Islet cell hyperexpression of HLA class I antigens: a defining feature in type 1 diabetes
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Human pancreatic beta cells may be complicit in their own demise in type 1 diabetes, but how this occurs remains unclear. One potentially contributing factor is hyperexpression of HLA class I antigens. This was first described approximately 30 years ago, but has never been fully characterised and was recently challenged as artefactual. Therefore, we investigated HLA class I expression at the protein and RNA levels in pancreases from three cohorts of patients with type 1 diabetes. The principal aims were to consider whether HLA class I hyperexpression is artefactual and, if not, to determine the factors driving it.
Pancreas samples from type 1 diabetes patients with residual insulin-containing islets (n = 26) from the Network for Pancreatic Organ donors with Diabetes (nPOD), Diabetes Virus Detection study (DiViD) and UK recent-onset type 1 diabetes collections were immunostained for HLA class I isoforms, signal transducer and activator of transcription 1 (STAT1), NLR family CARD domain containing 5 (NLRC5) and islet hormones. RNA was extracted from islets isolated by laser-capture microdissection from nPOD and DiViD samples and analysed using gene-expression arrays.
Hyperexpression of HLA class I was observed in the insulin-containing islets of type 1 diabetes patients from all three tissue collections, and was confirmed at both the RNA and protein levels. The expression of β2-microglobulin (a second component required for the generation of functional HLA class I complexes) was also elevated. Both ‘classical’ HLA class I isoforms (i.e. HLA-ABC) as well as a ‘non-classical’ HLA molecule, HLA-F, were hyperexpressed in insulin-containing islets. This hyperexpression did not correlate with detectable upregulation of the transcriptional regulator NLRC5. However, it was strongly associated with increased STAT1 expression in all three cohorts. Islet hyperexpression of HLA class I molecules occurred in the insulin-containing islets of patients with recent-onset type 1 diabetes and was also detectable in many patients with disease duration of up to 11 years, declining thereafter.
Islet cell HLA class I hyperexpression is not an artefact, but is a hallmark in the immunopathogenesis of type 1 diabetes. The response is closely associated with elevated expression of STAT1 and, together, these occur uniquely in patients with type 1 diabetes, thereby contributing to their selective susceptibility to autoimmune-mediated destruction.
KeywordsDiViD HLA class I HLA-F Islet cell nPOD Pancreas STAT1 Type 1 diabetes
Diabetes Virus Detection study
HLA class I
Mean fluorescence intensity
NLR family CARD domain containing 5
Network for Pancreatic Organ donors with Diabetes
Optimal cutting temperature
Signal transducer and activator of transcription 1
The incidence of type 1 diabetes is increasing rapidly worldwide [1, 2, 3], probably because of changes in the environment that ultimately impact the development, functional activity and longevity of pancreatic beta cells.
Against this background, the cellular and molecular events associated with the initiation and progression of type 1 diabetes remain poorly understood, largely because the disease process cannot be studied non-invasively in the pancreases of living individuals. Hence, deductions regarding pathogenic processes are made from analyses of tissue recovered either after death or by pancreas biopsy in living individuals [4, 5, 6, 7]. Collectively, such approaches have been applied in few cases worldwide, reflecting the paucity of accessible samples in which the tissue architecture has been preserved and the destructive process is still present and amenable to study [8, 9].
Despite these limitations, a consensus model has emerged in which type 1 diabetes is envisaged to result from the selective destruction of beta cells by immune cells infiltrating the islets of Langerhans [1, 2, 3, 8, 9]. In this scenario, CD8+ T cells are considered to be the major effectors of beta cell death, and are directed to the pancreatic islets to participate in the autoimmune assault against beta cells [10, 11, 12, 13]. It is also likely, however, that beta cells are complicit in these events by aberrantly processing and presenting cellular antigens, thereby becoming visible to autoreactive CD8+ T cells . This could be achieved in several ways, including via upregulated expression of the MHC (i.e. HLA class I [HLA-I]) molecules [15, 16, 17].
