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. 2014 Jan 13;211(1):89–104. doi: 10.1084/jem.20130301

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© 2014 Ramesh et al.

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Figure 4. — MDR1⁺ Th17.1 cells are enriched and activated in clinically inflamed tissue. (A) Mononuclear cells were isolated from CD patient peripheral blood (PBMC; left), uninvolved gut (middle), or involved gut (right), and were analyzed for expression of CD45RO and CCR7. Data shown are on CD3⁺CD4⁺CD25⁻ gated T cells and represent 5 experiments on cells from different patients. (B) Mononuclear cells from CD patient PBMC, uninvolved gut, or involved gut tissue (as in A) were loaded with Rh123, stained with antibodies against CD3, CD4, CD25, CD45RO, c-Kit, and CD161 after a 1-h Rh123 efflux period at 37°C, and analyzed by FACS. Data shown are on CD3⁺CD4⁺CD25⁻CD45RO⁺ gated memory T cells. Rh123 efflux versus c-Kit (top) or CD161 (bottom) expression is shown. Data represent 3 (CD161 staining) or 4 (c-Kit staining) experiments on cells isolated from different patients. (C) Percentages of total memory (CD45RO⁺) cells (top left), MDR1⁺/Rh123^lo memory cells (top right), c-Kit⁺MDR1⁺/Rh123^lo memory T cells (bottom left), or CD161⁺MDR1⁺/Rh123^lo memory cells (bottom right) were determined in CD patient PBMC, uninvolved gut, or involved gut tissue by FACS analysis as in B. Data are shown as mean percentages ± SD from 3–5 individual patients. *, P < 0.05; **, P < 0.01 by paired Student’s t test. (D) MDR1⁺ (Rh123^lo) or MDR1⁻ (Rh123^hi) CD3⁺CD4⁺CD25⁻CD45RO⁺ memory T cells were FACS-sorted from the PBMC of one HC donor or from mononuclear cells isolated from the involved gut of one CD patient. Sorted cells were lysed directly ex vivo, and RNA was isolated for microarray analysis. Relative (fold change) expression of ABCB1 (MDR1), KIT (c-Kit), KLRB1 (CD161), or IL17A is shown for the T cell subsets as indicated. (E) Relative ex vivo expression (Log₂ fold change) of pathogenic mouse Th17-signature genes (red; Lee et al., 2012), nonpathogenic Th17-signature genes (blue; Lee et al., 2012), or other notable (gray) genes was determined by microarray analysis of MDR1⁺ or MDR1⁻ memory T cells sorted from involved CD patient gut tissue as in D. (F and G) Mononuclear cells isolated from involved CD patient gut tissue were FACS-sorted into MDR1⁺ or MDR1⁻ CD3⁺CD4⁺CD25⁻CD45RO⁺ memory T cells, and expression of pathogenic (F) or nonpathogenic (G) mouse Th17-signature genes (Lee et al., 2012) was analyzed by nanostring. Data are shown as individual (color coded) and mean expression values (AU – arbitrary units) from 2 independent patients. Horizontal bars represent the mean values.