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In this article, Derry Roopenian relates the traditional view of minor histocompatibility (H) loci to recent advances in understanding of the tissue rejection process and the molecular nature of minor histocompatibility antigens. He proposes that minor H loci can be subdivided by the ability of their products to stimulate different T-cell subsets and discusses the implications of this concept in terms of the origins and behavior of minor H loci and their antigens, tumor immunology and autoimmunity.

Therapeutic monoclonal antibodies are widely recognized to be a most promising means to treat an increasing number of human diseases, including cancers and autoimmunity. To a large extent, the efficacy of monoclonal antibody treatment is because IgG antibodies have greatly extended persistence in vivo. However, conventional rodent models do not mirror human antibody pharmacokinetics. The key molecule responsible for the extended persistence antibodies is the major histocompatibility complex class I family Fc receptor, FcRn. We describe human FcRn transgenic mouse models and how they can be exploited productively for the preclinical pharmacokinetic evaluation of therapeutic antibodies.

Interleukin 21 (IL-21) is a pleiotropic cytokine produced by CD4 T cells that affects the differentiation and function of T, B, and NK cells by binding to a receptor consisting of the common cytokine receptor γ chain and the IL-21 receptor (IL-21R). IL-21, a product associated with IL-17-producing CD4 T cells (TH17) and follicular CD4 T helper cells (TFH), has been implicated in autoimmune disorders including the severe systemic lupus erythematosus (SLE)-like disease characteristic of BXSB-Yaamice. To determine whether IL-21 plays a significant role in this disease, we compared IL-21R-deficient and -competent BXSB-Yaamice for multiple parameters of SLE. The deficient mice showed none of the abnormalities characteristic of SLE in IL-21R-competentYaamice, including hypergammaglobulinemia, autoantibody production, reduced frequencies of marginal zone B cells and monocytosis, renal disease, and premature morbidity. IL-21 production associated with this autoimmune disease was not a product of TH17 cells and was not limited to conventional CXCR5+TFHbut instead was produced broadly by ICOS+CD4+splenic T cells. IL-21 arising from an abnormal population of CD4 T cells is thus central to the development of this lethal disease, and, more generally, could play an important role in human SLE and related autoimmune disorders.

Periodontal disease is one of the most common infectious diseases of humans. Immune responses to infection trigger loss of alveolar bone from the jaw and eventual tooth loss. We investigated the contribution of B cell IgD to alveolar bone loss by comparing the response of B cell normal BALB/cJ mice and IgD deficient BALB/c-Igh-5−/−Jmice to oral infection withPorphyromonas gingivalis, a gram-negative periodontopathic bacterium from humans.P. gingivalis-infected normal mice lost bone. Specific antibody toP. gingivaliswas lower and oral colonization was higher in IgD deficient mice; yet bone loss was completely absent. Infection increased the proportion of CD69+activated B cells and CD4+T cells in immune normal mice compared to IgD deficient mice. These data suggest that IgD is an important mediator of alveolar bone resorption, possibly through antigen-specific coactivation of B cells and CD4+T cells.

The purpose of this study was to determine whether humoral immunity prevents bacterially induced alveolar bone loss. BALB/cByJ mice were orally infected with the human periodontopathic bacterium Porphyromonas gingivalis, and were compared with sham-infected mice. Specific serum antibody titers to P. gingivalis were measured by enzyme-linked immunosorbent assay. Alveolar bone levels were measured as the distance from the cementoenamel junction to the alveolar bone crest and bone loss was defined as a change in bone levels over time or between infected and sham-infected animals. The specific humoral response was predominantly of the IgG isotype, although low levels of specific serum IgA were also present. Antibody titers in the infected animals were significantly different from those in the sham-infected animals by 18 days and remained at maximal levels at 47 days. Bone loss became significant by 26 days and continued to progress at 47 days. Thus the serum antibody response to oral infection with P. gingivalis preceded detectable bone loss and remained elevated while bone loss increased. The presence of specific antibody did not prevent the onset or progression of bone loss.

The neonatal Fc receptor for IgG (FcRn) is a distant member of the MHC class I protein family. It binds IgG and albumin in a pH-dependent manner and protects these from catabolism by diverting them from a degradative fate in lysosomes. In addition, FcRn-mediated IgG transport across epithelial barriers is responsible for the transmission of IgG from mother to infant and can also enhance IgG-mediated antigen uptake across mucosal epithelia. We now show a previously undescribed role for FcRn in mediating the presentation of antigens by dendritic cells when antigens are present as a complex with antibody by uniquely directing multimeric immune complexes, but not monomeric IgG, to lysosomes.

The fine immunogenetics of the chromosome 7 mouse minor histocompatibility (H) locus H-4 was investigated. Both class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) and class II MHC-restricted "helper" T cells (TH) specifically reactive with H-4 antigens were isolated as clones and were used as genetic probes for classical backcross segregation analysis. Results of a four point cross indicated that the H-4 locus was actually comprised of two genes, that have been designated H-46 and H-47. The former encodes antigens recognized by the TH and the latter encodes antigens recognized by the CTL. Moreover, these two genes could be separated from the gene pink-eyed dilution (p) which was found to be "sandwiched" between them. The functional significance of a minor H congenic strain differing by both TH-defined H-46 and CTL-defined H-47 was addressed using F1 complementation tests. Such studies indicated that immune responses against H-46 antigens was required for generation of H-47-specific CTL. Altogether, these results suggest selective presentation of different minor H gene products by class I or class II MHC proteins and that the minor H "locus" H-4 may have necessarily included both TH and CTL-defined genes because of requisite TH-CTL collaboration.

Interleukin-21 (IL-21) is a pleiotropic cytokine whose function is only now being unraveled. Abundant evidence indicates that activated CD4 T cells are the primary, if not the only, source of IL-21. While it is clear that IL-21 is actively transcribed by naive activated T cells, recent studies have shown that IL-21 potentially promotes a developmental shift of naive T cells toward the Th2 phenotype. BXSB-Yaa mice develop an autoimmune syndrome similar to systemic lupus erythematosus (SLE), affecting males earlier than females on account of the presence of the Yaa (Y-linked autoimmune acceleration) locus. Previous results indicate the elevation of IL-21 expression by BXSB-Yaa mice at an age when the early characteristics of autoimmune processes first become evident. We set out to determine whether IL-21 was necessary for disease progression in BXSB-Yaa mice. Mice were treated for 24 weeks with soluble IL-21R-Fc in order to therapeutically neutralize the IL-21 present. The results overall suggest a biphasic effect of IL-21, negatively influencing survival early on and positively influencing survival at later stages. We propose that IL-21 exerts a pleiotropic effect in which it promotes the protective effects of CD8+ suppressor cells in the early disease phase and then promotes the humoral components of SLE in the later disease stages. This experiment provides preliminary evidence for a role of IL-21 in modulating the severity of SLE in BXSB-Yaa mice.

