Radiation enhances regulatory T cell representation

Abstract

Purpose: Immunotherapy could be a useful adjunct to standard cytotoxic therapies such as radiation in patients with micrometastatic disease, although successful integration of immunotherapy into treatment protocols will require further understanding of how standard therapies affect the generation of antitumor immune responses. This study was undertaken to evaluate the impact of radiation therapy (RT) on immunosuppressive T regulatory (Treg) cells.

Methods and materials: Treg cells were identified as a CD4(+)CD25(hi)Foxp3(+) lymphocyte subset, and their fate was followed in a murine TRAMP C1 model of prostate cancer in mice with and without RT.

Results: CD4(+)CD25(hi)Foxp3(+) Treg cells increased in immune organs after local leg or whole-body radiation. A large part, but not all, of this increase after leg-only irradiation could be ascribed to radiation scatter and Treg cells being intrinsically more radiation resistant than other lymphocyte subpopulations, resulting in their selection. Their functional activity on a per-cell basis was not affected by radiation exposure. Similar findings were made with mice receiving local RT to murine prostate tumors growing in the leg. The importance of the Treg cell population in the response to RT was shown by systemic elimination of Treg cells, which greatly enhanced radiation-induced tumor regression.

Conclusions: We conclude that Treg cells are more resistant to radiation than other lymphocytes, resulting in their preferential increase. Treg cells may form an important homeostatic mechanism for tissues injured by radiation, and in a tumor context, they may assist in immune evasion during therapy. Targeting this population may allow enhancement of radiotherapeutic benefit through immune modulation.

Figure 1

Tregs increase temporarily in Foxp3^EGFP mice after local irradiation. Splenocytes, lymph nodes, blood and lung were stained for CD4 (FL3) and CD25 (FL2) and assessed by flow cytometry. A) Splenocyte dot blot of Foxp3 (FL-1) vs forward scatter. B) CD25 vs FoxP3 dot blot of CD4⁺ splenocytes. C) Box and whisker diagram summarizing the number of FoxP3⁺ cells and the number of CD4⁺/CD25^hi/Foxp3⁺ Tregs in the spleen. D) Correlation of CD4⁺ T cells that have high CD25 expression and FoxP3 in spleen, blood, lymph nodes and lung. E) Splenocytes dot blot of CD39 vs CD4. F) CD39 vs Foxp3 dot blot. G) Time course of Treg changes in spleen, lymph nodes, blood and lung after 10Gy gamma-irradiation of the right leg. Data are mean fold increases in all organs to control of n=4 ± s.d. *p<0.05.

Figure 1

Tregs increase temporarily in Foxp3^EGFP mice after local irradiation. Splenocytes, lymph nodes, blood and lung were stained for CD4 (FL3) and CD25 (FL2) and assessed by flow cytometry. A) Splenocyte dot blot of Foxp3 (FL-1) vs forward scatter. B) CD25 vs FoxP3 dot blot of CD4⁺ splenocytes. C) Box and whisker diagram summarizing the number of FoxP3⁺ cells and the number of CD4⁺/CD25^hi/Foxp3⁺ Tregs in the spleen. D) Correlation of CD4⁺ T cells that have high CD25 expression and FoxP3 in spleen, blood, lymph nodes and lung. E) Splenocytes dot blot of CD39 vs CD4. F) CD39 vs Foxp3 dot blot. G) Time course of Treg changes in spleen, lymph nodes, blood and lung after 10Gy gamma-irradiation of the right leg. Data are mean fold increases in all organs to control of n=4 ± s.d. *p<0.05.

Figure 2

The radiation-induced increase in Tregs in the spleen of C57Bl/6 mice is dose-dependent. The right leg was exposed to various doses of radiation 48h prior to analysis or left untreated. Splenocytes were stained for CD4, CD25 and Foxp3 and analyzed by FACScan. Dose-response of A) Treg changes, of B) CD4⁺ splenocyte levels and of C) total splenocyte numbers per spleen. Data are mean of n=4 ± s.d. of 2 independent experiments *p<0.05.

Figure 2

The radiation-induced increase in Tregs in the spleen of C57Bl/6 mice is dose-dependent. The right leg was exposed to various doses of radiation 48h prior to analysis or left untreated. Splenocytes were stained for CD4, CD25 and Foxp3 and analyzed by FACScan. Dose-response of A) Treg changes, of B) CD4⁺ splenocyte levels and of C) total splenocyte numbers per spleen. Data are mean of n=4 ± s.d. of 2 independent experiments *p<0.05.

Figure 3

Low radiation doses to the whole body reduced spleen size, weight and cell numbers while increasing the proportion of splenic Tregs. C57Bl/6 mice were exposed to X-irradiation ranging from 0.25 to 2Gy. 48h later spleens were weighed, splenocytes counted and analyzed as above. A) Dose-dependent fall in splenic weight and cell numbers. B) Photograph taken of spleens from 2Gy irradiated mice illustrating the change in size. C) CD4⁺/CD25^hi/Foxp3⁺ Treg increased with radiation dose. Data are mean of n=3 ± s.d. *p<0.05.

