Outcomes and Adverse Events in Patients with Cancer after Diagnosis of Immunotherapy-Associated Diabetes Mellitus: A Retrospective Cohort Study.

Immune checkpoint inhibitor (CPI)-induced diabetes mellitus (CPI-DM) is a rare immune-related adverse event (irAE). Patients and providers fear that continuing CPIs puts patients at risk for additional irAEs and thus may discontinue therapy. Currently, there are little data to inform this decision. Therefore, this study aims to elucidate whether discontinuing CPIs after diagnosis of CPI-DM impacts the development of future irAEs and cancer outcomes such as progression and death. Patients who developed CPI-DM during cancer treatment at UCSF from 1 July 2015 to 5 July 2023 were analyzed for cancer outcomes and irAE development. Fisher's exact tests, Student t-tests, Kaplan-Meier methods, and Cox regression were used as appropriate. Of the 43 patients with CPI-DM, 20 (47%) resumed CPIs within 90 days of the irAE, 4 (9%) patients restarted after 90 days, and 19 (44%) patients never restarted. Subsequent irAEs were diagnosed in 9 of 24 (38%) who resumed CPIs and 3 of 19 (16%) who discontinued CPIs (p = 0.17). There was no significant difference in death (p = 0.74) or cancer progression (p = 0.55) between these two groups. While our single-institution study did not show worse cancer outcomes after discontinuing CPIs, many variables can impact outcomes, which our study was not adequately powered to evaluate. A nuanced approach is needed to decide whether to continue CPI treatment after a severe irAE like CPI-DM.

Impact on in-depth immunophenotyping of delay to peripheral blood processing.

Peripheral blood mononuclear cell (PBMC) immunophenotyping is crucial in tracking activation, disease state, and response to therapy in human subjects. Many studies require shipping of blood from clinical sites to a laboratory for processing to PBMC, which can lead to delays that impact sample quality. We used an extensive cytometry by time-of-flight (CyTOF) immunophenotyping panel to analyze the impacts of delays to processing and distinct storage conditions on cell composition and quality of PBMC from seven adults across a range of ages, including two with rheumatoid arthritis. Two or more days delay to processing resulted in extensive red blood cell contamination and increased variability of cell counts. While total memory and naïve B and T cell populations were maintained, four days delay reduced frequencies of monocytes. Variation across all immune subsets increased with delays of up to seven days in processing. Unbiased clustering analysis to define more granular subsets confirmed changes in PBMC composition, including decreases of classical and non-classical monocytes, basophils, plasmacytoid dendritic cells, and follicular helper T cells, with each subset impacted at a distinct time of delay. Expression of activation markers and chemokine receptors changed by day two, with differential impacts across subsets and markers. Our data support existing recommendations to process PBMC within 36 hours of collection but provide guidance on appropriate immunophenotyping experiments with longer delays.

Aire mediates tolerance to insulin through thymic trimming of high-affinity T cell clones.

Insulin is a central autoantigen in the pathogenesis of T1D, and thymic epithelial cell expression of insulin under the control of the Autoimmune Regulator (Aire) is thought to be a key component of maintaining tolerance to insulin. In spite of this general working model, direct detection of this thymic selection on insulin-specific T cells has been somewhat elusive. Here, we used a combination of highly sensitive T cell receptor transgenic models for detecting thymic selection and sorting and sequencing of Insulin-specific CD4+ T cells from Aire-deficient mice as a strategy to further define their selection. This analysis revealed a number of unique t cell receptor (TCR) clones in Aire-deficient hosts with high affinity for insulin/major histocompatibility complex (MHC) ligands. We then modeled the thymic selection of one of these clones in Aire-deficient versus wild-type hosts and found that this model clone could escape thymic negative selection in the absence of thymic Aire. Together, these results suggest that thymic expression of insulin plays a key role in trimming and removing high-affinity insulin-specific T cells from the repertoire to help promote tolerance.

De novo identification of CD4+ T cell epitopes.