The neonatal FcR (FcRn) is a receptor that protects IgG from catabolism and is important in maintaining high serum Ab levels. A major site of expression of FcRn is vascular endothelial cells where FcRn functions to extend the serum persistence of IgG by recycling internalized IgG back to the surface. Because FcRn is expressed in other tissues, it is unclear whether endothelial cells are the only site of IgG protection. In this study, we used FcRn-deficient mice and specific antiserum to determine the tissue distribution of FcRn in the adult mouse. In addition to its expression in the vascular endothelium of several organs, we found FcRn to be highly expressed in bone marrow-derived cells and professional APCs in different tissues. Experiments using bone marrow chimeras showed that FcRn expression in these cells acted to significantly extend the half-life of serum IgG indicating that in addition to the vascular endothelium, bone marrow-derived phagocytic cells are a major site of IgG homeostasis.

The neonatal Fc receptor for IgG (FcRn) functions to transport maternal IgG to a fetus or newborn and to protect IgG from degradation. Although FcRn is expressed in a variety of tissues and cell types, the extent to which FcRn expression is regulated by immunological and inflammatory events remains unknown. Stimulation of intestinal epithelial cell lines, macrophage-like THP-1, and freshly isolated human monocytes with the cytokine TNF-α rapidly up-regulated FcRn gene expression. In addition, the TLR ligands LPS and CpG oligodeoxynucleotide enhanced the level of FcRn expression in THP-1 and monocytes. Treatment of TNF-stimulated THP-1 cells with the NF-κB-specific inhibitor or overexpression of a dominant negative mutant inhibitory NF-κB (IκBα; S32A/S36A) resulted in down-regulation of FcRn expression. By using chromatin immunoprecipitation we identified three NF-κB binding sequences within introns 2 and 4 of the human FcRn gene. An EMSA confirmed the p50/p50 and/or p65/p50 complex (s) bound to intron 2- or 4-derived oligonucleotides containing putative NF-κB binding sequences, respectively. The intronic NF-κB sequences in combination with the promoter or alone regulated the expression of a luciferase reporter gene in response to TNF-α stimulation or overexpression of NF-κB p65 and p50. DNA looping interactions potentially occurred after the stimulation between intronic NF-κB sequences and the FcRn promoter as shown by a chromosome conformation capture assay. Finally, TNF-α stimulations enhanced IgG transport across an intestinal Caco-2 epithelial monolayer. Together, these data provide the first evidence that NF-κB signaling via intronic sequences regulates FcRn expression and function.

Nonhomologous end joining (NHEJ) is a critical DNA repair pathway, with proposed tumor suppression functions in many tissues. Mutations in the NHEJ factor ARTEMIS cause radiation-sensitive severe combined immunodeficiency in humans and may increase susceptibility to lymphoma in some settings. We now report that deficiency for Artemis (encoded by Dclre1c/Art in mouse) accelerates tumorigenesis in several tissues in a Trp53 heterozygous setting, revealing tumor suppression roles for NHEJ in lymphoid and non-lymphoid cells. We also show that B-lineage lymphomas in these mice undergo loss of Trp53 heterozygosity by allele replacement, but arise by mechanisms distinct from those in Art Trp53 double null mice. These findings demonstrate a general tumor suppression function for NHEJ, and reveal that interplay between NHEJ and Trp53 loss of heterozygosity influences the sequence of multi-hit oncogenesis. We present a model where p53 status at the time of tumor initiation is a key determinant of subsequent oncogenic mechanisms. Because Art deficient mice represent a model for radiation-sensitive severe combined immunodeficiency, our findings suggest that these patients may be at risk for both lymphoid and non-lymphoid cancers.

Therapeutic monoclonal antibodies are widely recognized to be a most promising means to treat an increasing number of human diseases, including cancers and autoimmunity. To a large extent, the efficacy of monoclonal antibody treatment is because IgG antibodies have greatly extended persistence in vivo. However, conventional rodent models do not mirror human antibody pharmacokinetics. The key molecule responsible for the extended persistence antibodies is the major histocompatibility complex class I family Fc receptor, FcRn. We describe human FcRn transgenic mouse models and how they can be exploited productively for the preclinical pharmacokinetic evaluation of therapeutic antibodies.

The introduction of germ line modifications by gene targeting in mouse embryonic stem (ES) cells has proven a fundamental technology to relate genes to mammalian biology. Critical aspects required for successful gene targeting have traditionally been experimental enhancements that increase the frequency or detection of homologous recombination within ES cells; however, the utilization of such methods may still result in the failed isolation of a positively targeted ES cell clone. In this study, we discuss the current enhancement methods and describe an ES cell pooling strategy that maximizes the ability to detect properly targeted ES cells regardless of an inherent low targeting efficiency. The sensitivity required to detect correctly targeted events out of a pool of ES cell clones is provided by polymerase chain reaction (PCR), and only those pools containing positives need to be expanded and screened to find individually targeted clones. This method made it possible to identify targeted clones from a screen of approximately 2,300 ES cell colonies by performing only 123 PCR reactions. This technically streamlined approach bypasses the need to troubleshoot and re-engineer an existing targeting construct that is functionally suitable despite its low targeting frequency.

The identification of the neonatal FcR (FcRn) as an IgG homeostasis regulator has led to research aimed at delineating a role for FcRn in humorally mediated disease. FcRn is a class I-related molecule that prolongs the half-life of serum IgG by preferentially binding IgG at low pH and inhibiting its degradation. Its role in protective immunity to infectious organisms is unknown. We investigated the function of FcRn in the murine model of Lyme arthritis, caused by infection with Borrelia burgdorferi. We infected FcRn � /� and wild-type mice with B. burgdorferi and monitored the development of arthritis. Infected FcRn � /� mice demonstrated decreased serum levels of anti-B. burgdorferi antibodies and borreliacidal activity. Moreover, these mutant mice developed increased ankle swelling and joint histopathology following infection. Our data suggest that FcRn ameliorates murine Lyme arthritis by preventing the degradation of protective borreliacidal antibodies.

It is now understood that the nonclassical major histocompatibility complex-I molecule FcRn binds albumin and retrieves it from an intracellular degradative fate. Whether FcRn in the liver modulates albumin turnover through effects on biosynthesis and production is not known. Thus we quantified the appearance of biosynthetically labeled albumin in plasma after an intravenous bolus injection of [3H]leucine in FcRn-deficient mice. The production rates for both albumin (FcRn substrate) and transferrin (nonsubstrate) are increased by ∼20% in FcRn-deficient mice compared with normal mice, likely compensating for the lowered plasma oncotic pressure caused by hypoalbuminemia in FcRn-deficient mice. Determining the magnitude of FcRn-mediated effects on albumin turnover, we then measured the steady-state plasma concentrations of biosynthetically labeled albumin and transferrin during [3H]leucine infusion. The concentration of albumin was ∼40% lower in FcRn-deficient mice compared with normal mice. Furthermore, the ∼40% lower plasma albumin concentration in FcRn-deficient mice along with the ∼20% increase in albumin production indicate, by the mass-balance equation, that albumin degradation in FcRn-deficient mice is twice that of normal mice. These studies of biosynthetically labeled, and thus native, albumin support our previous finding that FcRn protects albumin from degradation. Permitting quantification of the magnitude of FcRn-mediated recycling, they further indicate that FcRn has extraordinary capacity: the amount of albumin saved from degradation by FcRn-mediated recycling is the same as that produced by the liver.

