One in seven pathogenic variants can be challenging to detect by NGS: an analysis of 450,000 patients with implications for clinical sensitivity and genetic test implementation.

PURPOSE: To evaluate the impact of technically challenging variants on the implementation, validation, and diagnostic yield of commonly used clinical genetic tests. Such variants include large indels, small copy-number variants (CNVs), complex alterations, and variants in low-complexity or segmentally duplicated regions. METHODS: An interlaboratory pilot study used synthetic specimens to assess detection of challenging variant types by various next-generation sequencing (NGS)-based workflows. One well-performing workflow was further validated and used in clinician-ordered testing of more than 450,000 patients. RESULTS: In the interlaboratory study, only 2 of 13 challenging variants were detected by all 10 workflows, and just 3 workflows detected all 13. Limitations were also observed among 11 less-challenging indels. In clinical testing, 21.6% of patients carried one or more pathogenic variants, of which 13.8% (17,561) were classified as technically challenging. These variants were of diverse types, affecting 556 of 1,217 genes across hereditary cancer, cardiovascular, neurological, pediatric, reproductive carrier screening, and other indicated tests. CONCLUSION: The analytic and clinical sensitivity of NGS workflows can vary considerably, particularly for prevalent, technically challenging variants. This can have important implications for the design and validation of tests (by laboratories) and the selection of tests (by clinicians) for a wide range of clinical indications.

Integrating oral and general health screening at senior centers for minority elders.

Racial/ethnic and socioeconomic disparities regarding untreated oral disease exist for older adults, and poor oral health diminishes quality of life. The ElderSmile program integrated screening for diabetes and hypertension into its community-based oral health activities at senior centers in northern Manhattan. The program found a willingness among minority seniors (aged ≥ 50 years) to be screened for primary care sensitive conditions by dental professionals and a high level of unrecognized disease (7.8% and 24.6% of ElderSmile participants had positive screening results for previously undiagnosed diabetes and hypertension, respectively). Dental professionals may screen for primary care-sensitive conditions and refer patients to health care providers for definitive diagnosis and treatment. The ElderSmile program is a replicable model for community-based oral and general health screening.

Upregulation of mucin4 in ER-positive/HER2-overexpressing breast cancer xenografts with acquired resistance to endocrine and HER2-targeted therapies.

We studied resistance to endocrine and HER2-targeted therapies using a xenograft model of estrogen receptor positive (ER)/HER2-overexpressing breast cancer. Here, we report a novel phenotype of drug resistance in this model. MCF7/HER2-18 xenografts were treated with endocrine therapy alone or in combination with lapatinib and trastuzumab (LT) to inhibit HER2. Archival tumor tissues were stained with hematoxylin and eosin and with mucicarmine. RNA extracted from tumors at early time points and late after acquired resistance were analyzed for mucin4 (MUC4) expression by microarray and quantitative reverse transcriptase-PCR. Protein expression of the MUC4, ER, and HER2 signaling pathways was measured by immunohistochemistry and western blotting. The combination of the potent anti-HER2 regimen LT with either tamoxifen (Tam + LT) or estrogen deprivation (ED + LT) can cause complete eradication of ER-positive/HER2-overexpressing tumors in mice. Tumors developing resistance to this combination, as well as those acquiring resistance to endocrine therapy alone, exhibited a distinct histological and molecular phenotype-a striking increase in mucin-filled vacuoles and upregulation of several mucins including MUC4. At the onset of resistance, MUC4 mRNA and protein were increased. These tumors also showed upregulation and reactivation of HER2 signaling, while losing ER protein and the estrogen-regulated gene progesterone receptor. Mucins are upregulated in a preclinical model of ER-positive/HER2-overexpressing breast cancer as resistance develops to the combination of endocrine and anti-HER2 therapy. These mucin-rich tumors reactivate the HER2 pathway and shift their molecular phenotype to become more ER-negative/HER2-positive.

Challenges with development of a pharmacokinetics assay to measure a variably glycosylated fusion protein.

