CD74 is highly expressed in human gastric and colon tumors and on isolated epithelial cells.

<p>CD74 mRNA levels measured by qRT PCR are increased in A) human gastric tumor samples and at higher levels in tissues from patients with nodal involvement, B) human colon tumor samples and at higher levels in tissues from patients with nodal involvement, and on epithelial cells isolated from human gastric and colon tumors in C) a representative flow cytometry plot and D) in compiled flow cytometry data from all samples. N = 8 for D and the mean ± standard error are shown as the results of duplicated in multiple experiments. *<i>p</i><0.05.</p

MIF induces proliferation of gastric and colon carcinoma cells.

<p>Tumor-derived gastric and colon supernatants induced proliferation of N87 and Caco-2 cells, which was decreased upon adding A) anti-MIF neutralizing antibodies or anti-CD74 blocking antibodies. B) Chronic exposure of HS738 or N87 cells with recombinant MIF increased proliferation that was sustained after returning cells to regular media for 8 weeks. N = 8 and the mean ± standard error are shown as the results of duplicated in multiple experiments. *<i>p</i><0.05.</p

MIF is highly expressed in gastric and colon tumors and human tissue-derived fibroblasts.

<p>MIF mRNA levels measured by qRT PCR are increased in A) human gastric tumor samples and at higher levels in tissues from patients with nodal involvement, B) human colon tumor samples and at higher levels in tissues from patients with nodal involvement, and C) in the supernatants tumor derived gastric and colon fibroblasts compared to matched normal as measured by Luminex singleplex assay. N = 8 for C and the mean ± standard error are shown as the results of duplicated in multiple experiments. *<i>p</i><0.05.</p

MIF induces pro-tumorigenic signaling.

<p>Chronic MIF treatment induces A) Akt, B) c-Jun, and C) Erk1/2 phosphorylation. N = 8 and the mean ± standard error are shown as the results of duplicated in multiple experiments. *<i>p</i><0.05.</p

Chronic MIF exposure induces HS738 cell transformation.

<p>HS738 chronic exposure to MIF induces A) upregulation of TERT gene expression so similar levels as N87 control cells and B) colony formation in a focus forming assay. N = 8 and the mean ± standard error are shown as the results of duplicated in multiple experiments. *<i>p</i><0.05.</p

Chronic MIF exposure induces mesenchymal epithelial transition.

<p>HS738 cells have A) a fibroblast morphology, which changes B) upon chronic exposure to recombinant MIF. MIF exposure also decreases expression of fibroblast markers by C) mRNA levels by qRT PCR compared to untreated cells and by D) flow cytometry, while increasing expression of epithelial markers EpCam and E-cadherin by E) mRNA levels by qRT PCR compared to untreated cells and by D) flow cytometry. N = 8 and the mean ± standard error are shown as the results of duplicated in multiple experiments. *<i>p</i><0.05.</p

Th17 development increases in culture with <i>H. pylori-</i>exposed and cancer gastric myofibrobroblasts.

<p><b>A</b>) N-GMF and Hp-GMF primed CD4⁺ T cells were analyzed for RORγt expression by flow cytometry. <b>B</b>) N-GMF and C-GMF primed CD4⁺ T cells were analyzed for RORγt expression by flow cytometry. <b>C</b>) Compiled data for all experiments of N-GMF, Hp-GMF, and C-GMF primed CD4⁺ T cells analyzed for RORγt expression by flow cytometry. <b>D</b>) IL-17A levels were measured in co-cultures of CD4⁺ T cells with GMF by Luminex bead array. The RNA levels from GMF primed CD4⁺ T cells were also analyzed by quantitative real time PCR for <b>E</b>) RORγ and IL-17A. The mRNA levels were normalized to 18S. Data represent mean of mRNA fold increase ± standard errors from three duplicate experiments from 3 sets of GMF (n = 18). <b>*</b><i>p</i><0.05, between CD4⁺ and CD4⁺ incubated with GMF, <b>**</b><i>p</i><0.05 between untreated and <i>H. pylori</i> treated or cancer.</p

IL-6 and TGF-β produced in tumors and by GMF contribute to Th17 development.

<p>The level of mRNA measured by quantitative real time RT-PCR in <i>H. pylori</i> infected and cancer tissues compared to normal tissues for <b>A</b>) IL-6, <b>B</b>) TGF-β, and <b>C</b>) IL-1β. Data represent mean of mRNA fold increase ± standard errors from triplicates compared to the levels in matched normal samples. Supernatants from GMF cultures were analyzed for <b>D</b>) IL-6 and <b>E</b>) TGF-β by Luminex bead array. <b>F</b>) RORγt expression by GMF primed CD4⁺ T cells was analyzed by flow cytometry. <b>G</b>) IL-17A production was measured in supernatants by Lunimex bead array. <b>H</b>) RORγ and IL-17A mRNA levels in cancer and <i>H. pylori-</i>exposed GMF primed CD4⁺ T cells was examined by quantitative real time RT-PCR, normalized to 18S, and compared to normal GMF. Data represent mean of mRNA fold increase ± standard errors from three duplicate experiments in triplicate from 3 sets of GMF (n = 18). <b>*</b><i>p</i><0.05 between normal and <i>H. pylori</i> treated or cancer, <b>**</b><i>p</i><0.05 <i>H. pylori</i> treated or cancer and TGF-β and IL-6 neutralization.</p

RORγt expressing cells proliferate in culture with GMF in a class II MHC dependent manner.

<p>CD4⁺ T cells labeled with CFSE and gated on RORγt⁺ cells for representative histograms for <b>A</b>) non-proliferating control, <b>B</b>) CD4⁺ T cells in culture with normal GMF <b>C</b>) CD4⁺ T cells in culture with <i>H. pylori-</i>exposed GMF, <b>D</b>) CD4⁺ T cells in culture with <i>H. pylori-</i>exposed GMF pre-incubated with class II MHC blocking antibodies <b>E</b>) CD4⁺ T cells in culture with cancer GMF <b>F</b>) CD4⁺ T cells in culture with cancer GMF pre-incubated with class II MHC blocking antibodies. <b>G</b>) Compiled data from 3 experiments in triplicate show the percent proliferating cells from CD4⁺ T cells in culture with normal GMF, <i>H. pylori-</i>exposed GMF, and cancer GMF with class II MHC blocking. (n = 9). <b>*</b><i>p</i><0.05 <i>H. pylori</i> treated or cancer and class II MHC blockade.</p

Gastric myofibroblast express CD90, α-smooth muscle actin, and class II MHC.

<p>Gastric myofibroblast express <b>A</b>) CD90 by flow cytometry compared to the solid peak isotype control. These cells also express <b>B</b>) α-smooth actin and class II MHC, where class II MHC is upregulated by exposure to <i>H. pylori</i> as shown by confocal microscopy. Class II MHC is also upregulated by <b>C</b>) incubation with CD4⁺ T cells and <b>D</b>) <i>H. pylori</i> exposure by flow cytometry. Figures are representative of 3 experiments in duplicate.</p