CD4+ T cells recognize peptide antigens presented on class II major histocompatibility complex (MHC-II) molecules to carry out their function. The remarkable diversity of T cell receptor sequences and lack of antigen discovery approaches for MHC-II make profiling the specificities of CD4+ T cells challenging. We have expanded our platform of signaling and antigen-presenting bifunctional receptors to encode MHC-II molecules presenting covalently linked peptides (SABR-IIs) for CD4+ T cell antigen discovery. SABR-IIs can present epitopes to CD4+ T cells and induce signaling upon their recognition, allowing a readable output. Furthermore, the SABR-II design is modular in signaling and deployment to T cells and B cells. Here, we demonstrate that SABR-IIs libraries presenting endogenous and non-contiguous epitopes can be used for antigen discovery in the context of type 1 diabetes. SABR-II libraries provide a rapid, flexible, scalable and versatile approach for de novo identification of CD4+ T cell ligands from single-cell RNA sequencing data using experimental and computational approaches.

Human inherited PD-L1 deficiency is clinically and immunologically less severe than PD-1 deficiency.

We previously reported two siblings with inherited PD-1 deficiency who died from autoimmune pneumonitis at 3 and 11 years of age after developing other autoimmune manifestations, including type 1 diabetes (T1D). We report here two siblings, aged 10 and 11 years, with neonatal-onset T1D (diagnosed at the ages of 1 day and 7 wk), who are homozygous for a splice-site variant of CD274 (encoding PD-L1). This variant results in the exclusive expression of an alternative, loss-of-function PD-L1 protein isoform in overexpression experiments and in the patients' primary leukocytes. Surprisingly, cytometric immunophenotyping and single-cell RNA sequencing analysis on blood leukocytes showed largely normal development and transcriptional profiles across lymphoid and myeloid subsets in the PD-L1-deficient siblings, contrasting with the extensive dysregulation of both lymphoid and myeloid leukocyte compartments in PD-1 deficiency. Our findings suggest that PD-1 and PD-L1 are essential for preventing early-onset T1D but that, unlike PD-1 deficiency, PD-L1 deficiency does not lead to fatal autoimmunity with extensive leukocytic dysregulation.

Neutralizing IFN-γ autoantibodies are rare and pathogenic in HLA-DRB1*15:02 or 16:02 individuals.

BACKGROUNDWeakly virulent environmental mycobacteria (EM) can cause severe disease in HLA-DRB1*15:02 or 16:02 adults harboring neutralizing anti-IFN-γ autoantibodies (nAIGAs). The overall prevalence of nAIGAs in the general population is unknown, as are the penetrance of nAIGAs in HLA-DRB1*15:02 or 16:02 individuals and the proportion of patients with unexplained, adult-onset EM infections carrying nAIGAs.METHODSThis study analyzed the detection and neutralization of anti-IFN-γ autoantibodies (auto-Abs) from 8,430 healthy individuals of the general population, 257 HLA-DRB1*15:02 or 16:02 carriers, 1,063 patients with autoimmune disease, and 497 patients with unexplained severe disease due to EM.RESULTSWe found that anti-IFN-γ auto-Abs detected in 4,148 of 8,430 healthy individuals (49.2%) from the general population of an unknown HLA-DRB1 genotype were not neutralizing. Moreover, we did not find nAIGAs in 257 individuals carrying HLA-DRB1* 15:02 or 16:02. Additionally, nAIGAs were absent in 1,063 patients with an autoimmune disease. Finally, 7 of 497 patients (1.4%) with unexplained severe disease due to EM harbored nAIGAs.CONCLUSIONThese findings suggest that nAIGAs are isolated and that their penetrance in HLA-DRB1*15:02 or 16:02 individuals is low, implying that they may be triggered by rare germline or somatic variants. In contrast, the risk of mycobacterial disease in patients with nAIGAs is high, confirming that these nAIGAs are the cause of EM disease.FUNDINGThe Laboratory of Human Genetics of Infectious Diseases is supported by the Howard Hughes Medical Institute, the Rockefeller University, the St. Giles Foundation, the National Institutes of Health (NIH) (R01AI095983 and U19AIN1625568), the National Center for Advancing Translational Sciences (NCATS), the NIH Clinical and Translational Science Award (CTSA) program (UL1 TR001866), the French National Research Agency (ANR) under the "Investments for the Future" program (ANR-10-IAHU-01), the Integrative Biology of Emerging Infectious Diseases Laboratory of Excellence (ANR-10-LABX-62-IBEID), ANR-GENMSMD (ANR-16-CE17-0005-01), ANR-MAFMACRO (ANR-22-CE92-0008), ANRSECTZ170784, the French Foundation for Medical Research (FRM) (EQU201903007798), the ANRS-COV05, ANR GENVIR (ANR-20-CE93-003), and ANR AI2D (ANR-22-CE15-0046) projects, the ANR-RHU program (ANR-21-RHUS-08-COVIFERON), the European Union's Horizon 2020 research and innovation program under grant agreement no. 824110 (EASI-genomics), the Square Foundation, Grandir - Fonds de solidarité pour l'enfance, the Fondation du Souffle, the SCOR Corporate Foundation for Science, the Battersea & Bowery Advisory Group, William E. Ford, General Atlantic's Chairman and Chief Executive Officer, Gabriel Caillaux, General Atlantic's Co-President, Managing Director, and Head of business in EMEA, and the General Atlantic Foundation, Institut National de la Santé et de la Recherche Médicale (INSERM) and of Paris Cité University. JR was supported by the INSERM PhD program for doctors of pharmacy (poste d'accueil INSERM). JR and TLV were supported by the Bettencourt-Schueller Foundation and the MD-PhD program of the Imagine Institute. MO was supported by the David Rockefeller Graduate Program, the Funai Foundation for Information Technology (FFIT), the Honjo International Scholarship Foundation (HISF), and the New York Hideyo Noguchi Memorial Society (HNMS).

