Flagellin is an immunodominant Ag in Crohn disease, with many patients showing anti-flagellin Abs. To study the clonality of flagellin-reactive CD4 cells in Crohn patients, we used a common CD154-based enrichment method following short-term Ag exposure to identify Ag-reactive CD4 cells. CD154 expression and cytokine production following Ag exposure compared with negative control responses (no Ag exposure) revealed that only a small fraction of CD154-enriched cells could be defined by Ag-reactive cytokine responses. This was especially true for low-frequency flagellin-reactive CD4 cells compared with polyclonal stimulation or Candida albicans Ag exposure. Moreover, we found that culture conditions used for the assay contributed to background CD40L (CD154) expression in the CD154-enriched CD4 cells. Using a cut-off rule based on flow cytometry results of the negative control CD154-enriched CD4 cells, we could reliably find the fraction of Ag-reactive cells in the CD154-enriched population. Ag-reactive CD4 cytokine production was restricted to CD4 cells with an effector memory phenotype and the highest levels of induced CD154 expression. This has important implications for identifying Ag-specific T cells of interest for single cell cloning, phenotyping, and transcriptomics.

Gut microbial Ag-specific CD4+ T cells play a central role in experimental murine colitis and are thought to mediate the chronic intestinal inflammation of Crohn disease (CD) (1, 2). The gut microbial Ag flagellin has been characterized as an immunodominant Ag in Crohn patients (3), and an aberrant adaptive immune response to flagellin proteins is well known in CD (4, 5). The diagnosis of Crohn is supported by the presence of serum Abs against flagellin Ags (CBir1, A4-Fla2, Fla3, and FlaX) (6), and this serologic marker is associated with worse disease outcomes (7, 8). Moreover, CD patients widely share a small number of TCR CDR3 regions, some of which are associated with flagellin reactivity (9). However, it is unknown whether these immune responses to flagellin Ags themselves primarily cause the inflammatory damage in CD. Gaining insight into flagellin Ag-specific T cell–mediated disease mechanisms could provide a rationale to target Ag-specific T cells as novel therapies.

To associate highly shared Crohn TCR clonotypes with Ag specificity and function, we aimed to identify flagellin-specific T cells in Crohn patients and characterize their cytokine profile and suitability for isolation. Several classic methods that depend on activation marker expression and cytokine production have been used to define microbe-reactive T cells, including T cell proliferation assays ([3H]thymidine incorporation and CFSE dilution), cytokine secretion assays (ELISA and ELISPOT), and the so-called activation-induced marker assay (1013). Each of these assays have their individual limitations in establishing specific details about Ag-reactive T cells because individually, they may not directly determine the original frequency of Ag-reactive T cells, cannot detect whether the same cell makes multiple cytokines, measure cytokine production based on the length of observation, or may not provide the chance for isolation of single cells of interest.

Previous studies have demonstrated that CD40L (CD154) is overexpressed on circulating T cells in CD, and the induction of CD40L is involved in pathogenic cytokine production in these patients (14, 15). Therefore, taking advantage of this transiently expressed ligand by using the previous described CD154+ (CD40L) enrichment assay, we aimed to directly enumerate and characterize Ag-specific cells after short-term stimulation, which lowers the risk of potential phenotypic and functional changes because of in vitro effects during prolonged culture ex vivo (16, 17).

Furthermore, Ag-reactive T cell enrichment technology (16) allows for accurate and sensitive assessment of CD4 cells and, relevant for our studies, does not require prior knowledge of the peptide–MHC complex (911). Cognate Ag-activated CD4 cells induce CD40L (CD154) surface expression, which is important for activation of APCs and further CD4 T cell priming. Previous studies have shown that CD154 expression can be detected effectively following 7-h stimulation of healthy donor PBMC with polyclonal stimulation (Staphylococcus aureus enterotoxins and PMA/I) and fungal (Candida albicans and Aspergillus fumigatus), viral (CMV and HIV Gag), and bacterial (Escherichia coli and Mycobacterium) Ags (13, 16). We now report the use of CD-associated flagellin Ag-driven CD154 expression together with intracellular cytokine production to identify small subsets of CD4 cells, which we take to be authentic Ag-reactive cells. These flagellin-reactive CD4 cells represent a plausible percentage of total circulating CD4 cells, are increased in patients with active CD, and can be differentiated from the increased background expression of CD154 induced by the culture conditions themselves.

For our study, we recruited patients from the Birmingham VA Medical Center and the University of Alabama at Birmingham outpatient clinics. Eligible patients had verified inflammatory bowel disease (IBD) diagnoses according to conventional clinical, endoscopic, and histologic criteria (18); current disease status was documented by clinical evaluation and endoscopic and/or imaging studies. Blood samples were obtained from CD (n = 38) and non-IBD healthy control patients (n = 5). Demographic and clinical characteristics are summarized in Table I. This study was approved by the Institutional Review Boards of the Birmingham VA Medical Center and the University of Alabama at Birmingham. All donors involved in the study signed an informed consent before their inclusion.

Blood samples were collected in heparin-coated tubes and processed within 2 h of procurement. The mononuclear cell fraction was isolated using a density gradient. Blood was diluted 1:1 with 1× PBS (Life Technologies), 0.5% human serum type AB (Corning), and 2 mM EDTA (Invitrogen) (PEB) and layered on top of Ficoll-Paque Plus (GE Healthcare) and centrifuged for 25 min at room temperature at 400 × g with acceleration and no brake. The mononuclear cell fraction was aspirated and washed with PEB and centrifuged for 20 min at room temperature at 300 × g. The cell pellet was then resuspended in PEB, and the number of viable cells was counted using trypan blue (Sigma-Aldrich). The cells were then resuspended in RPMI 1640 medium with l-glutamine (Life Technologies, HyClone Laboratories), supplemented with 5% (v/v) AB serum (Corning) and 25 mM HEPES (Life Technologies), and 5 × 106 PBMCs/well were plated in 24-well plates for overnight incubation at 37°C and 5% CO2.

