*3.11. Immunomodulation*

The immunomodulatory effect of radiation on immune cells within tumors and normal tissue is a well-documented phenomenon in other models of IR-induced damage involving infiltration of immune cells of both the innate and adaptive immune system, as well as differentiation and gene expression changes within irradiated immune cell populations [101–103]. Lombaert et al. recently reported that female CH3 mice receiving fractionated IR (5 × 6 Gy) showed increased fibrosis, inflammation and expression of innate and adaptive immune markers (i.e., *Clec12a, Cma1, Pld4,* and *Lyz2*) in irradiated SMGs 300 days post-IR [54]. This is the first published evidence of IR-induced immunomodulation in mouse salivary glands, despite being an important area of research for other tissues and models of IR-induced damage, such as pneumonitis and pulmonary fibrosis following thoracic irradiation [102]. Similar changes in the expression patterns of these markers of immunomodulation have been shown in 15 Gy irradiated parotid glands of minipigs at 16 weeks post-IR [54].

In humans, there also is limited research on the effect of RT on salivary gland immune responses. SMG biopsies from patients receiving fractionated radiotherapy (1.8–2 Gy per fraction, 5 days per week, for a total dose of 60–70.6 Gy) revealed lymphocytic infiltration (i.e., sialadenitis), where the majority of lymphocytic infiltrates were CD3<sup>+</sup> T cells with a 1:1.8 ratio of CD4<sup>+</sup> to CD8<sup>+</sup> T cells and significant numbers of granzyme B-stained cytotoxic T cells [33]. Macrophages and monocytes were also present, localized to the periductal and periacinar compartments.

Immunomodulation is recognized as a driver of IR-induced pneumonitis and pulmonary fibrosis [102,104]. During the acute phase, myeloid- and lymphoid-derived immune cells infiltrate lung tissue, leading to inflammation and the release of cytokines and chemokines [102]. In the chronic phase, interactions between IR-damaged tissue-resident cells, recruited immune cells and the microenvironment activate signaling pathways that promote immunomodulation, myofibroblast activation and fibrosis [102]. In IR-induced pulmonary fibrosis, CD4<sup>+</sup> T cells shift from pro-inflammatory (TH1 and TH17) during the pneumonitic phase to anti-inflammatory (TH2 and TREG) during the fibrotic phase [102,104]. In addition to TH2 and TREG cells, resident innate lymphoid cells (ILCs) are important regulators of fibrosis [105–107]. Notably, a unique subset of ILCs has been described in mouse SMGs [108,109], although their potential contributions to IR-induced fibrosis of the salivary gland are yet to be investigated. Nonetheless, the recent findings in CH3 mice [54], highlight the potential for future research to investigate the role of salivary gland immune cells in IR-induced hyposalivation.