Aim: Development of recombinant fusion proteins as drugs poses unique challenges for bioanalysis. This paper describes a case study of a glycosylated fusion protein, where variable glycosylation, matrix interference and high sensitivity needs posed unique challenges. Results: Six different assay configurations, across four different platforms were evaluated for measurement of drug concentrations. Two platforms that achieved the assay requirements were Simoa HD-1 and immune-capture LC-MS/MS-based assay. Conclusion: Both, Simoa HD-1 and the mass spectrometry-based methods were able to detect total drug by providing the adequate matrix tolerance, required sensitivity and detection of all the various glycosylated fusion proteins to support clinical sample analysis. The mass spectrometry-based method was selected due to robustness and ease of method transfer.

More on FOX News: FOXA1 on the horizon of estrogen receptor function and endocrine response.

Estrogen receptor α (ER) is a major driver of breast cancer and the target of endocrine therapy. Full disclosure of the cofactors regulating ER interactions with chromatin and its transcriptional regulatory activity is still elusive. Novel genome-wide profiling tools have mapped ER binding events in breast cancer cells and delineated cofactors important in ER activity. Among these, the Forkhead protein FOXA1 is emerging as a key factor dictating global chromatin structure and the transcriptional function of ER in breast and non-breast cancer cells. The significance of FOXA1 in the chromatin interactions and transcriptional regulation of both estrogen- and tamoxifen-bound ER, and in supporting tamoxifen-resistant cell growth, may impact current endocrine therapies.

ß1 integrin mediates an alternative survival pathway in breast cancer cells resistant to lapatinib.

INTRODUCTION: The overexpression of human epidermal growth factor receptor (HER)-2 in 20% of human breast cancers and its association with aggressive growth has led to widespread use of HER2-targeted therapies, such as trastuzumab (T) and lapatinib (L). Despite the success of these drugs, their efficacy is limited in patients whose tumors demonstrate de novo or acquired resistance to treatment. The ß1 integrin resides on the membrane of the breast cancer cell, activating several elements of breast tumor progression including proliferation and survival. METHODS: We developed a panel of HER2-overexpressing cell lines resistant to L, T, and the potent LT combination through long-term exposure and validated these models in 3D culture. Parental and L/T/LT-resistant cells were subject to HER2 and ß1 integrin inhibitors in 3D and monitored for 12 days, followed by quantification of colony number. Parallel experiments were conducted where cells were either stained for Ki-67 and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) or harvested for protein and analyzed by immunoblot. Results were subjected to statistical testing using analysis of variance and linear contrasts, followed by adjustment with the Sidak method. RESULTS: Using multiple cell lines including BT474 and HCC1954, we reveal that in L and LT resistance, where phosphorylation of EGFR/HER1, HER2, and HER3 are strongly inhibited, kinases downstream of ß1 integrin--including focal adhesion kinase (FAK) and Src--are up-regulated. Blockade of ß1 by the antibody AIIB2 abrogates this up-regulation and functionally achieves significant growth inhibition of L and LT resistant cells in 3D, without dramatically affecting the parental cells. SiRNA against ß1 as well as pharmacologic inhibition of FAK achieve the same growth inhibitory effect. In contrast, trastuzumab-resistant cells, which retain high levels of phosphorylated EGFR/HER1, HER2, and HER3, are only modestly growth-inhibited by AIIB2. CONCLUSIONS: Our data suggest that HER2 activity, which is suppressed in resistance involving L but not T alone, dictates whether ß1 mediates an alternative pathway driving resistance. Our findings justify clinical studies investigating the inhibition of ß1 or its downstream signaling moieties as strategies to overcome acquired L and LT resistance.

An integrated approach for characterizing immunogenic responses toward a bispecific antibody.

Bispecific antibodies (bsAbs) recognize and bind two different targets or two epitopes of the same antigen, making them an attractive diagnostic and treatment modality. Compared to the production of conventional bivalent monospecific antibodies, bsAbs require greater engineering and manufacturing. Therefore, bsAbs are more likely to differ from endogenous immunoglobulins and contain new epitopes that can increase immunogenic risk. Anti-A/B is a bsAb designed using a 'knobs-into-holes' (KIH) format. Anti-A/B exhibited an unexpectedly high immunogenicity in both preclinical and clinical studies, resulting in early termination of clinical development. Here, we used an integrated approach that combined in silico analysis, in vitro assays, and an in vivo study in non-human primates to characterize anti-A/B immunogenicity. Our findings indicated that the immunogenicity is associated with epitopes in the anti-B arm and not with mutations engineered through the KIH process. Our results showed the value of this integrated approach for performing immunogenicity risk assessment during clinical candidate selection to effectively mitigate risks during bsAb development.