Following an overnight incubation, the PBMCs were either left unstimulated or stimulated for 7 h with the following Ags: C. albicans lysate (20 μg/ml; Greer Laboratories), CytoStim (1 mg/ml; Miltenyi Biotec), and flagellin Ags CBir1, FlaX, and Fla2 (10 ng/ml), which were kindly provided by Dr. C.O. Elson (University of Alabama, Birmingham, AL), in the presence of 1 mg/ml anti-CD40, 1 mg/ml anti-CD28 functional grade pure mAb (both Miltenyi Biotec), and 1 mg/ml brefeldin A (BD Biosciences), which was added for the last 3 h of incubation. For MHC class II blocking, anti–HLA-DR, -DP, and -DQ (Tü39; BD Pharmingen) was added at 10 μg/ml to the culture 30 min before Ags and costimulatory molecules. Cells were kept at 37°C and 5% CO2 for the duration of the stimulation.

After stimulation, cells were separated using the CD154 MicroBead Kit (Miltenyi Biotec). The cells were indirectly magnetically labeled with CD154 biotin and antibiotin microbeads and enriched by using MS MACS columns (Miltenyi Biotec). The surface staining was performed on the first column, followed by fixation, permeabilization (Inside Stain Kit; Miltenyi Biotec), and intracellular cytokine staining on a second column.

For surface staining of enriched cells, we stained with different combinations of the following mAbs according to manufacturer’s protocols (clone names in parentheses): LIVE/DEAD Fix NIR-80 (L34975; Life Technologies), CD3-FITC (HIT3a), CD14–allophycocyanin (M5E2), CD20–allophycocyanin (2H7), CD8–allophycocyanin (RPA-T8), CD69-PE-Cy5 (FN50), CD4-PE (RPT-T4), CD4–Alexa Fluor 700 (RPA-T4), (all Becton Dickinson Pharmingen), CD69–Pacific blue (FN50), CD62L–Brilliant Violet 421 (DREG-56), mouse IgG1k–Brilliant Violet 421 (MPOC-21), CD45RO–Brilliant Violet 421 (UCHL1), mouse IgG2ak–Brilliant Violet 421 (MPOC-173), CD4-PECy7 (RPA-T4) (all BioLegend), CD4-PE-Vio615 (REA623; Miltenyi Biotec), and FcR blocking reagent (Miltenyi Biotec). After fixation and permeabilization (Inside Stain Kit; Miltenyi Biotec), we stained for CD154 expression (or isotype controls) alone using CD154–Pacific Blue (24-31; BioLegend), mouse IgG1k–Pacific blue (MPOC-21; BioLegend), CD154-PE (REA238), mouse IgG2a-PE, REA Control(S)-PE, CD154-VioBlue (5C8), mouse IGg2a-VioBlue, CD154-FITC (REA238), or recombinant human IgG1-FITC (REA Control(S)-FITC) (all Miltenyi Biotec) or in combination with different cytokines (or their isotype controls) using TNF-α–Alexa Fluor 700 (MP6-XT22; BioLegend), Rat IgG1k (RTK207; BioLegend), TNF-α PECy7 (Mab; MAB1; BD Biosciences), mouse IgG1K-PECy7 (MPOC-2; BD Biosciences), TNF-α–PE-Vio770 (cA2), human IGg-PE-Vio770 (Miltenyi Biotec), REA Control (I)-PE-Vio770, IFN-γ–PECy7 (4S.B3; eBioscience), IFN-γ–Alexa Fluor 700 (B27; BD Biosciences), mouse IgG1K–Alexa Fluor 700 (MPOC-21; BD Biosciences), IL-17A–PerCP-Cyanine5.5 (N49-653; BD Biosciences), mouse IGg1k-PerCP-Cyanine5.5 (MOPC-21; BD Biosciences), IL-17A–PerCP-Cyanine5.5 (eBio64DEC17; eBioscience), or mouse IgG1K-PerCP-Cyanine5.5 (P3.6.2.8.1; eBioscience). Stained cells were acquired on a LSR II Flow Cytometer (BD Biosciences) using FACSDiva software (version 8.0; BD Biosciences) within 24 h after staining and analyzed using FlowJo software (version 10.5.3; TreeStar).

Then, 10 × 106 PBMC were stimulated 0, 3, 7, 9, and 12 h with no Ag or with the following: C. albicans lysate (20 μg/ml; Greer Laboratories), CytoStim (1 mg/ml; Miltenyi Biotec), and combined flagellin Ags CBir1, Fla-X, and Fla-2 (10 ng/ml) in the presence of 1 mg/ml CD40 and 1 mg/ml CD28 functional grade pure Ab (both Miltenyi Biotec). For intracellular staining, 1 mg/ml brefeldin A (BD Biosciences) was added for the final 3 h (3-h time point) or 4 h (later time points) of incubation. Cells were kept at 37°C and 5% CO2 for the duration of the stimulation and then enriched for CD154 expression as mentioned above.

The software package GraphPad Prism (version 7; GraphPad Software) was used to analyze data and to perform statistical analyses. Statistical significance of differences was assessed using nonparametric Mann–Whitney U tests and Kruskal–Wallis with Dunn multiple comparison and two-way ANOVA with Dunnett multiple comparison: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.001.

To study how CD40L (CD154) expression could be used to identify flagellin-reactive circulating CD4+ T cells from CD patients, we stimulated PBMCs with Ags for 7 h and magnetically enriched for CD154 (Fig. 1A). To assess CD154 expression on enriched CD154+ cells, we first gated on viable cells and then excluded non–T cell lineages (CD14+, CD20+) and CD8+ cells to obtain viable CD4+ T cells (Fig. 1B). The viable CD8 CD4+ T cells were evaluated for CD154 expression. A significantly higher expression of CD154+CD4+ T cells was detected among the Ag-stimulated PBMC samples compared with unstimulated samples and CD154 isotype controls (Fig. 1C, Supplemental Fig. 1A). CD154 expression on the flow-through cells was similar to isotype control, showing that CD154+ cells have been efficiently retained on the column (Fig. 1C). Enriched cells ranged from 400–1000 (CytoStim), 30–1000 (C. albicans), 10–100 (CBir1), 10–500 (Fla2), and 10–400 (FlaX) CD154+ cells per 105 CD4+ T cells. These results show that CD154 enrichment is sensitive enough to detect circulating flagellin-reactive CD4+ T cells from CD patients.

FIGURE 1.