Upregulation of HLA-I expression (often cited as ‘hyperexpression’) in pancreatic islets has been studied in relatively few type 1 diabetes patients, and no previous attempts have been made to verify the phenomenon across multiple cohorts. Moreover, the concept has primarily been examined at the protein level using immunocytochemical approaches and has rarely been corroborated with gene-expression data to verify that the two are concordant. Indeed, in one recent study, it was argued that such concordance may not exist .
Therefore, in the present work, we have taken advantage of a unique collaborative strength achieved by combining three of the world’s most significant collections of pancreas samples from individuals with type 1 diabetes: an archival collection of postmortem samples from the UK ; the Network for Pancreatic Organ donors with Diabetes (nPOD) collection of organ donor pancreases (USA) [19, 20]; and pancreatic biopsy material from living individuals participating in the Norwegian Diabetes Virus Detection study (DiViD) . In this study, we interrogated these tissues to provide definitive evidence as to whether islet cell hyperexpression of HLA-I antigens is an early and defining feature of human type 1 diabetes. Collectively, these new data allow us to report the most comprehensive examination of HLA-I expression ever undertaken involving the human pancreas in type 1 diabetes.
Formalin-fixed, paraffin-embedded (FFPE) pancreas sections were available from three cohorts: the nPOD and DiViD collections, and an archival collection from the UK (electronic supplementary material [ESM] Tables 1, 2). Frozen tissue was also available from nPOD and DiViD. Analyses were performed with 17 control and 26 type 1 diabetic individuals for whom FFPE and frozen tissue were available. All samples were studied with appropriate ethical approval and, in the case of the DiViD study, participants provided written informed consent.
Immunohistochemistry and immunofluorescence
Immunohistochemistry was performed using a standard immunoperoxidase approach, as previously described . To examine multiple antigens within the same FFPE section, samples were probed in a sequential manner with up to three different antibodies (ESM Tables 3, 4). The mean fluorescence intensity (MFI) of stained antigens was measured using ImageJ Version 1.50b Java 1.8.0_77; https://imagej.nih.gov/ij/download.html. Some slides were processed with isotype control antisera to confirm the specificity of labelling (ESM Fig. 1). Frozen sections were stained using a standard immunofluorescence approach .
Islet microdissection and RNA collection
Optimal cutting temperature (OCT) slides of pancreatic tissue were used for laser-capture microscopy. This was conducted on an Arcturus Pixcell II laser capture microdissection system (Arcturus Bioscience, Mountain View, CA, USA). Islets were recognised by their natural autofluorescence . All islets visible in two to five sections from each sample were pooled, and RNA was extracted using the Arcturus PicoPure RNA Isolation Kit (Applied Biosystems, Grand Island, NY, USA). RNA quantity and quality were determined using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). RNA samples were subjected to gene-expression analysis using Affymetrix expression arrays (Thermo Fisher Scientific, Santa Clara, CA, USA), as previously described .
Affymetrix array analysis
Using the Affymetrix Human Gene 2.0 ST array, CEL files were generated from both control and type 1 diabetic donors, as previously described . Raw signal-intensity values from Affymetrix spike-in controls demonstrated that array hybridisation had been successful (i.e. bioB<bioC<bioD<Cre). Data quality was verified by measuring the positive vs negative area under the curve. Raw signal-intensity values from all arrays were robust multichip average background corrected, quantile normalised, median polish summarised and log2 transformed [25, 26, 27]. NetAffx-determined probe-set annotations for HLA genes (Affymetrix) were re-mapped according to RefSeq, release 73 (15 November 2015; see ftp://ftp.ncbi.nlm.nih.gov/refseq/release/release-catalog/archive/). For each HLA gene, where multiple mappings were possible (i.e. HLA-A, -B, -C and -F), probe sets were annotated according to eight major haplotypes incorporated into the human genome assembly, as previously described . Because probe sets shared mappings, it was not possible to identify HLA subtypes uniquely using this gene chip; rather, transcript clusters were used to examine changes in global gene expression. Processing was carried out using the Partek Genomics Suite, version 6.5 (Partek, St Louis, MO, USA). The resulting normalised expression data for specific genes of interest were then subjected to analysis as described below.
