Fresh duodenal tissue was obtained from the nPOD (Gainesville, FL, USA). All experiments were performed according to the guidelines of the University of Florida Institutional Review Board, with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by University of Florida Institutional Review Board. Included in the current study were 17 T1D and 13 non-T1D cases (Table S1 in Supplementary Material). When the proximal duodenum is included with pancreases donations, the duodenal tissue is harvested following the isolation of pancreatic tissues, pancreatic lymph nodes, spleen, and non-pancreatic lymph nodes. Specifically, the duodenum is opened and the mucosa cleaned of mucous and bacterial content with a moistened gauze pad after which a section is placed in transport media and immediately processed as follows. While the all organs are harvested according to the same protocol in every case, the time from death to organ harvest and thus confounders such as ischemia are not available. For each case, 8.0 g of duodenal tissue was processed as follows: (1) 0.3 g of tissue was preserved for (1a) protein extraction and (1b) RNA extraction to evaluate the soluble mediator profile of the duodenum by ELISA and real-time PCR, respectively. (2) The remaining tissue was processed for (2a) intestinal immune cell isolation and (2b) intestinal crypt isolation for characterization of resident immune cell population by flow cytometry and establishment of primary IEC cultures, respectively (15). (3) Following the establishment of confluent primary IEC cultures (3a) culture supernatants and (3b) cellular mRNA were collected to evaluate the soluble mediator profile of the primary cultures by ELISA and real-time PCR, respectively. (4) In order to evaluate the effect of the T1D and non-T1D-derived IEC-soluble mediator profile on T cell proliferation and polarization, T cells were isolated from a single systemically healthy individual (not part of the experimental cohort) and activated in the presence of supernatants from resting IEC cultures described in step 3, after which the subsequent T cell proliferation and polarization was characterized by flow cytometry. Please note while all samples (n = 17 T1D and n = 13 non-T1D) underwent evaluation for experiments outlined step 1, due to timing of receipt, only a subset of these samples (n = 8 and n = 10) underwent evaluation for experiments outlined in steps 1–4 (Table 1; Table S1 in Supplementary Material).

A total of 0.3 g of duodenal tissue was minced and homogenized by bead beating in cell extraction buffer (Life Technologies, Carlsbad, CA, USA) prepared with a protease inhibitor cocktail (cOmplete, Roche, Basel, Switzerland) and PMSF protease inhibitor (Life Technologies). Protein samples were stored at −80°C until ELISA could be performed. An additional 0.3 g of duodenal tissue was minced and homogenized by bead beating in Buffer RLT (Qiagen, Hilden, Germany) containing β-mercaptoethanol (β-ME) and total RNA harvested using an RNeasy extraction kit (Qiagen, Hilden, Germany) and also stored at −80°C until quantitative PCR (RT-qPCR) could be performed.

Crypts and resident immune cells were liberated using a protocol adapted from Booth and O’Shea (15–17). Human duodenal tissues were prepared by removing the longitudinal muscle layer and washing with ice-cold Mg²⁺- and Ca²⁺-free Hank’s Balanced Salt Solution (HBSS) (Mediatech, Manassas, VA, USA) containing 100 U penicillin, 100 μg/mL streptomycin (Mediatech), 25 μg/mL gentamycin (MP Biomedicals, Solon, OH, USA), and 0.5 mM dithiothreitol (DTT) (Thermo Fisher Scientific). After which the tissue was cut to yield 2 cm² pieces, suspended in 50 mL of HBSS wash solution, inverted vigorously 10 times, and the contents allowed to settle for 1 min. The supernatant was removed and the process repeated four times. After the fifth wash, the settled contents were removed, minced with a sterile surgical scalpel, and suspended in 50 mL of the HBSS wash solution. The resulting solution was passed over a 400 μm² filter, and the flow-through was collected for resident immune cells analysis (data not shown). The remaining tissue was digested in 50 mL of a buffer containing 75 U/mL collagenase type XI (Sigma-Aldrich, St. Louis, MO, USA), 20 μg/mL dispase neutral protease II (Roche, Indianapolis, IN, USA), 0.5 mM DTT, and 1% v/v fetal bovine serum (FBS) (Thermo Fisher Scientific) in Dubelcco’s Modification of Eagles Medium with 4.5 g/L glucose and l-glutamine, without sodium pyruvate (DMEM) (Corning, Corning, NY, USA). The digestion buffer containing the tissue was then evenly divided, placed in a 37°C incubator, and allowed to shake at 180 RPM for 3 h. The resulting digestion mixture was again passed over a 400 μm² filter, and the tissue fragments atop the filter were washed with 25 mL complete growth media {DMEM [8.5 g/L sodium pyruvate (Mediatech)], 2.5% v/v FBS, 0.25 U/mL insulin (Sigma-Aldrich), 100 U penicillin, 100 μg/mL streptomycin, 25 μg/mL gentamycin, 5 μg/mL transferrin (Sigma-Aldrich), and 10 ng/ml epidermal growth factor (Sigma-Aldrich) containing 2% w/v d-sorbitol (Sigma-Aldrich)}. The tissue debris remaining in the filter was discarded, and the flow-through containing proliferative crypt structures and liberated intestinal immune cells was centrifuged at 200 × g for 4 min. The resulting pellet of intestinal crypts was used for the establishment of IEC cultures as described below. The resulting supernatant was collected for resident immune cells, which were cryopreserved at 1 × 10⁶ cells/mL in cell freezing media (ScienCell Research Laboratories, Carlsbad, CA, USA) and stored in liquid nitrogen until flow cytometry could be performed.