Effects of Singulair (montelukast) treatment for capsular contracture.

BACKGROUND: Capsular contracture (CC) is one of the most common complications of breast augmentation surgery. Leukotrienes are implicated in the inflammatory cascade and have been postulated to be involved in the formation of CC. Therefore, leukotriene antagonists Accolate and Singulair have been prescribed by plastic surgeons off-label to treat and prevent CC. To date, there are no studies investigating the efficacy of Singulair on CC. OBJECTIVE: The authors retrospectively review a series of patients treated with Singulair to determine whether it improves CC after breast implant surgery. METHODS: Nineteen patients treated with Singulair by the senior surgeon (NH) after implant placement from March 2006 to November 2009 were included in this study. Follow-up on Singulair efficacy was obtained by a combination of office chart review and standardized telephone questionnaire. Results were characterized as complete improvement, improvement, no change, or worse. RESULTS: Seventeen patients presented with CC resulting from a variety of breast operations. Two patients who had a history of recurrent CC were prescribed Singulair prophylactically immediately after surgery. Twenty-one breasts with existing CC were included in the total. Two (11%) patients became worse, three (16%) patients had no change, five (26%) improved, seven (37%) completely improved, and two (11%) were prevented from having CC formation. CONCLUSION: Our preliminary study shows that Singulair improves CC. Breasts with mild CC (Baker score < III) appeared to have better improvement with Singulair compared to those with more severe contracture (Baker score III and IV). Singulair is well tolerated with minimal side effects and can be administered to patients after breast implant surgery to improve CC.

Injectable dendritic cell-carrying alginate gels for immunization and immunotherapy.

Dendritic cell vaccines, in which antigen-loaded dendritic cells (DCs) are injected directly into patients to trigger immune responses, are in development as a treatment for cancer and some infectious diseases. In this study, we tested the concept of delivering DCs in an injectable hydrogel matrix, with the aim of harboring dendritic cells for prolonged time periods at a defined site and trapping/concentrating factors secreted by DCs to establish an inflammatory milieu in situ. To achieve these goals, a self-gelling formulation of alginate was developed, obtained by mixing calcium-loaded alginate microspheres with soluble alginate solution and dendritic cells, a formulation that rapidly gelled in vivo. When injected subcutaneously in mice, these alginate 'vaccination nodes' containing activated DCs attracted both host dendritic cells and a large number of T cells to the injection sites over a week in vivo, while some of the inoculated DCs trafficked to the draining lymph nodes. Using an adoptive transfer model to track a defined population of T cells responding to immunization with antigen-loaded DCs, we show that DC/alginate immunization led to recruitment of activated antigen-specific T cells to the alginate matrix, in a manner dependent on the presence of the DCs. This gel/DC immunization system may thus be of interest for immunotherapy to direct the accumulation of immune cells at solid tumors or infection sites in the presence of supporting factors co-delivered by the hydrogel matrix.

In Trypanosoma brucei RNA editing, band II enables recognition specifically at each step of the U insertion cycle.

Trypanosome RNA editing is the posttranscriptional insertion and deletion of uridylate (U) residues, often to a massive extent, through cycles of cleavage, U addition or U removal, and ligation. These editing cycles are catalyzed by a complex that we purified to seven major proteins (bands I through VII). Here we analyze the role of band II using extracts of clonal band II RNA interference (RNAi) cell lines prepared by a rapid protocol that enables retention of activities that are lost during traditional extract preparation. By individually scoring each step of editing, we show that band II is critical for all steps of U insertion but is not important for any of the steps of U deletion or for their coordination into the U deletion cycle. This specificity supports the long- standing model that U-insertional and U-deletional activities are separated within the editing complex. Furthermore, by assaying the basic activities of the enzymes that catalyze the steps of U insertion, independent of their action in editing, we show that band II is not any of those enzymes. Rather, band II enables endonuclease action at authentic U insertion sites, terminal-uridylyl-transferase (TUTase) action at cleaved U insertion sites, and U-insertion-specific ligase (band V/IREL) action in the editing complex. Thus, band II facilitates each step of U insertion by providing proper RNA and/or protein recognition. We propose that band II (TbMP81) be called IRER, indicating its essential nature in U-insertional RNA editing recognition.

A cell-laden microfluidic hydrogel.

