Immune markers of severe acute pancreatitis.

PURPOSE OF REVIEW: Acute pancreatitis is a common acute inflammatory disorder of the pancreas, and its incidence has been increasing worldwide. Approximately 10% of acute pancreatitis progresses to severe acute pancreatitis (SAP), which carries significant morbidity and mortality. Disordered immune response to pancreatic injury is regarded as a key event that mediates systemic injury in SAP. In this article, we review recent developments in immune biomarkers of SAP and future directions for research. RECENT FINDINGS: Given the importance of the NLRP3-inflammasome pathway in mediating systemic inflammatory response syndrome and systemic injury, recent studies have investigated associations of SAP with systemic levels of activators of NLRP3, such as the damage associated molecular patterns (DAMPs) for the first time in human SAP. For example, circulating levels of histones, mitochondrial DNAs, and cell free DNAs have been associated with SAP. A panel of mechanistically relevant immune markers (e.g., panel of Angiopoeitin-2, hepatocyte growth factor, interleukin-8 (IL-8), resistin and sTNF-α R1) carried higher predictive accuracies than existing clinical scores and individual immune markers. Of the cytokines with established relevance to SAP pathogenesis, phase 2 trials of immunotherapies, including tumor necrosis factor (TNF)-alpha inhibition and stimulation of IL-10 production, are underway to determine if altering the immunologic response can reduce the severity of acute pancreatitis (AP). SUMMARY: Circulating systemic levels of various DAMPs and a panel of immune markers that possibly reflect activities of different pathways that drive SAP appear promising as predictive biomarkers for SAP. But larger multicenter studies are needed for external validation. Studies investigating immune cellular pathways driving SAP using immunophenotyping techniques are scarce. Interdisciplinary efforts are also needed to bring some of the promising biomarkers to the bedside for validation and testing for clinical utility. Studies investigating the role of and characterization of altered gut-lymph and gut-microbiota in severe AP are needed.

Abatacept increases T cell exhaustion in early RA individuals who carry HLA risk alleles.

Exhausted CD8 T cells (TEX) are associated with worse outcome in cancer yet better outcome in autoimmunity. Building on our past findings of increased TIGIT+KLRG1+ TEX with teplizumab therapy in type 1 diabetes (T1D), in the absence of treatment we found that the frequency of TIGIT+KLRG1+ TEX is stable within an individual but differs across individuals in both T1D and healthy control (HC) cohorts. This TIGIT+KLRG1+ CD8 TEX population shares an exhaustion-associated EOMES gene signature in HC, T1D, rheumatoid arthritis (RA), and cancer subjects, expresses multiple inhibitory receptors, and is hyporesponsive in vitro, together suggesting co-expression of TIGIT and KLRG1 may broadly define human peripheral exhausted cells. In HC and RA subjects, lower levels of EOMES transcriptional modules and frequency of TIGIT+KLRG1+ TEX were associated with RA HLA risk alleles (DR0401, 0404, 0405, 0408, 1001) even when considering disease status and cytomegalovirus (CMV) seropositivity. Moreover, the frequency of TIGIT+KLRG1+ TEX was significantly increased in RA HLA risk but not non-risk subjects treated with abatacept (CTLA4Ig). The DR4 association and selective modulation with abatacept suggests that therapeutic modulation of TEX may be more effective in DR4 subjects and TEX may be indirectly influenced by cellular interactions that are blocked by abatacept.

Recognition of mRNA Splice Variant and Secretory Granule Epitopes by CD4+ T Cells in Type 1 Diabetes.

A recent discovery effort resulted in identification of novel splice variant and secretory granule antigens within the HLA class I peptidome of human islets and documentation of their recognition by CD8+ T cells from peripheral blood and human islets. In the current study, we applied a systematic discovery process to identify novel CD4+ T cell epitopes derived from these candidate antigens. We predicted 145 potential epitopes spanning unique splice junctions and within conventional secretory granule antigens and measured their in vitro binding to DRB1*04:01. We generated HLA class II tetramers for the 35 peptides with detectable binding and used these to assess immunogenicity and isolate T cell clones. Tetramers corresponding to peptides with verified immunogenicity were then used to label T cells specific for these putative epitopes in peripheral blood. T cells that recognize distinct epitopes derived from a cyclin I splice variant, neuroendocrine convertase 2, and urocortin-3 were detected at frequencies that were similar to those of an immunodominant proinsulin epitope. Cells specific for these novel epitopes predominantly exhibited a Th1-like surface phenotype. Among the three epitopes, responses to the cyclin I peptide exhibited a distinct memory profile. Responses to neuroendocrine convertase 2 were detected among pancreatic infiltrating T cells. These results further establish the contribution of unconventional antigens to the loss of tolerance in autoimmune diabetes.

