FoxP3⁺ cells lose CCR7 expression upon antigen priming, and only antigen primed CCR7^low cells can efficiently migrate into tumors.

<p>(A) Loss of CCR7 on FoxP3⁺ T cells following antigen priming. OVA-specific FoxP3⁺ T cells isolated from RIP-mOVA×DO11.10 Rag2 (−/−)mice were cultured in the presence of the OVA_323–339 peptide and irradiated splenocytes as antigen presenting cells for 7 days, and CCR7 expression by FoxP3⁺ T cells was examined. (B) Comparison of the homing ability of antigen primed versus naïve FoxP3⁺ T cells. Fresh and antigen-primed FoxP3⁺ T cells were co-injected i.v. into 4T1-OVA tumor-bearing mice and the relative migration of the two FoxP3⁺ T cell populations into tumors and other organs were determined 36 h following the injection. (C) Comparison of antigen primed wild type and CCR7 (−/−) FoxP3⁺ T cells into tumors. CD4⁺ T cells, isolated from wild type and CCR7−/− mice were antigen primed in vitro for 5 days and injected into B16-tumor bearing mice. The relative migration of the two FoxP3⁺ T cell populations into tumors and other organs were determined 36 h following the injection. Representative and combined data obtained from 4–5 different experiments are shown. “*” indicates significant differences between the tumor and indicated organs (B) or between WT and CCR7 (−/−) FoxP3⁺ T cells (C).</p

Naïve FoxP3⁺ T cells that cannot migrate first to lymphoid tissues fail to populate tumors.

<p>(A) Population of wild type versus CCR7 (−/−) FoxP3⁺ T cells in tumors. Wild type and CCR7 (−/−) CD4⁺ T cells, which were freshly isolated from lymphoid tissues, were separately transferred into scurfy mice on day 2 following birth. B16 tumor cells were implanted into the scurfy mice at 5 weeks of age, and the mice were sacrificed 2 weeks later. (B) Competitive population of wild type and CCR7 (−/−) FoxP3⁺ T cells in tumors. Wild type CD45.1⁺ CD4⁺ T cells and CCR7 (−/−) CD45.2⁺ CD4⁺ T cells were co-transferred into FoxP3-deficient scurfy mice on day 2 following birth. B16 tumor cells were implanted into the scurfy mice at 5 weeks of age, and the mice were sacrificed 2 weeks later. (C) Expression of CCR7 and CD62L by wild type FoxP3⁺ T cells populating tumors or lymph nodes of scurfy mice. Representative and combined data obtained from 3–4 different experiments are shown. “*” indicates significant differences between wild type and CCR7 (−/−) FoxP3⁺ T cells.</p

Tumor-infiltrating FoxP3⁺ T cells express the Helios antigen.

<p>Mice harboring one of the four types of tumors (A20 B cell lymphoma, CT26 colon cancer, 4T1 breast cancer, and B16 melanoma) were examined for expression of Helios by FoxP3⁺ T cells. (A) Helios expression by FoxP3⁺ T cells in tumors and various secondary lymphoid tissues such as the draining lymph node (D-LN), other peripheral lymph nodes (PLN), and mesenteric lymph node (MLN). (B) Frequencies of Helios-negative FoxP3⁺ T cells in tumors and secondary lymphoid tissues. (C) Detection of Helios⁺ FoxP3⁺ T cells in tumors by confocal microscopy. Arrows indicate T cells that co-express FoxP3 and Helios in tumors. Representative and combined (n = 4) data are shown. “*” indicates significant differences from tumors.</p

Tumor-infiltrating Helios⁺ FoxP3⁺ T cells are down-regulated for CD62L and CCR7.

<p>(A) Expression of CCR7 and CD62L by Helios⁺ FoxP3⁺ T cells in indicated organs and tumors (A20). (B) Expression of CCR7 and CD62L by Helios⁺ FoxP3⁺ T cells in indicated tumors and organs. Representative and combined data obtained from 4–6 different experiments are shown. “*” indicates significant differences from the tumor.</p

Induction of FoxP3⁺ regulatory T cells in tumors is inefficient, and most tumor-infiltrating FoxP3⁺ T cells have the memory phenotype.

<p>(A) Splenocytes of DO11.10 Rag2−/− mice (containing naïve FoxP3⁻ CD4⁺ T cells but not FoxP3⁺ T cells) were injected i.v. into BALB/c mice, and A20 tumor cells expressing OVA were implanted 15 hours later. Mice were sacrificed 2 weeks later, and the conversion rate of naïve FoxP3⁻ CD4⁺ T cells into FoxP3⁺ T cells (represented by % FoxP3⁺ of KJ-1.26⁺ CD4⁺ T cells) was determined in various organs and tumors. (B) Memory FoxP3⁺ T cells are enriched in tumors. Expression of CD44 and CD62L by FoxP3⁺ T cells in tumors and peripheral lymph nodes were compared. Representative and combined data obtained from 3 (A) or 5–8 different experiments (B) are shown. “*” indicates significant differences between the tumor and indicated organs (A) or PLN (B).</p

Most tumor-infiltrating FoxP3⁺ cells retain FoxP3 expression in vivo.

