Immunoglobulin G4-related disease is a poorly understood systemic chronic inflammatory disorder in which it is not clear whether IgG4 promotes or inhibits autoimmunity, nor is it known how production of this Ab is stimulated through T cell-dependent and/or innate immune mechanisms. Autoimmune pancreatitis (AIP) is a common organ-specific manifestation of IgG4-related disease. In this issue, Arai et al. (p. 3033) identified major contributions of plasmacytoid dendritic cells (pDCs) to AIP in both mice and humans. In a mouse AIP model involving treatment of MRL/Mp mice with polyinosinic-polycytidylic acid [poly (I:C)], pDCs were increased in the pancreatic infiltrate, and levels of type I IFNs and BAFF were elevated in the serum. In addition, neutrophil extracellular traps (NETs), which can induce IFN-α production by pDCs, were observed in the pancreatic tissue of the poly (I:C)-treated mice. Depletion of pDCs or blockade of the IFNAR inhibited AIP development, IFN-α and BAFF production, and NET formation in these mice, indicating that pDC infiltration of the pancreas and IFN-α signaling were required for disease development. pDCs expressing IFN-α and BAFF were found in the inflamed pancreas of patients with IgG4-related AIP, and these pancreatic lesions also contained NETs that may have formed in response to anti-lactoferrin immune complexes. In vitro, NETs combined with pDCs from AIP patients, but not control pDCs, could induce IgG4 production by control B cells through a process requiring IFN-α. These results from both mice and patients suggest that NET-triggered activation of pDCs and subsequent IFN-α production is important for the development of AIP.

Leishmaniasis is a chronic tropical disease in which type 1 immune responses are associated with control of Leishmania infection and type 2 responses with worsening of disease. Coinfections, which can modulate the immune response to Leishmania, are likely to occur in leishmaniasis patients due to the long-term nature of parasite infection. Crosby et al. (p. 3301) hypothesized that infection of Leishmania major-infected mice with lymphocytic choriomeningitis virus (LCMV), which generally elicits high levels of IFN-γ production, would promote resolution of infection. Surprisingly, LCMV infection initiated 2 wk after L. major infection exacerbated leishmaniasis severity, which was associated with increased intralesion infiltration of cells including neutrophils, CD8+ T cells, and CD4+ T cells, but reduced infiltration of Th1-promoting inflammatory monocytes. However, 3 wk following viral infection, lymph node cells from coinfected mice produced increased basal levels of IFN-γ relative to controls, but CD4+ T cells in these mice showed impaired responses to L. major. Within the lesions of coinfected mice, infiltrating CD8+ T cells expressed granzyme B and upregulated expression of NKG2D. Depletion of these CD8+ T cells or blockade of NKG2D reduced disease to levels seen in mice infected with L. major alone, which was associated with reduced infiltration of neutrophils and increased infiltration of inflammatory monocytes into the lesions. These data reveal a mechanism by which viral infection might exacerbate cutaneous leishmaniasis and suggest that such mechanisms are more complicated than simple predictions of enhanced type 1 or type 2 immunity.

Human killer Ig-like receptors (KIRs) and the HLA class I molecules they bind show high degrees of variability which allow for very diverse NK cell responses. Hilton et al. (p. 3160) have now analyzed the genomic organization and functional properties of all of the known lineage III KIR alleles, which encode KIRs that consist of the KIR2DL2/3 molecules that mainly recognize the HLA-C1 (C1) epitope and KIR2DL1/KIR2DS1 molecules that recognize the HLA-C2 (C2) epitope. The human KIR haplotypes are divided into two functional groups: A, which mainly encode inhibitory receptors, and B, which encode both inhibitory and activating receptors. The authors found that the alleles encoding the C1-specific or C2-specific KIRs could each be classified into four phylogenetic clades which differentially mapped to A or B haplotypes in the centromeric (Cen) regions except for KIR2DS1 alleles, which were instead found in the telomeric (Tel) region of KIR B haplotypes. KIR2DL1 molecules encoded by Cen A bound C2 with higher avidity than those encoded by Cen B, and the residues responsible for binding and signaling differences between the two haplotypes were identified. Cen B-encoded KIR2DL2/3 molecules, in contrast, had higher avidity for C1 (and, although binding was weak overall, C2) than those encoded by Cen A. Thus, although inhibitory receptors for both C1 and C2 were found in both Cen A and Cen B, the different haplotypes showed preferences for C2 or C1 binding, respectively. Further analysis of the KIR2DL molecules determined requirements for the cell surface expression and proper folding of KIR2DL1 and the effects of variation in two residues on both specificity and avidity of KIR2DL2/3 alleles. The authors also analyzed how differences in the amino acid sequences of the ligand-binding domains of KIR2DS1 allotypes affected the avidity of their binding to C2. This study provides a great deal of structural insight into KIR function and suggests that although most KIR haplotypes include receptors capable of recognizing either HLA-C epitope, it is likely that only one of the KIR2DL loci in a given haplotype encodes a strong inhibitory receptor.