Background Recent developments in sequence databases provide the opportunity to relate the expression pattern of genes to their genomic position, thus creating a transcriptome map. Quantitative trait loci (QTL) are phenotypically-defined chromosomal regions that contribute to allelically variant biological traits, and by overlaying QTL on the transcriptome, the search for candidate genes becomes extremely focused. Results We used our novel data mining tool, ExQuest, to select genes within known diabesity QTL showing enriched expression in primary diabesity affected tissues. We then quantified transcripts in adipose, pancreas, and liver tissue from Tally Ho mice, a multigenic model for Type II diabetes (T2D), and from diabesity-resistant C57BL/6J controls. Analysis of the resulting quantitative PCR data using the Global Pattern Recognition analytical algorithm identified a number of genes whose expression is altered, and thus are novel candidates for diabesity QTL and/or pathways associated with diabesity. Conclusion Transcription-based data mining of genes in QTL-limited intervals followed by efficient quantitative PCR methods is an effective strategy for identifying genes that may contribute to complex pathophysiological processes.

NKT cell activation by α-galactosylceramide (α-GalCer) inhibits autoimmune diabetes in NOD mice, in part by inducing recruitment to pancreatic lymph nodes (PLNs) of mature dendritic cells (DCs) with disease-protective effects. However, how activated NKT cells promote DC maturation, and what downstream effect this has on diabetogenic T cells was unknown. Activated NKT cells were found to produce a soluble factor(s) inducing DC maturation. Initially, there was a preferential accumulation of mature DCs in the PLNs of α-GalCer-treated NOD mice, followed by a substantial increase in T cells. Adoptive transfer of a diabetogenic CD8 T cell population (AI4) induced a high rate of disease (75%) in PBS-treated NOD recipients, but not in those pretreated with α-GalCer (8%). Significantly, more AI4 T cells accumulated in PLNs of α-GalCer than PBS-treated recipients, while no differences were found in mesenteric lymph nodes from each group. Compared with those in mesenteric lymph nodes, AI4 T cells entering PLNs underwent greater levels of apoptosis, and the survivors became functionally anergic. NKT cell activation enhanced this process. Hence, activated NKT cells elicit diabetes protection in NOD mice by producing a soluble factor(s) that induces DC maturation and accumulation in PLNs, where they subsequently recruit and tolerize pathogenic T cells.

CD8 T lymphocytes (CTL) responsive to immunodominant minor histocompatibility (minor H) Ags are thought to play a disproportionate role in allograft rejection in MHC-identical solid and bone marrow transplant settings. Although many studies have addressed the mechanisms underlying immunodominance in models of infectious diseases, cancer immunotherapy, and allograft immunity, key issues regarding the molecular basis of immunodominance remain poorly understood. In this study, we exploit the minor H Ag system to understand the relationship of the various biochemical parameters of Ag presentation and recognition to immunodominance. We show that the duration of individual minor H Ag presentation and the avidity of T cell Ag recognition influence the magnitude and, hence, the immunodominance of the CTL response to minor H Ags. These properties of CTL Ag presentation and recognition that contribute to immunodominance have implications not only for tissue transplantation, but also for autoimmunity and tumor vaccine design.

Expression profiling promises to provide insight into normal biological and pathological processes (Alizadeh et al. 2000; Shaffer et al. 2001; van't Veer et al. 2002). The hope is that knowledge obtained from gene expression patterns will predict disease outcome or indicate individualized courses of therapy. The two technologies that have emerged as the most promising gene expression tools are hybridization-based microarrays and quantitative real-time RT-PCR (QPCR)analysis (Duggan et al. 1999; Lockhart and Winzeler 2000; Giulietti et al. 2001; Green et al. 2001). Microarrays permit the simultaneous analysis of a large number of genes, but extensive replicate sampling can be labor-intensive. Additionally, samples with limiting RNA (such as mouse peripheral blood or laser-capture microdissection samples)can only be used following cDNA amplification (Wang et al. 2000), which adds another processing step that could introduce bias. This makes longitudinal microarray analysis of peripheral blood samples from an experimental cohort technically challenging. QPCR platforms using gene-specific primers provide highly sensitive and reproducible expression quantification from small amounts of starting material (Gibson et al. 1996; Heid et al. 1996; Schmittgen et al. 2000), but have been limited in the number of genes analyzed practically. Therefore, we combined the multiple gene approach of microarrays with the sensitivity of QPCR to produce a high-throughput customized “ImmunoQuantArray” (IQA), the first generation of which consists of 96 gene-specific QPCRs designed to monitor genes associated with immune processes. QPCR instruments monitor gene-specific amplicons with fluorescent dye chemistry. The amplification curves typically have a sigmoidal shape in which the exponential amplification phase reveals the number of PCR cycles required to achieve a certain fluorescence intensity. A cycle threshold or Ct value for each reaction is the number of cycles at which the reaction crosses the fluorescence threshold. The fewer cycles required to reach a certain fluorescence intensity, the lower the Ct value and the greater the initial amount of input target cDNA. Genes that do not amplify during the 40-cycle PCR are considered “off” and are given a Ct value of 40 (Heid et al. 1996). QPCR data are usually interpreted as fold changes in gene expression. Changes in gene expression are derived by normalizing the expression of a gene to that of an appropriate “housekeeping” gene (assumed to be invariant; Livak and Schmittgen 2001). This relative normalization procedure is presently regarded to be the only practical option available for interpreting QPCR data. An alternative, accurate quantification of input RNA/cDNA is challenging when input RNA is of limiting quantities and impractical for scale-up (Morrison et al. 1998). To more reliably evaluate expression changes in QPCR data, we developed a novel statistical algorithm—Global Pattern Recognition (GPR)—to reveal significant changes in gene expression patterns. Inspired by triangulation techniques to determine positional information in cartography and astronomy, GPR goes through several iterations to compare the change of expression of a gene normalized to every other gene in the IQA. By comparing the expression of each gene to every other gene in the array, a global pattern is established, and significant changes are identified and ranked. Importantly, GPR takes advantage of biological replicates to extract significant changes in gene expression, thus providing a novel alternative to the use of relative normalization in QPCR experiments. We show here that the IQA/GPR approach is a reliable analytical tool that can be used to establish immunological gene expression profiles of normal mice and in mice developing rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and graft versus host disease (GVHD). Moreover, we show that one can obtain temporal expression profiles from unamplified blood cDNA samples from individual mice, thus making it possible to establish the relationship between gene expression pattern and individual disease severity.

There is a pressing need for interactive bioinformatics tools that empower investigators with the means to extract information and organize it in a simplified but meaningful format. A wealth of mammalian gene expression data is readily accessible, much of which is based on expressed sequence tags (ESTs). Many mammalian ESTs are derived from tissue-specific cDNA libraries in which the number of ESTs representing a specific gene approximates the transcriptional expression level in the source tissue. Our program ExQuest (Expressional Quantification of ESTs) organizes the public EST database (dbEST) into hierarchical tissue classes and reports tissue or developmental gene expression patterns for both mRNA and genomic sequences. ExQuest also displays tissue expression patterns of genes in the context of assembled chromosomes. These interactive “transcriptome” maps provide a novel tool for investigating the genomic basis of gene expression as well as prioritizing candidate genes within genetically mapped mutant and quantitative trait loci.