Detection and optimization of intracellular CD154 in CD4 T cells stimulated in the presence of brefeldin A (BFA), anti-CD28, and anti-CD40. (A) Experiment protocol overview. 5.0 × 106 PBMC from CD patients cultured with Ags and magnetically enriched for CD154 on magnetic columns. (B) Representative contour plots showing gating strategy for determining viable CD4+ T cell events. We gated on lymphocyte populations and then exclude cell aggregates, dead cells, non–T cell lineages, and CD8 T cells (CD14+, CD20+, and CD8+). (C) Histogram representation of intracellular CD154 expression for enriched and flow through with isotype control. (D) Kinetics of intracellular CD154 in CD4+ T cells following polyclonal and Ag stimulation. Timeline of CD154 expression after stimulation periods (0, 3, 7, 9, and 12 h postactivation) in the presence of BFA. The percentage of viable CD154+CD4+ T cells from Crohn patients (n = 3) exposed to C. albicans, CytoStim, and combined flagellin Ags (CBir1, Fla-2, and Fla-X) or left unexposed for 7 h. Bars represent the means ± SEM of three independent experiments. Statistical significance compared the mean for each time point for each condition using Kruskal–Wallis test with Dunn multiple comparison. *p < 0.05. (E) Histogram representation of CD154 surface expression following stimulation with flagellin Ags or no stimulation (No Ag).

FIGURE 1.

Detection and optimization of intracellular CD154 in CD4 T cells stimulated in the presence of brefeldin A (BFA), anti-CD28, and anti-CD40. (A) Experiment protocol overview. 5.0 × 106 PBMC from CD patients cultured with Ags and magnetically enriched for CD154 on magnetic columns. (B) Representative contour plots showing gating strategy for determining viable CD4+ T cell events. We gated on lymphocyte populations and then exclude cell aggregates, dead cells, non–T cell lineages, and CD8 T cells (CD14+, CD20+, and CD8+). (C) Histogram representation of intracellular CD154 expression for enriched and flow through with isotype control. (D) Kinetics of intracellular CD154 in CD4+ T cells following polyclonal and Ag stimulation. Timeline of CD154 expression after stimulation periods (0, 3, 7, 9, and 12 h postactivation) in the presence of BFA. The percentage of viable CD154+CD4+ T cells from Crohn patients (n = 3) exposed to C. albicans, CytoStim, and combined flagellin Ags (CBir1, Fla-2, and Fla-X) or left unexposed for 7 h. Bars represent the means ± SEM of three independent experiments. Statistical significance compared the mean for each time point for each condition using Kruskal–Wallis test with Dunn multiple comparison. *p < 0.05. (E) Histogram representation of CD154 surface expression following stimulation with flagellin Ags or no stimulation (No Ag).

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The optimal time for inducing CD154 expression among circulating flagellin-specific CD4+ T cells from CD patients is unknown. To determine the optimal duration of flagellin Ag stimulation for our assay, we evaluated the kinetics of intracellular CD154 expression. Overnight-rested PBMCs were incubated with anti-CD40, anti-CD28, and brefeldin A and stimulated with combined flagellin Ags and our positive controls (C. albicans and CytoStim) or left unstimulated, and the cells were removed at various time points (3, 7, 9, and 12 h) (Fig. 1D). CytoStim-stimulated CD4 T cells showed peak expression of intracellular CD154 from 3 to 7 h, whereas C. albicans and flagellin Ag-stimulated cells showed peak expression of intracellular CD154 at 7 h of stimulation. Of note, we observed an increased background expression of intracellular CD154 by unstimulated cells starting at the 3-h time point. Following stimulations, there was a significantly higher population of CD154+ expressing cells in comparison with background (isotype control and unstimulated cells) at 7-h stimulation (Fig. 1D, 1E). These data validate the use of 7-h incubation times to detect intracellular CD154 expression by flagellin Ag-specific CD4+ T cells.

Ag-reactive CD4+ T cells can produce cytokines upon exposure to their cognate Ag in the setting of APCs. Using a combination of CD154 enrichment and multiparametric analysis of cytokines by flow cytometry, we examined CD154+ enriched cells for cytokine production. Following short-term Ag stimulation, the surface-stained CD154-enriched cells were permeabilized, fixed, and intracellularly stained for CD154, IFN-γ, TNF-α, and IL-17A. Subsets of cytokine-producing CD154+CD4+ T cells were detected at varying numbers with the highest response to C. albicans (Supplemental Fig. 1B). The flagellin Ags CBir1, Fla2, and FlaX also induced cytokine production in smaller percentages of the CD154+CD4+ T cells (Fig. 2A). These data show that the CD4 T cells enriched for CD154 expression have both cytokine-producing and cytokine-nonproducing CD4 cells.

FIGURE 2.

Flagellin Ag-exposed CD4+ T cells analyzed for cytokine expression using the CD154+ enrichment assay. (A) Representative contour plots of TNF-α–, IL-17A–, and IFN-γ–producing cells among CBir1- and Fla-2–exposed CD154+CD4+ T cells with (CD154 and cytokine) isotype control. (B) Frequency of CBir1-, Fla2-, and FlaX-exposed IFN-γ+, IL-17A+, and TNF-α+ single-positive CD154+CD4+ T cells among Crohn patients (n = 10). (C) Frequency of CBir1-, Fla2-, and FlaX-exposed TNF-α+IL-17A+ and TNF-α+IFN-γ+ double-positive CD154+CD4+ T cells among Crohn patients (n = 10). Bars represent the means ± SEM of 10 independent experiments; nonparametric Kruskal–Wallis test with Dunn multiple comparison. *p < 0.05, **p < 0.01.

FIGURE 2.

Flagellin Ag-exposed CD4+ T cells analyzed for cytokine expression using the CD154+ enrichment assay. (A) Representative contour plots of TNF-α–, IL-17A–, and IFN-γ–producing cells among CBir1- and Fla-2–exposed CD154+CD4+ T cells with (CD154 and cytokine) isotype control. (B) Frequency of CBir1-, Fla2-, and FlaX-exposed IFN-γ+, IL-17A+, and TNF-α+ single-positive CD154+CD4+ T cells among Crohn patients (n = 10). (C) Frequency of CBir1-, Fla2-, and FlaX-exposed TNF-α+IL-17A+ and TNF-α+IFN-γ+ double-positive CD154+CD4+ T cells among Crohn patients (n = 10). Bars represent the means ± SEM of 10 independent experiments; nonparametric Kruskal–Wallis test with Dunn multiple comparison. *p < 0.05, **p < 0.01.

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Among these small fractions of cytokine-producing Ag-specific CD154+CD4+ T cells, there were significantly higher percentages of CytoStim-stimulated and CBir1-, Fla2-, and C. albicans–specific TNF-α, IFN-γ, and IL-17A, single-positive CD154+CD4+ T cells compared with unstimulated cells (Fig. 2B, Supplemental Fig. 1C). CD patients’ (n = 11) response to FlaX showed only a marginal increase in the fraction of TNF-α+, IL-17A+, and IFN-γ+ single-positive CD154+CD4+ T cells versus unstimulated cells (Fig. 2B).

