(#28) HLA class II can be expressed in the islets of type 1 diabetic patients

PRESENTED BY: Estefania Quesada-Masachs

First NameLast NameAffiliation/Institution
SamuelZilbermanLa Jolla Institute for Immunology
SakthiRajendranNationwide Children’s Hospital
Jae-Hyun MasonLeeLa Jolla Institute for Immunology
TiffanyChuLa Jolla Institute for Immunology
Matthiasvon HerrathLa Jolla Institute for Immunology


Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing β cells are damaged by the immune system. However, the immunological mechanisms that lead to T1D have remained incompletely understood. It has been suggested that HLA class II (HLA-II) can play a role in T1D pathogenesis, and certain mutations in genes encoding for HLA-II molecules are associated with different risks of developing T1D. HLA-II is known to be widely expressed by antigen-presenting cells (APCs), and macrophages were found to be the most predominant APC islet infiltrate in T1D donors. Additionally, it has been suggested that HLA-II can be upregulated in β-cells exposed to proinflammatory cytokines in vitro. However, its expression in the islet β-cells of T1D patients has been historically controversial and its specific role in T1D is still undefined. The aim of this project is to characterize and quantify the expression of HLA class II in human pancreatic tissue sections obtained from the network of pancreatic organ donors (nPOD), to investigate the cellular sources of HLA-II in the pancreas and to analyze the induction of HLA-II expression in isolated human islets (Integrated Islet Distribution Program, IIDP) and in human pancreatic organoids (InSphero).


Antibodies for HLA class II (DP, DQ, DR), HLA-I (A, B, C), CD31, CD68, and Insulin were independently optimized in Formalin Fixed Paraffin Embedded (FFPE) sections of human tonsils. Multicolor immunofluorescent imaging panels were optimized in FFPE sections of tonsils, pancreas, and in isolated human islets. Human pancreatic FFPE nPOD sections from six non-diabetic, five auto-antibody positive (Aab+), and five T1D donors were stained with HLA-II, CD68, and Insulin. Isolated human islets and human islet organoids were cultured for four days with proinflammatory cytokines (IFN-γ, TNF-α, IL-1β) of varying concentrations. After culture with cytokines, islet function was measured by glucose‐stimulated insulin secretion (GSIS), and islets were subsequently stained with HLA-II, HLA-I, and Insulin. Images of whole tissue sections were obtained using Zeiss AxioScan Z1 slide scanner and were analyzed using Qupath. High resolution images of randomly selected regions of interest from the tissue sections and of the stained isolated human islets and islet organoids were acquired with the Zeiss LSM780 confocal microscopy system. Image analysis was performed on confocal images with Zen Blue and ImageJ.

Summary of Results

First, we observed that of non-islet cells, CD68+ was a more important source of HLA-II than CD31. After that, we stained 16 nPOD cases with HLA-II, CD68 and Insulin. We analyzed the totality of the tissue sections retrieving data from 7416 islets (4034 islets from non-diabetic, 2951 from Aab+, and 431 islets from T1D cases). We observed a statistically significant higher percentage of HLA-II-positive area in the insulin-containing islets (ICIs) of T1D cases (24.31% of islet area) compared to non-diabetic (3.82%), Aab+ (2.31%), and T1D insulin-deficient islets (IDIs) (0.67%). Within T1D ICIs, many of the β-cells were found to express HLA-II, since 45.9% of the total Insulin signal colocalized with HLA-II. In the non-diabetic and Aab+ cases, most of the Insulin signal of the β-cells did not colocalized with HLA-II (5.9% and 4.1%, respectively). In the peri-islet region, T1D ICIs also exhibited a higher percentage of positive area for HLA class II (18.9%) when compared with the other groups (expressing 2.6-4.8%). Significantly higher CD68+ macrophage infiltration was observed within and around ICIs in T1D cases (5.06% of islet area and 6.55% of peri-islet area) when compared with the other cases, and 17.09% of the HLA-II signal within T1D ICIs colocalized with CD68. Interestingly, we also observed a higher expression of HLA-II and CD68 in the exocrine tissue of T1D patients when compared with the non-diabetic and Aab+ cases. Then we tested if pro-inflammatory cytokines were able to induce HLA-II expression in isolated human islets (IIDP) and in islet organoids (InSphero). Upon treatment of the islets with inflammatory cytokine stressors (combinations of IFN-γ, TNF-α +/- IL-1β) we observed an impairment in islet function assessed by GSIS, and the induction of HLA-II and HLA-I expression (which was not observed in control islets cultured with media). Different concentrations of TNF-α by itself did not impair the islet function or induce HLA class II expression. However, different concentrations of IFN-γ by itself produced altered GSIS responses and an increased expression of HLA-II and HLA-I, although this increase was not statistically significant.


HLA-II can be expressed by pancreatic β-cells in T1D patients. We observed a higher expression of HLA-II in the ICIs and the peri-islet regions of T1D donors when compared with non-diabetic and Aab+ donors. Aab+ cases did not exhibit higher levels of HLA-II than the controls in any pancreatic region. HLA-II was homogeneously expressed in the exocrine and endocrine pancreatic tissue of both non-diabetic and Aab+ donors. T1D cases also presented a higher expression of HLA-II in the exocrine pancreas when compared with the other groups, however, it was substantially higher in the islet and peri-islet regions. In general, a higher expression of CD68+ was present in the islet, peri-islet, and exocrine regions of the T1D cases. HLA-II expressing CD68+ macrophages are frequently found in and around T1D ICIs, and often in close contact with surrounding β-cells. Further studies may investigate the functional state of infiltrating macrophages in T1D cases, and investigate potential macrophage-islet cross-reaction in vitro. HLA-II expression is inducible in the β-cells of isolated human islets and human islet organoids in vitro by treating them with a combination of pro-inflammatory cytokines. Our findings support a potential pathogenic role for HLA-II in T1D.