The encapsulation of mammalian cells within the bulk material of microfluidic channels may be beneficial for applications ranging from tissue engineering to cell-based diagnostic assays. In this work, we present a technique for fabricating microfluidic channels from cell-laden agarose hydrogels. Using standard soft lithographic techniques, molten agarose was molded against a SU-8 patterned silicon wafer. To generate sealed and water-tight microfluidic channels, the surface of the molded agarose was heated at 71 degrees C for 3 s and sealed to another surface-heated slab of agarose. Channels of different dimensions were generated and it was shown that agarose, though highly porous, is a suitable material for performing microfluidics. Cells embedded within the microfluidic molds were well distributed and media pumped through the channels allowed the exchange of nutrients and waste products. While most cells were found to be viable upon initial device fabrication, only those cells near the microfluidic channels remained viable after 3 days, demonstrating the importance of a perfused network of microchannels for delivering nutrients and oxygen to maintain cell viability in large hydrogels. Further development of this technique may lead to the generation of biomimetic synthetic vasculature for tissue engineering, diagnostics, and drug screening applications.

Distinct functions of two RNA ligases in active Trypanosoma brucei RNA editing complexes.

Trypanosome RNA editing is a unique U insertion and U deletion process that involves cycles of pre-mRNA cleavage, terminal U addition or U removal, and religation. This editing can occur at massive levels and is directed by base pairing of trans-acting guide RNAs. Both U insertion and U deletion cycles are catalyzed by a single protein complex that contains only seven major proteins, band I through band VII. However, little is known about their catalytic functions, except that band IV and band V are RNA ligases and genetic analysis indicates that the former is important in U deletion. Here we establish biochemical approaches to distinguish the individual roles of these ligases, based on their distinctive ATP and pyrophosphate utilization. These in vitro analyses revealed that both ligases serve in RNA editing. Band V is the RNA editing ligase that functions very selectively to seal in U insertion (IREL), while band IV is the RNA editing ligase needed to seal in U deletion (DREL). In combination with our earlier findings about the cleavage and the U-addition/U-removal steps of U deletion and U insertion, these results show that all three steps of these editing pathways exhibit major differences and suggest that the editing complex could have physically separate regions for U deletion and U insertion.

Assembly and function of the RNA editing complex in Trypanosoma brucei requires band III protein.

Trypanosome RNA editing, the posttranscriptional insertion and deletion of U residues in mitochondrial transcripts, is catalyzed by a protein complex containing seven distinct proteins. In this study, we cloned the gene for band III, a 555-amino-acid protein with two separate zinc finger motifs. We prepared antibodies that showed band III protein cofractionates with the previously characterized band IV protein throughout the purification of the editing complex and is not found free or in other protein associations; therefore, it is a true constituent of the editing complex. Double-stranded RNA interference efficiently depleted band III protein and demonstrated that band III expression is essential for growth of procyclic trypanosomes and for RNA editing. These depleted cell extracts were deficient specifically in guide RNA-directed endonuclease cleavage at both U deletion and U insertion sites and in the activity of the band IV ligase, but they retained the 3'-U-exonuclease and terminal-U-transferase activities as well as band V ligase of the editing complex. Loss of band III protein also resulted in almost complete loss of the band IV ligase protein and altered sedimentation of the band V ligase. These data indicate that band III is either the RNA editing endonuclease or a factor critical for cleavage activity in the editing complex. They also demonstrate that band III is required for proper assembly of the editing complex.

Trypanosoma brucei RNA editing complex: band II is structurally critical and maintains band V ligase, which is nonessential.

Maturation of Trypanosoma brucei mitochondrial mRNA involves massive posttranscriptional insertion and deletion of uridine residues. This RNA editing utilizes an enzymatic complex with seven major proteins, band I through band VII. We here use RNA interference (RNAi) to examine the band II and band V proteins. Band II is found essential for viability; it is needed to maintain the normal structure of the editing complex and to retain the band V ligase protein. Previously, band III was found essential for certain activities, including maintenance of the editing complex and retention of the band IV ligase protein. Thus, band II and band V form a protein pair with features analogous to the band III/band IV ligase pair. Since band V is specific for U insertion and since band IV is needed for U deletion, their parallel organization suggests that the editing complex has a pseudosymmetry. However, unlike the essential band IV ligase, RNAi to band V has only a morphological but no growth rate effect, suggesting that it is stimulatory but nonessential. Indeed, in vitro analysis of band V RNAi cell extract demonstrates that band IV can seal U insertion when band V is lacking. Thus, band IV ligase is the first activity of the basic editing complex shown able to serve in both forms of editing. Our studies also indicate that the U insertional portion may be less central in the editing complex than the corresponding U deletional portion.