Murine orthotopic corneal allografts experience immune privilege and have good survival as compared with skin allografts. However, privilege is not complete, and some grafts are still rejected. Unexpectedly, corneas expressing minor histocompatibility (H) alloantigens are rejected at a higher rate than major histocompatibility complex (MHC) disparate grafts. We hypothesize that certain immunodominant minor H alloantigens are extremely immunogenic when expressed in corneal tissue, terminate ocular immune privilege, and initiate corneal allograft rejection.Corneal allograft survival and the role of CD4+ T cells and CD8+ T cells were examined in corneal transplants that expressed genetically defined minor H3 alloantigens. The H3 locus contains at least two minor H genes. H3a is presented by MHC class I and recognized exclusively by CD8+ T cells. H3b is presented by class II and recognized exclusively by CD4+ T cells. Congenic strains that differ from C57BL/10 at (1) H3a, (2) H3b, or (3) H3a+H3b were used for orthotopic corneal and skin transplants. Donor corneas expressing either H3a or H3a+H3b experienced immune privilege and survived longer than skin allografts. By contrast, donor corneas expressing H3b (recognized by CD4+ T cells) experienced vigorous rejection and were eliminated faster than skin allografts.There are minor H alloantigens that terminate ocular immune privilege and initiate corneal allograft rejection. These minor H alloantigens are more immunogenic when expressed in corneal tissue than when they are expressed in skin allografts.

Minor histocompatibility (H) antigens are a diverse assemblage of major histocompatibility complex (MHC)-bound peptides with the unifying property of acting as alloantigens that induce allogeneic tissue rejection. They are a consequence of any form of accumulated genetic variation that translates to differential MHC-presented peptide epitopes, the most common form of which is simple sequence polymorphisms. The universe of potential minor H antigens is large when transplantation is performed between genetically unrelated, MHC-matched individuals, especially considering the remarkable discriminative sensitivity of T cells. However, the phenomenon of immunodominance greatly simplifies immune responses that ensue. One mouse minor H antigen, H60, stands out in that the preponderance of the CD8 T cell response elicited in a complex alloantigenic setting is directed against this single minor H antigen epitope. Its immunodominance is because mice lacking H60 develop an unusually robust T cell repertoire dedicated to this single minor H antigen. The now well-characterized mouse minor H antigen system should provide a vehicle to assess the degree to which immunodominant alloantigens contribute to transplant rejection.

Although CD8 T cells are thought to be a principal effector population of graft-versus-host disease (GVHD), their dynamics and specificity remain a mystery. Using a mouse model in which donor and recipient were incompatible at many minor histocompatibility antigens (minor H Ags), the CD8 T-cell response was tracked temporally and spatially through the course of GVHD. Donor CD8 T cells in the circulation, spleen, lung, and liver demonstrated virtually identical kinetics: rapid expansion and then decline prior to morbidity. Remarkably, up to one fourth of the CD8 T cells were directed against a single minor antigen, H60. Extreme H60 immunodominance occurred regardless of sampling time, site, and genetic background. This study is the first to analyze the T cells participating in GVHD in “real-time,” demonstrates the exceptional degree to which immunodominance of H60 can occur, and suggests that such superdominant minor H Ags could be risk factors for GVHD.

Evidence suggests that there is a significant genetic component to susceptibility and resistance to chronic periodontal disease. Data from both clinical studies and studies using animal models are reviewed here. Also outlined are the genomic methods that are now available for identifying susceptibility and resistance loci.

Periodontal diseases are chronic inflammatory diseases that can result in resorption of the alveolar bone of the jaw. We have developed a murine model in which alveolar bone loss is induced by oral infection with Porphyromonas gingivalis, an oral anaerobic bacterium associated with periodontal disease in humans. Here we compared a strain of immunocompetent mice (C57BL/6J) to the same strain of mice made T cell deficient by genetic deletion of the alpha chain of their T cell receptors (C57BL/6J-Tcra). T cell deficiency did not affect the ability of P. gingivalis to implant in the oral cavity. The two strains of mice had equal percentages of P. gingivalis among their cultivable anaerobes 7 weeks after infection. The same bacterial load led to much less bone resorption in the T cell deficient mice than in the immune normal mice, measured as either the number of sites with significant loss, or as the total amount of bone resorbed. T cell deficient mice lost bone at only three out of 14 measurement sites, compared with eight out of 14 sites in the wild-type mice. The total amount of bone lost was 70% less in the T cell deficient mice. T cell deficient mice had lower titers of P. gingivalis-specific IgG than the wild-type mice after oral infection did, but the same titers of specific IgA. Lower titers did not correlate with greater bone loss. Antigen-activated T lymphocytes are known to induce osteoclastogenesis; here we demonstrate that T cell deletion decreases the amount of alveolar bone loss induced by infection of the murine oral cavity.

PURPOSE: The neonatal Fc receptor (FcRn) plays a critical role in the homeostasis and degradation of immunoglobulin G (IgG). It mediates the transport of IgG across epithelial cell barriers and recycles IgG in endothelial cells back into the bloodstream. These functions critically depend on the binding of FcRn to the Fc domain of IgG. The half-life and distribution of intravitreally injected anti-VEGF molecules containing IgG-Fc domains might therefore be affected by FcRn expressed in the eye. In order to establish whether FcRn-Fc(IgG) interactions may occur in the eye, we studied the mRNA and protein distribution of FcRn in postmortem ocular tissue. \ud \ud METHODS: We used qPCR to study mRNA expression of the transmembrane chain of FcRn (FCGRT) in retina, optic nerve, RPE/choroid plexus, ciliary body/iris plexus, lens, cornea, and conjunctiva isolated from mouse, rat, pig, and human postmortem eyes and used immunohistochemistry to determine the pattern of FcRn expression in FCGRT-transgenic mouse and human eyes.\ud \ud RESULTS: In all four tested species, Fcgrt mRNA was expressed in the retina, RPE/choroid, and the ciliary body/iris, while immunohistochemistry documented FcRn protein expression in the ciliary body epithelium, macrophages, and endothelial cells in the retinal and choroidal vasculature.\ud \ud CONCLUSIONS: Our results demonstrate that FcRn has the potential to interact with IgG-Fc domains in the ciliary epithelium and retinal and choroidal vasculature, which might affect the half-life and distribution of intravitreally injected Fc-carrying molecules.

It has been difficult to genetically map the genes encoding tumor Ags because they arise as a consequence of somatic mutational events. CTL-mediated immunoselection can impose potent immunoselective pressure against tumor cells, resulting in the survival of rare tumor Ag-loss variants. We subjected a heterozygous 3-methylcholanthrene-induced murine sarcoma cell line to CTL immunoselection, selecting for the loss of a tumor-specific Ag, recognized antigen from MCA-induced tumor 1 (Ram1). Several variants eluded CTL recognition by genetic loss of the hemizygously expressed tumor-specific Ag epitope. A frequently observed genetic escape mechanism was spontaneous mitotic recombination resulting in loss of heterozygosity on chromosome 4. Higher density genetic analyses along with functional confirmation with an independently produced chromosome 4 loss of heterozygosity variant positioned the Ram1 locus to a distal 7.1 cM interval on chromosome 4. This region of the mouse genome is rich in tumor-modifier genes and this positioning of Ram1 may thus provide insight into the genetic basis of 3-methycholanthrene-induced tumor Ags.

