Issue published May 1, 2007

T cell–mediated autoimmune diseases such as multiple sclerosis and rheumatoid arthritis are driven by autoaggressive Th cells. The pathogenicity of such Th cells has, in the past, been considered to be dictated by their cytokine polarization profile. The polarization of such effector T cells relies critically upon the actions of cytokines secreted by APCs. While Th1 polarization has long been associated with the pathogenesis of autoimmune diseases, recent data obtained in gene-targeted mice and the discovery of Th17 cell involvement in autoimmunity conflict with this hypothesis. In light of these recent developments, we discuss in this review the actions of APC-derived cytokines and their emerging roles in T cell polarization in the context of autoimmune inflammatory responses.

A major challenge for the immune system is to recognize and eliminate cells undergoing carcinogenesis. Immune defense against tumors is complex. It can be mediated early by the innate immune system (i.e., phagocytes, NK cells, NKT cells, cytokines, and complement proteins) and later by the adaptive immune system (i.e., B cells and T cells). The eight articles in this Review series on tumor immunology discuss the mechanisms underlying immune surveillance of tumors, the regulation of carcinogenesis by immune inflammatory mediators, current approaches to controlling tumor growth through immunotherapy, and novel targets of immunotherapy.

T he ability of the immune system to identify and destroy nascent tumors, and to thereby function as a primary defense against cancer, has been debated for many decades. Recent findings by a number of investigators in both mouse models of cancer and humans with cancer now offer compelling evidence that particular immune cell types, effector molecules, and pathways can sometimes collectively function as extrinsic tumor suppressor mechanisms. This work provides the basis for further study of natural immunity to cancer and for rational use of this information in the design of immunotherapies in combination with other conventional cancer treatments.

T umors arise from normal cells of the body through genetic mutation. Although such genetic mutation often leads to the expression of abnormal antigens, the immune system fails to respond effectively to these antigens; that is, it is tolerant of these antigens. This acquired state of tolerance must be overcome for cancer immunotherapy to succeed. Indoleamine 2,3-dioxygenase (IDO) is one molecular mechanism that contributes to tumor-induced tolerance. IDO helps create a tolerogenic milieu in the tumor and the tumor-draining lymph nodes, both by direct suppression of T cells and enhancement of local Treg-mediated immunosuppression. It can also function as an antagonist to other activators of antitumor immunity. Therefore, strategies to block IDO might enhance the effectiveness of tumor immunotherapy.

T umors require a constant influx of myelomonocytic cells to support the angiogenesis and stroma remodeling needed for their growth. This is mediated by tumor-derived factors, which cause sustained myelopoiesis and the accumulation and functional differentiation of myelomonocytic cells, most of which are macrophages, at the tumor site. An important side effect of the accumulation and functional differentiation of these cells is that they can induce lymphocyte dysfunction. A complete understanding of the complex interplay between neoplastic and myelomonocytic cells might offer novel targets for therapeutic intervention aimed at depriving tumor cells of important growth support and enhancing the antitumor immune response.

T umors express antigens that should induce immune-mediated rejection, but spontaneous rejection of established tumors is rare. Recent work demonstrates that one reason for the lack of tumor rejection is that tumors actively defeat host immunity. This concept forces us to rethink current approaches to harnessing potent, specific host immunity to battle cancer, most of which are based on the paradigm that inducing more antitumor immune cells alone is therapeutic. However, as I discuss in this Personal Perspective, a newer paradigm predicts that reducing tumor-driven immune suppression will be clinically beneficial. CD4⁺CD25⁺ Tregs are one mechanism of tumor-driven immune evasion that provide prototypical targets for testing novel anticancer treatment strategies within the newer paradigm.

I t has been established that cancer can be promoted and/or exacerbated by inflammation and infections. Indeed, chronic inflammation orchestrates a tumor-supporting microenvironment that is an indispensable participant in the neoplastic process. The mechanisms that link infection, innate immunity, inflammation, and cancer are being unraveled at a fast pace. Important components in this linkage are the cytokines produced by activated innate immune cells that stimulate tumor growth and progression. In addition, soluble mediators produced by cancer cells recruit and activate inflammatory cells, which further stimulate tumor progression. However, inflammatory cells also produce cytokines that can limit tumor growth. Here we provide an overview of the current understanding of the role of inflammation-induced cytokines in tumor initiation, promotion, and progression.