<p>FoxP3-GFP-Cre×Rosa-tdTomato mice were implanted with B16 cells to form tumors. Expression of GFP on tdTomato⁺ cells was examined by flow cytometry. GFP⁺ tdTomato⁺ T cells are current FoxP3⁺ T cells, whereas GFP⁻ tdTomato⁺ T cells are ex-FoxP3⁺ T cells. Representative (A) and combined (B) data obtained from 4 different experiments are shown.</p

Memory type FoxP3⁺ regulatory T cells are more efficient than naïve type FoxP3⁺ T cells in migration into tumors.

<p>Pooled mononuclear cells including FoxP3⁺ T cells prepared from spleen, MLN and PLN were injected into BALB/c mice bearing A20 tumors, and the mice were sacrificed 36 hours later. (A) Detection of the migration of FoxP3⁺ cells into tumors. (B) Frequencies of naïve versus memory FoxP3⁺ T cells that migrated into various organs and tumors. Naïve FoxP3⁺ T cells are defined as CD62L⁺ (CD44⁻) cells, and memory FoxP3⁺ T cells are defined as CD62L⁻ (CD44^+/−) cells. Representative and combined data obtained from 4 different experiments are shown. “*” indicates significant differences between frequencies of naïve and memory FoxP3⁺ T cells.</p

Expression of memory T cell trafficking receptors by tumor infiltrating FoxP3⁺ T cells.

<p>(A) Expression of CCR7, CCR8 and CXCR4 by tumor versus PLN-residing FoxP3⁺ T cells. (B) Chemotaxis of tumor versus PLN-residing FoxP3⁺ T cells to CCL19 and CCL21 (CCR7 ligands), CCL1 (CCR8), and CXCL12 (CXCR4). Cells from A20 tumor-bearing mice were examined. Representative (A) or combined (B) data obtained from 4–7 different experiments are shown.</p

Succinylated Chitosan Derivative Has Local Protective Effects on Intestinal Inflammation

We have previously reported on the anti-inflammatory effects of a water-soluble chitosan derivative, zwitterionic chitosan (ZWC). In the present study, we hypothesized that orally administered ZWC would provide local anti-inflammatory effects in the intestinal lumen. ZWC indeed showed anti-inflammatory effects in various in vitro models including peritoneal macrophages, engineered THP1 monocytes, and Caco-2 cells. In Caco-2 cells, ZWC applied before the lipopolysaccharide (LPS) challenge was more effective than when it was applied after it in preventing LPS-induced cell damage. When administered to mice via drinking water as a prophylactic measure, ZWC protected the animals from 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis, helping them to recover the body weight, restore the gross and histological appearance of the colon, and generate FoxP3⁺ T cells. In contrast, orally administered ZWC did not protect the animals from LPS-induced systemic inflammation. These results indicate that orally administered ZWC reaches the colon with minimal absorption through the upper gastrointestinal tract and provides a local anti-inflammatory effect

Anti-4-1BB pre-activated PDCA-1⁺ B cells inhibit in vivo humoral responses.

<p><b>A</b> Cartoon showing the steps involved in purification of PDCA-1⁺ B cells, activation with IL-7 or anti-4-1BB, adoptive transfer, and serum analysis. <b>B</b>, <b>C</b>, PDCA-1⁺ B cells were purified from spleens of naïve B6 mice, cultured for 3d with IL-7 (100 ng/ml) or soluble anti-4-1BB (5 µg/ml), and pulsed with 0.5 mg/ml NP⁴⁰-AECM Ficoll overnight. Ag-pulsed cells (2×10⁶) were injected (i.v.) into syngeneic hosts. Sera collected on d7 were analyzed for the presence of anti-NP IgM <b>B</b>, and IgG3 <b>C</b> Abs by ELISA. The extent of anti-NP Ab production (absorbance) is shown by the bar graphs (mean ± SD). The results of four pooled independent experiments are shown (<i>n</i> = 4). (D–I) PDCA-1⁺ B cells were purified from the spleens of naïve B6 mice, cultured for 3d with IL-7 (100 ng/ml) or soluble anti-4-1BB (5 µg/ml), pulsed overnight with 0.5 mg/ml NP²³-CGG and adoptively transferred (2×10⁶; i.v.) into syngeneic hosts. Sera collected on d7 were analyzed for the presence of anti-NP IgM <b>D</b>, IgG1 <b>E</b>, IgG2a <b>F</b>, IgG2b <b>G</b>, IgG3 <b>H</b>, and IgA <b>I</b> Abs by ELISA. The extent of anti-NP Ab production (absorbance) is shown by the bar graphs (mean ± SD). The results of four pooled independent experiments are shown (<i>n</i> = 4). * <i>p</i><0.05.</p