Assessing the Pathophysiology of Hyperglycemia in the Diabetes RElated to Acute Pancreatitis and Its Mechanisms Study: From the Type 1 Diabetes in Acute Pancreatitis Consortium.

OBJECTIVES: The metabolic abnormalities that lead to diabetes mellitus (DM) after an episode of acute pancreatitis (AP) have not been extensively studied. This article describes the objectives, hypotheses, and methods of mechanistic studies of glucose metabolism that comprise secondary outcomes of the DREAM (Diabetes RElated to Acute pancreatitis and its Mechanisms) Study. METHODS: Three months after an index episode of AP, participants without preexisting DM will undergo baseline testing with an oral glucose tolerance test. Participants will be followed longitudinally in three subcohorts with distinct metabolic tests. In the first and largest subcohort, oral glucose tolerance tests will be repeated 12 months after AP and annually to assess changes in ß-cell function, insulin secretion, and insulin sensitivity. In the second, mixed meal tolerance tests will be performed at 3 and 12 months, then annually, and following incident DM to assess incretin and pancreatic polypeptide responses. In the third, frequently sampled intravenous glucose tolerance tests will be performed at 3 months and 12 months to assess the first-phase insulin response and more precisely measure ß-cell function and insulin sensitivity. CONCLUSIONS: The DREAM study will comprehensively assess the metabolic and endocrine changes that precede and lead to the development of DM after AP.

Severe acute pancreatitis exhibits distinct cytokine signatures and trajectories in humans: a prospective observational study.

Severe acute pancreatitis (SAP) is associated with substantial morbidity and mortality. Several cytokines have been identified to have pathophysiological significance in SAP, but studies characterizing their early trajectories are lacking. Here we characterize the early trajectories of seven key cytokines associated with SAP and compare them with non-SAP subjects. Five proinflammatory cytokines (angiopoietin-2, interleukin-6, interleukin-8, monocyte chemoattractant protein-1, resistin) and two anti-inflammatory cytokines (hepatocyte growth factor, and soluble tumor necrosis factor-α receptor-1A) were measured in a prospective cohort of acute pancreatitis subjects (2012-2016) at the time of enrollment and then every 24 h for 5 days or until discharge. The cytokines' levels and trajectories were calibrated based on date of pain onset and were compared between healthy controls and three severity categories (mild, moderate, and severe). The cohort (n = 170) consisted of 27 healthy controls, 65 mild, 38 moderate, and 40 SAP. From day 1 of symptom onset, SAP subjects exhibited significantly higher levels of both pro- and anti-inflammatory cytokines compared with non-SAP and healthy subjects. But in SAP subjects, all proinflammatory cytokines' levels trended downward after day 2 (except for a flat slope for angiopoeitin-2) whereas for non-SAP subjects, the trajectory was upward: this trajectory difference between SAP versus non-SAP subjects resulted in narrowing of the differences initially seen on day 1 for proinflammatory cytokines. For anti-inflammatory cytokines, the trajectories were uniformly upward for both SAP and non-SAP subjects. Proinflammatory cytokine response is an early and time-sensitive event in SAP that should be accounted for when designing future biomarker studies and/or therapeutic trials.NEW & NOTEWORTHY In this study, we showed that the proinflammatory cytokine response in SAP is an early event, with subsequent downregulation of proinflammatory cytokines beginning at day 1 of symptom onset. Our findings underscore the importance of enrolling subjects very early in the disease course when conducting studies to investigate early immune events of SAP; this current study also serves as an important reference for the design of future biomarker studies and therapeutic trials in SAP.

Characterizing T cell responses to enzymatically modified beta cell neo-epitopes.