Aire in Autoimmunity.

The role of the autoimmune regulator (Aire) in central immune tolerance and thymic self-representation was first described more than 20 years ago, but fascinating new insights into its biology continue to emerge, particularly in the era of advanced single-cell genomics. We briefly describe the role of human genetics in the discovery of Aire, as well as insights into its function gained from genotype-phenotype correlations and the spectrum of Aire-associated autoimmunity-including insights from patients with Aire mutations with broad and diverse implications for human health. We then highlight emerging trends in Aire biology, focusing on three topic areas. First, we discuss medullary thymic epithelial diversity and the role of Aire in thymic epithelial development. Second, we highlight recent developments regarding the molecular mechanisms of Aire and its binding partners. Finally, we describe the rapidly evolving biology of the identity and function of extrathymic Aire-expressing cells (eTACs), and a novel eTAC subset called Janus cells, as well as their potential roles in immune homeostasis. Expected final online publication date for the Annual Review of Immunology, Volume 42 is April 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Tracking the role of Aire in immune tolerance to the eye with a TCR transgenic mouse model.

Roughly one-half of mice with partial defects in two immune tolerance pathways (AireGW/+Lyn-/- mice) spontaneously develop severe damage to their retinas due to T cell reactivity to Aire-regulated interphotoreceptor retinoid-binding protein (IRBP). Single-cell T cell receptor (TCR) sequencing of CD4+ T cells specific for a predominate epitope of IRBP showed a remarkable diversity of autoantigen-specific TCRs with greater clonal expansions in mice with disease. TCR transgenic mice made with an expanded IRBP-specific TCR (P2.U2) of intermediate affinity exhibited strong but incomplete negative selection of thymocytes. This negative selection was absent in IRBP-/- mice and greatly defective in AireGW/+ mice. Most P2.U2+/- mice and all P2.U.2+/-AireGW/+ mice rapidly developed inflammation of the retina and adjacent uvea (uveitis). Aire-dependent IRBP expression in the thymus also promoted Treg differentiation, but the niche for this fate determination was small, suggesting differences in antigen presentation leading to negative selection vs. thymic Treg differentiation and a stronger role for negative selection in preventing autoimmune disease in the retina.

Validation of a murine proteome-wide phage display library for identification of autoantibody specificities.

Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq) in profiling mouse autoantibodies. This library was validated using 7 genetically distinct mouse lines across a spectrum of autoreactivity. Mice deficient in antibody production (Rag2-/- and µMT) were used to model nonspecific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor-restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate 3 distinct autoimmune disease models. First, serum from Lyn-/- IgD+/- mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities. Second, serum from nonobese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, was enriched in peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire-/- mouse sera were used to identify numerous autoantigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 carrying recessive mutations in AIRE. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity profiling.

Immunosuppression causes dynamic changes in expression QTLs in psoriatic skin.