Intestinal epithelial cell cultures were established from isolated crypts as previously described (15, 17) and maintained in 24-well collagen-coated culture dishes (Greiner Bio-One, Monroe, NC, USA) in 1 mL complete IEC (cIEC) media [DMEM, 5 g/L sodium pyruvate (Mediatech), 2.5% v/v FBS, 0.25 U/mL insulin (Sigma-Aldrich), 100 U penicillin, 100 µg/mL streptomycin, 25 µg/mL gentamicin, 5 µg/mL transferrin (Sigma-Aldrich), 10 ng/mL epidermal growth factor (Sigma-Aldrich)]. Cultures were left unstimulated for 24 h after which supernatants were collected and stored at −80°C. In addition, cellular total RNA was harvested using an RNeasy extraction kit (Qiagen, Hilden, Germany) and stored at −80°C until RT-qPCR could be performed.

Reverse transcription and RT-qPCR was performed according to MIQE guidelines (18). Synthesis of cDNA was performed using SuperScript^® Reverse Transcriptase (Life Technologies), whereby RT-qPCR was performed using primer sets (Table S2 in Supplementary Material), and SsoAdvanced™ SYBR Green Supermix was used according to manufacturer recommendations. Data were collected and analyzed using CFX Connect™ and CFX Manager™ (Bio-Rad Laboratories, Berkeley, CA, USA) according to the ddCT algorithm using 18S as a reference gene and are presented as fold change. ELISA technology (EMD Millipore, Billerica, MA, USA) was used according to manufacturer protocol to assess whole tissue and primary IEC production of interleukin 10 (IL-10). Data were analyzed using a standard curve, positive, and negative control. For whole tissue expression, all data were normalized to total protein, while media-only levels were subtracted from primary IEC expression, which was normalized to 18S RNA levels from each primary cell culture.

Cryopreserved intestinal immune cells were thawed from liquid nitrogen at 37°C, washed, and suspended in PBS prior to incubation with a fixable Live/Dead Yellow viability dye (Life Technologies) for 10 min at RT. Following Fc receptor blocking (Human TruStain FcX™, BioLegend), surface staining was performed in FACS buffer [PBS, 1% FBS, 4 mM EDTA, and antibiotics (penicillin, streptomycin, and amphotericin B)] interrogating expression of lineage markers (CD3, CD14, CD19, CD20), CD56, CD127, CD117, NKp44, CD45, CD3, αβ TCR, γδ TCR, CD4, CD8, CCR6, CXCR3 (Table S3 in Supplementary Material). Intracellular staining for FOXP3 and HELIOS was performed using FOXP3 Fix/Perm Buffer Set (BioLegend). All antibodies were used at manufacturer-recommended concentrations. Fluorescence minus one or isotype controls were used as indicated. Data were acquired using a BD LSR Fortessa (BD Biosciences, Franklin Lakes, NJ, USA) cytometer and analyzed using FlowJo data analysis software (FlowJo, LLC, Ashland, OR, USA). All data were normalized to 1,000,000 total cells collected in the lymphocyte gate and are presented as frequencies and total cell numbers.