Most of the patient samples also contained double-cytokine positive Ag-reactive cells. The percentage of TNF-α+ IFN-γ+ and TNF-α+ IL-17A+ double-positive CD154+CD4+ T cells following exposure to CBir1, Fla2, CytoStim and C. albicans was significantly increased in the CD patients (n = 11) (Fig. 2C, Supplemental Fig. 1D). A significantly high percentage of Fla2, C. albicans, and CytoStim-exposed triple-cytokine positive (TNF-α, IL-17A, and IFN-γ) CD154+CD4+ T cells were also detected in the CD patients. Triple-cytokine positive CD154+CD4+ T cells were detected in only one of the patients stimulated by FlaX and three of the patients stimulated by CBir1. These data show that cytokine-producing flagellin-specific CD4+ T cells are a heterogeneous minority fraction of the total CD154+CD4+ T cells population within Crohn peripheral blood.

We found that a large proportion of the Ag-exposed CD154+ enriched cells failed to produce cytokines during the incubation. It is unclear whether these cytokine-nonproducing cells are Ag-reactive clonotypes with less affinity for the Ag, are Ag-reactive clonotypes at different phases of reactivity regardless of affinity, have pre-existing CD154 expression, or have Ag-independent CD154 expression. To determine Ag-specific reactivity independent of cytokine production, we analyzed the effects of culture conditions on CD154 expression among negative control cells. PBMCs from a cohort of Crohn patients (n = 4) were incubated for 7 h in the presence or absence of anti-CD40, anti-CD28, and brefeldin A (added 3 h before harvest). Cells were enriched and stained for CD154 expression as discussed. All four donors showed that CD154 expression was significantly induced in comparison with isotype control. Moreover, CD4 cells that were cultured in the presence of anti-CD40 and anti-CD28 also had a significantly higher expression of CD154 in comparison with cells that were cultured in the absence of anti-CD40 and anti-CD28 (Fig. 3A, 3B). In contrast, brefeldin A on its own did not appear to affect CD154 detection (Fig. 3C). The addition of anti-CD40 and anti-CD28 Ab to our assay has been shown to be necessary to prevent surface CD154 downregulation and to enhance short-term T cell activation, respectively (16). Our data indicate that the addition of these factors contribute to an increased background CD154 expression enough to capture these cells on the enrichment column.

FIGURE 3.

Identifying factors that contribute to increased CD154 background on unstimulated cells. (A) Unstimulated PBMCs were incubated for 7 h in the presence or absence of anti-CD40/anti-CD28 with brefeldin A (BFA). Cells were enriched and stained for CD154 expression. Histogram plots of four donors showing CD154 expression among viable CD4+ T cells. (B) Scatter plot represents geometric mean fluorescence intensity (gMFI) of CD154 expression in CD14CD20CD8CD4+ T cells incubated with (black circle) or without anti-CD40/anti-CD28 (red circle) in the presence of BFA. Bars represent the means ± SEM of five independent experiments using different Crohn patients (n = 5); Mann–Whitney U comparison. *p < 0.05. (C) Histogram of CD154 expression of a representative Crohn unstimulated CD4+ T cells incubated for 7 h with or without anti-CD40/anti-CD28 in the presence or absence of BFA. (D) Ag-exposed PBMCs from one active Crohn patient cultured in the presence of anti–HLA-DP-DR-DQ.

FIGURE 3.

Identifying factors that contribute to increased CD154 background on unstimulated cells. (A) Unstimulated PBMCs were incubated for 7 h in the presence or absence of anti-CD40/anti-CD28 with brefeldin A (BFA). Cells were enriched and stained for CD154 expression. Histogram plots of four donors showing CD154 expression among viable CD4+ T cells. (B) Scatter plot represents geometric mean fluorescence intensity (gMFI) of CD154 expression in CD14CD20CD8CD4+ T cells incubated with (black circle) or without anti-CD40/anti-CD28 (red circle) in the presence of BFA. Bars represent the means ± SEM of five independent experiments using different Crohn patients (n = 5); Mann–Whitney U comparison. *p < 0.05. (C) Histogram of CD154 expression of a representative Crohn unstimulated CD4+ T cells incubated for 7 h with or without anti-CD40/anti-CD28 in the presence or absence of BFA. (D) Ag-exposed PBMCs from one active Crohn patient cultured in the presence of anti–HLA-DP-DR-DQ.

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To examine whether CD154 expression in our system has Ag-dependent and -independent contributions, we repeated our in vitro stimulation during the blockade of APC interaction with CD4 cells. C. albicans and flagellin Ags-exposed PBMCs treated with the pan-HLA-DR blocking Ab showed drastically decreased CD154 expression; the response to flagellin Ags Fla2 (Fig. 3D), CBir1, and FlaX (data not shown) was eliminated. A similar decrease was not seen for activation by CytoStim, which does not depend on Ag presentation by an APC. These results suggest that although there is CD154 expression because of culture conditions, the majority of CD154 expression does seem to be related to Ag exposure and reveals a heterogeneity of CD4 response.

CD154-enriched cells following Ag exposure contain a large proportion of cells that overlap with CD154-enriched cells that were not incubated with Ags despite our exclusion criteria to remove doublets, dead cells, CD8+ T cells, and nonlineage (CD14+ and CD20+) cells prior to gating on CD4+ lymphocytes (Fig. 4A). This was particularly important for studying flagellin-reactive T cells, which were typically of a lower number although equivalently high CD154 mean expression. We developed a gating strategy to eliminate these nontarget cells for optimal detection of presumed Ag-induced CD154+CD4+ events using the powerful stimulation by C. albicans Ag. Using a cohort of Crohn patients (n = 3), CD154 expression on C. albicans–exposed CD154+-enriched cells far exceeded that of the respective isotype-stained control samples (without and with Ag exposure), confirming that little CD154 expression is due to nonspecific Ab staining (Fig. 4, Supplemental Fig. 2A). Histogram plots of CD154 expression by C. albicans–exposed CD4+ T cells often show a bimodal distribution; we designated these two distinct CD154-expressing populations as CD154dim and CD154highCD4+ (Fig. 4A). Further examination of CD154 expression using histogram plot overlays for C. albicans–exposed and unexposed CD4+ T cells (and respective isotype controls) show that the C. albicans–exposed CD154dimCD4+ T cell population was completely overlapped by the Ag-unexposed CD4+ T cells population. However, a relatively small amount of Ag-unexposed CD4+ T cells expressing CD154 (above the 97.5% gradient for CD154 staining) overlapped with the C. albicans–exposed CD154highCD4+ cell population. This suggested that following Ag exposure, CD4 cells could be divided into strata based on CD154 expression in the negative control cells (no added Ag). We have defined the CD154dim cells as those cells below the 97.5% proportion in Ag-unexposed CD154+CD4+ T cells and the CD154hi cells, which are cells above the 97.5% proportion of the Ag-unexposed CD154+CD4+ T cells. Analysis of histogram plot overlays of CD154 expression by CytoStim-stimulated and -unstimulated CD4+ T cells (and respective isotype controls) showed that there was also some overlap of CD154 expression between CytoStim-stimulated and -unstimulated CD4+ T cells (Supplemental Fig. 2A).