Introduction: Previous studies verify the formation of enzymatically post-translationally modified (PTM) self-peptides and their preferred recognition by T cells in subjects with type 1 diabetes (T1D). However, questions remain about the relative prevalence of T cells that recognize PTM self-peptides derived from different antigens, their functional phenotypes, and whether their presence correlates with a specific disease endotype. Methods: To address this question, we identified a cohort of subjects with T1D who had diverse levels of residual beta cell function. Using previously developed HLA class II tetramer reagents, we enumerated T cells that recognize PTM GAD epitopes in the context of DRB1*04:01 or PTM IA2 epitopes in the context of DQB1*03:02 (DQ8). Results: Consistent with prior studies, we observed higher overall frequencies and a greater proportion of memory T cells in subjects with T1D than in HLA matched controls. There were significantly higher numbers of GAD specific T cells than IA2 specific T cells in subjects with T1D. T cells specific for both groups of epitopes could be expanded from the peripheral blood of subjects with established T1D and at-risk subjects. Expanded neo-epitope specific T cells primarily produced interferon gamma in both groups, but a greater proportion of T cells were interferon gamma positive in subjects with T1D, including some poly-functional cells that also produced IL-4. Based on direct surface phenotyping, neo-epitope specific T cells exhibited diverse combinations of chemokine receptors. However, the largest proportion had markers associated with a Th1-like phenotype. Notably, DQ8 restricted responses to PTM IA2 were over-represented in subjects with lower residual beta cell function. Neo-epitope specific T cells were present in at-risk subjects, and those with multiple autoantibodies have higher interferon gamma to IL-4 ratios than those with single autoantibodies, suggesting a shift in polarization during progression. Discussion: These results reinforce the relevance of PTM neo-epitopes in human disease and suggest that distinct responses to neo-antigens promote a more rapid decline in beta cell function.

Sensitive detection of multiple islet autoantibodies in type 1 diabetes using small sample volumes by agglutination-PCR.

Islet autoantibodies are predominantly measured by radioassay to facilitate risk assessment and diagnosis of type 1 diabetes. However, the reliance on radioactive components, large sample volumes and limited throughput renders radioassay testing costly and challenging. We developed a multiplex analysis platform based on antibody detection by agglutination-PCR (ADAP) for the sample-sparing measurement of GAD, IA-2 and insulin autoantibodies/antibodies in 1 µL serum. The assay was developed and validated in 7 distinct cohorts (n = 858) with the majority of the cohorts blinded prior to analysis. Measurements from the ADAP assay were compared to radioassay to determine correlation, concordance, agreement, clinical sensitivity and specificity. The average overall agreement between ADAP and radioassay was above 91%. The average clinical sensitivity and specificity were 96% and 97%. In the IASP 2018 workshop, ADAP achieved the highest sensitivity of all assays tested at 95% specificity (AS95) rating for GAD and IA-2 autoantibodies and top-tier performance for insulin autoantibodies. Furthermore, ADAP correctly identified 95% high-risk individuals with two or more autoantibodies by radioassay amongst 39 relatives of T1D patients tested. In conclusion, the new ADAP assay can reliably detect the three cardinal islet autoantibodies/antibodies in 1µL serum with high sensitivity. This novel assay may improve pediatric testing compliance and facilitate easier community-wide screening for islet autoantibodies.

Characterization of regulatory T cells in obese omental adipose tissue in humans.

Obesity-associated visceral adipose tissue (AT) inflammation promotes insulin resistance and type 2 diabetes (T2D). In mice, lean visceral AT is populated with anti-inflammatory cells, notably regulatory T cells (Tregs) expressing the IL-33 receptor ST2. Conversely, obese AT contains fewer Tregs and more proinflammatory cells. In humans, however, there is limited evidence for a similar pattern of obesity-associated immunomodulation. We used flow cytometry and mRNA quantification to characterize human omental AT in 29 obese subjects, 18 of whom had T2D. Patients with T2D had increased proportions of inflammatory cells, including M1 macrophages, with positive correlations to body mass index. In contrast, Treg frequencies negatively correlated to body mass index but were comparable between T2D and non-T2D individuals. Compared to human thymic Tregs, omental AT Tregs expressed similar levels of FOXP3, CD25, IKZF2, and CTLA4, but higher levels of PPARG, CCR4, PRDM1, and CXCL2. ST2, however, was not detectable on omental AT Tregs from lean or obese subjects. This is the first comprehensive investigation into how omental AT immunity changes with obesity and T2D in humans, revealing important similarities and differences to paradigms in mice. These data increase our understanding of how pathways of immune regulation could be targeted to ameliorate AT inflammation in humans.

DRB4*01:01 Has a Distinct Motif and Presents a Proinsulin Epitope That Is Recognized in Subjects with Type 1 Diabetes.