Peripheral blood mononuclear cells (PBMC) were isolated from the buffy coat of a single healthy donor (not part of the experimental cohort) by centrifugation (400 × g, 30 min, no brake) on Ficoll-Paque Premium (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Cells were washed twice with HBSS and plated at a density of 1 × 10⁶ cells/mL with either 50% cRMPI/50% cIEC, or 50% cRPMI/50% 24-h supernatants from T1D- or non-T1D-derived IEC cultures. T cells were activated with Dynabeads^® Human T-Activator CD3/CD28 (Life Technologies) at a 1:1 bead to cell ratio with IL-2 (100 U/mL) for 4 days. CellTrace™ Violet-cell proliferation kit (Life Technologies) was used according to manufacturer protocols to evaluate CD3+ proliferation. Proliferation index was defined as the average number of divisions of each T cell as calculated by 1/sum[p_n/(2ⁿ)].

Hematoxylin- and eosin-stained sections of duodenum from all cases were obtained from nPOD and blindly evaluated by an experienced histopathologist (Jian Li) according to Marsh–Oberhuber classification (19) (Table S4 in Supplementary Material). Additional 5 µm sectioned slides of duodenum from all cases were used to assess goblet cell frequency by Periodic-acid Schiff staining (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer protocol. Additional 5-µm tissue sections of duodenum from all cases were used to assess expression of HLA class II. Specifically, sections were deparaffinized and rehydrated prior to antigen retrieval in a sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0; 96.5°C, 20 min). Slides were rinsed with PBS and permeabilized in a blocking buffer [1% (w/v) BSA, 10% (v/v) donkey serum, 0.1% (v/v) Triton X-100 in PBS]. Primary antibodies were used to probe for HLA-DR (TAL.1B5, Dako, Carpinteria, CA, USA) (16 h, 4°C, 1:20 in blocking buffer). Following three washes in PBS containing 3% (w/v) BSA and 0.1% (v/v) Triton X-100, tissue sections were probed with donkey anti-mouse AlexaFluor^® 488 (Life Technologies) (3 h RT, 1:400 in PBS). Following three washes in PBS, sections were mounted with VECTASHIELD^® HardSet Antifade (Vector Laboratories, Burlingame, CA, USA). Images were acquired with an EVOS^® FL (Life Technologies) and analyzed and quantified using ImageJ software (20). Data are presented as the mean fluorescence intensity difference of the region of interest and background. Exposure time and brightness and contrast adjustments were applied identically across all groups. A rainbow LUT was applied for visualization.

Data are expressed as mean ± SEM unless otherwise noted. Differences between groups were analyzed by the one-tailed Mann–Whitney t test using GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA). Differences in fold change of expression were analyzed by Wilcoxon matched-pairs signed rank test. Results were considered statistically significant at p ≤ 0.05.

Previous studies using small intestinal biopsy samples from relatively small cohorts of children with T1D have suggested intestinal immune activation in some individuals with T1D (7, 8). In order to determine if this altered intestinal microenvironment in TID extended to that of the IEC-specific compartment, the expression of various IEC-specific soluble mediators associated with immunoregulation and inflammation and were assessed in duodenal tissues from donors with or without T1D (Table 1).

In duodenal tissues derived from individuals with T1D, a 21-fold higher level of mRNA expression of the antimicrobial peptide beta-defensin 2 (BD-2) and a 61-fold higher level of mRNA expression of IL-17C were observed (Figures 1A,B). Importantly, these mediators were significantly elevated in the absence of similar mRNA elevations in the regulatory cytokines thymic stromal lymphopoietin (TSLP) and protein expression of IL-10 (Figures 1A–C). As BD-2 and IL-17C are known to be TLR5-response genes (21, 22), expression levels of TLR5 were also evaluated, whereby no significant differences in the mRNA levels of TLR5 were observed (Figures 1A,B).

The inflammatory state of the intestinal tract is heavily regulated by and has a direct effect on the recently described ILC lineage (9). Thus, the frequency and phenotype of ILC within the duodenal tissues were also evaluated. Although no significant differences were observed in the frequency of total NK or ILC populations (Figures 2A,B), a significantly higher frequency of CD127+NKp44−CD117− cells (ILC1) concomitant with a lower frequency of CD127+CD117+NKp44+/− cells (ILC3) was observed in the T1D cohort compared to that of the non-T1D cohort (Figures 2C–E).

