In vivo Expansion of Naïve CD4+CD25high FOXP3+ Regulatory T Cells in Patients with Colorectal Carcinoma after IL-2 Administration

Regulatory T cells (Treg cells) are increased in context of malignancies and their expansion can be correlated with higher disease burden and decreased survival. Initially, interleukin 2 (IL-2) has been used as T-cell growth factor in clinical vaccination trials. In murine models, however, a role of IL-2 in development, differentiation, homeostasis, and function of Treg cells was established. In IL-2 treated cancer patients a further Treg-cell expansion was described, yet, the mechanism of expansion is still elusive. Here we report that functional Treg cells of a naïve phenotype - as determined by CCR7 and CD45RA expression - are significantly expanded in colorectal cancer patients. Treatment of 15 UICC stage IV colorectal cancer patients with IL-2 in a phase I/II peptide vaccination trial further enlarges the already increased naïve Treg-cell pool. Higher frequencies of T-cell receptor excision circles in naïve Treg cells indicate IL-2 dependent thymic generation of naïve Treg cells as a mechanism leading to increased frequencies of Treg cells post IL-2 treatment in cancer patients. This finding could be confirmed in naïve murine Treg cells after IL-2 administration. These results point to a more complex regulation of Treg cells in context of IL-2 administration. Future strategies therefore might aim at combining IL-2 therapy with novel strategies to circumvent expansion and differentiation of naïve Treg cells

T helper type 1 memory cells disseminate postoperative ileus over the entire intestinal tract

Localized abdominal surgery can lead to disruption of motility in the entire gastrointestinal tract (postoperative ileus). Intestinal macrophages produce mediators that paralyze myocytes, but it is unclear how the macrophages are activated, especially those in unmanipulated intestinal areas. Here we show that intestinal surgery activates intestinal CD103(+)CD11b(+) dendritic cells (DCs) to produce interleukin-12 (IL-12). This promotes interferon-γ (IFN-γ) secretion by CCR9(+) memory T helper type 1 (T(H)1) cells which activates the macrophages. IL-12 also caused some T(H)1 cells to migrate from surgically manipulated sites through the bloodstream to unmanipulated intestinal areas where they induced ileus. Preventing T cell migration with the drug FTY720 or inhibition of IL-12, T-bet (T(H)1-specific T box transcription factor) or IFN-γ prevented postoperative ileus. CCR9(+) T(H)1 memory cells were detected in the venous blood of subjects 1 h after abdominal surgery. These findings indicate that postoperative ileus is a T(H)1 immune-mediated disease and identify potential targets for disease monitoring and therap

T helper type 1 memory cells disseminate postoperative ileus over the entire intestinal tract

Localized abdominal surgery can lead to disruption of motility in the entire gastrointestinal tract (postoperative ileus). Intestinal macrophages produce mediators that paralyze myocytes, but it is unclear how the macrophages are activated, especially those in unmanipulated intestinal areas. Here we show that intestinal surgery activates intestinal CD103(+)CD11b(+) dendritic cells (DCs) to produce interleukin-12 (IL-12). This promotes interferon-γ (IFN-γ) secretion by CCR9(+) memory T helper type 1 (T(H)1) cells which activates the macrophages. IL-12 also caused some T(H)1 cells to migrate from surgically manipulated sites through the bloodstream to unmanipulated intestinal areas where they induced ileus. Preventing T cell migration with the drug FTY720 or inhibition of IL-12, T-bet (T(H)1-specific T box transcription factor) or IFN-γ prevented postoperative ileus. CCR9(+) T(H)1 memory cells were detected in the venous blood of subjects 1 h after abdominal surgery. These findings indicate that postoperative ileus is a T(H)1 immune-mediated disease and identify potential targets for disease monitoring and therap

Functional analysis of CD4⁺CD25^highFOXP⁺ T_reg cells.

<p>(<b>A</b>) CD4⁺ cells were separated by flow cytometric cell sorting into conventional CD4⁺CD25⁻ and regulatory CD4⁺CD25^high T cells as defined by their expression of CD25. (<b>B</b>) Re-analysis of FOXP3 expression in CD4⁺CD25⁻ T_conv (left, grey fill) and CD4⁺CD25^high T_reg cells (right, grey fill) post cell sorting. Isotype control (black line). (<b>C</b>) Reduction of proliferation of CD4⁺CD25⁻ T_conv cells stimulated with beads coated with CD3 and CD28 mAbs (black bar) by highly purified CD4⁺CD25^highFOXP3⁺ T_reg cells from healthy donors (white bar) or colorectal cancer patients before (dark grey bar) and after therapy (light grey bar).</p

Replicative history of CD4⁺CD25^highFOXP3⁺ T_reg cell populations defined by the expression of CD45RA and CCR7.

