Expansion of CD8+ T cells with regulatory function after interaction with intestinal epithelial cells  Matthieu Allez, Jens Brimnes, Iris Dotan, Lloyd Mayer  Gastroenterology  Volume 123, Issue 5, Pages 1516-1526 (November 2002) DOI: 10.1053/gast.2002.36588 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Quantitative analysis of T-cell proliferation in an IEC:T-cell coculture and calculation of the proportion of the initial population that has responded by dividing. T cells were labeled with CFSE before addition to the culture with irradiated IECs. Cells were stained at day 5 with anti-CD3 PE. The analysis was gated on CD3+ cells. (A) The percentage of dividing cells (cells that have undergone more than one division) is shown. The proportion of the original population induced into cell division after contact with IECs can be determined by using the method reported by Lyons.23 (B) The number of events was measured in each cell division cohort peak. To calculate the percentage of original cells from which they arose, the percentage of events in a given cycle, n, is divided by 2n. The number resulting from this division is referred to as the undivided cohort number. The sum of these undivided cohort numbers (from division 2 to more than 6) represents the percentage of cells that started to divide after interaction with IECs (0.6% in this experiment). This is representative of 10 experiments. Gastroenterology 2002 123, 1516-1526DOI: (10.1053/gast.2002.36588) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Comparison of proliferation of CD8+ vs. CD4+ T cells in IEC:T-cell cocultures and in MLRs. Proliferation analysis at day 7 of CFSE-labeled T cells cultured (A) alone, (B) with IECs, and (C) with non–T cells. Cells were stained with anti-CD3 PE and anti-CD8 PerCP. Analysis was gated on CD3+ cells. Percentages represent the number of cells in each quadrant. The percentage of CD8+ and CD4+ T cells that were induced to divide from the initial population was calculated as described in Figure 1. It was 0.9% and 0.2% in the IEC:T-cell coculture and 0.6% and 0.7 % in the MLR culture, respectively. This is representative of 5 independent experiments. Gastroenterology 2002 123, 1516-1526DOI: (10.1053/gast.2002.36588) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 CD4+ T cells are not necessary for the CD8+ T-cell proliferation in IEC:T-cell cocultures. CD8+ T cells were negatively selected by using the RosetteSep procedure, CFSE labeled, and cultured (A) alone or (B) with IECs. Analysis was gated on lymphocytes by forward-scatter and side-scatter; the percentage of CD8+ proliferating T cells is shown. (C) Purity of CD8+ T cell suspension is shown. This is representative of 3 independent experiments. Gastroenterology 2002 123, 1516-1526DOI: (10.1053/gast.2002.36588) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Expansion of a CD8+ CD28− subset in IEC:T-cell cocultures. Analysis at day 7 of CFSE-labeled T cells cultured with IECs. Cells were stained with anti-CD3 PE, anti-CD8 PerCP, and anti-CD28 APC. Analysis was gated on CD3+ cells. CD28 expression is shown on CD8+ (A) proliferating and (B) nonproliferating cells, and on CD8− (C) proliferating and (D) nonproliferating T cells in an IEC:T-cell coculture. The percentage of CD28− cells is indicated. This is representative of 5 independent experiments. Gastroenterology 2002 123, 1516-1526DOI: (10.1053/gast.2002.36588) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 IEC-activated CD8+ T cells express CD101 and CD103. Analysis at day 7 of CFSE-labeled T cells cultured with IECs. Cells were incubated with anti-CD101 (18F7) or anti-CD103 (28C12), washed with PBS, stained with PE-conjugated goat anti-mouse antibody, washed and stained with anti-CD8 PerCP and anti-CD3 APC. Analysis was gated on CD3+ cells. The figure shows CD101 and CD103 expression of CD8+ proliferating (A and B, respectively) and CD8+ nonproliferating (C and D, respectively) T cells in IEC:T-cell cocultures. The percentage of CD101+ and CD103+ cells is indicated. This is representative of 5 independent experiments. Gastroenterology 2002 123, 1516-1526DOI: (10.1053/gast.2002.36588) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 (A) The proliferation of CD8+ T cells is decreased when IECs are preincubated with anti-gp180 mAb B9. IECs were preincubated with mAb B9 or with an isotype control, and washed before addition to the coculture with T cells. Analysis of CFSE-labeled T