I n vertebrates, the TLRs are a family of specialized immune receptors that induce protective immune responses when they detect highly conserved pathogen-expressed molecules. Synthetic agonists for several TLRs, including TLR3, TLR4, TLR7, TLR8, and TLR9, have been or are being developed for the treatment of cancer. TLR9 detects the unmethylated CpG dinucleotides prevalent in bacterial and viral DNA but not in vertebrate genomes. As discussed in this Review, short synthetic oligodeoxynucleotides containing these immune stimulatory CpG motifs activate TLR9 in vitro and in vivo, inducing innate and adaptive immunity, and are currently being tested in multiple phase II and phase III human clinical trials as adjuvants to cancer vaccines and in combination with conventional chemotherapy and other therapies.

B ecause of the large preexisting antigenic load and immunosuppressive environment within a tumor, inducing therapeutically useful antitumor immunity in cancer patients requires the development of powerful vaccination protocols. An approach gaining increasing popularity in the tumor vaccine field is to immunize cancer patients with their own DCs loaded ex vivo with tumor antigens. The underlying premise of this approach is that the efficiency and control over the vaccination process provided by ex vivo manipulation of the DCs generates an optimally potent APC and a superior method for stimulating antitumor immunity in vivo compared with the more conventional direct vaccination methods, offsetting the added cost and complexity associated with this form of customized cell therapy.

T he transfusion of T cells, also called adoptive T cell therapy, is an effective treatment for viral infections and has induced regression of cancer in early-stage clinical trials. However, recent advances in cellular immunology and tumor biology are guiding new approaches to adoptive T cell therapy. For example, use of engineered T cells is being tested as a strategy to improve the functions of effector and memory T cells, and manipulation of the host to overcome immunotoxic effects in the tumor microenvironment has led to promising results in early-stage clinical trials. Challenges that face the field and must be addressed before adoptive T cell therapy can be translated into routine clinical practice are discussed.

I n the 40 years since Harvard medical student Gilbert Omenn first described a rare, inherited disorder producing a paradoxical combination of immunodeficiency and immune dysregulation, the pathogenesis of Omenn syndrome (OS) has remained mysterious. In separate studies reported in this issue of the JCI, two mouse models bearing mutations in the V(D)J recombinase analogous to those causing human OS have been shown to recapitulate the disease and provide insight into the genesis of immunodeficiency combined with autoimmunity and atopy in OS and other disease settings (see the related articles beginning on pages 1260 and 1270).

A utosomal recessive cutaneous disorders, including various types of epidermolysis bullosa (EB), usually manifest shortly after birth. The clinical course of these diseases is often characterized by severe complications, limited therapeutic options, and a poor prognosis. A study by Pasmooij et al. reported in this issue of the JCI unravels the molecular mechanisms by which germline mutations in non-Herlitz junctional EB can be corrected in vivo by multiple spontaneously occurring somatic mutational events, a phenomenon known as revertant mosaicism (see the related article beginning on page 1240). These insights open new avenues of thinking for the design of future gene therapy strategies for skin diseases.

D iabetic foot ulcers (DFUs), a leading cause of amputations, affect 15% of people with diabetes. A series of multiple mechanisms, including decreased cell and growth factor response, lead to diminished peripheral blood flow and decreased local angiogenesis, all of which can contribute to lack of healing in persons with DFUs. In this issue of the JCI, Gallagher and colleagues demonstrate that in diabetic mice, hyperoxia enhances the mobilization of circulating endothelial progenitor cells (EPCs) from the bone marrow to the peripheral circulation (see the related article beginning on page 1249). Local injection of the chemokine stromal cell–derived factor–1α then recruits these EPCs to the cutaneous wound site, resulting in accelerated wound healing. Thus, Gallagher et al. have identified novel potential targets for therapeutic intervention in diabetic wound healing.

P osttranslational modification is critical for the function of the gene products of ras oncogenes, which are frequently mutated in cancer. Ras proteins are modified by farnesyltransferase (FTase), but many related small GTPases that also end in a CAAX motif (where C is cysteine, A is often an aliphatic amino acid, and X is any amino acid) are modified by a closely related enzyme known as geranylgeranyltransferase type I (GGTase-I). Accordingly, inhibitors for both of these enzymes have been developed, and those active against FTase are in clinical trials. In this issue of the JCI, Sjogren et al. report the development of a mouse strain homozygous for a conditional allele of the gene that encodes GGTase-I (see the related article beginning on page 1294). They found that ablation of the GGTase-I–encoding gene in cells destined to produce lung tumors driven by oncogenic K-Ras resulted in delayed onset and decreased severity of disease, validating in a genetic model the theory that GGTase-I is a good target for anti-cancer drug development.