The truth about over-the-counter topical anti-aging products: a comprehensive review.

LEARNING OBJECTIVES: The reader is presumed to have knowledge of the basic concepts of skin aging. After studying this article, the participant should be able to: Physicians may earn 1 AMA PRA Category 1 credit by successfully completing the examination based on material covered in this article. The examination begins on page 413. ASAPS members can also complete this CME examination online by logging onto the ASAPS Members-Only Web site (http://www.surgery.org/members) and clicking on "Clinical Education" in the menu bar. One of the main objectives for an aesthetic surgery patient seeking consultation is a desire to look younger and reverse the appearance of aging. Most of these patients also use topical creams in addition to undergoing surgical procedures. Over-the-counter (OTC) anti-aging products are a billion-dollar industry to which even young patients who wish to prevent the aging process contribute. Many OTC products advertise dramatic results, but there have been relatively little scientific data to support these claims. We reviewed the literature on ingredients commonly found in OTC anti-aging creams. We conclude that although many different compounds are marketed as anti-aging products, studies proving their efficacy are limited. Vitamin C and alpha-hydroxy acids have been the most extensively researched products, and their anti-aging capabilities have been demonstrated in the literature. There have also been some promising studies on vitamin A and vitamin B derivatives. Moisturizers have been shown to increase skin hydration and improve the overall appearance of skin. Studies also indicate that pentapeptides can be effective in decreasing facial wrinkles and roughness. However, botanicals, which have become popular over the last few years, require significantly more research to formulate any positive conclusions for their topical application. As aesthetic surgeons, it behooves us to educate ourselves on the most common ingredients found in topical anti-aging products and their efficacy.

Use of Biosynthetic Controls as Performance Standards for Next-Generation Sequencing Assays of Somatic Tumors: A Multilaboratory Study.

BACKGROUND: Next-generation sequencing (NGS) assays are highly complex tests that can vary substantially in both their design and intended application. Despite their innumerous advantages, NGS assays present some unique challenges associated with the preanalytical process, library preparation, data analysis, and reporting. According to a number of professional laboratory organization, control materials should be included both during the analytical validation phase and in routine clinical use to guarantee highly accurate results. The SeraseqTM Solid Tumor Mutation Mix AF10 and AF20 control materials consist of 26 biosynthetic DNA constructs in a genomic DNA background, each containing a specific variant or mutation of interest and an internal quality marker at 2 distinct allelic frequencies of 10% and 20%, respectively. The goal of this interlaboratory study was to evaluate the Seraseq AF10 and AF20 control materials by verifying their performance as control materials and by evaluating their ability to measure quality metrics essential to a clinical test. METHODS: Performance characteristics were assessed within and between 6 CLIA-accredited laboratories and 1 research laboratory. RESULTS: Most laboratories detected all 26 mutations of interest; however, some discrepancies involving the internal quality markers were observed. CONCLUSION: This interlaboratory study showed that the Seraseq AF10 and AF20 control materials have high quality, stability, and genomic complexity in variant types that are well suited for assisting in NGS assay analytical validation and monitoring routine clinical applications.

Multiplexed Reference Materials as Controls for Diagnostic Next-Generation Sequencing: A Pilot Investigating Applications for Hypertrophic Cardiomyopathy.

Diagnostic next-generation sequencing (NGS)-based gene panels are increasingly used for prevalent disorders with genetic and clinical heterogeneity. Clinical development, validation, and quality management of these panels ideally includes reference samples containing prevalent pathogenic variants; however, clinical domain expertise to select appropriate variants may not be present, samples are often not publicly available, and their inclusion is associated with added cost. Expert-designed, multiplexed controls can remedy some of these challenges. One approach relies on spiking biosynthetic fragments carrying desired variants into human genomic DNA. We piloted the utility of this approach for hypertrophic cardiomyopathy. Data from >3000 previously sequenced probands were used to select 10 common pathogenic and/or technically challenging variants in the top hypertrophic cardiomyopathy genes. Multiplexed controls were constructed across a range of ideal and realistic allelic fractions for heterozygous germline variants. NGS was performed in quadruplicate, and results were compared with diagnostic NGS data for the source patient samples. Overall, results were indistinguishable from patient-derived data with variants being detected at or reasonably close to the targeted allelic fraction ratios. The exception was a common 25-bp deletion in MYBPC3, underscoring the importance of including such variants in test development. These controls may be an attractive addition to the repertoire of materials for development, validation, and quality monitoring of clinical NGS assays.

