Bone marrow–derived antigen-presenting cells (APCs) take up cell-associated antigens and present them in the context of major histocompatibility complex (MHC) class I molecules to CD8+ T cells in a process referred to as cross-priming. Cross-priming is essential for the induction of CD8+ T cell responses directed towards antigens not expressed in professional APCs. Although in vitro experiments have shown that dendritic cells (DCs) and macrophages are capable of presenting exogenous antigens in association with MHC class I, the cross-presenting cell in vivo has not been identified. We have isolated splenic DCs after in vivo priming with ovalbumin-loaded β2-microglobulin–deficient splenocytes and show that they indeed present cell-associated antigens in the context of MHC class I molecules. This process is transporter associated with antigen presentation (TAP) dependent, suggesting an endosome to cytosol transport. To determine whether a specific subset of splenic DCs is involved in this cross-presentation, we negatively and positively selected for CD8− and CD8+ DCs. Only the CD8+, and not the CD8−, DC subset demonstrates cross-priming ability. FACS® studies after injection of splenocytes loaded with fluorescent beads showed that 1 and 0.6% of the CD8+ and the CD8− DC subsets, respectively, had one or more associated beads. These results indicate that CD8+ DCs play an important role in the generation of cytotoxic T lymphocyte responses specific for cell-associated antigens.

The minor histocompatibility antigen (mHag) HA-1 is the only known mHag for which mismatching is correlated with the development of severe graft versus host disease (GvHD) after human leukocyte antigen-identical bone marrow transplantation. HA-1 was found to be a nonapeptide derived from an allele of the KIAA0223 gene. The HA-1-negative allelic counterpart encoded by KIAA0223 had one amino acid difference from HA-1. Family analysis with HA-1 allele-specific polymerase chain reaction showed an exact correlation between this allelic polymorphism and the HA-1 phenotype. HA-1 allele typing of donor and recipient should improve donor selection and allow the determination of bone marrow transplantation recipients with high risk for HA-1-induced GvHD development.

Murine splenic dendritic cells (DCs) can be divided into two subsets based on CD8α expression, but the specific role of each subset in stimulation of T cells is largely unknown. An important function of DCs is the ability to take up exogenous antigens and cross-present them in the context of major histocompatibility complex (MHC) class I molecules to CD8+ T cells. We previously demonstrated that, when cell-associated ovalbumin (OVA) is injected into mice, only the CD8+ DC subset cross-presents OVA in the context of MHC class I. In contrast to this selectivity with cell-associated antigen, we show here that both DC subsets isolated from mice injected with OVA/anti-OVA immune complexes (OVA-IC) cross-present OVA to CD8+ T cells. The use of immunoglobulin G Fc receptor (FcγR) common γ-chain–deficient mice revealed that the cross-presentation by CD8− DCs depended on the expression of γ-chain–containing activating FcγRs, whereas cross-presentation by CD8+ DCs was not reduced in γ-chain–deficient mice. These results suggest that although CD8+ DCs constitutively cross-present exogenous antigens in the context of MHC class I molecules, CD8− DCs only do so after activation, such as via ligation of FcγRs. Cross-presentation of immune complexes may play an important role in autoimmune diseases and the therapeutic effect of antitumor antibodies.

H-Y is a transplantation antigen that can lead to rejection of male organ and bone marrow grafts by female recipients, even if the donor and recipient match at the major histocompatibility locus of humans, the HLA (human leukocyte antigen) locus. However, the origin and function of H-Y antigens has eluded researchers for 40 years. One human H-Y antigen presented by HLA-B7 was identified as an 11-residue peptide derived from SMCY, an evolutionarily conserved protein encoded on the Y chromosome. The protein from the homologous gene on the X chromosome, SMCX , differs by two amino acid residues in the same region. The identification of H-Y may aid in transplantation prognosis, prenatal diagnosis, and fertilization strategies.