Histocompatibility (H) Ags are responsible for chronic graft rejection and graft vs host disease in solid tissue and bone marrow transplantation among MHC-matched individuals. Here we defined the molecular basis of self-nonself discrimination for the murine chromosome 7 encoded H47 histocompatibility locus, known by its trait of graft-rejection for over 40 years. H47 encodes a novel, highly conserved cell surface protein containing the SCILLYIVI (SII9) nonapeptide in its transmembrane region. The p7 isoleucine-to-phenylalanine substitution in SII9 defined the antigenic polymorphism and T cell specificity. Despite absence of the canonical consensus motif and weak binding to Db MHC I, both H47 peptides were presented to CTLs. However, unlike all the other known H loci, the relative immunogenicity of both H47 alleles varied dramatically and was profoundly influenced by neighboring H loci. The results provide insights into the peptide universe that defines nonself and the basis of histoincompatibility.

Minor histocompatibility Ags (minor H Ags) are substantial impediments to MHC-matched solid tissue and bone marrow transplantation. From an antigenic standpoint, transplantation between MHC-matched individuals has the potential to be remarkably complex. To determine the extent to which the immune response is simplified by the phenomenon of immunodominance, we used peptide/MHC tetramers based on recently discovered minor H Ags (H60, H13, and HY) and monitored in vivo CD8 T cell responses of female C57BL/6 mice primed with MHC-matched, but background-disparate, male BALB.B cells. CD8 T cells against H60 overwhelmed responses to the H13 and HY throughout primary and secondary challenge. H60 immunodominance was an inherent quality, overcoming a lower memory precursor frequency compared with that of H13 and evoking a T cell response with diverse TCRVβ usage. IFN-γ staining examining congenically defined minor H Ags extended H60 dominance over additional minor H Ags, H28, H4, and H7. These four minor H Ags accounted for up to 85% of the CD8 T cell response, but H60 stood out as the major contributor. These findings show that immunodominance applies to antigenically complex transplantation settings in vivo and that the responses to the H60 minor H Ag dominates in this model. We suggest that immunodominant minor H Ags are those that result from the absence of a self analog.

Alveolar bone resorption can be induced in specific-pathogen-free mice by oral infection withPorphyromonas gingivalis(P. J. Baker, R. T. Evans, and D. C. Roopenian, Arch. Oral Biol. 39:1035–1040, 1994). Here we used a mouse strain, C57BL/6J, which is relatively resistant toP. gingivalis-induced bone loss to examine whether partial or complete deletion of various adhesion molecules would increase susceptibility. Complete deletion of P-selectin or nearly complete lack of expression of intercellular adhesion molecule 1 (ICAM-1) led to increased susceptibility to bone resorption after oral infection, while a hypomorphic defect in β2-integrins did not. Both the total amount of bone lost and the number of sites at which there was significant loss were increased in mice deficient in either ICAM-1 or P-selectin. Each of the three adhesion molecule deficiencies was sufficient to decreaseP. gingivalis-specific serum immunoglobulin G responses, but lower antibody titers did not lead to increased bone loss in partially β2-integrin-deficient mice. In conclusion, P-selectin and ICAM-1 deficiencies increase susceptibility to and severity of alveolar bone loss afterP. gingivalisinfection. This finding underscores the importance of innate immunity in protection againstP. gingivalis-induced alveolar bone resorption.

Previous work has indicated that an important component for the initiation of autoimmune insulin-dependent diabetes mellitus (IDDM) in the NOD mouse model entails MHC class I-restricted CD8 T cell responses against pancreatic β cell Ags. However, unless previously activated in vitro, such CD8 T cells have previously been thought to require helper functions provided by MHC class II-restricted CD4 T cells to exert their full diabetogenic effects. In this study, we show that IDDM development is greatly accelerated in a stock of NOD mice expressing TCR transgenes derived from a MHC class I-restricted CD8 T cell clone (designated AI4) previously found to contribute to the earliest preclinical stages of pancreatic β cell destruction. Importantly, these TCR transgenic NOD mice (designated NOD.AI4αβ Tg) continued to develop IDDM at a greatly accelerated rate when residual CD4 helper T cells were eliminated by introduction of the scid mutation or a functionally inactivated CD4 allele. In a previously described stock of NOD mice expressing TCR transgenes derived from another MHC class I-restricted β cell autoreactive T cell clone, IDDM development was retarded by elimination of residual CD4 T cells. Hence, there is variability in the helper dependence of CD8 T cells contributing to the development of autoimmune IDDM. The AI4 clonotype represents the first CD8 T cell with a demonstrated ability to progress from a naive to functionally activated state and rapidly mediate autoimmune IDDM development in the complete absence of CD4 T cell helper functions.

Nonobese diabetic (NOD) mice develop insulin-dependent diabetes mellitus due to autoimmune T lymphocyte-mediated destruction of pancreatic β cells. Although both major histocompatibility complex class I-restricted CD8+and class II-restricted CD4+T cell subsets are required, the specific role each subset plays in the pathogenic process is still unclear. Here we show that class I-dependent T cells are required for all but the terminal stages of autoimmune diabetes development. To characterize the diabetogenic CD8+T cells responsible, we isolated and propagatedin vitroCD8+T cells from the earliest insulitic lesions of NOD mice. They were cytotoxic to NOD islet cells, restricted to H-2Kd, and showed a diverse T cell receptor β chain repertoire. In contrast, their α chain repertoire was more restricted, with a recurrent amino acid sequence motif in the complementarity-determining region 3 loop and a prevalence of Vα17 family members frequently joined to the Jα42 gene segment. These results suggest that a number of the CD8+T cells participating in the initial phase of autoimmune β cell destruction recognize a common structural component of Kd/peptide complexes on pancreatic β cells, possibly a single peptide.

Current knowledge of histocompatibility (H) loci located outside of the major histocompatibility complex - i.e. those encoding the so-called minor H antigens - was surveyed at a recent meeting ∗ ∗The 1st International Symposium on Minor Histocompatibility Antigens was held at Bar Harbor, ME, USA, on 14–17 September 1997..

MRL/Mp-lpr/lpr (MRL-lpr) mice develop an aggressive autoimmune disorder characterized by arthritis, vasculitis, and glomerulonephritis. Renal injury is associated with increased expression of major histocompatibility complex (MHC) molecules, as well as cytokines, adhesion molecules (intracellular adhesion molecule-1, vascular cell adhesion molecule-1), and autoantibodies. By using either MHC Class I (MRL-lpr B2m-/-) or MHC Class II deficient (MRL-lpr Ab-/-) kidneys in a transplant model, we tested the role of renal expression of these molecules in the development of autoimmune renal injury. Kidneys from MRL-lpr B2m-/- or MRL-lpr Ab-/- mice as well as control wild-type mice transplanted into MRL-lpr wt/- recipients developed nephritis, CD4+ and CD8+ T cell infiltration, and heavy glomerular deposition of immunoglobulin. Spontaneously proliferating autoreactive T cells were found in wild-type MRL-lpr and MRL-lpr B2m-/- but not MRL-lpr Ab-/- mice. These results suggest that the absence of renal expression of either Class I or Class II molecules does not provide marked protection from autoimmune lupus nephritis and supports the possibility that protection from autoimmune disease in MRL-lpr Ab-1- mice is related to the loss of autoreactive MHC Class II-dependent CD4+ T cells.

