Altered chemokine production and accumulation of regulatory T cells in intestinal adenomas of APCMin/+ mice

Abstract

Tumor progression in the colon moves from aberrant crypt foci to adenomatous polyps to invasive carcinomas. The composition of the tumor-infiltrating leukocyte population affects the ability of the immune system to fight the tumor. T cell infiltration into colorectal adenocarcinomas, particularly T helper 1 (Th1) type T cells as well as increased regulatory T cell (Treg) frequencies, is correlated with improved prognosis. However, whether Th1 cells and Tregs are already present at the adenoma stage is not known. In this study, the APC(Min/+) mouse model of intestinal adenomatous polyposis was used to investigate tumor-associated lymphocyte subsets and the mechanisms of their accumulation into gastrointestinal adenomas. Compared to unaffected tissue, adenomas accumulated CD4(+)FoxP3(+) putative Treg in parallel with lower frequencies of conventional T cells and B cells. The accumulation of Treg was also observed in human adenomatous polyps. Despite high Treg numbers, the function of conventional T cells present in the APC(Min/+) adenomas was not different from those in the unaffected tissue. Adenomas displayed an altered chemokine balance, with higher CCL17 and lower CXCL11 and CCL25 expression than in the unaffected tissue. In parallel, CXCR3(+) Tregs were largely absent from adenomas. The data indicate that already in early stages of tumor development, the balance of lymphocyte-recruiting chemokines is altered possibly contributing to the observed shift toward higher frequencies of Treg.

Regulatory T cells control endothelial chemokine production and migration of T cells into intestinal tumors of APCmin/+ mice

“…Lamina propria cells from small intestinal mouse tumors and unaffected intestine were isolated as previously described [11, 30]. Briefly, epithelial cells were removed by HBSS medium containing EDTA, FCS, and HEPES.…”

“…Total RNA was isolated and used for cDNA synthesis as previously described [11]. Each real-time PCR reaction mixture contained cDNA, Power SYBR Green master mix (Applied Biosystems) and oligonucleotide primers to detect CXCL9 (forward: 5′-GAC CTT AAA CAA TTT GCC CCA AG-3′, reverse: 5′-TCC TTC ACC CCC ATC TGC TGA ATC TGG-3′), CXCL10 (forward: 5′-GAA ATT ATT CCT GCA AGC CAA TTT-3′, reverse: 5′-TCA CCC TTC TTT TTC ATG TAG CA-3′), CXCL11 (forward: 5′-GCA GAT ATT GAG AAA GCC TCC A-3′, reverse: 5′-TGG GAT TTA GGC ATC GTT GT-3′) and 18S RNA (forward, 5′-CGG C-GT TAT TCC CAT GAC-3′, reverse, 5′-AAG TTT CAG CTT TGC AAC CA-3′).…”

“…Tumor-infiltrating lymphocytes (TIL), like natural killer (NK) cells, CD8 + cytotoxic T cells and CD4 + T helper (Th) cells have all been found to promote anti-tumor immunity [2, 6]. Previous studies from both our group and others have demonstrated an accumulation of regulatory T cells (Treg) in both human [7–9] and mouse [10, 11] intestinal tumors. Treg can control TIL function [12], but their role in CRC progression is currently unclear.…”

“…These mice develop tumors along the entire intestine and can be used to study early events of CRC since this is a non-invasive, non-metastasising model [26]. However, immunologically the APC min/+ tumors mimic the human counterpart well since they both show accumulation of Treg, a shift in lymphocyte composition and changed chemokine expression compared to unaffected intestine [7, 9, 11, 27]. By breeding APC min/+ mice with depletion of regulatory T cell (DEREG) mice, which harbour a high affinity diphtheria toxin (DT) receptor under the control of the FOXP3 promoter [28], we were able to deplete Treg by DT injections in tumor-bearing mice.…”

Status Quo and Prospect of Colorectal Animal Models: Application of Transgenic Technology

“…Niho et al [16] have used the APC Δ1309 mouse model to confirm that APC gene mutations can inhibit the formation of small intestinal adenoid tumors when in a state of hyperlipidemia. Moreover, Akeus et al [17] have successfully set up an APC Min/+ knockout gene mouse model and identified the relationship between cancer-associated lymphocytes and the accumulation of gastrointestinal adenoma. They draw a preliminary conclusion that mutations and the deletion of APC Min/+ in Wnt signaling pathways may affect the development of lymphatic system tumors, the number of T cells and the accumulation of adenoma.…”