Dengue virus (DENV) is a major global health burden and vaccine development is complicated by the existence of four distinct serotypes (DENV1-4) and the fact that non-neutralizing Abs induced to one serotype may enhance infection upon exposure to another serotype. Therefore, detailed understanding of the humoral response to DENV is needed to develop protective vaccines. Traditional ELISpots using plates coated with Ag make identification of individual cells making cross-reactive Abs difficult. In contrast, the Quad-Color FluoroSpot (QCF) designed by Hadjilaou et al. (p. 3490) determines B cell DENV specificity and cross-reactivity for all four serotypes at the single-cell level. Plates were coated with an anti-human IgG to capture all secreted Abs before culture with hybridomas or stimulated cells and then 1 μg of each DENV serotype was added to each well. Spots were then detected by a mixture of serotype-specific human and mouse mAbs labeled with distinct Qdot fluorophores. Control experiments using a single DENV serotype or individual mAb detection ensured that using all four viral serotypes and a mixture of detecting mAbs did not skew or hinder detection when used in combination. QCF analysis of stimulated cells from a convalescent primary DENV3-infected patient showed some cross-reactivity, with half of the cells specific for only DENV3, whereas simulated cells from a patient with secondary DENV3 revealed that 96% of the cells were cross-reactive to all four DENV serotypes. The QCF can provide a useful tool not only for dengue researchers, but also for those studying immune responses to other infectious diseases that exhibit multiple serotypes.

A variety of genetic mutations can result in different forms of SCID, which are categorized based on the presence of T, B, and/or NK cells. Swine can serve as a useful large animal model for humans, and a naturally occurring T- B- NK+ SCID was recently reported in a line of Yorkshire pigs at Iowa State University. Waide et al. (p. 3171) have now identified the genetic basis for immunodeficiency in these pigs. Relative to healthy littermates, SCID pigs had dramatically increased numbers of immature double negative thymocytes, suggesting that a developmental arrest occurred before TCR rearrangement but late enough in development that NK cells were unaffected. The authors proposed that these SCID pigs had a defect in the DNA damage repair machinery, as their fibroblasts were significantly more sensitive to ionizing radiation than cells of control animals. A genome-wide association study identified a region on chromosome 10 that contained the Artemis gene and was associated with autosomal recessive transmission of SCID in these pigs, corresponding to observations that mutations in human and mouse Artemis can cause T- B- NK+ SCID. Two mutations were identified in porcine Artemis, of which one disrupted exon splicing and the other resulted in a frameshift and a premature stop codon. In pigs with SCID, these mutations were present in either a homozygous or compound heterozygous state. Transfection of porcine SCID fibroblasts with human Artemis could restore their resistance to ionizing radiation, further confirming the role of Artemis in SCID. In addition to veterinary immunological studies, these SCID pigs could serve as a useful preclinical model for a variety of human studies.

Humans with chemokine receptor polymorphisms affecting monocyte chemotaxis are protected from cardiovascular events, identifying monocyte recruitment as a pathway that could be a useful therapeutic target. Mukovozov et al. (p. 3334) examined the effect of Slit2, a secreted neurorepellent, on monocyte adhesion and chemotaxis. Both murine and human monocytes expressed the Slit2 receptor Robo-1, and Slit2 was able to inhibit monocyte chemotaxis to CCL2 and CXCL12 in transwell assays by inhibiting Rac1, Cdc42, and phosphorylation of AKT. Exposure to N-Slit2, a bioactive N-terminal fragment of Slit2 that retains binding ability to Robo-1, also decreased monocyte adhesion to immobilized ICAM-1 and VCAM-1. Exposure to N-Slit2 did not inhibit monocyte rolling or tethering to activated endothelial cells in vitro, but resulted in enhanced monocyte detachment upon exposure to increasing shear force, indicating that Slit2 interferes with the later stages of monocyte recruitment. Cells expressing a constitutively active form of Rac1 were resistant to N-Slit2, confirming that Rac1 mediated the Slit2-induced inhibition of postadhesion stabilization. N-Slit2 treatment of low density lipoprotein receptor deficient mice, a common atherosclerosis model, reduced proliferating CD45+ cells in nascent atherosclerotic lesions. Compared to PBMCs from healthy control subjects, PBMCs from patients with coronary artery disease had lower levels of Robo-1, and the ability of Slit2 to inhibit CCL2 chemotaxis was blunted. These data suggest that the Slit-2/Robo-1 axis may be an appealing therapeutic target for vascular inflammatory diseases.