<p>CD4⁺CD25^high T_reg cells were isolated by flow cytometric cell sorting according to their expression of CD25 as well as CD45RA and CCR7 in three T_reg-cell subsets, namely T_naïve (CD45RA⁺CCR7⁺), T_CM (CD45RA⁻CCR7⁺), and T_EM cells (CD45RA⁻CCR7⁻). (<b>A</b>) Strategy of flow cytometric analysis of CD4 and CD25 expression on the surface of CD4⁺ T cells as exemplified for a colorectal cancer patient. (<b>B</b>) Re-analysis of FOXP3 and CD25 expression (left) as well as CCR7 and CD45RA expression (right) in CD4⁺CD25^high T_reg cells. (<b>C</b>) Naïve, central and effector memory CD4⁺CD25^high T_reg cells from healthy donors and colorectal cancer patients before and after chemoimmunotherapy were assessed for TREC (T-cell receptor excision circle) content. Genomic DNA of sorted subsets was isolated, and the number of TREC was determined by quantitative real-time PCR. Data are shown as the mean values obtained for 2 independent healthy donors and 2 colorectal cancer patients. Error bars represent SD.</p

patient characteristics.

<p>patient characteristics.</p

Increase of naïve CD4⁺CD25^highFOXP3⁺ T_reg cells in colorectal cancer patients after chemoimmunotherapy.

<p>(<b>A</b>) Strategy of flow cytometric analysis of CCR7 and CD45RA expression on the surface of CD4⁺CD25^highFOXP3⁺ T_reg cells as exemplified for a representative healthy donor (left) and a representative colorectal cancer patient (right). Frequencies of (<b>B</b>) CCR7⁺CD45RA⁺ naïve T_reg cells (T_naive), (<b>C</b>) CCR7⁺CD45RA⁻ central memory T_reg cells (T_CM), and (<b>D</b>) CCR7⁻CD45RA⁻ effector memory T_reg cells (T_EM) were assessed in peripheral blood of colorectal cancer patients (n = 15) before (light grey bars) and after therapy (dark grey bars) as well as healthy individuals (white bars, n = 22). Significant differences (p<0.05, Student's <i>t</i> test) between healthy donors and colorectal cancer patients before and after chemoimmunotherapy are marked by an asterisk (*). Error bars represent SD. (<b>E</b>) Assessment of regulatory function of naïve and memory CD4⁺CD25^high T_reg cells sorted according to their CD45RA expression from colorectal cancer patients. Reduction of proliferation of CD4⁺CD25⁻ T_conv cells stimulated with beads coated with CD3 and CD28 mAbs by highly purified naïve and memory CD4⁺CD25^high T_reg cells from colorectal cancer patients before and after therapy.</p

Frequency of CD4⁺CD25^highFOXP3⁺ T_reg cells.

<p>(<b>A</b>) Flow cytometric analysis of CD25 and FOXP3 on peripheral blood derived CD4⁺ T cells from a representative healthy individual (left panel) and a representative colorectal cancer patient before treatment (right panel). Numbers represent percentage of events within the gate. (<b>B</b>) Frequency of CD4⁺CD25^highFOXP3⁺ T_reg cells in 22 healthy donors and 15 colorectal cancer patients (CRC) before treatment. Each dot represents a single individual assessed in the respective group; mean expression (line) of all samples in each group is also shown (*, p<0.05, Student's <i>t</i> test). (<b>C</b>) CTLA4 (top) and GITR expression (bottom) in CD4⁺CD25^highFOXP3⁺ T_reg cells of healthy donors (left, grey fill) and colorectal cancer patients (right, grey fill). Isotype control (black line). (<b>D</b>) Frequency of CTLA4 (left) and GITR (right) expressing CD4⁺CD25^highFOXP3⁺ T_reg cells in healthy donors (white) and colorectal cancer patients (grey, CRC) before treatment. Shown here are median, 25^th and 75^th percentile (box), 10^th and 90^th percentile (whiskers) and outliers (dots), (*, p<0.05, Student's <i>t</i> test).</p

IL-2 administration leads to an expansion of “naïve” CD4⁺CD25^highFOXP3⁺ T_reg cells in C57BL/6 mice.

<p>(<b>A</b>) Flow cytometric analysis of CD4 and FOXP3 expression in CD4⁺ T cells from untreated as well as IL-2-treated animals in spleen, peripheral and mesenteric lymph nodes, peripheral blood, thymus, and liver. Significant differences (p<0.05, Student's <i>t</i> test) between untreated and IL-2 treated animals are marked by an asterisk (*). (<b>B</b>) Analysis of “naïve” CD45RB^high CD4⁺CD25^highFOXP3⁺ T_reg cells in spleen, peripheral and mesenteric lymph nodes, peripheral blood, thymus, and liver. Significant differences (p<0.05, Student's <i>t</i> test) between untreated and IL-2 treated animals are marked by an asterisk (*). Similar results were obtained in two independent experiments. (<b>C</b>) CD45RB⁺CD44^lowCD62L⁺ naïve CD4⁺CD25⁻ T_conv and CD4⁺CD25^high T_reg cells were isolated by flow cytometric cell sorting and assessed for TREC content. Genomic DNA of sorted subsets was isolated, and the number of TREC was determined by quantitative real-time PCR (n = 3, p<0.05, Student's <i>t</i> test). Error bars represent SD. Similar results were obtained in three independent experiments.</p