Partial Depletion of Regulatory T Cells Does Not Influence the Inflammation Caused by High Dose Hemi-Body Irradiation

<div><p>There is clinical interest in the modulation of regulatory T cells for cancer therapy. The safety of these therapies in combination with conventional anti-cancer therapies, including radiation therapy, can be studied in animal models. The effects of partial depletion of regulatory T (Treg) cells with an anti-CD25 antibody in conjunction with ionizing radiation on inflammation and tissue injury were analyzed in C57BL/6 mice. An anti-CD25 antibody (PC61) was administered 3 days prior to 13 Gy lower-half hemi-body irradiation (HBI). The blood, spleen, mesenteric lymph nodes (mLNs) and inguinal lymph nodes (iLNs) were harvested at various times thereafter. Alterations in the proportion of leukocyte subsets including CD4⁺ T cells, CD8⁺ T cells, Treg cells, B cells, NK cells, NK1.1⁺ T cells, macrophages and granulocytes were analyzed by FACS. The lungs, liver, pancreas, stomach, jejunum, duodenum, ileum, colon and kidney were harvested and studied by H&E staining. Expression of inflammatory mediators in plasma and tissue were investigated by ELISA. HBI significantly decreased the leukocyte pool though the various leukocyte subsets had different sensitivities to HBI. The administration of PC61 significantly decreased the proportion of Treg cells in spleen, iLN, mLN and blood (reduction of approximately 60%). Irradiation significantly increased the proportion of Treg cells in the spleen, iLN and mLN. HBI induced a systemic inflammatory reaction as demonstrated by increased plasma levels of IL-6, KC/CXCL1 and circulating granulocytes in the blood. Neutrophils also infiltrated the small bowel. The same general patterns were observed whether or not Treg cells were partially depleted with PC61 prior to HBI. These data demonstrate that partial depletion of Treg cells in these mice does not influence HBI-induced inflammatory response and tissue injury, and that combining anti-CD25 therapy with radiation may be safe and well tolerated in a clinical setting.</p> </div

Inflammatory mediators in plasma.

<p>Plasma levels of (A) IL-6 and (B) KC/CXCL1 were determined at different time points after treatment. Data are shown as Mean±SD (n = 4). Two way ANOVA showed significant interactions over time between treatments (all p<0.01). No differences were detected between PC61+irradiation compared to Rat IgG+irradiation treatment group profiles over time in the levels of IL-6 (p≥0.099) and KC/CXCL1 (p≥0.475). No significant differences were found between the PC61+sham irradiation compared to Rat IgG+sham irradiation treatment group profiles over time (all p>0.05). Differences between all other pair-wise comparisons of treatment group profiles over time were significant (p<0.05). All post hoc pairwise comparisons were performed with Tukey's multiple comparisons. This is one representative experiment of three performed.</p

Leukocyte subsets in the spleen and blood.

<p>In spleen, irradiation reduced proportions of B cells, CD4⁺ T cells and CD8⁺ T cells; increased proportions of NK1.1⁺ T cells and granulocytes. In blood, B cells were most radiosensitive; NK cells were most resistant; granulocytes increased on days 7 and 14. Data are expressed as Mean±SD (n = 4). Two way ANOVA showed significant interactions over time between treatments in all cell subsets (p<0.01) except NK1.1⁺ T cells in blood (p = 0.288). No significant differences were detected between PC61+irradiation compared to Rat IgG+irradiation treatment group profiles over time for any of the subsets in the spleen (p≥0.322) and blood (p≥0.095). No significant differences were found between the PC61+sham irradiation compared to Rat IgG+sham irradiation treatment group profiles over time (all p>0.05). Differences between all other pair-wise comparisons of treatment group profiles over time were significant (p<0.05). All post hoc pairwise comparisons were performed with Tukey's multiple comparisons. This is one representative experiment of three performed.</p

The proportion of CD4⁺FoxP3⁺ cells in the spleens, LNs and peripheral blood.

<p>(A) The proportion of CD4⁺FoxP3⁺ Treg cells and (B) the proportion of CD4⁺CD25⁺ cells. PC61 administration reduced the proportion of CD4⁺FoxP3⁺ Treg cells and CD4⁺CD25⁺ cells in spleen, iLN, mLN and blood, irradiation increased their proportion in spleen, iLN, and mLN but not in blood. Data are shown as Mean±SD (n = 4). Two way ANOVA showed significant interactions over time between treatments in the proportion of CD4⁺FoxP3⁺ Treg cells and in the proportion of CD4⁺CD25⁺ cells in all compartments (all p<0.01). Differences between all pairs (PC61+sham irradiation <i>vs</i> Rat IgG+sham irradiation, PC61+irradiation <i>vs</i> PC61+sham irradiation, Rat IgG+sham irradiation compare to Rat IgG+irradiation, Rat IgG+sham irradiation compare to PC61+irradiation, PC61+sham irradiation compared to Rat IgG+irradiation and PC61+sham irradiation compared to PC61+irradiation treatment groups) of treatment group profiles over time were significant (p<0.05) except the proportion of Treg cells between PC61+sham irradiation <i>vs</i> PC61+irradiation in blood (p = 0.948), and the proportion of CD4⁺CD25⁺ cells between PC61+sham irradiation <i>vs</i> PC61+irradiation in all compartments (p≥0.105). All post hoc pairwise comparisons were performed with Tukey's multiple comparisons. This is one representative experiment of three performed.</p

The absolute number of CD4⁺FoxP3⁺ Treg cells in the spleens and LNs.

