1. Volume 125, Issue 2, Pages 345-356 (August 2003)
Human small-intestinal epithelium contains functional natural killer lymphocytes 
Francisco León, Ernesto Roldán, Laura Sanchez, Cristina Camarero, Alfredo Bootello, Garbiñe Roy 
Gastroenterology 
Volume 125, Issue 2, Pages (August 2003)
DOI: /S (03)

2. Figure 1
Markers associated with ontogeny and immaturity in the IEL compartment. IEL were first identified and gated in region R on the basis of their low side-scatter (SSC) and CD45 expression (A ). Shown are representative dot plots of CD3ϵ intracytoplasmic staining (icCD3ϵ fluorescein isothiocyanate [FITC]) on surface CD3+ and CD3− IEL subsets (smCD3 PerCP) (B). Second, smCD3− were gated in and analyzed for the expression of NK markers (C, D, and E ) and CD8α (F ). Panels G–J are representative of the expression of precursor-cell markers on CD3+ and CD3− IEL subsets, and panels K and L show the expression of CD8α and CD8β on these subsets, evidencing the predominant expression of the CD8αα homodimer in the NK-like subset.
Gastroenterology  , DOI: ( /S (03) )

3. Figure 2
Expression of co-stimulatory markers on CD3+ and CD3− IEL subsets. CD4+ IEL expressed CD28 (B), unlike CD3− IEL (A ). (C ) Expression of CD86 on a small fraction of both CD3+ and CD3− IEL. By contrast, CD80 was not found on IEL (D). (F ) Intracellular detection of CTLA-4 (icCTLA4), expressed in only a small fraction of CD3+ IEL (intracellular isotype matched IgG control in panel E; icIgG-PE). Analyzed cells were selected on gate R, as in Figure 1.
Gastroenterology  , DOI: ( /S (03) )

4. Figure 3
Cytolytic capacity of freshly isolated CD3+ IEL; (A ) granularity and size (forward scatter [FSC]/side scatter [SSC]) of cocultured CD3+ IEL and K562 cells in a 1:1 ratio. (B) Fluorescence of DiO-labeled K562 vs. the PI staining of the IEL (DiO−) and K562 coculture in an ungated acquisition (upper right quadrant, DiO+PI+ compromised K562 cells; lower right quadrant, DiO+PI− intact K562 cells). (C ) FL2/PI histograms analyzed on gated DiO+ K562 cells. The results of this representative experiment are shown as percentage of lysis at 0:1, 1:1, and 5:1 effector-target cell ratios. FL, fluorescence.
Gastroenterology  , DOI: ( /S (03) )

5. Figure 4
Cytotoxic capacity of CD3+ and CD3− IEL long-term cultured with IL-2. (A ) Granularity and CD3 expression of purified IEL cultured with IL-2 before magnetic separation of the CD3+ and CD3− fractions. (B) Morphology of cocultured K562 (target) and IEL (effector) in a 1:1 ratio. (C ) Autofluorescence in the FL1 channel (FL1-AF) of mixed effector and target cells, showing how the difference between these populations allows for their discrimination during FCM analysis. The dot plots on the right represent the analysis of PI incorporation into K562 cells, previously selected on the basis of their light scatter parameters (region R in panel B). The effector cells were peripheral blood (PB) NK cells (D, E ), ileal expanded CD3− IEL (F, G), or CD3+ IEL (H, I ). The upper right quadrants show the percentage of compromised K562 PI+ at the effector-target ratios of 2:1 and 1:1 in this representative experiment. SSC, side scatter; FSC, forward scatter. FL, fluorescence.
Gastroenterology  , DOI: ( /S (03) )

6. Figure 5
Flow cytometric analysis of some potential cytolytic mechanisms of IEL. (A ) Representative analysis of intracellular perforin content of freshly isolated IEL from a duodenal biopsy, with the corresponding isotype-matched IgG-PE control (B). Intracellular perforin content on CD3− (C ) and CD3+ (D) IEL subsets after in vitro IL-2 expansion for 2–3 weeks. Representative dot plots of CD95L (FasL) (E ), CD95 (Fas) (F ), CD120a (G), and CD120b (H ) expression on CD3+ and CD3− IEL subsets. The histograms on the right suggest the specificity of the fluorometric CD95L-PE signal by its partial blockage with recombinant human CD95L (rh-CD95L) protein. PE, phycoerythrin.
Gastroenterology  , DOI: ( /S (03) )

7. Figure 6
Cytokine production by freshly isolated IEL. IEL isolated from duodenal biopsies were cultured for 12 hours with or without PMA and ionomycin in the presence of monensin, surface-labeled for different antigens (CD3 and CD45), and fixed and permeabilized before intracellular cytokine staining. Representative dot plots are shown.
Gastroenterology  , DOI: ( /S (03) )

8. Figure 7
Cytokine production by IL-2-expanded IEL. IEL were obtained by spontaneous detachment from small-intestinal mucosa and cultured with IL-2 (10 ng/mL) for 2–3 weeks. Before cytokine detection, IEL cultures were stimulated with PMA and ionomycin for 5 hours in the presence of monensin. Representative dot plots show IFN-γ and IL-2 production with (B, D) or without (A, C ) PMA with Io stimulation. IO, ionomycin.
Gastroenterology  , DOI: ( /S (03) )