T ype 2 diabetes mellitus affects 9.6% of the adults in the United States and more than 200 million people worldwide. Diabetes can be a devastating disease, but it can now be treated with nine classes of approved drugs (insulins, sulfonylureas, glinides, biguanides, α-glucosidase inhibitors, thiazolidinediones, glucagon-like peptide 1 mimetics, amylin mimetics, and dipeptidyl peptidase 4 inhibitors), in addition to diet and exercise regimens. Choosing which drug to give a patient is based on efficacy and also availability, cost, safety, tolerability, and convenience. Personalized medicine promises a path for individually optimized treatment choices, but realizing this promise will require a more comprehensive characterization of disease and drug response. In this issue of the JCI, Shu et al. make significant progress by integrating diverse data supporting the hypothesis that genetic variation in organic cation transporter 1 (OCT1) affects the response to the widely used biguanide metformin (see the related article beginning on page 1422). We discuss metformin, OCT1, pharmacogenetics, and how the integrative genomics revolution is likely to change our understanding and treatment of diabetes.

M utations in presenilins are responsible for approximately 40% of all early-onset familial Alzheimer disease (FAD) cases in which a genetic cause has been identified. In addition, a number of mutations in presenilin-1 (PS1) have been suggested to be associated with the occurrence of frontal temporal dementia (FTD). Presenilins are highly conserved transmembrane proteins that support cleavage of the amyloid precursor protein by γ-secretase. Recently, we discovered that presenilins also function as passive ER Ca²⁺ leak channels. Here we used planar lipid bilayer reconstitution assays and Ca²⁺ imaging experiments with presenilin-null mouse embryonic fibroblasts to analyze ER Ca²⁺ leak function of 6 FAD-linked PS1 mutants and 3 known FTD-associated PS1 mutants. We discovered that L166P, A246E, E273A, G384A, and P436Q FAD mutations in PS1 abolished ER Ca²⁺ leak function of PS1. In contrast, A79V FAD mutation or FTD-associated mutations (L113P, G183V, and Rins352) did not appear to affect ER Ca²⁺ leak function of PS1 in our experiments. We validated our findings in Ca²⁺ imaging experiments with primary fibroblasts obtained from an FAD patient possessing mutant PS1-A246E. Our results indicate that many FAD mutations in presenilins are loss-of-function mutations affecting ER Ca²⁺ leak activity. In contrast, none of the FTD-associated mutations affected ER Ca²⁺ leak function of PS1, indicating that the observed effects are disease specific. Our observations are consistent with the potential role of disturbed Ca²⁺ homeostasis in Alzheimer disease pathogenesis.

R evertant mosaicism due to in vivo reversion of an inherited mutation has been described in the genetic skin disease epidermolysis bullosa (EB) for the genes KRT14 and COL17A1. Here we demonstrate the presence of multiple second-site mutations, all correcting the germline mutation LAMB3:c.628G→A;p.E210K, in 2 unrelated non-Herlitz junctional EB patients with revertant mosaicism. Both probands had a severe reduction in laminin-332 expression in their affected skin. Remarkably, the skin on the lower leg of patient 078-01 (c.628G→A/c.1903C→T) became progressively clinically healthy, with normal expression of laminin-332 on previously affected skin. In the other proband, 029-01 (c.628G→A/c.628G→A), the revertant patches were located at his arms, shoulder, and chest. DNA analysis showed different second-site mutations in revertant keratinocytes of distinct biopsy specimens (c.565-3T→C, c.596G→C;p.G199A, c.619A→C;p.K207Q, c.628+42G→A, and c.629-1G→A), implying that there is not a single preferred mechanism for the correction of a specific mutation. Our data offer prospects for EB treatment in particular cases, since revertant mosaicism seems to occur at a higher frequency than expected. This opens the possibility of applying revertant cell therapy in mosaic EB of the LAMB3 gene by using autologous naturally corrected keratinocytes, thereby bypassing the recombinant gene correction phase.

E ndothelial progenitor cells (EPCs) are essential in vasculogenesis and wound healing, but their circulating and wound level numbers are decreased in diabetes. This study aimed to determine mechanisms responsible for the diabetic defect in circulating and wound EPCs. Since mobilization of BM EPCs occurs via eNOS activation, we hypothesized that eNOS activation is impaired in diabetes, which results in reduced EPC mobilization. Since hyperoxia activates NOS in other tissues, we investigated whether hyperoxia restores EPC mobilization in diabetic mice through BM NOS activation. Additionally, we studied the hypothesis that impaired EPC homing in diabetes is due to decreased wound level stromal cell–derived factor–1α (SDF-1α), a chemokine that mediates EPC recruitment in ischemia. Diabetic mice showed impaired phosphorylation of BM eNOS, decreased circulating EPCs, and diminished SDF-1α expression in cutaneous wounds. Hyperoxia increased BM NO and circulating EPCs, effects inhibited by the NOS inhibitor N-nitro-l-arginine-methyl ester. Administration of SDF-1α into wounds reversed the EPC homing impairment and, with hyperoxia, synergistically enhanced EPC mobilization, homing, and wound healing. Thus, hyperoxia reversed the diabetic defect in EPC mobilization, and SDF-1α reversed the diabetic defect in EPC homing. The targets identified, which we believe to be novel, can significantly advance the field of diabetic wound healing.