FIGURE 4.

Defining the cytokine-producing subset within the CD154+ CD4+ T cells following Ag exposure. (A) Representative histogram overlay plots for CD154 expression among Crohn patients (n = 3). Histogram overlay plots are shown with isotype controls for Ag-exposed cells (gray dotted line) and unexposed cells (blue solid line). CD154 expression for Ag-unexposed cells (dark gray solid line) and Ag-exposed cells (black solid line). (B) Representative flow plots of cytokine production by CD154-expressing cells. Histogram overlay plots are shown with unexposed cells (dark gray solid lines) and the Ag-exposed cells (solid black line). Contour plots of TNF-α, IL-17A, and IFN-γ production by CD154dim/hiCD4+ T cells following C. albicans or Fla-2 exposure. (C) The cumulative frequency of TNF-α, IFN-γ, and IL-17A produced by CD154dim (gray circle) or CD154hi (black circle) CD4+ T cells following Ag exposure among Crohn patients (n = 15). Bars represent the means ± SEM of 15 independent experiments; Mann–Whitney U test. **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 4.

Defining the cytokine-producing subset within the CD154+ CD4+ T cells following Ag exposure. (A) Representative histogram overlay plots for CD154 expression among Crohn patients (n = 3). Histogram overlay plots are shown with isotype controls for Ag-exposed cells (gray dotted line) and unexposed cells (blue solid line). CD154 expression for Ag-unexposed cells (dark gray solid line) and Ag-exposed cells (black solid line). (B) Representative flow plots of cytokine production by CD154-expressing cells. Histogram overlay plots are shown with unexposed cells (dark gray solid lines) and the Ag-exposed cells (solid black line). Contour plots of TNF-α, IL-17A, and IFN-γ production by CD154dim/hiCD4+ T cells following C. albicans or Fla-2 exposure. (C) The cumulative frequency of TNF-α, IFN-γ, and IL-17A produced by CD154dim (gray circle) or CD154hi (black circle) CD4+ T cells following Ag exposure among Crohn patients (n = 15). Bars represent the means ± SEM of 15 independent experiments; Mann–Whitney U test. **p < 0.01, ***p < 0.001, ****p < 0.0001.

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Therefore, we defined a positive Ag-specific response as that level of CD154 expression following Ag exposure above the 97.5% distribution of CD154 expression in the Ag-unexposed cells. Using a gating scheme in which we exclude the cells below the 97.5% proportion of unexposed CD154+ cells, we next examined flagellin-exposed cells among a group of CD patients (n = 3). Histogram plot overlays of unexposed and flagellin Ag-exposed cells show that above the 97.5% proportion of unstimulated CD154+ cells were a small distinct population of CD154high cells (Fig. 4A). Crohn patients had variable responses to CBir1, Fla2, and FlaX with reactivity to at least two of the three flagellin Ags. Overall, using a gating scheme that excludes cells below the 97.5% proportion of unstimulated CD154+ cells allowed for the detection of a cell subset in which flagellin Ag-specific CD154+CD4+ T cells likely reside.

To further validate the exclusion of cells that fall below the 97.5% proportion of Ag-unexposed CD154+ cells, we examined cytokine production by Ag-exposed cells that fall below the 97.5% (CD154dim) and above 97.5% (CD154hi) portion of unexposed CD154+CD4+ T cells (Fig. 4B; C. albicans and Fla2) among 15 active Crohn patients; we found that the Ag-exposed CD154hi cells produce significantly higher percentages of TNF-α, IFN-γ, and IL-17A in comparison with CD154dim cells (Fig. 4C). Interestingly, CytoStim-stimulated CD154dimCD4+ T cells and CD154hiCD4+ T cells displayed similar levels of IFN-γ and IL-17A expression (Fig. 2C, Supplemental Fig. 2B).

Using another activation marker, CD69, along with CD154 induction, did not improve the identification of strictly cytokine-producing cells following Ag exposure. We found that following stimulation, CD154dimCD4+ T cells and CD154hiCD4+ T cells highly expressed CD69 (Supplemental Fig. 3A). However, flagellin- and C. albicans–exposed CD69+CD154hiCD4+ T cells produce substantially higher TNF-α, IFN-γ, and IL-17A (data not shown) cytokines in comparison with CD69+CD154dimCD4+ T cells but only in a subset of the CD69+CD154hi CD4+ T cells. In contrast, CytoStim-exposed CD69+CD154hiCD4+ T cells produce nearly similar levels of cytokines, particularly IFN-γ and IL-17A (data not shown) in comparison with CD69+CD154dimCD4+ T cells (Supplemental Fig. 3B). Together, these data demonstrate that including a criterion that removes the cells that fall below the 97.5% CD154 expression level in the negative control allowed us to focus on a subset of T cells that included the cytokine-producing CD4 cells. Including an additional activation marker did not help in further defining this set of cytokine-producing cells.

We also studied whether the CD154-enriched cells were uniformly effector memory cells and whether cytokine production was confined to a particular CD4 phenotype effector memory T cells (TEM) (CD45RO+ and CD62L), central memory T cells (CD45RO+ and CD62L+), or naive T cells (CD45RO and CD62L+) (Fig. 5). We compared our Ag-exposed CD154dim to CD154high populations and found that TEM cells dominated the CD4CD154high subset following exposure to Ag and that naive T cells and central memory T cells were the most abundant in the CD154dim population (Fig. 5A). Cytokine production by the Ag-exposed CD154hiCD4+ T cells was almost exclusively limited to the TEM (Fig. 5B). In contrast, following polyclonal stimulation, we found no difference in the percentage of central memory, effector memory, and naive cell phenotypes among the CD154hiCD4+ and CD154dimCD4+ T cells (Supplemental Fig. 2D). In addition, we found no difference in cytokine production by CytoStim-stimulated CD154hiCD4+ T cells among the different CD4 subsets (Fig. 5B).