Psoriasis is a chronic, systemic inflammatory condition primarily affecting skin. While the role of the immune compartment (e.g., T cells) is well established, the changes in the skin compartment are more poorly understood. Using longitudinal skin biopsies (n = 375) from the "Psoriasis Treatment with Abatacept and Ustekinumab: A Study of Efficacy"(PAUSE) clinical trial (n = 101), we report 953 expression quantitative trait loci (eQTLs). Of those, 116 eQTLs have effect sizes that were modulated by local skin inflammation (eQTL interactions). By examining these eQTL genes (eGenes), we find that most are expressed in the skin tissue compartment, and a subset overlap with the NRF2 pathway. Indeed, the strongest eQTL interaction signal - rs1491377616-LCE3C - links a psoriasis risk locus with a gene specifically expressed in the epidermis. This eQTL study highlights the potential to use biospecimens from clinical trials to discover in vivo eQTL interactions with therapeutically relevant environmental variables.

Ikaros is a principal regulator of Aire+ mTEC homeostasis, thymic mimetic cell diversity, and central tolerance.

Mutations in the gene encoding the zinc-finger transcription factor Ikaros (IKZF1) are found in patients with immunodeficiency, leukemia, and autoimmunity. Although Ikaros has a well-established function in modulating gene expression programs important for hematopoietic development, its role in other cell types is less well defined. Here, we uncover functions for Ikaros in thymic epithelial lineage development in mice and show that Ikzf1 expression in medullary thymic epithelial cells (mTECs) is required for both autoimmune regulator-positive (Aire+) mTEC development and tissue-specific antigen (TSA) gene expression. Accordingly, TEC-specific deletion of Ikzf1 in mice results in a profound decrease in Aire+ mTECs, a global loss of TSA gene expression, and the development of autoimmunity. Moreover, Ikaros shapes thymic mimetic cell diversity, and its deletion results in a marked expansion of thymic tuft cells and muscle-like mTECs and a loss of other Aire-dependent mimetic populations. Single-cell analysis reveals that Ikaros modulates core transcriptional programs in TECs that correlate with the observed cellular changes. Our findings highlight a previously undescribed role for Ikaros in regulating epithelial lineage development and function and suggest that failed thymic central tolerance could contribute to the autoimmunity seen in humans with IKZF1 mutations.

Non-coding sequence variation reveals fragility within interleukin 2 feedback circuitry and shapes autoimmune disease risk.

Genetic variants associated with human autoimmune diseases commonly map to non-coding control regions, particularly enhancers that function selectively in immune cells and fine-tune gene expression within a relatively narrow range of values. How such modest, cell-type-selective changes can meaningfully shape organismal disease risk remains unclear. To explore this issue, we experimentally manipulated species-conserved enhancers within the disease-associated IL2RA locus and studied accompanying changes in the progression of autoimmunity. Perturbing distinct enhancers with restricted activity in conventional T cells (Tconvs) or regulatory T cells (Tregs)-two functionally antagonistic T cell subsets-caused only modest, cell-type-selective decreases in IL2ra expression parameters. However, these same perturbations had striking and opposing effects in vivo , completely preventing or severely accelerating disease in a murine model of type 1 diabetes. Quantitative tissue imaging and computational modelling revealed that each enhancer manipulation impinged on distinct IL-2-dependent feedback circuits. These imbalances altered the intracellular signaling and intercellular communication dynamics of activated Tregs and Tconvs, producing opposing spatial domains that amplified or constrained ongoing autoimmune responses. These findings demonstrate how subtle changes in gene regulation stemming from non-coding variation can propagate across biological scales due to non-linearities in intra- and intercellular feedback circuitry, dramatically shaping disease risk at the organismal level.

ABCC8-Related Monogenic Diabetes Presenting Like Type 1 Diabetes in an Adolescent.