Individual comparisons of MFI, RNA or protein were performed using either a Satterthwaite corrected two-sample test or paired/unpaired Student’s t test and considered significant if p < 0.05. In the case of multiple comparisons, statistical significance was indicated at a Bonferroni-corrected nominal α level of 0.025. Correlations were evaluated and considered strong if p < 0.05 and the Spearman’s rank correlation coefficient (r) was >0.80. All reported p values are two-tailed and unadjusted. Statistical analyses were performed using SAS version 9.4 (SAS Institute, Cary, NC, USA).
Lobular hyperexpression of HLA-A, -B and -C (HLA-ABC) in type 1 diabetes
Classification of donors based on HLA-ABC expression
Since islet hyperexpression of HLA-ABC has been claimed to be artefactual , we monitored the levels of HLA-ABC in a subset of nPOD donors in two independent laboratories using pancreas sections preserved by different methods (frozen vs FFPE). Staining for HLA-ABC was performed using either an immunoperoxidase method coupled with a mouse primary antiserum in FFPE tissue (ESM Fig. 2a) or via immunofluorescence in OCT sections (ESM Fig. 2b) from the same donor, using a different primary antiserum. A blinded analysis was conducted with donors classified into three categories: normal, elevated and hyperexpression (i.e. at least one islet with extremely high expression of HLA-ABC affecting all endocrine cells) (ESM Fig. 2). Unblinding of the analysis revealed a 100% concordance rate between laboratories (ESM Fig. 2c).
Further confirmation of the staining specificity in FFPE tissue was obtained by staining serial islet sections with two different HLA-ABC antibodies. In all cases where hyperexpression of HLA-ABC was detected with one antiserum, this was confirmed in the same islet on the serial section with the second antiserum (ESM Fig. 3, ESM Tables 3, 4).
Examination of patients with increasing disease duration revealed that HLA-ABC hyperexpression was not restricted only to patients with recent-onset type 1 diabetes, but was also observed in individuals with longer-term disease (i.e. up to 11 years) when ICIs were retained (ESM Fig. 4). However, the proportion of ICIs hyperexpressing HLA-ABC decreased as the duration of type 1 diabetes increased (r = –0.883, p < 0.0001; ESM Fig. 4). HLA-I hyperexpression was not found in patients lacking residual ICIs. It was also absent from the ICIs of patients with still longer disease durations (>11 years), even among those who retained insulin immunopositivity (three nPOD patients with a total of 110 ICIs) after this time (ESM Fig. 4).
β2-Microglobulin (β2M) is elevated in islets in type 1 diabetes
Expression of RNA transcripts encoding HLA or β2M in laser-captured, microdissected islets
HLA-F expression is also elevated in the ICIs of individuals with recent-onset type 1 diabetes
NLR family CARD domain containing 5 (NLRC5) expression does not correlate with HLA-ABC hyperexpression
Signal transducer and activator of transcription 1 (STAT1) expression correlates positively with HLA-ABC hyperexpression in type 1 diabetes
Analysis of the expression of STAT1 at the mRNA level in laser-captured islets confirmed the data obtained at the protein level. Moreover, in common with HLA-ABC (ESM Fig. 4), the extent of this increase declined with disease duration (Fig. 6c). Consistent with this, the most pronounced rise was seen in islets harvested by laser-capture microdissection from the six DiViD patients (p = 0.0263; Fig. 6d, ESM Fig. 7), who were studied very close to disease onset.