The spleen is the lymphoid organ that induces immune responses toward blood-borne pathogens. Specialized macrophages in the splenic marginal zone are strategically positioned to phagocytose pathogens and cell debris, but are not known to play a role in the activation of T-cell responses. Here we demonstrate that splenic marginal metallophilic macrophages (MMM) are essential for crosspresentation of blood-borne antigens by splenic dendritic cells (DCs). Our data demonstrate that antigens targeted to MMM as well as blood-borne adenoviruses are efficiently captured by MMM and exclusively transferred to splenic CD8 + DCs for cross-presentation and for the activation of cytotoxic T lymphocytes. Depletion of macrophages in the marginal zone prevents cytotoxic T-lymphocyte activation by CD8 + DCs after antibody targeting or adenovirus infection. Moreover, we show that tumor antigen targeting to MMM is very effective as antitumor immunotherapy. Our studies point to an important role for splenic MMM in the initial steps of CD8 + T-cell immunity by capturing and concentrating blood-borne antigens and the transfer to cross-presenting DCs which can be used to design vaccination strategies to induce antitumor cytotoxic T-cell immunity.antigen presentation | infection T he spleen is essential for the induction of immune responses toward blood-borne antigens and has a unique architecture. Arterial blood flow terminates in marginal sinuses situated in the marginal zone (MZ) that surrounds the white pulp containing Bcell follicles and T-cell zones. Marginal sinuses are lined by reticular cells and contain marginal zone B cells and two types of macrophages (Mφ) (1, 2). Marginal metallophilic macrophages (MMM), characterized by the expression of sialic acid-binding Iglike lectin-1 (Siglec-1, Sialoadhesin, CD169) (3, 4), are located as a tight network in the inner part of the MZ near the white pulp, whereas marginal zone macrophages (MZM), which specifically express the C-type lectin SIGN-R1, can be found in the outer MZ toward the red pulp (5). Both MZM and a subset of MMM express the type I scavenger receptor MARCO (6). Although MMM and MZM efficiently take up particulate antigens (Ag) present in the blood (7-9), they are hitherto considered not to be important for the generation of T-cell responses (8-10).In contrast to Mφ, dendritic cells (DCs) are specialized Agpresenting cells that have a dominant role in initiating primary Tcell responses. Murine splenic DCs can be divided into two different subsets based on the expression of phenotypic markers: localization and function (11). CD8 + DCs express the C-type lectin DEC205 and are found in the T-cell zone and the outer marginal zone (12). They are specialized in cross-presentation of Ag and in the activation and tolerization of cytotoxic T cells (CTLs) (13-16). Furthermore, they are important for the generation of antitumor specific immune responses and the elimination of tumors in vivo (17). In contrast, CD8− DCs are specialized in the activation of CD4 + T cells. CD8 − DCs are...

Minor histocompatibility antigen disparities between human leukocyte antigen (HLA)-matched bone marrow donors and recipients are a major risk factor for graft versus host disease (GVHD). An HLA-A2.1-restricted cytotoxic T cell clone that recognized the minor histocompatibility antigen HA-2 was previously isolated from a patient with severe GVHD after HLA-identical bone marrow transplantation. The HLA-A2.1-bound peptide representing HA-2 has now been identified. This peptide appears to originate from a member of the non-filament-forming class I myosin family. Because HA-2 has a phenotype frequency of 95 percent in the HLA-A2.1-positive population, it is a candidate for immunotherapeutic intervention in bone marrow transplantation.

A peptide recognized by two cytotoxic T cell clones specific for the human minor histocompatibility antigen H-Y and restricted by HLA-A*0201 was identified. This peptide originates from SMCY, as do two other H-Y epitopes, supporting the importance of this protein as a major source of H-Y determinants in mice and humans. In naturally processed peptides, T cells only recognize posttranslationally altered forms of this peptide that have undergone modification of a cysteine residue in the seventh position. One of these modifications involves attachment of a second cysteine residue via a disulfide bond. This modification has profound effects on T cell recognition and also occurs in other class I MHC-associated peptides, supporting its general importance as an immunological determinant.

Sialic acids are negatively charged nine-carbon carboxylated monosaccharides that often cap glycans on glycosylated proteins and lipids. Because of their strategic location at the cell surface, sialic acids contribute to interactions that are critical for immune homeostasis via interactions with sialic acid-binding Ig-type lectins (siglecs). In particular, these interactions may be of importance in cases where sialic acids may be overexpressed, such as on certain pathogens and tumors. We now demonstrate that modification of antigens with sialic acids (Sia-antigens) regulates the generation of antigenspecific regulatory T (Treg) cells via dendritic cells (DCs). Additionally, DCs that take up Sia-antigen prevent formation of effector CD4 + and CD8 + T cells. Importantly, the regulatory properties endowed on DCs upon Sia-antigen uptake are antigen-specific: only T cells responsive to the sialylated antigen become tolerized. In vivo, injection of Sia-antigen-loaded DCs increased de novo Treg-cell numbers and dampened effector T-cell expansion and IFN-γ production. The dual tolerogenic features that Sia-antigen imposed on DCs are Siglec-E-mediated and maintained under inflammatory conditions. Moreover, loading DCs with Sia-antigens not only inhibited the function of in vitro-established Th1 and Th17 effector T cells but also significantly dampened ex vivo myelinreactive T cells, present in the circulation of mice with experimental autoimmune encephalomyelitis. These data indicate that sialic acid-modified antigens instruct DCs in an antigen-specific tolerogenic programming, enhancing Treg cells and reducing the generation and propagation of inflammatory T cells. Our data suggest that sialylation of antigens provides an attractive way to induce antigen-specific immune tolerance. sialic acids | regulatory T cells | dendritic cells | tolerance | glycans