21Encephalitozoon cuniculi is an intracellular pathogen that stablishes a balanced relationship 22 with immunocompetent individuals, which is dependent of T lymphocytes activity. We 23 previously showed X-linked immunodeficiency (XID -B cell deficient) mice are more 24 susceptible to encephalitozoonosis and B-1 cells presence influences in the immune response. 25 Because XID mice are deficient both in B-1 and B-2 cells, here we investigate the role of these 26 cells against E. cuniculi infection using cyclophosphamide (Cy) immunosuppressed murine 27 model to exacerbate the infection. XID mice presented lethargy and severe symptoms, 28 associated with encephalitozoonosis and there was an increase in the peritoneal populations of 29 CD8 + and CD4 + T lymphocytes and macrophages and also in the proinflammatory cytokines 30 IFN-γ, TNF-α and IL-6. In BALB/c mice, no clinical signs were observed and there was an 31 increase of T lymphocytes and macrophages in the spleen, showing an effective immune 32 response. B-2 cells transfer to XID mice resulted in reduction of symptoms and lesion area with 33 increase of B-2 and CD4 + T populations in the spleen. B-1 cells transfer increased the peritoneal 34 populations of B-2 cells and macrophages and also reduced the symptoms. Therefore, the 35 immunodeficiency of B cells associated to Cy immunosuppression condition leads to 36 disseminated and severe encephalitozoonosis in XID mice with absence of splenic immune 37 response and ineffective local immune response, evidencing the B-1 and B-2 cells role against 38 microsporidiosis.39 40 lymphocytes 42 43 3 44 Author summary 45 46 The adaptive immune response plays a key role against Encephalitozoon cuniculi, an 47 opportunistic fungus for T cells immunodeficient patients. The role of B cells and antibody play 48 in natural resistance to Encephalitozoon cuniculi remains unresolved. Previously, we 49 demonstrated that B-1 deficient mice (XID), an important component of innate immunity, were 50 more susceptible to encephalitozoonosis, despite the increase in the number of CD4 + and CD8 + 51 T lymphocytes. In order to better understand the role of B-1 and B-2 cells and the relationship 52 with the other cells of the immune response in encephalitozoonosis, we infected with E. cuniculi 53 in cyclophosphamide immunosuppressed mice. Here we demonstrate that infected XID mice 54 showed reduction of T cells and macrophages and increase of proinflammatory cytokines 55 associated with disseminated and severe encephalitozoonosis with presence of abdominal 56 effusion and lesions in multiple organs. This pattern of infection observed in mice with genetic 57 deficiency in T cells, so we suggest that the absence of B-1 cells affects the cytotoxic capacity 58 of these lymphocytes. When we transfer B-2 cells to XID mice, the lesion areas caused by the 59 fungus, the populations of T lymphocytes in the peritoneum and the proinflammatory cytokines 60 decrease, indicating a better resolution of the infection. We speculate that B-1 and B-2 cells...