Immune cell plasticity within the adaptive immune cell compartments is also heavily regulated by the inflammatory state of the intestinal tract, thus the frequency and phenotype of T helper cells (CD4+) and cytotoxic T-cells (CD8+), within the duodenal tissues, were also evaluated (23). Similar to that of the total ILC populations, no significant difference in the frequencies of total CD3+, αβ+ nor γδ+ T-cells were observed between the TID and non-TID cohorts (Figures 3A–C). Furthermore, within the αβ+ T-cell populations, similar frequencies of CD4+ and CD8+ T-cells were observed (Figures 3A,B). Surprisingly, no significant differences in the frequency of duodenal CD4+CXCR3+ (Th1), CD4+CCR6+ (Th17) or CD4+CXCR3+CCR6+ (Th1/Th17), or CD4+FOXP3+ T-cells (Tregs) was observed (Figures 3A,C,E–G). Conversely, mirroring the observed phenotypic skewing of ILCs, the T1D cohort presented with a significantly higher frequency of duodenal CD8+CXCR3+ (Tc1) T-cells as well as a significantly lower frequency of CD8+CCR6+ (Tc17) T-cells (Figures 3A,D), although the frequency of CD8+CXCR3+CCR6+ (Tc1/Tc17) was similar between the cohorts (Figures 3A,E) (24, 25).

Appropriate communication of the intestinal tract with the environment is required for local and systemic immunologic homeostasis, which is in part regulated by the IEC through its physical and biochemical interactions with innate and adaptive immune populations (13). Thus, to determine if the phenomenon observed at the whole tissue level could be a result of altered IEC-innate immune function, primary IEC cultures were established from the crypts of duodenal tissues using our previously published method (15) (Table S1 in Supplementary Material). Here, the soluble mediator gene expression profile of resting T1D-derived IEC cultures was similar to that observed in the whole tissue, whereby 5- and 3.6-fold higher mRNA levels of BD-2 and IL-17C, respectively, were observed in T1D-derived cultures, when compared to non-TID derived cultures (Figures 4A,B), without similar upregulation in immunoregulatory mediators such as TSLP or IL-10 (Figures 4A–C). Again, this altered soluble mediator expression was in the absence of significant differences in the mRNA levels of TLR5 (Figures 4A,B).

To determine if the T1D-associated IEC-soluble mediator milieu could directly contribute to expansion and/or polarization of the T-cell compartment observed at the whole tissue level, the proliferative capacity and polarization of PBMC from a single healthy donor was assessed following exposure to IEC-conditioned supernatants. Here, the proliferation of CD3+ T-cells was significantly enhanced when exposed to conditioned supernatants from T1D-derived IEC cultures, mostly as a result of CD8+ T-cell expansion (Figures 5A,B). While conditioned supernatants from T1D-derived IEC cultures induced a similar polarization profile of CD4+ T-cell subsets, as did conditioned supernatants from non-T1D-derived IEC cultures (Figure 5D), there was a statistically significant difference in the effects on the CD8+ T-cell populations (Figures 5C,E). Specifically, the frequency of CD8+CXCR3+CCR6+/− T-cells (Tc1) was higher following exposure to conditioned supernatants from T1D-derived cultures, whereby the expression level of CXCR3+ was also significantly elevated on a per cell basis (Figures 5C,E). Similarly, while both sources of conditioned supernatants induced an expansion of CD4+FOXP3+ T-cells (Tregs) (Figure 5F), supernatants from T1D-derived IEC cultures polarized Tregs to that of a more Th1-like phenotype as measured by a higher frequency of CD4+FOXP3+CXCR3+CCR6+/− T cells (Figure 5F).

As previous studies had indicated intestinal inflammation inherent in T1D cohorts, the duodenal samples of this cohort were also evaluated for overt intestinal pathology and markers of intestinal inflammation. Specifically, all tissues were evaluated for gross pathology, goblet cell and intraepithelial lymphocyte (IEL) frequency, and expression of HLA-DR. Interestingly, the absence and presence of intestinal pathology were observed in both cohorts with similar incidence (Figure 6A), whereby no statistical differences in the frequency of goblet cells (Figure 6B), IELs (Figure 6C), or HLA-DR expression (Figure 6D) was observed.