R ag enzymes are the main players in V(D)J recombination, the process responsible for rearrangement of TCR and Ig genes. Hypomorphic Rag mutations in humans, which maintain partial V(D)J activity, cause a peculiar SCID associated with autoimmune-like manifestations, Omenn syndrome (OS). Although a deficient ability to sustain thymopoiesis and to produce a diverse T and B cell repertoire explains the increased susceptibility to severe infections, the molecular and cellular mechanisms underlying the spectrum of clinical and immunological features of OS remain poorly defined. In order to better define the molecular and cellular pathophysiology of OS, we generated a knockin murine model carrying the Rag2 R229Q mutation previously described in several patients with OS and leaky forms of SCID. These Rag2^R229Q/R229Q mice showed oligoclonal T cells, absence of circulating B cells, and peripheral eosinophilia. In addition, activated T cells infiltrated gut and skin, causing diarrhea, alopecia, and, in some cases, severe erythrodermia. These findings were associated with reduced thymic expression of Aire and markedly reduced numbers of naturally occurring Tregs and NKT lymphocytes. In conclusion, Rag2^R229Q/R229Q mice mimicked most symptoms of human OS; our findings support the notion that impaired immune tolerance and defective immune regulation are involved in the pathophysiology of OS.

P atients with Omenn syndrome (OS) have hypomorphic RAG mutations and develop varying manifestations of severe combined immunodeficiency. It is not known which symptoms are caused directly by the RAG mutations and which depend on other polymorphic genes. Our current understanding of OS is limited by the lack of an animal model. In the present study, we identified a C57BL/10 mouse with a spontaneous mutation in, and reduced activity of, RAG1. Mice bred from this animal contained high numbers of memory-phenotype T cells and experienced hepatosplenomegaly and eosinophilia, had oligoclonal T cells, and demonstrated elevated levels of IgE, major symptoms of OS. Depletion of CD4⁺ T cells in the mice caused a reduction in their IgE levels. Hence these “memory mutant” mice are a model for human OS; many symptoms of their disease were direct results of the Rag hypomorphism and some were caused by malfunctions of their CD4⁺ T cells.

M utations in LMNA, which encodes nuclear Lamins A and C cause diseases affecting various organs, including the heart. We have determined the effects of an Lmna H222P mutation on signaling pathways involved in the development of cardiomyopathy in a knockin mouse model of autosomal dominant Emery-Dreifuss muscular dystrophy. Analysis of genome-wide expression profiles in hearts using Affymetrix GeneChips showed statistically significant differences in expression of genes in the MAPK pathways at the incipience of the development of clinical disease. Using real-time PCR, we showed that activation of MAPK pathways preceded clinical signs or detectable molecular markers of cardiomyopathy. In heart tissue and isolated cardiomyocytes, there was activation of MAPK cascades and downstream targets, implicated previously in the pathogenesis of cardiomyopathy. Expression of H222P Lamin A in cultured cells activated MAPKs and downstream target genes. Activation of MAPK signaling by mutant A-type lamins could be a cornerstone in the development of heart disease in autosomal dominant Emery-Dreifuss muscular dystrophy.

P rotein geranylgeranyltransferase type I (GGTase-I) is responsible for the posttranslational lipidation of CAAX proteins such as RHOA, RAC1, and cell division cycle 42 (CDC42). Inhibition of GGTase-I has been suggested as a strategy to treat cancer and a host of other diseases. Although several GGTase-I inhibitors (GGTIs) have been synthesized, they have very different properties, and the effects of GGTIs and GGTase-I deficiency are unclear. One concern is that inhibiting GGTase-I might lead to severe toxicity. In this study, we determined the effects of GGTase-I deficiency on cell viability and K-RAS–induced cancer development in mice. Inactivating the gene for the critical β subunit of GGTase-I eliminated GGTase-I activity, disrupted the actin cytoskeleton, reduced cell migration, and blocked the proliferation of fibroblasts expressing oncogenic K-RAS. Moreover, the absence of GGTase-I activity reduced lung tumor formation, eliminated myeloproliferative phenotypes, and increased survival of mice in which expression of oncogenic K-RAS was switched on in lung cells and myeloid cells. Interestingly, several cell types remained viable in the absence of GGTase-I, and myelopoiesis appeared to function normally. These findings suggest that inhibiting GGTase-I may be a useful strategy to treat K-RAS–induced malignancies.