The common class I alleles (e.g., JC1 and Db) within the H2g7 major histocompatibility complex (MHC) clearly contribute to autoimmune IDDM in NOD mice, but the mechanism by which this occurs has been controversial. One laboratory has reported that the peptide transporter encoded by the Tapl gene within H2g7 is defective, and this contributes to IDDM by impairing MHC class I-mediated antigen presentation. If true, defective MHC class I-mediated antigen presentation should segregate with the H2g7 haplotype. NOD mice, related congenic stocks, and other control strains were used to test this hypothesis. H2g7-positive strains did not differ from those expressing other MHC haplotypes in ability to present MHC class I-restricted H3aa or H3ab minor histocompatibility (H) antigens to cytotoxic T-lymphocytes (CTL). The H2g7 haplotype was found to have a reduced capacity to mediate MHC class I-restricted presentation of the H47a minor H antigen. However, MHC class I-restricted presentation of H47a was found to be Tap independent. NOD mice and control strains also did not differ in ability to activate adenovirus-specific MHC class I restricted CTL. Thus, the H2g7 haplotype is not characterized by a Tap gene defect that only impairs the inductive phase of the immune response. In addition, MHC class I-restricted presentation of either minor H or adenoviral antigens was equivalent in male and female NOD mice. Therefore, while the class I alleles of the H2g7 haplotype exert diabetogenic functions in NOD mice, this is not elicited through a Tap gene defect. The absence of female-specific Tap gene defects also indicates this cannot account for the reduced male incidence of IDDM in some NOD mouse colonies.

Abstract 1666 NF-κB activation has been linked to the promotion of an assortment of malignancies. While in vitro studies have supported a tumor-promoting role for NF-κB in leukemias, in vivo evidence has not been collected. NF-κB has also been proposed as a therapeutic target for Philadelphia chromosome-positive leukemias including chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL). In this study, we investigate the role the Nfkb1 gene plays in CML and B-ALL using mouse models for these leukemias induced by the BCR-ABL oncogene. Contrary to conventional thinking, we find a tumor-suppressing role for Nfkb1 in BCR-ABL leukemias. To induced CML, we transduced bone marrow cells from 5-FU-primed wild type or Nfkb1−/− mice with BCR-ABL-GFP retrovirus, followed by transplantation of the transduced cells into lethally irradiated recipient mice. We show that recipients of BCR-ABL-transduced Nfkb1−/− donor cells developed CML more rapidly than the wild type recipients, correlating to elevated percentages (82.3% vs 38.5%) and total numbers of Gr-1+GFP+ cells in peripheral blood of the mice. We next tested whether the loss of Nfkb1 also accelerated B-ALL development under the B-ALL-inducing conditions, and show that recipients of BCR-ABL-transduced Nfkb1−/− donor cells succumbed to B-ALL more quickly than the wild type recipients. These results suggest that Nfkb1 does not stimulate leukemia growth, and instead, it plays a tumor-suppressing role in CML and B-ALL. Because these findings are opposite from what people believe in the field, we confirmed our findings by conducting a rescue experiment. The Nfkb1 gene encodes the p105 protein; roughly half of p105 product is selectively proteolyzed to yield the p50 transcription factor. In this rescue experiment, we transduced Nfkb1−/− donor cells with BCR-ABL alone or BCR-ABL and p50 of Nfkb1 to induce leukemia, and we show that acceleration of leukemia development caused by the Nfkb1 loss is partially reversed by p50. To study the underlying mechanism, we focused on CARD11, because it plays a critical role in mediating NF-κB signaling in lymphocytes. We find that CARD11 expression is induced in myeloid and increased in lymphoid cell lines expressing BCR-ABL. Furthermore, we induced B-ALL using Card1−/− mice. We find that similar to the loss of Nfkb1, the loss of Card11 expression also accelerates the development of B-ALL. To increase human relevance of our findings, we overexpressed p50 in human K562 cells by retroviral transduction with p50-IRES-GFP or empty vector, and find that FACS-sorted p50/GFP K562 cells grow significantly slower than empty vector-transduced cells. These findings support an idea that Nfkb1 signaling suppresses BCR-ABL-induce leukemia at least partially through a CARD11-dependent mechanism. In summary, our results demonstrate that Nfkb1 plays a tumor-suppressing role in BCR-ABL-induced leukemias, arguing that we should be careful when targeting of Nfkb1 in leukemia treatment. Disclosures: No relevant conflicts of interest to declare.

The purpose of this study was to elucidate the genetic origin of minor histocompatibility (H) antigens. Toward this end common inbred mouse strains, distinct subspecies, and species of the subgenus Mus were examined for expression of various minor H antigens. These antigens were encoded by the classical minor H loci H-3 and H-4 or by newly identified minor H antigens detected as a consequence of mutation. Both minor H antigens that stimulate MHC class I-restricted cytotoxic T cells (Tc) and antigens that stimulate MHC class II-restricted helper T cells (Th) were monitored. The results suggested that strains of distinct ancestry commonly express identical or cross-reactive antigens. Moreover, a correlation between the lack of expression of minor H antigens and ancestral heritage was observed. To address whether the antigens found on unrelated strains were allelic with the sensitizing minor H antigens or a consequence of antigen cross-reactivity, classical genetic segregation analysis was carried out. Even in distinct subspecies and species, the minor H antigens always mapped to the site of the appropriate minor H locus. Together the results suggest: 1) minor H antigen sequences are evolutionarily stable in that their pace of antigenic change is slow enough to predate subspeciation and speciation; 2) the minor H antigens originated in the inbred strains as a consequence of a rare polymorphism or loss mutation carried in a founder mouse stock that caused the mouse to perceive the wild-type protein as foreign; 3) there is a remarkable lack of antigenic cross-reactivity between the defined minor H antigens and other gene products.