DRB4*01:01 (DRB4) is a secondary HLA-DR product that is part of the high-risk DR4/DQ8 haplotype that is associated with type 1 diabetes (T1D). DRB4 shares considerable homology with HLA-DR4 alleles that predispose to autoimmunity, including DRB1*04:01 and DRB1*04:04. However, the DRB4 protein sequence includes distinct residues that would be expected to alter the characteristics of its binding pockets. To identify high-affinity peptides that are recognized in the context of DRB4, we used an HLA class II tetramer-based approach to identify epitopes within multiple viral Ags. We applied a similar approach to identify antigenic sequences within glutamic acid decarboxylase 65 and pre-proinsulin that are recognized in the context of DRB4. Seven sequences were immunogenic, eliciting high-affinity T cell responses in DRB4+ subjects. DRB1*04:01-restricted responses toward many of these peptides have been previously described, but responses to a novel pre-proinsulin 9-28 peptide were commonly observed in subjects with T1D. Furthermore, T cells that recognized this peptide in the context of DRB4 were present at significantly higher frequencies in patients with T1D than in healthy controls, implicating this as a disease-relevant specificity that may contribute to the breakdown of ß cell tolerance in genetically susceptible individuals. We then deduced a DRB4 motif and confirmed its key features through structural modeling. This modeling suggested that the core epitope within the pre-proinsulin 9-28 peptide has a somewhat unusual binding motif, with tryptophan in the fourth binding pocket of DRB4, perhaps influencing the availability of this complex for T cell selection.

Single-Cell RNA Sequencing Reveals Expanded Clones of Islet Antigen-Reactive CD4+ T Cells in Peripheral Blood of Subjects with Type 1 Diabetes.

The significance of islet Ag-reactive T cells found in peripheral blood of type 1 diabetes (T1D) subjects is unclear, partly because similar cells are also found in healthy control (HC) subjects. We hypothesized that key disease-associated cells would show evidence of prior Ag exposure, inferred from expanded TCR clonotypes, and essential phenotypic properties in their transcriptomes. To test this, we developed single-cell RNA sequencing procedures for identifying TCR clonotypes and transcript phenotypes in individual T cells. We applied these procedures to analysis of islet Ag-reactive CD4+ memory T cells from the blood of T1D and HC individuals after activation with pooled immunodominant islet peptides. We found extensive TCR clonotype sharing in Ag-activated cells, especially from individual T1D subjects, consistent with in vivo T cell expansion during disease progression. The expanded clonotype from one T1D subject was detected at repeat visits spanning >15 mo, demonstrating clonotype stability. Notably, we found no clonotype sharing between subjects, indicating a predominance of "private" TCR specificities. Expanded clones from two T1D subjects recognized distinct IGRP peptides, implicating this molecule as a trigger for CD4+ T cell expansion. Although overall transcript profiles of cells from HC and T1D subjects were similar, profiles from the most expanded clones were distinctive. Our findings demonstrate that islet Ag-reactive CD4+ memory T cells with unique Ag specificities and phenotypes are expanded during disease progression and can be detected by single-cell analysis of peripheral blood.

Antigen-Specific T Cell Analysis Reveals That Active Immune Responses to ß Cell Antigens Are Focused on a Unique Set of Epitopes.

CD38 is an activation marker that is present on recently activated T cells, but absent on resting memory T cells. In this study, we show that CD45RO+CD38+ ß cell Ag-specific CD4+ T cells were present at higher frequencies in type 1 diabetes subjects compared with those in healthy subjects. These results imply an ongoing ß cell immunity years after onset of diabetes and suggest these activated T cells have an active role in the disease process. The Ag specificities of these activated T cells were determined by a novel CD154 T cell epitope mapping assay. Although each patient usually had a unique set of epitopes recognized by these T cells, two epitopes, DR0401-restricted modified preproinsulin peptide 78-90K88S and zinc transport 8 266-285, were repeatedly identified in multiple subjects. Identifying these T cells and their specific antigenic epitopes might provide immunotherapeutic targets for personalized therapies.

Measurement of Pro-Islet Amyloid Polypeptide (1-48) in Diabetes and Islet Transplants.