Figure 4

Foxp3⁺ splenocytes are relatively more radioresistant than other spleen cells. Splenocytes were isolated from Foxp3^EGFP C57Bl/6 mice, irradiated in vitro with 0, 0.5, 1, 2, and 5Gy. Cell death was measured at 0, 6, 12, 24, and 48 hrs by 7-AAD/Annexin-V-staining A) Examples of 7-AAD vs Annexin-V gating for splenocytes 24hrs after 2 Gy showing decreased viability in whole splenocyte populations (left). In contrast, Foxp3⁺-gated splenocytes showed little radiation-induced cell death. (the increase in viability is due to loss of dead cells during incubation). B) Dose- and time-dependent changes in the fraction of all viable splenocytes as Annexin-V⁻/7-AAD⁻ (lower left quadrants in 4A). C) time-dependent but dose-independent changes in the fraction of Foxp3⁺ viable splenocytes. D) Dose- and time dependent rise in Foxp3⁺ splenic cells.

Figure 4

Foxp3⁺ splenocytes are relatively more radioresistant than other spleen cells. Splenocytes were isolated from Foxp3^EGFP C57Bl/6 mice, irradiated in vitro with 0, 0.5, 1, 2, and 5Gy. Cell death was measured at 0, 6, 12, 24, and 48 hrs by 7-AAD/Annexin-V-staining A) Examples of 7-AAD vs Annexin-V gating for splenocytes 24hrs after 2 Gy showing decreased viability in whole splenocyte populations (left). In contrast, Foxp3⁺-gated splenocytes showed little radiation-induced cell death. (the increase in viability is due to loss of dead cells during incubation). B) Dose- and time-dependent changes in the fraction of all viable splenocytes as Annexin-V⁻/7-AAD⁻ (lower left quadrants in 4A). C) time-dependent but dose-independent changes in the fraction of Foxp3⁺ viable splenocytes. D) Dose- and time dependent rise in Foxp3⁺ splenic cells.

Figure 5

CD4⁺/CD25⁺ Tregs suppressed cell proliferation of CD4⁺CD25⁻ T effector cells (T_eff) in vitro. Splenic Tregs were isolated from 10Gy leg-irradiated C57Bl/6 mice 48h post treatment. Tregs from healthy mice’s spleen served as control. A) Data are FL3(CD4) vs FL2(CD25) dot blots of whole spleens (left panel), CD4⁺/CD25⁻ fraction (middle panel) and CD4⁺/CD25⁺ fraction (right panel). B) aCD3/aCD28 − stimulated T_eff from control mice were mixed with Tregs from control or from irradiated animals and proliferation was assessed according to ³H-Thymidine incorporation after 3 days. T_eff alone were incubated separately without CD3/CD28 stimulation. Data are mean counts per minute (cpm) of n=4–6 ± sem.

Figure 6

Tumor control following local irradiation benefits from concurrent, systemic inhibition of Tregs. A) Local irradiation of TRAMP C1 tumors increased Tregs in the tumor and outside. Mice bearing tumors of 4.5–6.5mm in size were given radiation treatment with 10 and 20Gy and their spleen and tumors analyzed 48h later. CD4⁺/CD25^hi/Foxp3⁺ Tregs are shown as mean of n=4 ± s.d. B) Treatment of TRAMPC1 tumors with anti-CD25 antibody (aCD25 Ab) in combination with 10Gy local irradiation caused tumor growth delay. Tumor-bearing mice were given aCD25 Ab or PBS i.p. on day −4, −1, +7 and +14. Tumors were irradiated on day 0 with 10Gy or left untreated. Tumor size as mean mm diameter (2 dimension) of n = 2–6 ± s.d. C) Survival data with % of mice per treatment group alive. *p<0.05.

Figure 6

Tumor control following local irradiation benefits from concurrent, systemic inhibition of Tregs. A) Local irradiation of TRAMP C1 tumors increased Tregs in the tumor and outside. Mice bearing tumors of 4.5–6.5mm in size were given radiation treatment with 10 and 20Gy and their spleen and tumors analyzed 48h later. CD4⁺/CD25^hi/Foxp3⁺ Tregs are shown as mean of n=4 ± s.d. B) Treatment of TRAMPC1 tumors with anti-CD25 antibody (aCD25 Ab) in combination with 10Gy local irradiation caused tumor growth delay. Tumor-bearing mice were given aCD25 Ab or PBS i.p. on day −4, −1, +7 and +14. Tumors were irradiated on day 0 with 10Gy or left untreated. Tumor size as mean mm diameter (2 dimension) of n = 2–6 ± s.d. C) Survival data with % of mice per treatment group alive. *p<0.05.