<p>Both PC61 administration and irradiation reduced the number of CD4⁺FoxP3⁺ Treg cells in spleen, iLN and mLN. Irradiation resulted in a more rapid restoration of Treg cells after day 7. Data are shown as Mean±SD (n = 4). Two way ANOVA showed significant interactions over time between treatments in the Treg absolute numbers in spleen, iLN and mLN (all p<0.01). Differences between all pairs (PC61+sham irradiation <i>vs</i> Rat IgG+sham irradiation, PC61+irradiation <i>vs</i> PC61+sham irradiation, Rat IgG+sham irradiation compare to Rat IgG+irradiation, Rat IgG+sham irradiation compare to PC61+irradiation, PC61+sham irradiation compared to Rat IgG+irradiation and PC61+sham irradiation compared to PC61+irradiation treatment groups) of treatment group profiles over time were significant (p<0.05). All post hoc pairwise comparisons were performed with Tukey's multiple comparisons. This is one representative experiment of three performed.</p

Cell numbers in the spleens and lymph nodes.

<p>Mice were injected with PC61 or rat IgG MAb 3 days prior to receiving 13 Gy of HBI or sham irradiation. Cells in the (A) spleen, (B) iLN and (C) mLN were counted on the days indicated. Data are shown as Mean±SD (n = 4). Two way ANOVA showed significant interactions over time between treatments in spleen, iLN and mLN cell count (all p<0.01). No differences were detected between PC61+irradiation compared to Rat IgG+irradiation treatment group profiles over time in the spleen, iLN and mLN (p≥0.128). No significant differences were found between the PC61+sham irradiation compared to Rat IgG+sham irradiation treatment group profiles over time (all p≥0.072). Differences between all other pair-wise comparisons of treatment group profiles over time were significant (p<0.05). All post hoc pairwise comparisons were performed with Tukey's multiple comparisons. This is one representative experiment of three performed.</p

Production of inflammatory mediators in the lungs.

<p>Levels of (A) IL-6 and (B) KC/CXCL1 were measured in lysates of lung by ELISA. Data are shown as Mean±SD (n = 4). Two way ANOVA showed significant interactions over time between treatments (all p<0.01). No differences were detected between PC61+irradiation compared to Rat IgG+irradiation treatment group profiles over time in the levels of IL-6 and KC/CXCL1 (p≥0.089). No significant differences were found between the PC61+sham irradiation compared to Rat IgG+sham irradiation treatment group profiles over time (all p>0.05). Differences between all other pair-wise comparisons of treatment group profiles over time were significant (p<0.05). All post hoc pairwise comparisons were performed with Tukey's multiple comparisons. This is one representative experiment of three performed.</p

GI injury.

<p>Tissues were fixed, thin sectioned and studied with H&E staining. (A) Neutrophil infiltration in jejunum at 3 day after radiation. 200× magnification. The arrow on the image shows a neutrophil cluster. 200× magnification. (B) Crypt hyperplasia at 7 days after radiation. 100× magnification.</p

GI tract hyperplasia at 7 and 14 days after radiation treatment.

<p>Note: H&E stained tissues were observed under a microscope with 100× magnification. Villous length and crypt depth was measured, no. of crypt branching were calculated in ten random fields. Data are expressed as Mean±SD. For villous length, two way ANOVA showed significant interactions over time between treatments in jejunum and ileum (all p<0.01), no interaction over time between treatments in duodenum (p = 0.058). No differences were detected between PC61+irradiation compared to Rat IgG+irradiation treatment groups (p≥0.059). No significant differences were found between groups treated with PC61+sham irradiation compared to Rat IgG+sham irradiation (all p>0.05). Differences between all other pair-wise comparisons of treatment group profile over time were significant (p<0.05). For crypt depth, two way ANOVA showed no interactions over time between treatments (p≥0.108). No differences were detected between PC61+irradiation compared to Rat IgG+irradiation treatment groups (p≥0.174). No significant differences were found between groups treated with PC61+sham irradiation compared to Rat IgG+sham irradiation (all p>0.05). Differences between all other pair-wise comparisons of treatment group profile over time were significant (p<0.05). For crypt branching, two way ANOVA showed significant interactions over time between treatments (p<0.05), no interactions over time between treatments in duodenum (p = 0.868) and jejunum (p = 0.463). No differences were detected between PC61+irradiation compared to Rat IgG+irradiation treatment groups (p≥0.249). No significant differences were found between groups treated with PC61+sham irradiation compared to Rat IgG+sham irradiation (all p>0.05). Differences between all other pair-wise comparisons of treatment group profile over time were significant (p<0.05). All post hoc pairwise comparisons were performed with Tukey's multiple comparisons.</p

Production of inflammatory mediators in the jejunum, duodenum and ileum.

<p>Jejunum, duodenum and ileum were collected and levels of IL-1β and KC/CXCL1 were measured by ELISA of tissue lysates. (A) IL-1β and (B) KC/CXCL1 in jejunum, (C) IL-1β and (D) KC/CXCL1 in duodenum, (E) IL-1β and (F) KC/CXCL1 in ileum. Data are shown as Mean±SD (n = 4). Two way ANOVA showed significant interactions over time between treatments (all p<0.01). No differences were detected between PC61+irradiation compared to Rat IgG+irradiation treatment group profiles over time in the levels of IL-1 β (p≥0.119) and KC/CXCL1 (p≥0.084). No significant differences were found between the PC61+sham irradiation compared to Rat IgG+sham irradiation treatment group profiles over time (all p>0.05). Differences between all other pair-wise comparisons of treatment group profiles over time were significant (p<0.05). All post hoc pairwise comparisons were performed with Tukey's multiple comparisons. This is one representative experiment of three performed.</p