Context: Islet amyloid is a feature of ß-cell failure in type 2 diabetes (T2D) and type 1 diabetes (T1D) recipients of islet transplants. Islet amyloid contains islet amyloid polypeptide (IAPP; amylin), a circulating peptide that is produced in ß cells by processing of its precursor, proIAPP1-67, via an intermediate form, proIAPP1-48. Elevated proinsulin to C-peptide ratios in the plasma of persons with diabetes suggest defects in ß-cell prohormone processing. Objective: Determine whether plasma levels of precursor forms of IAPP are elevated in diabetes. Design, Setting, and Patients: We developed an immunoassay to detect proIAPP1-48 in human plasma, and we determined the ratio of proIAPP1-48 to mature IAPP in subjects with T1D, T2D, recipients of islet transplants, and healthy controls. Results: The proIAPP1-48 immunoassay had a limit of detection of 0.18 ± 0.06 pM and cross-reactivity with intact proIAPP1-67 <15%. Healthy individuals had plasma concentrations of proIAPP1-48 immunoreactivity of 1.5 ± 0.2 pM and a proIAPP1-48 to total IAPP ratio of 0.28 ± 0.03. Plasma concentrations of proIAPP1-48 immunoreactivity were not significantly different in subjects with T2D but were markedly increased in T1D recipients of islet transplants. Children and adults with T1D had reduced mature IAPP levels relative to age-matched controls but an elevated ratio of proIAPP1-48 to total IAPP. Conclusion: The ß cells in T1D and islet transplants have impaired processing of the proIAPP1-48 intermediate. The ratio of proIAPP1-48-to-IAPP immunoreactivity may have value as a biomarker of ß-cell stress and dysfunction.

Modular transcriptional repertoire analyses identify a blood neutrophil signature as a candidate biomarker for lupus nephritis.

Objective: LN is a severe complication of SLE. Non-invasive biomarkers are needed for identifying patients at risk of a renal flare, for differentiating proliferative from non-proliferative forms and for assessing prognoses for LN. Methods: We assessed the link between blood transcriptional signatures and LN using blood samples from patients with biopsy-proven LN, extra-renal SLE flares or quiescent SLE. Healthy controls, and control patients with glomerular diseases or bacterial sepsis were included. Modular repertoire analyses from microarray data were confirmed by PCR. Results: A modular neutrophil signature (upregulation of module M5.15) was present in 65% of SLE patients and was strongly associated with LN. M5.15 activity was stronger in LN than in extra-renal flares (88 vs 17%). M5.15 was neither correlated to IFN modules, nor to SLEDAI or anti-dsDNA antibodies, but moderately to CS dose. M5.15 activity was associated with severity of LN, was stronger when proliferative, and decreased in patients responding to treatment. M5.15 activation was not caused by higher CS dose because it correlated only moderately to neutrophil count and was also observed among quiescent patients. Among quiescent patients, those with a past history of LN had higher M5.15 activity (50 vs 8%). M5.15 activation was present in patients with bacterial sepsis or ANCA-associated vasculitis, but not in patients with other glomerular diseases. Overall, M5.15 activation was associated with past, present or future flares of LN. Conclusion: Modular neutrophil signature could be a biomarker for stratifying LN risk and for monitoring its response to treatment. Trial registration: ClinicalTrials.gov, http://clinicaltrials.gov , NCT00920114.

The Relationship of Immune Cell Signatures to Patient Survival Varies within and between Tumor Types.

Enhancing pre-existing anti-tumor immunity leads to therapeutic benefit for some patients, but why some tumors are more immunogenic than others remains unresolved. We took a unique systems approach to relate patient survival to immune gene expression in >3,500 tumor RNAseq profiles from a dozen tumor types. We found significant links between immune gene expression and patient survival in 8/12 tumor types, with tumors partitioned by gene expression comprising distinct molecular subtypes. T/NK cell genes were most clearly survival-related for melanoma, head and neck, and bladder tumors, whereas myeloid cell genes were most clearly survival-related with kidney and breast tumors. T/NK or myeloid cell gene expression was linked to poor prognosis in bladder and kidney tumors, respectively, suggesting tumor-specific immunosuppressive checkpoints. Our results suggest new biomarkers for existing cancer immunotherapies and identify targets for new immunotherapies.

Copy number loss of the interferon gene cluster in melanomas is linked to reduced T cell infiltrate and poor patient prognosis.

While immunotherapies are rapidly becoming mainstays of cancer treatment, significant gaps remain in our understanding of how to optimally target them, alone or in combination. Here we describe a novel method to monitor levels of immune cells and pathways in expression data from solid tumors using pre-defined groups or modules of co-regulated immune genes. We show that expression of an interconnected sub-network of type I interferon-stimulated genes (ISGs) in melanomas at the time of diagnosis significantly predicted patient survival, as did, to a lesser extent, sub-networks of T helper/T regulatory and NK/T Cytotoxic cell genes. As a group, poor prognosis tumors with reduced ISG and immune gene levels exhibited significant copy number loss of the interferon gene cluster located at chromosome 9p21.3. Our studies demonstrate a link between type I interferon action and immune cell levels in melanomas, and suggest that therapeutic approaches augmenting both activities may be most beneficial.