FIGURE 5.

The expression of CD62L and CD45RO by Ag-exposed CD154dim/hiCD4+ T cells was analyzed by flow cytometry. (A) The percentage of CD62L and CD45RO expression by Ag-exposed CD154dim/hiCD4+ T cells. Bars represent the means ± SEM of eight independent experiments; two-way ANOVA Dunnett multiple comparison tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (B) Representative contour plots of cytokine production by CD154hi cells among different CD4 subsets following C. albicans exposure. (C) Cumulative frequency of TNF-α, IFN-γ, and IL-17A produced by CD154hiCD4+ T cells subsets following Ag exposure among active Crohn patients (n = 8). Bars represent the means ± SEM of eight independent experiments; nonparametric Kruskal–Wallis test with Dunn multiple comparison. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 5.

The expression of CD62L and CD45RO by Ag-exposed CD154dim/hiCD4+ T cells was analyzed by flow cytometry. (A) The percentage of CD62L and CD45RO expression by Ag-exposed CD154dim/hiCD4+ T cells. Bars represent the means ± SEM of eight independent experiments; two-way ANOVA Dunnett multiple comparison tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (B) Representative contour plots of cytokine production by CD154hi cells among different CD4 subsets following C. albicans exposure. (C) Cumulative frequency of TNF-α, IFN-γ, and IL-17A produced by CD154hiCD4+ T cells subsets following Ag exposure among active Crohn patients (n = 8). Bars represent the means ± SEM of eight independent experiments; nonparametric Kruskal–Wallis test with Dunn multiple comparison. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Close modal

Among patients with active CD (n = 22), significantly high numbers and percentages of CD154hiCD4+ T cells were observed following flagellin Ag exposure (Fig. 6A, 6B). Similar data were found for C. albicans–exposed and CytoStim-stimulated CD154hiCD4+ T cells (Supplemental Fig. 4A). The absolute frequencies of CD154hiCD4+ T cells in the peripheral blood of active Crohn (based on the total number of CD4+ T cells applied on the column and excluding the nontarget cells) ranged between 1 and 100 (CBir1), 200 (Fla2), and 200 (FlaX) CD154hi cells per 1 × 105 CD4+ T cells (Fig. 6C). As expected, there were much higher frequencies for C. albicans–reactive (8–900) and CytoStim-stimulated (32–3200) CD154hi cells per 105 CD4+ T cells (Supplemental Fig. 4C). Reactivity to the tested flagellin Ag was detected in a majority of the active CD patients. To determine whether flagellin T cells response was CD specific, we evaluated the frequencies of flagellin-reactive CD154hiCD4+ T cells among non-IBD healthy control patients (n = 5) and active Crohn patients (n = 22). The frequencies of flagellin-reactive CD154hiCD4+ T cells were higher in active CD in comparison with healthy control subjects (Fig. 6C). In addition, these flagellin-specific CD154hiCD4+ T cells displayed a trend toward the elevated percentage of TNF-α, IFN-γ, and IL-17A compared with those from the healthy donors (Fig. 6D).

FIGURE 6.

Flagellin-specific CD4+ T cells can be detected in Crohn and healthy patients. (A) CD4+ T cell responses to Ag exposure were analyzed in active Crohn (n = 22). Number of enriched CD154hi cells active CD patients (n = 22). (B) Percentage of CD154hi cells among CD4+ T cells from active Crohn (n = 22) with expression above the background level of ∼2.50%. (C) Frequencies of CD154hiCD4+ T cells in peripheral blood of active Crohn (black dots; n = 22), and healthy (red dots; n = 5) patients were calculated from the number of CD154hi cells obtained after enrichment normalized to the total number of CD4+ T cells applied on the column. (D) Frequency of cytokine-producing cells among active Crohn (n = 22) and healthy (n = 5) patients were calculated from the total number of cytokine-positive cells normalized to the total number of CD154hi cells obtained after enrichment. Bars represent the means ± SEM of 522 independent experiments; Mann–Whitney U test. *p < 0.05, **p < 0.01, ****p < 0.0001. (E) The frequency of flagellin-specific CD4 T cells (stimulated sample minus Ag-unexposed control for CD154hi cells; background subtracted). (F) Comparison of CD154hiCD4+ T cells cytokine reactivity to different flagellin Ags within the same active Crohn patient (n = 22). Each symbol represents one patient (lines link result within the same active Crohn patient).

FIGURE 6.

Flagellin-specific CD4+ T cells can be detected in Crohn and healthy patients. (A) CD4+ T cell responses to Ag exposure were analyzed in active Crohn (n = 22). Number of enriched CD154hi cells active CD patients (n = 22). (B) Percentage of CD154hi cells among CD4+ T cells from active Crohn (n = 22) with expression above the background level of ∼2.50%. (C) Frequencies of CD154hiCD4+ T cells in peripheral blood of active Crohn (black dots; n = 22), and healthy (red dots; n = 5) patients were calculated from the number of CD154hi cells obtained after enrichment normalized to the total number of CD4+ T cells applied on the column. (D) Frequency of cytokine-producing cells among active Crohn (n = 22) and healthy (n = 5) patients were calculated from the total number of cytokine-positive cells normalized to the total number of CD154hi cells obtained after enrichment. Bars represent the means ± SEM of 522 independent experiments; Mann–Whitney U test. *p < 0.05, **p < 0.01, ****p < 0.0001. (E) The frequency of flagellin-specific CD4 T cells (stimulated sample minus Ag-unexposed control for CD154hi cells; background subtracted). (F) Comparison of CD154hiCD4+ T cells cytokine reactivity to different flagellin Ags within the same active Crohn patient (n = 22). Each symbol represents one patient (lines link result within the same active Crohn patient).