Abstract 687 Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality in allogeneic stem cell transplantation. We previously showed that recipient antigen-presenting cells (APCs) are required for CD8-dependent GVHD, while donor APCs promote GVHD in a MHC matched (C3H.SW (right arrow) B6; both H-2b) model (Shlomchik et al, Science 1999; Matte et al., Nat Med 2004). However, how donor APCs promote maximal GVHD was not addressed. The LTFNYRNL peptide from H60 is a dominant minor histocompatibility antigen (miHA) presented by H-2Kb. To study cross-presentation of H60, we crossed B6 mice congenic for H60 (CH60; hematopoietically restricted) or transgenic for H60 driven by an actin promoter (actH60; H60 is ubiquitously expressed) with B6 Kb-/- mice. These mice express H60 but cannot directly present it to donor CD8 cells as they do not express Kb. CH60C*Kb-/-and actH60*Kb-/- were irradiated and reconstituted with C3H.SW (H60-) bone marrow,106(6 superscript) CD8 T cells and 2*105( 5 superscript) CD4 (to promote the CD8 response to H60). Using H60-MHC tetramers, we detected H60-specific CD8 T cell expansion as early as day 8, with a peak at day14, demonstrating cross-priming by donor C3H.SW APCs. Intracellular IFN-γ staining and an in vivo CTL assay showed that these cross-primed CD8 T cells had effector functions. Surprisingly, accumulation of H60-tetramer+ cells was greater when it was exclusively cross-presented. SIINFEKL, a peptide derived from ovalbumin (OVA), is also presented by Kb. Therefore to confirm our findings we used B6 mice transgenic for ovalbumin crossed to Kb-/- mice (ova*Kb-/-) as recipients in the same model. SIINFEKL-tetramer+ T cells expansion was also observed in ova*Kb-/- recipients, demonstrating cross-priming. The source of miHA did not affect the cross-priming as similar SIINFEKL-reactive T cell expansion occurred in retransplanted (right arrow)ova*Kb-/-, ova*Kb-/-(right arrow) Kb-/- bone marrowγKb-/- chimeras. Cross-priming of SIINFEKL-reactive CD8 cells even occurred when BALB/c mice transgenic for OVA (BALB/c-ova; (H-2d)) were transplanted with B6 BM and a mix of B6 CD4 and CD8 cells. SIINFEKL-reactive cells produced IFN-γ and killed SIINFEKL-pulsed B6 cells in vivo. Because of the availability of knockout/transgenic mice backcrossed to B6, we used this system to explore mechanisms of cross-presentation. Donor CD11c+ dendritic cells (DCs) were required as cross-priming was abrogated when BALB/c-OVA mice were transplanted with BM from mice constitutively lacking CD11c+ DCs (Birnberg et al, Immunity 2008). CD4 help has been reported to be important for cross-priming. Surprisingly, however, cross-priming by donor APCs was unaffected when BALB/c-OVA mice were transplanted with B6 MHCII-/- BM but was greatly reduced in recipients of B6 CD40-/- BM. Thus, while CD40L activation of cross-priming DCs is important, CD4 cells which are likely the source of the CD40L need not actually make T cell receptor:MHC contacts with the cross-presenting DC. CD40L-conditioning of donor APCs is not required to cross-prime memory cells, as sort-purified memory CD8 cells from SIINFEKL-vaccinated mice expanded robustly in actOVA*Kb-/- but not Kb-/- mice. Cross-priming also occurred in recipients of IFNAR1-/- BM, indicating that Type I IFN activation of donor APCs is not required as has been reported in nontransplant settings. Taken together, our data demonstrate that cross-presentation by donor DCs occurs in MHC-matched and -mismatched transplants, and this cross-presentation likely explains the reduced GVHD we observed in recipients of MHCI- donor bone marrow. That T cells can be cross-primed to nonhematopoietic antigens provides a basis for persistent GVHD and for the generation of CD8 responses against antigens not initially targeted. We also found transplantation to be a permissive environment for cross-priming in that CD4 help could be delivered in trans, type I IFN APC activation was not required and memory cells could be activated without CD4 help. These data provide further rationale for targeting donor DCs and pathways required for cross-presentation to prevent and treat GVHD. Disclosures: No relevant conflicts of interest to declare.

IL-21 is a member of the IL-2 cytokine family that exerts potent biological effects on multiple hemopoietic cell types. It is produced by activated CD4 T cells, while its receptor, IL-21R, is found on cells including NK cells, dendritic, T cells and B cells. To investigate the role of IL-21 signaling in autoimmune disease, we addressed whether this cytokine contributes to the severe systemic lupus erythematosus (SLE)-like syndrome developed by BXSB-Yaa mice. We show that BXSB-Yaa CD4 T cells produce IL-21 but lack discriminative T follicular helper cell markers. We also show that BXSB-Yaa mice completely lacking the IL-21R or Igh6 are remarkably resistant to multiple aspects of the prototypic BXSB-Yaa autoimmune disease. Finally, we show that mixed bone marrow chimeric BXSB-Yaa mice lacking IL-21R specifically in the B-cell compartment are similarly resistant. These results suggest that IL-21 produced by a novel population of BXSB-Yaa CD4 T-cells acts through B-cells to evoke multiple features characteristic of severe SLE. The results indicate that IL-21 signaling through B-cells is an essential step in the pathogenesis of this SLE-like autoimmune disease and potentially others. Funded by an Arthritis Foundation grant to JB.

The purpose of this work was to elucidate the genetic fine structure of the central portion of mouse chromosome (Chr) 2. Seven Chr 2 congenic mouse strains [B10.PA(L)-pa we un at, B10.PA(L)-pa Aw, B10.PA(L)-we un at, B10.PA(J)-pa a, B10.FS-we Aw, B10.C-we Aw, and B10.YBR-a] were produced. Breeding studies were carried out using strains B10.PA(L)-pa we un at and B10.LP-H-13b to accurately determine the recombination frequencies between marker genes pa and we (1.9% +/- 0.3), we and un (8.8% +/- 0.5), and un and at (4.5% +/- 0.4) of strain B10.PA(L)-pa we un at. These strains and other Chr 2 congenic strains were typed for immunologically defined loci using monoclonal antibody (mAb) C23 reactive with the gene product of B2mb T-lymphocyte clone C1 reactive with the gene product of H-3a and H-3c, and lymphocyte clone H1.8 reactive with the gene product of Hd-1a. B2m and H-3 typing located a recombinational event separating [pa B2m H-3] from we (the order of bracketed genes is not known). Hd-1 typing indicated that Hd-1 maps distal to [H-42, H-44] and proximal to un. The gene order [pa, B2m, H-3], we, [H-42, H-45], Hd-1, un, H-13, at, with H-44 mapping centromeric to Hd-1, is indicated by the data.

Mouse models of SLE are considered pivotal in understanding the genetics of SLE along with identifying targets for therapy. Male mice of the BXSB/MpJ recombinant inbred strain, first described by Murphy and Roths in 1978, develop a spontaneous and severe SLE-like syndrome resulting in death averaging ~6 months of age, while females show only a very chronic syndrome. A mutant locus found on the Y chromosome is, therefore, appropriately named the Y-linked autoimmune accelerator (Yaa). Previous analysis have suggested that Yaa fails to induce an SLE-like disease in B6 mice, the caveat to this being that the SLE criteria analyzed were limited to serological analysis, histopathology, and mortality on mice up to 8–12 months of age. We report here that when mice of a significantly older age, 15–24 months, are compared to age-matched B6 controls, the B6.SB-Yaa/J mice clearly developed an SLE-like disease. This is characterized by significant splenomegaly, monocytosis, immune cells with an activation phenotype, the presence of homogeneous anti-nuclear antibody staining, glomerulonephritis, decreased weight and survival as well as a gene expression signature similar to BXSB/MpJ mice. These results suggest that Yaa alone is sufficient to induce an SLE-like syndrome in aged B6 mice. Funded by NIH R21DK074463 and DK56597 to DCR, Arthritis Foundation to JAB.