W e investigated whether TGF-β induced by anticancer therapies accelerates tumor progression. Using the MMTV/PyVmT transgenic model of metastatic breast cancer, we show that administration of ionizing radiation or doxorubicin caused increased circulating levels of TGF-β1 as well as increased circulating tumor cells and lung metastases. These effects were abrogated by administration of a neutralizing pan–TGF-β antibody. Circulating polyomavirus middle T antigen–expressing tumor cells did not grow ex vivo in the presence of the TGF-β antibody, suggesting autocrine TGF-β is a survival signal in these cells. Radiation failed to enhance lung metastases in mice bearing tumors that lack the type II TGF-β receptor, suggesting that the increase in metastases was due, at least in part, to a direct effect of TGF-β on the cancer cells. These data implicate TGF-β induced by anticancer therapy as a prometastatic signal in tumor cells and provide a rationale for the simultaneous use of these therapies in combination with TGF-β inhibitors.

E wing sarcoma gene EWS encodes a putative RNA-binding protein with proposed roles in transcription and splicing, but its physiological role in vivo remains undefined. Here, we have generated Ews-deficient mice and demonstrated that EWS is required for the completion of B cell development and meiosis. Analysis of Ews^–/– lymphocytes revealed a cell-autonomous defect in precursor B lymphocyte (pre–B lymphocyte) development. During meiosis, Ews-null spermatocytes were deficient in XY bivalent formation and showed reduced meiotic recombination, resulting in massive apoptosis and complete arrest in gamete maturation. Inactivation of Ews in mouse embryonic fibroblasts resulted in premature cellular senescence, and the mutant animals showed hypersensitivity to ionizing radiation. Finally, we showed that EWS interacts with lamin A/C and that loss of EWS results in a reduced lamin A/C expression. Our findings reveal essential functions for EWS in pre–B cell development and meiosis, with proposed roles in DNA pairing and recombination/repair mechanisms. Furthermore, we demonstrate a novel role of EWS in cellular senescence, possibly through its interaction and modulation of lamin A/C.

C ytoskeletal proteins have been implicated in the pathogenesis of cardiomyopathy, but how the cytoskeleton influences the transcriptional alterations associated with adverse cardiac remodeling remains unclear. Striated muscle activator of Rho signaling (STARS) is a muscle-specific actin-binding protein localized to the Z disc that activates serum response factor–dependent (SRF-dependent) transcription by inducing nuclear translocation of the myocardin-related SRF coactivators MRTF-A and -B. We show that STARS expression is upregulated in mouse models of cardiac hypertrophy and in failing human hearts. A conserved region of the STARS promoter containing an essential binding site for myocyte enhancer factor–2 (MEF2), a stress-responsive transcriptional activator, mediates cardiac expression of STARS, which in turn activates SRF target genes. Forced overexpression of STARS in the heart sensitizes the heart to pressure overload and calcineurin signaling, resulting in exaggerated deterioration in cardiac function in response to these hypertrophic stimuli. These findings suggest that STARS modulates the responsiveness of the heart to stress signaling by functioning as a cytoskeletal intermediary between MEF2 and SRF.

W e examined the role of p38α MAPK in mediating cardiomyopathy in mice overexpressing β₁-adrenergic receptor (β₁-AR) or β₂-AR by mating them with dominant-negative p38α (DNp38α) MAPK mice. Both β₁-AR and β₂-AR Tg mice had enhanced LV ejection fraction (LVEF) as young adults and developed similar cardiomyopathy at 11–15 months, characterized by reduced LVEF, myocyte hypertrophy, fibrosis, and apoptosis. We inhibited p38α MAPK by mating β₁-AR Tg and β₂-AR Tg mice with DNp38α MAPK mice, which rescued the depressed LVEF and reduced apoptosis and fibrosis in bigenic β₂-AR × DNp38α MAPK mice, but not bigenic β₁-AR × DNp38α MAPK mice, and failed to reduce myocyte hypertrophy in either group. G_sα was increased in both β₁-AR Tg and β₂-AR Tg mice and was still present in bigenic β₁-AR × DNp38α MAPK mice, but not bigenic β₂-AR × DNp38α MAPK mice. This suggests that p38α MAPK is one critical downstream signal for the development of cardiomyopathy following chronic β₂-AR stimulation, but other kinases may be more important in ameliorating the adverse effects of chronic β₁-AR stimulation.