An elevation in the expression of HLA-I antigens in the islet cells of patients with recent-onset type 1 diabetes was first reported approximately 30 years ago [15, 17]. However, neither the significance of this response with respect to disease aetiology nor the mechanism by which it is achieved has been revealed. Moreover, the concept of islet cell HLA-I hyperexpression in type 1 diabetes has recently been challenged, largely on the basis that immunostaining protocols are subject to artefact . In the current study, we counter this by providing firm evidence that the phenomenon occurs in each of the three cohorts of patients we examined, using multiple different HLA-ABC antisera and testing both FFPE and frozen sections. In considering the differing conclusions reached by ourselves and Skog et al , we note that the respective immunohistochemistry data are similar, but that Skog et al did not find an elevation at the RNA level in isolated islets from type 1 diabetes patients vs controls. They acknowledged, however, that this is not definitive, since downregulation of HLA-I might occur as a consequence of islet isolation. The studies also differ in that Skog et al  studied RNA expression in laser-captured islets from only two patients with type 1 diabetes, whereas we examined islets from many more patients. Therefore, we conclude that hyperexpression of HLA-I is a characteristic feature of islets in human type 1 diabetes, usually linked to remaining insulin, and that it is not an artefact unique to any particular geographical region, mode of tissue preservation or mechanism of pancreas retrieval. These conclusions are supported by evidence of concurrent induction of β2M production with HLA-I, and were confirmed at both the protein level (for all three cohorts) and in RNA studies for the nPOD and DiViD collections, where frozen material was available. The increase at the RNA level was most marked in the DiViD patients, who were all studied soon after disease onset.
We also found that the increased transcription of HLA-I isoforms was not restricted solely to the commonly studied HLA-ABC isoforms, but that another, atypical, isoform (HLA-F) was also enhanced. This was especially pronounced in the DiViD cohort and was confirmed at the protein level. Of interest, HLA-F expression was localised to the cell surface rather than intracellularly . It has been demonstrated that HLA-F can interact with open-conformation HLA-I heavy chains (without bound peptide), facilitating migration of the complex to the cell surface [33, 34].
Extending these novel data, we also discovered that islet cell hyperexpression of HLA-I can persist, at the protein level, beyond the initial phases of the disease, since it was also seen in patients who had been diagnosed with type 1 diabetes for 11 years prior to death (although it was lost beyond this time). Thus, while there was a tendency towards normalised HLA-I expression over time in the disease course, residual beta cells with elevated HLA-I expression could still be found in some patients long after disease onset. It follows, therefore, that the surviving beta cells in these patients had apparently evaded elimination despite displaying an altered HLA-I phenotype; possibly over many years. The reasons for this are unclear and will require further study. It is also possible that the elevated expression of HLA-I occurs at different times in the disease course and that the islets we examined were at various stages of this progression. However, earlier studies do not support this, since the proportion of ICIs displaying HLA-I hyperexpression in children with recent-onset disease has been reported to be extremely high, suggesting that essentially all such islets hyperexpress HLA-I at diagnosis . We also conclude that beta cells are necessary to initiate and sustain the response, since HLA-I hyperexpression was not present in islets devoid of beta cells in the plane of the section. This may reflect the egress of immune cells from IDIs , but it is also possible that elaboration of a diffusible molecule, such as one of the IFNs, by beta cells might drive islet HLA-I hyperexpression in surrounding cells [35, 36], since HLA-I hyperexpression does not correlate directly with insulitis.
Elevated expression of HLA-I has been observed in mouse models of type 1 diabetes, where IFN-γ release from infiltrating immune cells has been shown to be the driver [37, 38]. As noted above, however, we emphasise that, contrary to the situation in mice, hyperexpression of HLA-I can occur in human islets without evidence of insulitis [15, 22, 39, 40]. Of course, this does not exclude the likelihood that, in inflamed islets, the response might be enhanced or sustained by cytokines produced by immune cells.
An important additional new finding in the present work is the striking correlation between the levels of STAT1 measured in the beta cells of patients with type 1 diabetes and HLA-I hyperexpression. This was confirmed in multiple individual islets across a range of different patients and was demonstrated at the protein and RNA levels. STAT1 is a critical protein involved in mediating antiviral responses to IFNs, and its early upregulation in the progression of type 1 diabetes would be expected to place beta cells in a heightened state of responsiveness to these cytokines [41, 42, 43].
