IN THIS ISSUE

doi:10.4049/jimmunol.178.5.2605

SAXSy MBL Conformations

⇓ The pattern recognition molecule mannan-binding lectin (MBL) facilitates C-mediated pathogen clearance through recognition of conserved motifs on microbial surfaces. Structural analyses of MBL fragments by electron microscopy or x-ray crystallography indicate that it is a complex molecule. To determine its complete three-dimensional structure and any changes that occur during binding, Dong et al. (p. 3016 ) first applied small-angle x-ray scattering (SAXS) to MBL in solution under physiological conditions. They determined that nine MBL monomers formed a large MBL complex with a 3-fold rotational symmetry axis; three trimers of collagen-like stalks with carbohydrate recognition domains (CRDs) joined in a common hub. MBL molecules used in SAXS were immobilized on a surface by nonspecific electrostatic interactions and subjected to atomic force microscopy to obtain resolution at the single molecule level. MBL oligomers assumed a bagel-like appearance with the globular CRDs forming a circular ring. MBL assumed a conformation with greatly stretched stalks after binding to mannosamine-, but not to galactosamine-, coated surfaces in the wet or dry state. This combined use of SAXS and atomic force microscopy demonstrate that the diameter of ligand-bound MBL is ∼3-fold larger than oligomers in solution. The authors propose that the increase is due to unbending of the stalk curvature after CRD binding to surface mannosamine.

Protein Transfer to Kill Tumors

Studies show that immunostimulatory or inhibitory molecules are able to increase antitumor immunity. To overcome the limited efficacy of these treatments applied singly, Liu et al. (p. 3301 ) used their protein transfer method to inject mouse tumors with two chemotactic molecules to increase infiltration of neutrophils, dendritic cells (DCs) and T cells, followed by injection with two costimulatory molecules to increase functions of DCs and T cells. Injection with the two chemotactic molecules or with all four proteins increased infiltration of neutrophils and CD4⁺ and CD8⁺ T cells compared with controls, whereas an increase in DCs was seen only after injection with the four proteins. Injection with the chemotactic proteins or all four proteins reduced intratumoral regulatory T cells by >80%. The cytokine profile of cells isolated from tumors treated with all four proteins was predominantly Th1 with high IL-12 and IFN-γ and low IL-10 levels. Although growth of some established lymphomas was delayed or occasionally regressed after injection with either the chemotactic or costimulatory molecules, complete tumor regression was seen in 65% of tumors treated with all four pro-teins. Mice whose tumors regressed were protected against i.p. tumor challenge 2 or 6 mo later, and their CTLs had strong in vitro activity against the tumor cells 4-6 wk after rechallenge. The authors demonstrate that a combination of defined amounts of immunostimulatory molecules delivered to a mouse lymphoma by protein transfer enhances antitumor responses.

Role of IGF-1R in Graves’ Disease

Smith and collaborators reported expression of insulin-like growth factor-1R (IGF-1R) by a large fraction of fibroblasts from patients with autoimmune Graves’ disease (GD). However, it is not known how lymphocytes are recruited to the orbit to cause thyroid-associated ophthalmopathy (TAO). In a follow-up to their earlier work, Douglas et al. (p. 3281 ) in the Smith laboratory measured substantially higher expression of IGF-1R on peripheral blood and orbital CD4⁺ and CD8⁺ memory T cells from GD patients vs controls. In contrast, controls and a GD patient had similar high IGF-1R levels on bone marrow T cells. Anti-CD3 Ab plus IGF-1, or an IGF-1 analog that selectively activates IGF-1R, expanded IGF-1R⁺ T cells in culture greater than GD T cells exposed to anti-CD3 Ab alone. T cells from healthy donors did not respond to IGF-1 or the analog. Only GD T cells proliferated in response to GD-IgG, and IGF-1 reduced apoptosis only in GD T cells. The authors conclude that up-regulation of IGF-1R is responsible for proliferation and reduced apoptosis of inflammatory memory T cells recruited to orbital tissue in patients with GD.