SECTM1 produced by tumor cells attracts human monocytes via CD7-mediated activation of the PI3K pathway.

Tumor-associated macrophages (TAMs) have essential roles in tumor progression and metastasis. Tumor cells recruit myeloid progenitors and monocytes to the tumor site, where they differentiate into TAMs; however, this process is not well studied in humans. Here we show that human CD7, a T-cell and NK cell receptor, is highly expressed by monocytes and macrophages. Expression of CD7 decreases in M-CSF-differentiated macrophages and in melanoma-conditioned medium-induced macrophages (MCMI/Mφ) in comparison to monocytes. A ligand for CD7, SECTM1 (secreted and transmembrane protein 1), is highly expressed in many tumors, including melanoma cells. We show that SECTM1 binds to CD7 and significantly increases monocyte migration by activation of the PI3K (phosphatidylinositol 3'-kinase) pathway. In human melanoma tissues, tumor-infiltrating macrophages expressing CD7 are present. These melanomas, with CD7-positive inflammatory cell infiltrations, frequently highly express SECTM1, including an N-terminal, soluble form, which can be detected in the sera of metastatic melanoma patients but not in normal sera. Taken together, our data demonstrate that CD7 is present on monocytes and tumor macrophages and that its ligand, SECTM1, is frequently expressed in corresponding melanoma tissues, possibly acting as a chemoattractant for monocytes to modulate the melanoma microenvironment.

PK assays for antibody-drug conjugates: case study with ado-trastuzumab emtansine.

BACKGROUND: Antibody-drug conjugates (ADCs) combine the characteristics of large-molecule biologics and small-molecule drugs and are heterogeneous mixtures that can biotransform in vivo, resulting in additional complexity. ADC bioanalytical strategies require novel analytical methods, as well as existing large- and small-molecule methods. Because ADCs in late-stage clinical development are relatively new, regulatory guidelines and standard industry best practices for developing strategies for bioanalytical PK assays are still being established. RESULTS: A PK assay strategy was developed that included comprehensive novel reagent and assay characterization approaches for the ADC ado-trastuzumab emtansine (T-DM1). CONCLUSION: The bioanalytical strategy was successfully applied to the drug development of T-DM1 and ensured that key analytes were accurately measured in support of nonclinical and clinical development.

K12/SECTM1, an interferon-γ regulated molecule, synergizes with CD28 to costimulate human T cell proliferation.

CD7 is a cell-surface molecule, expressed on T lymphocytes and NK cells, which functions as a costimulatory receptor for T cell proliferation. SECTM1 has been proposed as a ligand for CD7. However, the expression pattern of this molecule in human immune cells and role in human T cell function remain unclear. In the present study, using human rSECTM1, we demonstrate that SECTM1 strongly costimulates CD4 and CD8 T cell proliferation and induces IFN-γ production, likely via a CD7-dependent mechanism. In addition, SECTM1 synergizes with suboptimal anti-CD28 to strongly augment T cell functions. We found a robust induction of IL-2 production when SECTM1 and anti-CD28 signals were present with TCR ligation. Furthermore, addition of SECTM1 into a MLR significantly enhanced proliferation of alloantigen-activated T cells, whereas blockade of SECTM1 inhibited T cell proliferation in a two-way MLR assay. Simultaneously blocking the effect of SECTM1, along with CTLA-4/Fc, diminishes two-way MLR. Finally, we demonstrated that expression of SECTM1 is not detected in monocytes and imMoDCs at the protein level. However, it is strongly induced by IFN-γ in monocytes and imMoDCs, and this induction is STAT1-dependent. These results indicate that SECTM1 is a broadly expressed, IFN-γ-inducible molecule, which functions as a potent costimulatory ligand for T cell activation and is synergistic with anti-CD28.