against the antimicrobial peptide LL-37, a member of the cathelicidin family, inhibits skin wound healing, participation of LL-37 in the healing process has not been established. Tokumaru et al. (p. 4662 ) found that normal human keratinocytes exposed to 1 μg/ml synthetic LL-37 had increased migration in culture and in Boyden chambers compared with untreated controls. The epidermal growth factor receptor (EGFR) was phosphorylated at 10 min, and STAT3 was phosphorylated at 15 min in LL-37-exposed cells; four inhibitors that act at different steps of the EGFR transactivation pathway blocked keratinocyte migration and STAT3 phosphorylation in response to LL-37. A STAT3 dominant negative mutant expressed in keratinocytes also blocked STAT3 phosphorylation and migration, whereas transfection of a construct expressing either suppressor of cytokine signaling 1 or 3 blocked migration. The authors present a model of skin wound healing whereby LL-37 induces keratinocyte migration by EGFR transactivation and STAT3 phosphorylation.

The bacterium Tropheryma whipplei is the causative agent of Whipple’s disease, a rare systemic disease characterized by large foamy macrophages with bacterial inclusions. The mechanism by which T. whipplei survives in macrophages is unknown. Desnues et al. (p. 4575 ) detected more bacterial DNA in macrophages than monocytes from healthy humans up to 8 h after in vitro infection. Bacterial DNA copy number, as determined by real-time quantitative PCR, increased in macrophages up to 12 days postinfection but disappeared from infected monocytes by day 3. Infected macrophages, but not monocytes, underwent apoptosis and secreted IL-16; release of thioredoxin and expression of other redox-associated molecules were up-regulated only in infected monocytes. Bacterial replication was inhibited in macrophages cultured with thioredoxin for 12 days before infection. T. whipplei replication was increased in both cell types by exposure to IL-16 for 18 h before infection, whereas anti-IL-16 Ab prevented bacterial replication in infected macrophages. IL-16 pretreatment abolished infection-stimulated up-regulation of thioredoxin genes in monocytes and repressed thioredoxin gene expression in macrophages but induced up-regulation of IL-16 in macrophages. Antibiotic therapy of patients with Whipple’s disease reduced bacterial copies in macrophages and plasma IL-16 levels to those of healthy controls. The data indicate that IL-16 supports increased T. whipplei replication in macrophages and that monocyte killing of T. whipplei is dependent on thioredoxin.

Shiku and coworkers serologically identified tumor-derived self-Ags recognized by CD4+CD25+ regulatory T cells and CD4+ helper T cells. Enhanced pulmonary metastases after i.v. challenge with syngeneic tumor cells occurred in BALB/c mice immunized with plasmids expressing any of four of the most common self-Ags. However, coimmunization with a plasmid expressing a tumor-specific CTL epitope resulted in increased resistance to challenge with syngeneic tumors expressing the CTL epitope. In further studies from the same laboratory, Nishikawa et al. (p. 4433 ) found enhanced pulmonary metastases after tumor challenge in naive mice that received CD4+CD25+ T cells from syngeneic animals immunized with a self-Ag compared with controls receiving cells from mice immunized with the self-Ag plus a CTL epitope. CD4+CD25+ T cells from the former animals suppressed peptide-specific proliferation of CD4+CD25 and CD8+ T cells in vitro and had increased Foxp3 mRNA expression. SCID mice reconstituted with naive invariant NKT, CD4+CD25 T, and CD8+ T cells and immunized with the self-Ag plus the CTL Ag had more CTL Ag-specific CD8+ T cells than controls. Mice immunized with a plasmid encoding the self-Ag plus one encoding IFN-γ did not have enhanced pulmonary metastases, and their CD4+CD25+ T cells did not suppress peptide-specific proliferation of CD4+CD25 T or CD8+ T cells. Pulmonary metastases were enhanced in SCID mice reconstituted with a mixture of wild-type invariant NKT and CD4+ T cells plus CD8+ T cells from IFN-γ−/− mice and immunized with self-Ag and CTL epitope. The data indicate that in vivo generation or activation of CD4+CD25+ regulatory T cells by tumor-derived self-Ags can be blocked by IFN-γ.

Mast cell degranulation occurs after clathrin-mediated endocytosis of the IgE/FcεRI complex and is a central feature of an allergic reaction. It is not known whether ubiquitin ligase Cbl, which targets receptor subunits for degradation, and the multidomain adaptor protein CIN85 (Cbl-interacting protein of 85 kDa) cooperate to control internalization of engaged FcεRIs. Molfetta et al. (p. 4208 ) used anti-CIN85 Abs to coprecipitate CIN85 and Cbl from rat basophilic leukemia (RBL) cells treated with anti-DNP IgE mAb and stimulated with a multivalent DNP compound. The CIN85/Cbl association directly correlated with c-Cbl tyrosine phosphorylation. Stably transfected RBL cells overexpressing wild-type CIN85 had enhanced FcεRI down-modulation 30 min after stimulation compared with stimulated cells transfected with an empty vector or CIN85 mutants unable to form a trimolecular complex with Cbl and endophilin. Rapid receptor redistribution from the cell surface to early endosomes was seen in 31% of stimulated cells overexpressing wild-type CIN85 that had been fixed, permeabilized, and treated with FITC-labeled anti-ligand Ab compared with 9.5% of control cells. By 90 min after stimulation, 55% of the receptors colocalized with late endosomes and lysosomes in cells overexpressing wild-type CIN85 compared with 28% in control cells. Only stimulated wild-type CIN85-overexpressing cells showed decreased degranulation as measured by release of β-hexosaminidase. The results support a role for CIN85/Cbl in internalization and degradation of FcεRI following Ag stimulation of RBL cells.

Summaries written by Dorothy L. Buchhagen, Ph.D.