1. Volume 141, Issue 2, Pages 610-620.e2 (August 2011)
Tolerance to Ingested Deamidated Gliadin in Mice is Maintained by Splenic, Type 1 Regulatory T Cells 
M. Fleur Du Pré, Anne E. Kozijn, Lisette A. van Berkel, Mariëtte N.D. ter Borg, Dicky Lindenbergh–Kortleve, Lise Torp Jensen, Yvonne Kooy–Winkelaar, Frits Koning, Louis Boon, Edward E.S. Nieuwenhuis, Ludvig M. Sollid, Lars Fugger, Janneke N. Samsom 
Gastroenterology 
Volume 141, Issue 2, Pages e2 (August 2011)
DOI: /j.gastro
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2. Figure 1
Transgenic mice express HLA-DQ2 on APC and gliadin-TCR on CD4+ T cells. FACS analysis: (A and B) DQ2.gliadinTCR splenocytes stained for HLA-DQ2 (SPV-L3) and (A) B220 or (B) CD11c. (C) Murine CD3 and human TCR Vβ1 expression on DQ2.gliadinTCR splenocytes. (D) CD4 and CD8 expression, gated on CD3+ splenocytes from DQ2.gliadinTCR mice. (E) CD4 and CD8 expression, gated on CD3+ splenocytes from gliadinTCR mice.
Gastroenterology  , e2DOI: ( /j.gastro )
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3. Figure 2
CD4+ gliadin-TCRtg T cells respond to deamidated but not to nondeamidated gliadin. (A) A total of 5 × 105 splenocytes from DQ2.gliadinTCR mice and MHCIIΔ/Δ mice were cultured with or without deamidated gliadin (TG2-gliadin), and 3H-thymidine incorporation was measured. (B and C) Gliadin-specific CD4+Vβ1+ T cells were stimulated with DQ2+ SPL-DCs loaded with gliadin, TG2-gliadin, OVA, or medium. (B) Release of IL-2 at 48 hours. (C) Release of IFN-γ, IL-4, IL-17, and IL-21 at 96 hours of culture. n.d., nondetectable. *P < .05.
Gastroenterology  , e2DOI: ( /j.gastro )
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4. Figure 3
Oral deamidated gliadin induces a dominant T-cell proliferation in the spleen but not in the MLN. (A) DQ2 mice received 7 × 106 CFSE-labeled CD4+Vβ1+ cells intravenously and 100 mg TG2-gliadin orally the next day. BALB/c mice received 1 × 107 CFSE-labeled CD4+KJ1-26+ cells intravenously and the next day OVA intragastrically (tolerance) or 400 μg OVA intramuscularly (immunity). Spleens and draining lymph nodes were isolated 72 hours after antigen administration and analyzed by FACS. For each group, a representative histogram plot and dot plot showing CFSE dilution (gated on transgenic CD4+ Vβ1+ or CD4+KJ1-26+ cells) is depicted. The percentages of CD4+Vβ1+ or CD4+KJ1-26+ cells in each peak of division were calculated and are represented as the mean ± SD for 5 mice (TG2-gliadin intragastrically) and 3 mice (OVA intragastrically/intramuscularly). (B) DQ2 mice enriched with CFSE-labeled CD4+Vβ1+ T cells were given 100 mg of nondeamidated gliadin or deamidated gliadin orally or 500 μg intramuscularly. At 72 hours after gliadin administration, spleens (for gliadin intragastrically) or draining popliteal lymph nodes (for gliadin intramuscularly) were isolated and CD4+Vβ1+ T cells were analyzed by flow cytometry for CFSE dilution.
Gastroenterology  , e2DOI: ( /j.gastro )
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5. Figure 4
Dividing gliadin-TCRtg T cells have an inflammatory phenotype. (A and B) DQ2 mice and BALB/c mice were enriched with CFSE-labeled gliadin-specific or OVA-specific T cells and challenged as described in Figure 3. At 72 hours after antigen administration, the phenotype of dividing transferred T cells was analyzed in draining lymph nodes or spleen. (A) Representative dot plot for CD62L and Foxp3. (B) Percentage of CD62Lhi and Foxp3+ cells calculated and expressed as mean ± SD for at least 3 mice. (C) DQ2 mice were enriched with CFSE-labeled gliadin-TCRtg T cells and, the next day, received gliadin or TG2-gliadin intragastrically. At 72 hours, gliadin-TCRtg T cells were FACS sorted from the spleens and restimulated in vitro with TG2-gliadin–loaded DQ2+ BM-DC. After 48 hours of restimulation, supernatants were analyzed for secreted cytokines. n.d., nondetectable. *P < .05.
Gastroenterology  , e2DOI: ( /j.gastro )
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6. Figure 5
Gliadin-TCRtg T cells secrete IL-10. (A) Dividing gliadin-TCRtg T cells were FACS sorted and restimulated as described in Figure 4C. (B) Restimulation of splenic gliadin-TCRtg T cells with phorbol myristate acetate/CaI for 14 hours. Relative expression of IL-10 messenger RNA by real-time polymerase chain reaction. (C) Splenic gliadin-TCRtg T cells were obtained and restimulated with TG2-gliadin as described in Figure 4C. After 48 hours of restimulation, cells were harvested and stained intracellularly for Vβ1, IFN-γ, and IL-10. One representative dot plot is shown (gated on Vβ1+ cells, n = 3). (D) Naive gliadin-TCRtg T cells (5 × 105) were stimulated with deamidated gliadin-loaded DQ2+ SPL-DCs (2 × 104) for 72 hours, harvested, and restimulated with fresh deamidated gliadin-loaded DQ2+ BM-DC in the presence or absence of a neutralizing αIL-10R antibody (1B1.2) or isotype control (GL113). Release of IFN-γ at 48 hours of culture was measured by enzyme-linked immunosorbent assay. *P < .05.
Gastroenterology  , e2DOI: ( /j.gastro )
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7. Figure 6
Transfer of gliadin-TCRtg T cells induces tolerance. Dividing CD4+ gliadin-TCRtg T cells were FACS sorted as described in Figure 4C. A total of 2.5 × 105 cells were adoptively transferred to DQ2.gliadinTCR mice. Two mice received 2.5 × 105 purified in vitro–generated gliadin-specific Foxp3+ Treg cells. Control mice did not receive any transferred cells. One day after adoptive transfer, both groups were sensitized subcutaneously with 100 μg TG2-gliadin. Five days later, mice were challenged with 10 μg TG2-gliadin in both ears and, after 24 hours, the increase in ear thickness was determined and compared with values before challenge. *p < .05.
Gastroenterology  , e2DOI: ( /j.gastro )
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