cells was performed at days 5 and 7. Cells were stained with anti-CD3 PE and anti-CD8 PerCP. Analysis was gated on CD3+ CD8+ cells; the % of proliferating CD8+ T cells is indicated. (B) Preincubation of IECs with anti-gp180 mAb (B9) decreases the proliferation of CD28−, CD101+, and CD103+ CD8+ T cells. Cells were prepared at day 7 as in Figure 5A and stained with anti-CD3 PE, anti-CD8 PerCP and anti-CD28 APC, or with anti-CD101 and CD103 and GAM PE Ab, and with anti-CD8 PerCP and anti-CD3 APC. The analysis was performed on proliferating CD8+ T cells, with a gate on CD3+ CD8+ CFSE low cells. The percentage of CD28±, CD101+, and CD103+ T cells among proliferating CD8+ T cells in cultures with IECs preincubated with an Ig control or with mAb B9 is shown. These are representative of 4 independent experiments. (C) Preincubation of non–T cells with mAb B9 does not decrease the proliferation of CD8+ T cells as well as CD28− CD101+ CD103+ T cells in MLRs. Cells were prepared at day 7 as in Figures 6A and 6B. The analysis was gated on CD8+ T cells; the percentage of CD28−/CD28+, CD101+, and CD103+ among CD8+ proliferating T cells after preincubation with (A) isotype control or (B) mAb B9 is shown. This is representative of 3 independent experiments. Gastroenterology 2002 123, 1516-1526DOI: (10.1053/gast.2002.36588) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 Lines of IEC-activated CD8+ T cells express CD103. CFSE-labeled T cells were cultured alone or with IECs. CD3+ CD8+ T cells cultured (A) alone or (B) proliferating in culture with IECs were sorted at day 10 (boxed in area). These CD8 cells were expanded by using IL-2/-7/-15, PHA, and feeder cells, and restimulated every 3 weeks. The phenotype of these CD8+ T-cell lines was studied after 3 cycles of stimulation. CD103 expression of (A) nonactivated and (B) IEC-activated CD8+ T-cell lines is shown. Gastroenterology 2002 123, 1516-1526DOI: (10.1053/gast.2002.36588) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 IEC-activated CD8+ T-cell lines are suppressive. (A) IEC-activated and nonactivated CD8+ T-cell lines were added to PBMCs stimulated with pokeweed mitogen (1 μg/mL) at a ratio of 1:5 (1 CD8+ T cell to 5 PBMCs). Ig secretion, measured by ELISA, was significantly reduced in the presence of IEC-activated CD8+ T-cell lines. In contrast, nonactivated CD8+ T-cell lines had no effect. (B) IEC-activated CD8+ T-cell lines were placed directly in the same well as an unrelated MLR (in which T cells were CFSE labeled before culture with irradiated allogeneic non–T cells) at a ratio of 1:5. Proliferation analysis was gated on CD3+ CD4+ cells. The percentage of CD4 cells that underwent more than 2 divisions is shown. The proliferation of CD4 cells was significantly reduced in the presence of IEC-activated CD8+ T-cell lines. This is representative of 4 independent experiments. Gastroenterology 2002 123, 1516-1526DOI: (10.1053/gast.2002.36588) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 9 IEC-activated CD8+ T cells that express CD101 and/or CD103, in contrast to CD101−/CD103− IEC-activated CD8+ T cells, are suppressive in function. CD101+/CD103+ and CD101−/CD103− CD8+ T cells proliferating (CFSElo) in an IEC:T-cell coculture, as well as nonactivated CD8+ T cells, were sorted and added to PBMCs stimulated with pokeweed mitogen (1 μg/mL) at a ratio of 1:10 (1 CD8+ T cells to 10 PBMCs). Ig secretion, measured by ELISA, was significantly reduced in the presence of IEC-activated CD8+ T cells expressing CD101 and/or CD103 (P < 0.007). In contrast, CD101−/CD103− IEC-activated CD8+ T cells, as well as nonactivated CD8+ T cells, had no significant effect. Gastroenterology 2002 123, 1516-1526DOI: (10.1053/gast.2002.36588) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 10 LP CD3+ CD8+ T cells, in contrast to unfractionated and CD3+ CD8− LP cells, can suppress Ig secretion by PBMCs stimulated with pokeweed mitogen. LP cells, LP CD3+ CD8+ and LP CD3+ CD8− cells (sorted by using a Cytomation Moflo as described earlier), were added to PBMCs stimulated with pokeweed mitogen (1 μg/mL) at a ratio of 1:4 (1 LP cell to 4 PBMCs). Ig secretion, measured by ELISA, was significantly reduced in the presence of CD3+ CD8+ LP T cells (P < 0.03). In contrast, unfractionated and CD3+ CD8− LP cells had no significant effect. Gastroenterology 2002 123, 1516-1526DOI: (10.1053/gast.2002.36588) Copyright © 2002 American Gastroenterological Association Terms and Conditions