S tudies with isolated membrane fractions have shown that calmodulin (CaM) inhibits the activity of cardiac muscle cell Ca²⁺ release channel ryanodine receptor 2 (RyR2). To determine the physiological importance of CaM regulation of RyR2, we generated a mouse with 3 amino acid substitutions (RyR2-W3587A/L3591D/F3603A) in exon 75 of the Ryr2 gene, which encodes the CaM-binding site of RyR2. Homozygous mutant mice showed an increased ratio of heart weight to body weight, greatly reduced fractional shortening of the left ventricle, and lethality at 9–16 days of age. Biochemical analysis of hearts of 7- and 10-day-old homozygous mutant mice indicated an impaired CaM inhibition of RyR2 at micromolar Ca²⁺ concentrations, reduction in RyR2 protein levels and sarcoplasmic reticulum Ca²⁺ sequestration, and upregulation of genes and/or proteins associated with class II histone deacetylase/myocyte enhancer factor-2 and calcineurin signaling pathways. Sustained Ca²⁺ transients, often displaying repeated periods of incomplete Ca²⁺ removal, were observed in homozygous cardiomyocytes. Taken together, the data indicate that impaired CaM inhibition of RyR2, associated with defective sarcoplasmic reticulum Ca²⁺ release and altered gene expression, leads to cardiac hypertrophy and early death.

T he adipose-derived hormone, leptin, acts via its receptor (LRb) to convey the status of body energy stores to the brain, decreasing feeding and potentiating neuroendocrine energy expenditure. The failure of high levels of leptin in most obese individuals to promote weight loss defines a state of diminished responsiveness to increased leptin, termed leptin resistance. Leptin stimulates the phosphorylation of several tyrosine residues on LRb to mediate leptin action. We homologously replaced LRb in mice with a receptor with a mutation in one of these sites (Tyr985) in order to examine its role in leptin action and signal attenuation in vivo. Mice homozygous for this mutation are neuroendocrinologically normal, but females demonstrate decreased feeding, decreased expression of orexigenic neuropeptides, protection from high-fat diet–induced obesity, and increased leptin sensitivity in a sex-biased manner. Thus, leptin activates autoinhibitory signals via LRb Tyr985 to attenuate the anti-adiposity effects of leptin, especially in females, potentially contributing to leptin insensitivity in obesity.

W e have developed a model of autoimmunity to investigate autoantibody-mediated cross-presentation of self antigen. RIP-mOVA mice, expressing OVA in pancreatic β cells, develop severe autoimmune diabetes when given OT-I cells (OVA-specific CD8⁺ T cells) and anti-OVA IgG but not when given T cells alone. Anti-OVA IgG is not directly injurious to the islets but rather enhances cross-presentation of apoptotic islet antigen to the OT-I cells, leading to their differentiation into potent effector cells. Antibody-driven effector T cell activation is dependent on the presence of activating Fc receptors for IgG (FcγRs) and cross-priming DCs. As a consequence, diabetes incidence and severity was reduced in mice lacking activating FcγRs. An intact complement pathway was also required for disease development, as C3 deficiency was also partially protective. C3-deficient animals exhibited augmented T cell priming overall, indicating a proinflammatory role for complement activation after the T cell priming phase. Thus, we show that autoreactive antibody can potently enhance the activation of effector T cells in response to cross-presented self antigen, thereby contributing to T cell–mediated autoimmunity.

B reast cancers lacking estrogen and progesterone receptor expression and Her2 amplification exhibit distinct gene expression profiles and clinical features, and they comprise the majority of BRCA1-associated tumors. Here we demonstrated that the p53 family member p63 controls a pathway for p73-dependent cisplatin sensitivity specific to these “triple-negative” tumors. In vivo, ΔNp63 and TAp73 isoforms were coexpressed exclusively within a subset of triple-negative primary breast cancers that commonly exhibited mutational inactivation of p53. The ΔNp63α isoform promoted survival of breast cancer cells by binding TAp73 and thereby inhibiting its proapoptotic activity. Consequently, inhibition of p63 by RNA interference led to TAp73-dependent induction of proapoptotic Bcl-2 family members and apoptosis. Breast cancer cells expressing ΔNp63α and TAp73 exhibited cisplatin sensitivity that was uniquely dependent on TAp73. Thus, in response to treatment with cisplatin, but not other chemotherapeutic agents, TAp73 underwent c-Abl–dependent phosphorylation, which promoted dissociation of the ΔNp63α/TAp73 protein complex, TAp73-dependent transcription of proapoptotic Bcl-2 family members, and apoptosis. These findings define p63 as a survival factor in a subset of breast cancers; furthermore, they provide what we believe to be a novel mechanism for cisplatin sensitivity in these triple-negative cancers, and they suggest that such cancers may share the cisplatin sensitivity of BRCA1-associated tumors.