Close modal

Although Hegazy et al. (19) recently reported that there are reduced frequencies of circulating C. albicans–reactive CD154+CD4+ T cells and increased frequencies of S. aureus enterotoxins–stimulated CD154+CD4+ T cells in Crohn patients compared with healthy controls, we found no significant difference in the frequencies of C. albicans–reactive CD154hiCD4+ T cells between Crohn and healthy control patients (Table I). We, however, did observe a higher frequency of CytoStim-stimulated CD154hiCD4+ T cells among active Crohn patients compared with those from healthy donors (Supplemental Fig. 4C, 4D).

Table I.
Demographics and clinical characteristics of studied individuals
CharacteristicsAll Patients (n = 43)CD (n = 38)Non-IBD HC (n = 5)
Male/female 25/17 23/15 3/2 
Median (range) age 31 (19–76) 30 (19–76) 31 (26–71) 
Concomitant medications    
 5-Aminosalicylates   
 Infliximab/adalimumab  20  
 Vedolizumab/Ustekn/Certolizumab   
 Corticosteroids   
 Azathioprine/6-meracaptopurine   
 None  
CharacteristicsAll Patients (n = 43)CD (n = 38)Non-IBD HC (n = 5)
Male/female 25/17 23/15 3/2 
Median (range) age 31 (19–76) 30 (19–76) 31 (26–71) 
Concomitant medications    
 5-Aminosalicylates   
 Infliximab/adalimumab  20  
 Vedolizumab/Ustekn/Certolizumab   
 Corticosteroids   
 Azathioprine/6-meracaptopurine   
 None  

HC, healthy control.

We also noted that Crohn patients did not have uniform patterns of response to all Ags. To determine whether a patient with high T cell reactivity to one flagellin Ag had a similar high reactivity to another flagellin Ag, we examined the responses to CBir1, FlaX, and Fla2 among active Crohn subjects (Fig. 6E, 6F). Even though several patients had a strong response to all the tested flagellin Ags, there were some patients that had a strong response to only one or two of the tested flagellin Ags. High levels of TNF-α, IFN-γ, and IL-17A were not seen in response to all tested flagellin Ags among patients reactive to all these Ags (Fig. 6F). Likewise, patients reactive to one or two flagellin Ags did not produce equally high levels of TNF-α, IFN-γ, and IL-17A in response to those Ags (Supplemental Fig. 4E). These results demonstrate that flagellin-specific CD4+ T can be detected in Crohn as well as some healthy patients.

Gut microbial Ag-specific CD4 T cells play a key role in intestinal inflammation in experimental models (1, 2). The association of Abs against flagellin Ags in CD is well known, but less is known about aberrant T cell responses to the same immunodominant Ags. A few translational studies have attempted to functionally characterize microbial-reactive CD4+ T cells in IBD patients (10, 20). Recently, Hegazy et al. (19) used the CD154 expression in CD4 cells to demonstrate that microbial-reactive CD4+ T cells can be detected in the peripheral blood and intestinal tissues of healthy adults as well as IBD patients. These studies used whole bacterial lysates and not purified recombinant bacterial Ags, including flagellins, so specific Ag reactivity could not be reported (19). Other studies characterizing flagellin-reactive CD4+ T cells have used other methods that rely on Ag-induced proliferation (CFSE and [3H]thymidine incorporation) or induction of activation markers like OX40 and CD25 (10, 20). The differences in these assays include an extended Ag exposure time (16 h–7 d) necessary for stimulation, which can miss early events like cytokine production and possibly overlook the earliest-responding CD4 clonotypes. So, some of the gaps in the knowledge of microbial-reactive CD4 cells in CD (and healthy controls) include identifying candidate clonotypes reactive to purified immunodominant microbial Ags relevant to CD and characterizing the phenotype of these CD4 cells and the heterogeneity of the response itself among Crohn patients.

In this report, we have measured the presence and functionality of flagellin-reactive CD4+ T cells in CD patients with the help of an Ag-reactive T cell enrichment approach. PBMCs from donors were stimulated for only 7 h, and cells were enriched for CD154 expression. CD154 is upregulated on T cells when T cells recognize peptide Ag/MHC complexes through αβ-TCR and receive costimulation through CD28 binding to CD80 and CD86. CD154 is transiently expressed on T cells, but its interaction with CD4 is necessary for generating Th1 CD4+ memory T cells. CD154 enrichment facilitates the ex vivo analysis of the Ag-specific T cells using CD154 expression only or in combination with cytokine secretion (21). By using this method, we show that short-term induction of (intracellular) CD154 is sensitive enough to detect small numbers of circulating flagellin Ag-reactive memory CD4+ T cells from CD patients.

However, our study revealed an increase in CD154 expression in CD4 cells not exposed to added Ags using this method. Only one other group has reported CD154 background expression in CD4 cells that were not exposed to exogenous Ag during incubation; this group used CD154 staining during culture conditions along with anti-CD40 and anti-CD49d Abs rather than our CD154 enrichment strategy (22). Using an intracellular CD154 staining approach, Frentsch et al. (23) found no increase in CD154 expression in control cultures (no added Ag) following a 6-h culture with anti-CD40 mAb alone. However, as shown by our data, we found that adding anti-CD40 and anti-CD28 mAb leads to an “Ag-independent” stimulation of CD154 expression within our control cultures. The addition of anti-CD40 Ab to our assay is necessary the block the binding of CD154 (CD40L) with CD40, which prevents CD154 internalization and degradation. CD28 has a role in initiating T cell activation by binding CD80/86 on APC. The added anti-CD28 Ab in our assay will bind CD28 and stimulate T cells without CD80/86 on APC, leading to enhanced short-term T cell activation. Perhaps the addition of anti-CD40 and anti-CD28 Ab to our culture enhances the activation of T cells that interact with APCs that are preloaded with other Ags and are stimulated but are unable to make sufficient cytokines. These CD154-expressing cells need to be accounted for when trying to identify actual Ag-specific clonotypes.

One way to account for Ag-reactive CD4 cells among the CD154 expression increased by the culture conditions themselves was to measure another cell response that was associated with Ag exposure alone; in this case, intracellular cytokine production was a useful discriminating factor. Using an additional activation marker like CD69 along with CD154 expression did not improve the ability to identify the specific subset of cytokine-producing Ag-reactive cells. We found that the cytokine-producing CD4 cells could always be found in that fraction of CD154-positive cells above the top 2.5% of CD154 mean fluorescence intensity in the control cultures. No cytokine coexpression was ever found below this gate. In fact, we took this as the gating strategy to identify the Ag-reactive cells in the Ag-added cultures; the percentage of CD154-expressing cells from these Ag-exposed cultures ranged from 3.26 to 26.7% CBir1-reactive, 4.38–19.8% Fla2-reactive, 3.1–21.6% FlaX-reactive, 43–77.1% C. albicans–reactive, and 35.8–83.4% CytoStim-stimulated cells.