IgG antibodies modified for high binding affinity for their receptor FcRn have emerged as a major treatment for a wide array of both neoplastic and autoimmune diseases. The goal of this study was to analyze the effect of human IgG1 antibodies with enhanced in vitro binding to FcRn on their in vivo half-life in mice deficient in mouse FcRn and expressing human FcRn. Two mutants (N434A and T307A/E380A/N434A) of the humanized monoclonal antibody Hu4D5, directed against epidermal growth factor receptor 2 (p185 HER2), show increased pH-dependent binding to human FcRn in vitro. They also have an extended half-life in vivo compared to wild-type antibody in FcRn transgenic mice but not in mice expressing endogenous mouse FcRn. When injected into FcRn-humanized mice at a concentration sufficient to partially saturate human FcRn, the mutants with an extended serum half-life were most effective in reducing the half-life of a tracer antibody. Finally, we use a serum-transfer model of arthritis and FcRn-humanized mice to test whether mutant antibodies could have enhanced therapeutic potential by promoting accelerated clearance of arthritogenic antibodies. Results indicate that the engineered mutant T307A/E380A/N434A is most effective in vivo in ameliorating the inflammatory arthritic lesions. In summary, FcRn-humanized mice are a promising animal model for human IgG therapeutic development.

Adoptive immunotherapy of human Philadelphia chromosome-positive (Ph+) leukemia has not been effective due to difficulties of identifying tumor-specific antigens for the disease. It has been reported that T lymphocytes activated by some minor histocompatibility antigens (mHAs) can efficiently treat mice receiving HL4 cells (a leukemia cell line) without causing graft-versus-host disease (GVHD). We investigated whether this anti-mHA immunotherapeutic strategy is applicable to treating Ph+ leukemia that includes B-ALL (B-cell acute lymphoblastic leukemia) and CML (chronic myeloid leukemia). Because the mHA, H60, is an immunodominant mHA among H7, H28, and H58 in mice (Choi et al, Immunity 17:593–603, 2002), we tested whether H60 can serve as an immunotherapeutic target in the treatment of B-ALL and CML in our retroviral bone marrow transduction/transplantation disease models (Li et al, J Exp Med189:1399–1412, 1999). We used C57BL/6-H60 congenic mouse splenocytes to immunize wild type C57BL/6 (B6) mice that do not have the H60 gene, and the activated T lymphocytes from the immunized B6 mice were injected into mice with BCR-ABL-induced B-ALL or CML (3×107 cells/mouse). We found that H60-specific T lymphocytes at a single dose completely eliminated leukemic cells within 10 days after the injection of the cells and cured B-ALL and CML, however, in mice receiving donor bone marrow cells transduced with empty vector and treated with H60-specific T lymphocytes, the non-BCR-ABL-expressing donor marrow cells lasted for more than 4 weeks, suggesting that H60-activated T lymphocytes have much stronger inhibitory effect on BCR-ABL-expressing leukemic cells than on normal marrow cells. In contrast, leukemia mice receiving naïve T lymphocytes died within 3 to 4 weeks post BCR-ABL induction of leukemia. Six months later, no residual leukemia cells were detected in the spleen and bone morrow of the leukemia mice receiving H60-activated T lymphocytes. In addition, no GVHD was observed in the mice. We also use H7/H28/H60 triple congenic B6 mice in our study, and found that H7/H28/H60-primed T lymphocytes were more effective in elimination of leukemia cells in mice compared with when the single H60 antigen was used in similar experiment. Especially, the H7/H28/H60-primed T lymphocytes were highly effective in treating B-ALL mice, and did not induce GVHD. These results indicate that mHAs are promising targets for immunotherapy of BCR-ABL-induced B-ALL and CML.

Gene expression profiling of human and mouse B cell lineage neoplasms using microarrays has provided a powerful means for:identification of previously unrecognized subsets of a single histologically-defined disease category;relating a specific disease to a previously unrecognized cell of origin; andestablishing disease subsets defined by characteristic host-tumor interactions. Other anticipated returns including the rapid identification of new therapeutic targets have fallen short of expectations. In addition, discrepancies among results obtained using different platforms pose a major obstacle to bringing this approach into common clinical practice. Quantitation of gene expression by qPCR provides an attractive alternative approach but has been limited by the lack of high-throughput methodologies. We have approached this issue by developing qPCR methods that assay 384 “cancer genes” in a single plate, covering a wide range of targets from pathways implicated in the development of hematopoietic as well as solid tumors. We used this system to compare RNA prepared from normal spleens, mouse PCT and two lymphoma types with strikingly similar cytologies but differing origins - high-grade splenic marginal zone lymphomas (MZL) and centroblastic DLBCL (CBL). Terminally differentiated PCT were readily distinguished from MZL and CBL and MZL from CBL. Statistical analyses identified subsets of genes that distinguished each normal or tumor set from all the others and these are being validated against new groups of each tumor type. The same initial cases were also evaluated using oligonucleotide microarrays, providing instructive similarities and differences in measures of shared genes. From these comparisons, we have identified potentially important signaling pathways, such as Notch, Wnt and IL6, as well as drugable targets. These are being evaluated in studies of cultured cell lines and can be extended to in vivo tests of efficacy in mice bearing transplanted or primary tumors.

Endoplasmic reticulum (ER)-associated aminopeptidase (ERAP)1 has been implicated in the final proteolytic processing of peptides presented by major histocompatibility complex (MHC) class I molecules. To evaluate the in vivo role of ERAP1, we have generated ERAP1-deficient mice. Cell surface expression of the class Ia molecules H-2Kb and H-2Db and of the class Ib molecule Qa-2 was significantly reduced in these animals. Although cells from mutant animals exhibited reduced capacity to present several self- and foreign antigens to Kb-, Db-, or Qa-1b–restricted CD8+ cytotoxic T cells, presentation of some antigens was unaffected or significantly enhanced. Consistent with these findings, mice generated defective CD8+ T cell responses against class I–presented antigens. These findings reveal an important in vivo role of ER-associated peptidase activity in tailoring peptides for presentation by MHC class Ia and class Ib molecules.

Purpose The present study was conducted to investigate healing of alkali-burned corneas in MRL/MpJ (MRL) mice. Methods Gross, clinical, and histologic criteria were used to compare healing of alkali-burned corneas in MRL and control C57BL/6J (B6) mice. Effects of neutrophil depletion of B6 mice and allogeneic reconstitution of B6 mice with MRL bone marrow on wound healing were evaluated. Gene expression patterns in normal and wounded corneas were surveyed with array-based quantitative real-time RT-PCR (AQPCR). Results MRL mice showed accelerated reepithelialization and decreased corneal opacity compared with B6 mice after alkali wounding. Marked inflammatory cell infiltration and fibrosis were evident in the corneas and anterior chambers of B6 mice. MRL mice showed less severe lesions, except for stromal edema. Rapid reepithelialization and reduced keratitis/iritis were also observed in neutrophil-depleted B6 mice, but not in B6 mice reconstituted with MRL bone marrow. AQPCR showed transcriptional changes of fewer genes associated with inflammation and corneal tissue homeostasis in alkali-burned corneas from MRL mice. Increased expression of an anti-inflammatory gene, Socs1, and a gene associated with healing, Mmp1a, were evident in MRL corneas. Conclusions Alkali-burned corneas heal faster and more completely in MRL mice than in B6 mice, by means of rapid reepithelialization, reduced inflammation, and reduced fibrosis. Reduced inflammation, including decreased neutrophil infiltrates and the lack of a robust proinflammatory gene expression signature correlates with the rapid healing. However, the rapid-healing phenotype is not intrinsic to MRL hematopoietic progenitor cells.