G lucocorticoids (GCs) are widely used in the treatment of allergic skin conditions despite having numerous side effects. Here we use Cre/loxP-engineered tissue- and cell-specific and function-selective GC receptor (GR) mutant mice to identify responsive cell types and molecular mechanisms underlying the antiinflammatory activity of GCs in contact hypersensitivity (CHS). CHS was repressed by GCs only at the challenge phase, i.e., during reexposure to the hapten. Inactivation of the GR gene in keratinocytes or T cells of mutant mice did not attenuate the effects of GCs, but its ablation in macrophages and neutrophils abolished downregulation of the inflammatory response. Moreover, mice expressing a DNA binding–defective GR were also resistant to GC treatment. The persistent infiltration of macrophages and neutrophils in these mice is explained by an impaired repression of inflammatory cytokines and chemokines such as IL-1β, monocyte chemoattractant protein-1, macrophage inflammatory protein-2, and IFN-γ–inducible protein 10. In contrast TNF-α repression remained intact. Consequently, injection of recombinant proteins of these cytokines and chemokines partially reversed suppression of CHS by GCs. These studies provide evidence that in contact allergy, therapeutic action of corticosteroids is in macrophages and neutrophils and that dimerization GR is required.

R eceptor-mediated airway smooth muscle (ASM) contraction via G_αq, and relaxation via G_αs, underlie the bronchospastic features of asthma and its treatment. Asthma models show increased ASM G_αi expression, considered the basis for the proasthmatic phenotypes of enhanced bronchial hyperreactivity to contraction mediated by M₃-muscarinic receptors and diminished relaxation mediated by β₂-adrenergic receptors (β₂ARs). A causal effect between G_i expression and phenotype has not been established, nor have mechanisms whereby G_i modulates G_q/G_s signaling. To delineate isolated effects of altered G_i, transgenic mice were generated overexpressing G_αi2 or a G_αi2 peptide inhibitor in ASM. Unexpectedly, G_αi2 overexpression decreased contractility to methacholine, while G_αi2 inhibition enhanced contraction. These opposite phenotypes resulted from different crosstalk loci within the G_q signaling network: decreased phospholipase C and increased PKCα, respectively. G_αi2 overexpression decreased β₂AR-mediated airway relaxation, while G_αi2 inhibition increased this response, consistent with physiologically relevant coupling of this receptor to both G_s and G_i. IL-13 transgenic mice (a model of asthma), which developed increased ASM G_αi, displayed marked increases in airway hyperresponsiveness when G_αi function was inhibited. Increased G_αi in asthma is therefore a double-edged sword: a compensatory event mitigating against bronchial hyperreactivity, but a mechanism that evokes β-agonist resistance. By selective intervention within these multipronged signaling modules, advantageous G_s/G_q activities could provide new asthma therapies.

H ow the fetus escapes rejection by the maternal immune system remains one of the major unsolved questions in transplantation immunology. Using a system to visualize both CD4⁺ and CD8⁺ T cell responses during pregnancy in mice, we find that maternal T cells become aware of the fetal allograft exclusively through “indirect” antigen presentation, meaning that T cell engagement requires the uptake and processing of fetal/placental antigen by maternal APCs. This reliance on a relatively minor allorecognition pathway removes a major threat to fetal survival, since it avoids engaging the large number of T cells that typically drive acute transplant rejection through their ability to directly interact with foreign MHC molecules. Furthermore, CD8⁺ T cells that indirectly recognize fetal/placental antigen undergo clonal deletion without priming for cytotoxic effector function and cannot induce antigen-specific fetal demise even when artificially activated. Antigen presentation commenced only at mid-gestation, in association with the endovascular invasion of placental trophoblasts and the hematogenous release of placental debris. Our results suggest that limited pathways of antigen presentation, in conjunction with tandem mechanisms of immune evasion, contribute to the unique immunological status of the fetus. The pronounced degree of T cell ignorance of the fetus also has implications for the pathophysiology of immune-mediated early pregnancy loss.