Although STAT1 was elevated in beta cells soon after disease diagnosis, its expression declined with disease duration, thereby correlating with a similar decline in HLA-I, as discussed above. On this basis, it seems possible that the two may be coordinately regulated or that increased production of HLA-I in beta cells occurs as a consequence of enhanced STAT1 expression. In support of this (and in accord with others ), we found no significant increase in levels of the putative HLA-I transcriptional regulator NLRC5 in islets hyperexpressing HLA-I. Thus, an alternative transcriptional regulator must exist in beta cells and this could be STAT1. Importantly, however, cytosolic STAT1 expression was not increased in the non-beta islet endocrine cells in type 1 diabetes, despite these having elevated HLA-I. This implies that a separate mechanism may control HLA expression in these cells, although it is also possible that a modest level of STAT1 activation might occur in the absence of a dramatically altered cytosolic protein level and, in support of this, we did detect nuclear STAT1 in some non-beta cells (Fig. 6a).
Taken together, our observations provide solid evidence that islet cell HLA-I hyperexpression is a genuine pathological feature in type 1 diabetes, and this raises important questions about the role of this phenomenon in disease progression in humans. One hypothesis, which is consistent with our data, proposes that enhanced expression of HLA-I antigens is critical for early disease progression, promoting the effective engagement of influent CD8+ cytotoxic T cells specific to defined islet antigens. Finally, our findings also emphasise the complicity of beta cells in their own demise in type 1 diabetes.
We are pleased to acknowledge support from the organ procurement organizations partnering with nPOD to provide research resources (listed at www.jdrfnpod.org/our-partners.php). The authors also thank the surgeons of the DiViD study, B. Edwin (The Intervention Centre, Oslo University Hospital, Rikshospitalet, Oslo, Norway) and T. Buanes (Department of Hepato-Pancreato-Biliary Surgery, Oslo University Hospital-Rikshospitalet, Oslo, Norway), and specialist nurse T. Roald (Paediatric Department, Oslo University Hospital, Oslo, Norway), whose invaluable efforts were essential to the success of the DiViD study. We are grateful to A. Foulis for access to, and the transfer of, the UK Type 1 Diabetes Biobank to the University of Exeter Medical School. This biobank is now called the Exeter Archival Diabetes Biobank (http://foulis.vub.ac.be/).
We are pleased to acknowledge financial support from the European Union’s Seventh Framework Programme PEVNET (FP7/2007-2013, grant agreement number 261441). The participants of the PEVNET consortium are described at www.uta.fi/med/pevnet/publications.html. Additional support was provided by a Diabetes Research Wellness Foundation Non-Clinical Research Fellowship and, since 2014, a JDRF Career Development Award (5-CDA-2014-221-A-N) to SJR. KD-J has received grants from the South-Eastern Norway Regional Health Authority, the Novo Nordisk Foundation and through the PEVNET Consortium. The research was also performed with the support of nPOD, a collaborative type 1 diabetes research project sponsored by JDRF International (JDRF 25-2013-268 to MAA, including a subcontract to JSK), and of JDRF research grants awarded to the nPOD-V Consortium (JDRF 25-2012-516 to AP and JDRF 25-2012-770 to MAA). Additional funding was provided by JDRF to CEM (JDRF 47-2013-520) and from the National Institutes of Health (UC4 DK104155) to ICG and MvH (R01 AI092453).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
SJR and NGM designed the study, performed data analysis and interpretation, and drafted, revised and approved the manuscript. TR-C, ICG, MZ, MAR and PL performed data collection, analysis and interpretation, and revised and approved the manuscript. LK and KD-J collected patient material, and revised and approved the manuscript. ICG and CEM designed the Affymetrix array component of the study, and revised and approved the manuscript. JSK provided statistical expertise, and revised and approved the manuscript. MvH, AP and MAA provided critical interpretation of the data, and revised and approved the manuscript.
SJR and NGM are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
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