Background: Identifying cases of diabetes caused by single gene mutations between the more common type 1 diabetes (T1D) and type 2 diabetes (T2D) is a difficult but important task. We report the diagnosis of ATP-binding cassette transporter sub-family C member 8 (ABCC8)-related monogenic diabetes in a 35-year-old woman with a protective human leukocyte antigen (HLA) allele who was originally diagnosed with T1D at 18 years of age. Case Report: Patient A presented with polyuria, polydipsia, and hypertension at the age of 18 years and was found to have a blood glucose > 500 mg/dL (70-199 mg/dL) and an HbA1C (hemoglobin A1C) >14% (4%-5.6%). She had an unmeasurable C-peptide but no urine ketones. She was diagnosed with T1D and started on insulin therapy. Antibody testing was negative. She required low doses of insulin and later had persistence of low but detectable C-peptide. At the age of 35 years, she was found to have a protective HLA allele, and genetic testing revealed a pathogenic mutation in the ABCC8 gene. The patient was then successfully transitioned to sulfonylurea therapy. Discussion: Monogenic diabetes diagnosed in adolescence typically presents with mild to moderate hyperglycemia, positive family history and, in some cases, other organ findings or dysfunction. The patient in this report presented with very high blood glucose, prompting the diagnosis of T1D. When she was found to have a protective HLA allele, further investigation revealed the mutation in the sulfonylurea receptor gene, ABCC8. Conclusion: Patients suspected of having T1D but with atypical clinical characteristics such as negative autoantibodies, low insulin requirements, and persistence of C-peptide should undergo genetic testing for monogenic diabetes.

T cell deletional tolerance restricts AQP4 but not MOG CNS autoimmunity.

Aquaporin-4 (AQP4)-specific Th17 cells are thought to have a central role in neuromyelitis optica (NMO) pathogenesis. When modeling NMO, only AQP4-reactive Th17 cells from AQP4-deficient (AQP4-/-), but not wild-type (WT) mice, caused CNS autoimmunity in recipient WT mice, indicating that a tightly regulated mechanism normally ensures tolerance to AQP4. Here, we found that pathogenic AQP4 T cell epitopes bind MHC II with exceptionally high affinity. Examination of T cell receptor (TCR) α/ß usage revealed that AQP4-specific T cells from AQP4-/- mice employed a distinct TCR repertoire and exhibited clonal expansion. Selective thymic AQP4 deficiency did not fully restore AQP4-reactive T cells, demonstrating that thymic negative selection alone did not account for AQP4-specific tolerance in WT mice. Indeed, AQP4-specific Th17 cells caused paralysis in recipient WT or B cell-deficient mice, which was followed by complete recovery that was associated with apoptosis of donor T cells. However, donor AQP4-reactive T cells survived and caused persistent paralysis in recipient mice deficient in both T and B cells or mice lacking T cells only. Thus, AQP4 CNS autoimmunity was limited by T cell-dependent deletion of AQP4-reactive T cells. In contrast, myelin oligodendrocyte glycoprotein (MOG)-specific T cells survived and caused sustained disease in WT mice. These findings underscore the importance of peripheral T cell deletional tolerance to AQP4, which may be relevant to understanding the balance of AQP4-reactive T cells in health and in NMO. T cell tolerance to AQP4, expressed in multiple tissues, is distinct from tolerance to MOG, an autoantigen restricted in its expression.

Autoantigen profiling reveals a shared post-COVID signature in fully recovered and long COVID patients.

Some individuals do not return to baseline health following SARS-CoV-2 infection, leading to a condition known as long COVID. The underlying pathophysiology of long COVID remains unknown. Given that autoantibodies have been found to play a role in severity of SARS-CoV-2 infection and certain other post-COVID sequelae, their potential role in long COVID is important to investigate. Here, we apply a well-established, unbiased, proteome-wide autoantibody detection technology (T7 phage-display assay with immunoprecipitation and next-generation sequencing, PhIP-Seq) to a robustly phenotyped cohort of 121 individuals with long COVID, 64 individuals with prior COVID-19 who reported full recovery, and 57 pre-COVID controls. While a distinct autoreactive signature was detected that separated individuals with prior SARS-CoV-2 infection from those never exposed to SARS-CoV-2, we did not detect patterns of autoreactivity that separated individuals with long COVID from individuals fully recovered from COVID-19. These data suggest that there are robust alterations in autoreactive antibody profiles due to infection; however, no association of autoreactive antibodies and long COVID was apparent by this assay.

An Accounting of Contamination Control Requirements, Implementation, and Verification of the Sample Tubes for the Mars 2020 Mission and Future Return Sample Science.