R etinoic acid receptors (RARs) are members of the nuclear hormone receptor family and regulate the proliferation and differentiation of multiple different cell types, including promyelocytic leukemia cells. Here we describe a biochemical/functional interaction between the Ca²⁺/calmodulin–dependent protein kinases (CaMKs) and RARs that modulates the differentiation of myeloid leukemia cells. We observe that CaMKIIγ is the CaMK that is predominantly expressed in myeloid cells. CaMKII inhibits RAR transcriptional activity, and this enzyme directly interacts with RAR through a CaMKII LxxLL binding motif. CaMKIIγ phosphorylates RARα both in vitro and in vivo, and this phosphorylation inhibits RARα activity by enhancing its interaction with transcriptional corepressors. In myeloid cell lines, CaMKIIγ localizes to RAR target sites within myeloid gene promoters but dissociates from the promoter upon retinoic acid–induced myeloid cell differentiation. KN62, a pharmacological inhibitor of the CaMKs, enhances the terminal differentiation of myeloid leukemia cell lines, and this is associated with a reduction in activated (autophosphorylated) CaMKII in the terminally differentiating cells. These observations reveal a significant cross-talk between Ca²⁺ and retinoic acid signaling pathways that regulates the differentiation of myeloid leukemia cells, and they suggest that CaMKIIγ may provide a new therapeutic target for the treatment of certain human myeloid leukemias.

M etformin is among the most widely prescribed drugs for the treatment of type 2 diabetes. Organic cation transporter 1 (OCT1) plays a role in the hepatic uptake of metformin, but its role in the therapeutic effects of the drug, which involve activation of AMP-activated protein kinase (AMPK), is unknown. Recent studies have shown that human OCT1 is highly polymorphic. We investigated whether OCT1 plays a role in the action of metformin and whether individuals with OCT1 polymorphisms have reduced response to the drug. In mouse hepatocytes, deletion of Oct1 resulted in a reduction in the effects of metformin on AMPK phosphorylation and gluconeogenesis. In Oct1-deficient mice the glucose-lowering effects of metformin were completely abolished. Seven nonsynonymous polymorphisms of OCT1 that exhibited reduced uptake of metformin were identified. Notably, OCT1-420del (allele frequency of about 20% in white Americans), previously shown to have normal activity for model substrates, had reduced activity for metformin. In clinical studies, the effects of metformin in glucose tolerance tests were significantly lower in individuals carrying reduced function polymorphisms of OCT1. Collectively, the data indicate that OCT1 is important for metformin therapeutic action and that genetic variation in OCT1 may contribute to variation in response to the drug.

A MP-activated protein kinase (AMPK) responds to impaired cellular energy status by stimulating substrate metabolism for ATP generation. Mutation of the γ2 regulatory subunit of AMPK in humans renders the kinase insensitive to energy status and causes glycogen storage cardiomyopathy via unknown mechanisms. Using transgenic mice expressing one of the mutant γ2 subunits (N488I) in the heart, we found that aberrant high activity of AMPK in the absence of energy deficit caused extensive remodeling of the substrate metabolism pathways to accommodate increases in both glucose uptake and fatty acid oxidation in the hearts of γ2 mutant mice via distinct, yet synergistic mechanisms resulting in selective fuel storage as glycogen. Increased glucose entry in the γ2 mutant mouse hearts was directed through the remodeled metabolic network toward glycogen synthesis and, at a substantially higher glycogen level, recycled through the glycogen pool to enter glycolysis. Thus, the metabolic consequences of chronic activation of AMPK in the absence of energy deficiency is distinct from those previously reported during stress conditions. These findings are of particular importance in considering AMPK as a target for the treatment of metabolic diseases.

T he Fanconi anemia (FA) pathway maintains genomic stability in replicating cells. Some sporadic breast, ovarian, pancreatic, and hematological tumors are deficient in FA pathway function, resulting in sensitivity to DNA-damaging agents. FA pathway dysfunction in these tumors may result in hyperdependence on alternative DNA repair pathways that could be targeted as a treatment strategy. We used a high-throughput siRNA screening approach that identified ataxia telangiectasia mutated (ATM) as a critical kinase for FA pathway–deficient human fibroblasts. Human fibroblasts and murine embryonic fibroblasts deficient for the FA pathway were observed to have constitutive ATM activation and Fancg^–/–Atm^–/– mice were found to be nonviable. Abrogation of ATM function in FA pathway–deficient cells resulted in DNA breakage, cell cycle arrest, and apoptotic cell death. Moreover, Fanconi anemia complementation group G– (FANCG-) and FANCC-deficient pancreatic tumor lines were more sensitive to the ATM inhibitor KU-55933 than isogenic corrected lines. These data suggest that ATM and FA genes function in parallel and compensatory roles to maintain genomic integrity and cell viability. Pharmaceutical inhibition of ATM may have a role in the treatment of FA pathway–deficient human cancers.