One of the major problems derived from the exposure to ionizing radiation is the impairment of the immune system. The consequent immune-depression increases the risk of infections and may lead to immune-mediated disorders. The intensity and duration of the immune-compromised phase and its recovery depend on the dose, dose-rate and quality of radiation. In recent years, there has been a great interest in the effects induced by protons, both for a better assessment of the health risks in astronauts exposed to solar wind and cosmic radiations and for a better understanding of their effects in radiotherapy for oncologic patients. In the present study, we investigated the effects of the in vivo exposure to 2 Gy of integral dose absorbed by medium energy proton beams on mouse lymphoid spleen cells. The TOP-IMPLART accelerator was used as proton source. Irradiations were performed in air with pulsed (3.4 ms, 10Hz) 27 MeV proton beams. During the exposure, mice were anesthetized in order to keep them in the right position. Sham-exposed anesthetized age/gender/strain-matched mice were used as controls. Twenty-four hours and 1 week after irradiation, each mouse was individually analyzed for several parameters (5 mice/group). Results showed that the number of nucleated cells in the spleen was not significantly affected. Flow cytometry analyses revealed that the percentages of helper T (CD4), cytotoxic T (CD8) and B (CD19) cells within the spleen lymphocytes were not altered 24 hours after the exposure. At variance, 1 week after the exposure the frequency of CD4 (14% vs. 9%) and CD19 (37% vs. 26%) cells reduced. Spleen cells were stimulated with an anti-CD3 antibody and LPS to induce T cell and B cell activation, respectively. Both T and B cells were functionally impaired by the exposure. Twenty-four hours after irradiation, T cell proliferation was indeed reduced by 50% in exposed mice compared with controls. B cells also displayed a reduced cell proliferation in response to the mitogenic stimulus (-33%). Interestingly, 1 week after irradiation proliferative responses of T and B cells were still compromised. This first study allowed the conclusion that, in vivo local exposure to protons induced small changes in total spleen cell number, the frequency of CD4 and B cells being reduced 1 week after the exposure. More interesting, functional responses, such as T and B cell proliferation were partially compromised. These effects, in spite of the limited area of exposure, were not recovered after 1 week.

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