The Sample Tubes on the Mars 2020 Perseverance rover were required to meet strict cleanliness standards for possible organic and inorganic contamination introduction to collected samples. There were also strict planetary protection cleanliness standards required to limit possible biological contamination. Together, these sets of standards also applied to associated hardware, like the Sample Tube hermetic seals. This created unique challenges to manufacturing, cleaning, and verifying the final cleanliness state of the Sample Tubes, which are the main focus of this publication. Documenting the final cleanliness state of the Sample Tubes is critical for future analysis of collected martian samples, of significant interest to the scientific community, and will have implications for possible future missions like Mars Sample Return. An accounting of events that led to the final delivered state of the Sample Tubes on Earth with regard to contamination control cleanliness requirements, precision cleaning, processing, and verification are provided.

Lateral force separation of biopolymers using an atomic force microscope.

The lateral force separation of long chain biomolecules is demonstrated using an atomic force microscope (AFM). This is achieved by using an AFM tip to pull molecules away from the edge of a nanofluidic solution. By monitoring the torsion on the AFM cantilever, a characteristic force-distance signal is produced when long chain molecules separate and detach from the solvent edge. This lateral force separation using AFM (LFS-AFM) is demonstrated on egg albumin proteins and synthetic DNA strands. The detected length of the protein and nucleotide biopolymers was consistent with their calculated molecular contour length. LFS AFM provides separation and detection of single polymer strands that has potential applications in biochemical analysis, paleontology, and life detection.

A genomic data archive from the Network for Pancreatic Organ donors with Diabetes.

The Network for Pancreatic Organ donors with Diabetes (nPOD) is the largest biorepository of human pancreata and associated immune organs from donors with type 1 diabetes (T1D), maturity-onset diabetes of the young (MODY), cystic fibrosis-related diabetes (CFRD), type 2 diabetes (T2D), gestational diabetes, islet autoantibody positivity (AAb+), and without diabetes. nPOD recovers, processes, analyzes, and distributes high-quality biospecimens, collected using optimized standard operating procedures, and associated de-identified data/metadata to researchers around the world. Herein describes the release of high-parameter genotyping data from this collection. 372 donors were genotyped using a custom precision medicine single nucleotide polymorphism (SNP) microarray. Data were technically validated using published algorithms to evaluate donor relatedness, ancestry, imputed HLA, and T1D genetic risk score. Additionally, 207 donors were assessed for rare known and novel coding region variants via whole exome sequencing (WES). These data are publicly-available to enable genotype-specific sample requests and the study of novel genotype:phenotype associations, aiding in the mission of nPOD to enhance understanding of diabetes pathogenesis to promote the development of novel therapies.

Validation of a murine proteome-wide phage display library for the identification of autoantibody specificities.

Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq), to profile mouse autoantibodies. This system and library were validated using seven genetic mouse models across a spectrum of autoreactivity. Mice deficient in antibody production (Rag2-/- and µMT) were used to model non-specific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor (BCR)-restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate three distinct autoimmune disease models. First, serum from Lyn-/- IgD+/- mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities, lending support to the hypothesis that apoptosis is a shared origin of these antigens. Second, serum from non-obese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, enriched peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire-/- mouse sera were used to identify numerous auto-antigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 (APS1) carrying recessive mutations in AIRE. Among these were peptides derived from Perilipin-1, a validated autoimmune biomarker of generalized acquired lipodystrophy in humans. Autoreactivity to Perilipin-1 correlated with lymphocyte infiltration in adipose tissue and underscores the approach in revealing previously unknown specificities. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity.

Concurrent surface enhanced infrared and Raman spectroscopy with single molecule sensitivity.

Surface enhanced infrared absorption (SEIRA) and surface enhanced Raman Spectroscopy (SERS) were simultaneously measured from the same location on plasmonically active substrates. The spectra were acquired using an optical photothermal infrared spectrometer coupled with a Raman spectrometer. The sensitivity of this approach enables exceptionally small quantities of molecules to be interrogated while providing complementary information from both infrared and Raman spectroscopy. This arrangement provides additional improvement of SEIRA through the enhancement of both the optical photothermal detector signal and the infrared absorption. The plasmonic substrates tested were silver nanospheres and a gold coated atomic force microscope tip. The concurrent acquisition of SEIRA and SERS is further demonstrated by nano-sampling material onto an atomic force microscope tip. The analytes, Buckminsterfullerene and 1,2-bis(4-pyridyl) ethylene, were analyzed individually and as mixtures. The concurrent acquisition of SERIA and SERS is a unique approach. It has general applications in trace surface analysis and for the analysis of returned planetary samples.