FcγRIIA/Lipid Raft Association

⇓ Although activated immunoreceptor FcγRIIA is known to associate with lipid rafts to initiate a series of intracellular signaling events, the roles played by different receptor domains have not been delineated. García-García et al. (p. 3048 ) generated human FcγRIIA mutants defective in palmitoylation (Pal⁻), in phosphorylation of the ITAM (ITAM⁻), in the cytoplasmic domain (Cyt⁻), or in the transmembrane domain (TM⁻). Stable transfectants were created by introduction of these mutants into human cells lacking endogenous FcγRIIA. Analysis of detergent-resistant membrane (DRM) fractions from receptor-activated cells showed that ITAM⁻, Pal⁻, Cyt⁻, or most of the TM⁻ mutants were as capable as wild-type FcγRIIA of associating with lipid rafts. One TM⁻ mutant was not found in the DRM fraction, and two TM⁻ mutants were found in both the nonlipid raft and DRM fractions after activation. All TM⁻ mutants induced Syk activation, but two had kinetics slightly different from that of the wild-type receptor. DRM association was required for activation of NF-κB and for reduction in ERK phosphorylation; NF-κB activation and ERK phosphorylation were greater in mutants constitutively associated with DRM. Phagocytosis was efficient in wild-type FcγRIIA-transfected cells but was at lower levels in cells transfected with constitutively DRM-associated mutants. Phagocytosis in all cells was reduced in the presence of a Syk inhibitor. The authors conclude that only the TM domain of human FcγRIIA regulates its association with lipid rafts following ligand-induced cross-linking.

Investigating CIA Loci

⇓ Standard F2 crosses between susceptible and resistant strains of mice have identified a large number of quantitative trait loci for a variety of diseases. However, finer dissection of major loci is necessary to sort out genes involved in complex diseases. Ahlqvist et al. (p. 3084 ) used a partial advanced intercross (PAI) in combination with subcongenic strains to map new loci involved in collagen-induced arthritis (CIA), the mouse model for rheumatoid arthritis. The parents of the PAI were CIA-resistant C57BL/10 mice, one carrying a chromosome 7 and the other a chromosome 15 fragment from CIA-susceptible CBA/2 mice. Both parents had increased CIA susceptibility and anticollagen IgG production compared with the resistant C57BL/10 parent. After seven generations of PAI, the chromosome 15 linkage to CIA was retained and found to be higher in males. Two new loci were uncovered on chromosome 7; one interacted with a chromosome 15 locus to affect Ab production, whereas the other protected against arthritis. Subcongenic strains obtained by backcrossing PAI animals confirmed five new chromosome 15 subloci. Two loci increased CIA and Ab production in male mice, only one of which had an effect in female mice; a third locus increased IgG3 production. Two additional subloci were protective in that they decreased both CIA susceptibility and Ab production. Thus, the experiments show that the greater number of recombinations occurring in PAI vs standard F2 crosses permits detection of seven new disease-modifying loci for the complex disease CIA.

Understanding Immunodominance

Factors that determine immunodominance hierarchies of primary and secondary virus-specific CD8⁺ T cell responses are not fully understood. However, suggestions that TCR repertoire size and Ag load are important prompted Thomas et al. (p. 3091 ) to look at compensatory expansion of CD8⁺ T cells of other specificities in the absence of dominant epitopes in influenza virus infection. C57BL/6 mice infected i.p. with influenza virus were boosted intranasally (i.n.) 6 wk later with a serologically distinct influenza virus strain. Mice primed/boosted with viruses containing double knockouts (DKO) of two major MHC class I epitopes generated increased splenic CD8⁺ T cell responses specific for a third epitope after a secondary DKO virus challenge. Mice primed/boosted with triple knockout (TKO) viruses did not have an enhanced recall response. Mice infected i.n. with the TKO variant cleared the infection more rapidly and had higher virus-specific serum Ig levels 5 days later, whereas TKO-infected IgM^−/− or MHCII^−/− mice had worse clinical symptoms and lower virus clearance than controls infected i.n. with DKO or wild-type virus. However, only DKO virus-infected IgM^−/− or MHCII^−/− mice developed compensatory responses to other epitopes after DKO virus challenge. Secondary responses to minor epitopes were seen in both DKO and TKO virus-infected MHCII^−/− animals. The authors suggest a complex interplay between CD8⁺ T cell-mediated and humoral immunity in the development of immunodominance hierarchies during influenza virus infections in mice.

Aire-Regulated MTEC Development

⇓ Two conflicting models propose that autoimmune regulator (aire) either depresses structural tissue-restricted Ag (TRA) expression in terminally differentiated medullary thymic epithelial cells (MTECs) or affects TRA expression and negative selection by regulating differentiation of immature thymic epithelial cells. To distinguish between these models, Gillard et al. (p. 3007 ) determined that three transcriptional factors of multipotentiality that they had previously shown to be expressed in murine aire^+/+ MTECs were expressed at reduced levels in aire^−/− MTECs. Reduced expression of five structural genes and five transcription factors associated with endocrine portions of the pancreas also was seen in aire^−/− MTECs. Alteration of MTEC organization in the aire^−/− thymus was visualized by immunohistochemistry using mAbs specific for molecules expressed in medullary, cortical, and epithelial compartments. In particular, the aire^−/− thymus had a reduced medullary compartment with more prominent epithelial cysts and dispersed globular MTECs compared with the aire^+/+ thymus. The authors propose that aire regulation of the general program of immature MTEC development influences both organization of medullary epithelium and the range of TRAs expressed in the mouse thymus.