Trogocytosis is an intracellular transfer of membrane and membrane-associated proteins in a cell–cell contact manner. Trogocytosis plays an important role in immune regulation. We hypothesized that trogocytosis may play role in isotype switching and affinity maturation in the germinal center.
Trogocytosis of MHC-II by T follicular helper (Tfh) (CD4+CXCR5+CD45RA-) from dendritic cells (DCs) results in MHC-II-dressed Tfh to present antigen and stimulate B cells, resulting in B cell activation and proliferation with an important role in isotype switching and affinity maturation.
Monocyte-derived DCs (moDCs) were prepared by culturing purified CD14+ monocytes isolated from peripheral blood mononuclear cells (PBMCs), enriched by immunomagnetic positive selection and addition of GM-CSF (50 ng/ml) and IL-4 (20 ng/ml) for 6 days. Purified CD4+T cells were enriched by immunomagnetic negative selection and were activated using anti-CD3/CD28 beads for 24 hours. MoDCs were co-cultured with activated CD4+T cells (1:1) for 4 hrs. Harvested co-cultured cells were stained with antihuman monoclonal antibodies and isotype controls. The cells were washed and acquired on FACS Calibur and analyzed using FlowJo.
Expression of MHC-II, co-stimulatory molecule (CD86), and mean fluorescence intensity (MFI) was measured on activated CD4+T, Tfh, and their subpopulations. Activated CD4+T cells and moDCs without co-culture served as controls. Statistical analysis was performed by a parametric t test, and a value of p<0.05 was considered significant.
Our data show that following trogocytosis, MHC-II expression was significantly increased on activated CD4+T (p = 0.002), Tfh (p = 0.0002), Tfh1 (p = 0.0002), Tfh2 (p = 0.0001), and Tfh17 (p = 0.0004) cells. The MFI was also higher on total CD4+T (p = 0.01), Tfh (p = 0.0007), Tfh1 (p = 0.001), and Tfh2 (p = 0.03). The expression and MFI of CD86 were higher on total CD4+T, Tfh cells, and its subsets.
Trogocytosis results in MHC-II-dressed Tfh cells with Tfh1 showing a higher proportion of trogocytosed class MHC-II, followed by Tfh17 and Tfh2. Also, there is trogocytosis of CD86 on CD4T, Tfh, and its subsets. Future directions involve the exploration of the functional role of MHC-II-dressed Tfh cells as potent APCs and their contribution to isotype switching, affinity maturation, and restoration of immune function in patients with inborn errors of immunity.
