This suggests that increased metabolic capacity in MIM?/? cells could compensate for the effects of compromised BCR signaling upon B cell activation and differentiation. of B cell compartments in the periphery. Interestingly, we found that MIM?/? B cells are defected in BCR signaling in response to surface-bound antigens but, on the other hand, show increased metabolic activity after stimulation with LPS or CpG. gene were found in 6% of sequenced cancer samples and, depending on the cancer type, both diminished or increased gene expression profiles are seen (17). Regarding hematopoietic malignancies, MIM is upregulated, for example, in hairy cell and mantle cell lymphomas as well as in chronic lymphocytic leukemia (CLL). In CLL, interestingly, the good prognosis samples exhibit highest levels of MIM while the poor prognosis samples show lower MIM levels in comparison to good prognosis samples (17). In mice, it has been Laninamivir (CS-8958) reported that upon aging, MIM knockout animals develop lymphomas resembling diffuse large B cell lymphoma (DLBCL) (12). Moreover, a degenerative kidney disease, potentially linked to impaired cellCcell junction formation, as well as a defected dendritic spine formation and neuronal alterations have been reported in MIM knockout mice (18, 19). These findings illustrate the complexity of MIM function, the basis of Laninamivir (CS-8958) which remains enigmatic due to the lack of understanding about the molecular mechanisms and connected pathways. Despite the reported high expression in B cells and the association with hematopoietic malignancies, nothing is known about the role of MIM in activation of adaptive immune responses. In this study, we took advantage of a MIM knockout mouse model (MIM?/?, MIM-KO) (18) to explore the physiological role of MIM in B cell compartment, specifically in early B cell activation and mounting of the antibody responses. While we found no defects in B cell development, MIM-deficiency caused a variety of changes in mature B cells. MIM?/? B cells showed significantly reduced signaling upon stimulation with surface-bound antigens mimicking activation via immunological synapse. T cell-independent IgM responses were reduced in MIM?/? mice, while on the other hand, T cell-dependent immune responses appeared normal. Unlike BCR stimulation, MIM?/? B cells were robustly activated by TLR agonists that, interestingly, also led to increased metabolic activity in cells lacking MIM. Our study highlights the complex role of MIM in different cellular functions and can serve as a stepping stone for unveiling the role of MIM in hematopoietic cancers. Materials and Methods Antibodies and Chemicals List of antibodies and reagents used in the study can be found in Table 1. Table 1 Key reagents table. gene in 129/Sv ES-cells. Chimeric mice were backcrossed to C57Bl/6J background for several generations and the colony in Turku was established by breedings of heterozygote founder animals. RAB11FIP4 All experiments were done with age- and sex-matched animals and WT littermate controls were used whenever possible. Immunizations At the age of 3C4 months, groups of WT and for 1 min with no break and left for 1 h at 37C to attach to coated wells in a humidified incubator without Laninamivir (CS-8958) CO2 to avoid medium acidification. Seahorse XF96 plate (101085-004, Agilent) was used following the manufacturer’s instructions for XF Cell Mito Stress Test Kit (103015-100, Agilent). In this test, sequentially, 1 M oligomycin, 2 M FCCP, and 0.5 M rotenone/antimycin A were added to the media. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) data were recorded by WAVE software (Agilent). OCR and ECAR data were normalized to cell count and first baseline measurement of WT cells. Basal, maximum, and spare respiratory capacities were extracted with area.