After accounting for nonspecific CD154 expression and using cytokine production to characterize Ag reactivity within this enriched population, we can identify distinct populations of flagellin Ag-specific CD4+ T cells that range between 1 and 200 CD154hi cells per 105 CD4+ T cells among the total enriched CD154 cells. The cytokine profile of flagellin Ag-reactive CD154hiCD4+ T cells in Crohn patients is consistent with previous studies reporting that FlaX- and A4-Fla2–specific CD4+ T cells in Crohn patients can display Th1 (IFN-γ), Th17 (IL-17A), and Th1/Th17 (IFN-γ+ IL-17-A+) cytokine profiles (10, 20, 24). We demonstrated that a higher frequency of flagellin Ag-reactive CD4+ T CD154hi–expressing cells were found among active Crohn patients compared with non-IBD control donors. We also see a higher percentage of cytokine-producing cells among some of the active Crohn compared with non-IBD control patients. Unlike previous studies in which background (CD154 cells among control) was subtracted to show Ag-specific (CD154+) T cell responses only among Ag-stimulated cells, we have shown the frequencies of CD154 without background subtraction. Interestingly, PBMC from Crohn patients that were not exposed to any of our tested Ags exhibited a higher frequency of CD154hiCD4+ T cells compared with Ag-unexposed cells from healthy controls. This could imply that there are more CD4 T cells primed by other Ags or stimuli, possibly related to increased gut permeability, other than those we have tested in the active Crohn compared with healthy patients. Further examination of cytokine production by these nonspecifically activated CD154hi CD4 T cells from CD and healthy control patients showed that there is no significant difference in cytokine production associated with increased CD154 expression.

Among the Ag-stimulated CD154-expressing T cells that produced cytokines, we observed a higher percentage of cytokine-producing cells among the active Crohn compared with non-IBD control patients. The frequency of cytokine-producing flagellin-specific CD4 T cells ranged from 0.00 to 0.08% (TNF-α), 0.000–0.05% (IFN-γ), and 0.00–0.035% (IL-17A). These frequencies are well within the expected range of microbial Ag-specific T memory cells, which has been shown to be typically below 1% in the absence of acute infections (16, 20). Hegazy et al. (19), using the CD154 approach, previously demonstrated that the frequency of microbiota-reactive memory CD154+CD4+ TNF-α+ cells within PBMC of healthy donors and IBD patients can range from 0.0 to 0.025% (Salmonella typhimurium) and 0.0–0.08% (Clostridium difficile).

Not all Ag-stimulated CD154-expressing T cells from our donors produced cytokines (TNF-α, IFN-γ, and IL-17A), and in fact, there was a higher percentage of CD154 cells that did not produce these cytokines. It is possible that these CD154+ cells in our gating strategy may produce other effector cytokines because we were able to measure IL-13, IL-5, IL-4, IL-22, and IL-10 by multiplex cytokine assay of culture supernatant (data not shown), and other cytokines may need more time to be produced (16, 24). It is also possible that these are flagellin-specific cells but with lower affinity TCR clonotypes to different peptide–MHC complexes or are at a different status in terms of their overall ability to respond to Ag.

Furthermore, the magnitude and consistency of responses to the different flagellin Ags (CBir1, Fla2, and FlaX) is not predictable among patients. Although some patients responded to all three flagellins, there are patients that responded to only one flagellin Ag. Among patients with high reactivity to all Ags, we see that all the flagellins do not produce high levels of cytokine. These findings suggest that there may not be a strict hierarchy of the strength of response for a particular flagellin Ag within and among patients.

In summary, our data show that among the distinct CD154hi flagellin-specific T cells are a subset of cells that produce the relevant TNF-α, Th1, and Th17 cytokines that are involved in CD inflammation (25). Among the key points of this study is that in the search for Ag-specific CD4 clonotypes, especially when single cell isolation is required for study, attention to defining Ag-reactive (as opposed to nonspecifically activated) T cells can require additional features. Another point is that the cytokine-producing cells that we are identifying as Ag-specific may be only a subset of actual flagellin-reactive CD4 cells. The others that we can see by flow cytometry may be producing other cytokines, needing more time to produce their cytokine, may have TCRs with varying affinities for the Ag peptide–MHC complex, or may be at different cell phases that affect their ability to respond.

Future studies evaluating the clonality, phenotypes, transcriptomes, and gene signatures of these cytokine-producing CD4 cells and the cytokine-negative CD154hi flagellin-specific T cells from PBMC and intestinal lamina propria mononuclear cell populations will be valuable toward the understanding of functional differences between flagellin-reactive cells in Crohn and healthy controls. Our previous work had found TCRβ CDR3 sequences in both CD4 cells from PBMC and intestinal lamina propria mononuclear cell populations in the same patient, supporting shared clonotypes in the blood and lamina propria in CD. Our current report was an approach to interrogate the CD154 expression method more thoroughly for identifying Ag-reactive cells within PBMC as a way to identify these cells. Unfortunately, the CD154 assay requires many more CD4 T cells (16, 2224) than we can generally obtain from multiple endoscopic biopsies (we prefer to use biopsies to surgically resected bowel specimens as the latter typically come from patients who have been concurrently exposed to excessive immunosuppression). We are working on improving methods to compare flagellin reactivity between blood and gut mucosa CD4 cells within and among Crohn and control subjects to further extend our knowledge of the interplay of flagellin-reactive cells and their role in disease severity, activity, and potential for therapeutic targeting.

We thank Dr. Charles O. Elson and L. Wayne Duck (University of Alabama at Birmingham) for providing the flagellin Ags, Dr. Frances E. Lund (University of Alabama at Birmingham) for granting access to the MagPix instrumentation, and Dr. Davide Botta (University of Alabama at Birmingham) for running the Luminex multiplex assays. We thank the Department of Medicine and the Program in Immunology at the University of Alabama at Birmingham.

This work was supported by U.S. Department of Veterans Affairs Grant CX0001530 and Impact funds from University of Alabama at Birmingham.

The online version of this article contains supplemental material.

Abbreviations used in this article:

CD

Crohn disease

IBD

inflammatory bowel disease

TEM

effector memory T cell.

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The authors have no financial conflicts of interest.

Supplementary data