The maturation of DCs was induced by adding to the medium one of the following stimulants: 100 ng/ml LPS (Sigma-Aldrich, St-Louis, MO), 0.5?g/ml mouse IgG2a anti-human monoclonal HLA class I antibody clone Mouse monoclonal to CD3.4AT3 reacts with CD3, a 20-26 kDa molecule, which is expressed on all mature T lymphocytes (approximately 60-80% of normal human peripheral blood lymphocytes), NK-T cells and some thymocytes. CD3 associated with the T-cell receptor a/b or g/d dimer also plays a role in T-cell activation and signal transduction during antigen recognition W6/32 (Sigma-Aldrich), 0.5?g/ml mouse IgG2a anti-human monoclonal HLA-DR, DP, DQ clone T39 (BD Biosciences, San Diego, CA), or 0.5?g/ml mouse IgG2a isotype control (Sigma-Aldrich and BD Biosciences). of circulating Tfh PFE-360 (PF-06685360) cells than those with anti-HLA-I DSAs. Moreover, there was a predominance of lymphoid aggregates containing Tfh cells in biopsies from patients with antibody-mediated rejection and anti-HLA-II DSAs. Collectively, these data suggest that alloantibodies against HLA class II specifically promote the differentiation of naive T cells to Tfh cells following contact with DCs, a process that might appear in human allografts and constitutes a therapeutic target. Introduction Although the premature graft loss can be due to various causes, including infection, nephrotoxicity or recurrence of the primary renal disease1,2, alloimmunity remains the most common mechanism2,3. A report based on sensitive methods for detecting circulating anti-HLA antibodies suggested that up to 64% of graft losses could be due to rejection, mostly in the form of antibody-mediated rejection (ABMR)3. The most important physiopathologic component of ABMR is the presence of donor-specific antibodies (DSA), which often develop following transplantation. Alloantibodies against HLA class II antigens are associated with high levels of endothelial-associated transcripts following tissue injury, and ABMR is mostly associated with this class of alloantibodies4. We and others have reported that antibodies against HLA class II are not only more commonly associated with chronic ABMR than antibodies against HLA class I, but are also predictive of graft loss5C8. Thus far, the reason that antibodies against HLA class II are associated with negative graft outcomes has not been elucidated. B cells are responsible for producing anti-HLA antibodies; however, they need the help of T follicular helper lymphocytes (Tfh) to achieve this role9. In 2000, Tfh cells were first described as CD4+ T cells in human tonsils that express the chemokine receptor CXCR510C12. In the lymph node, Tfh cells support B cell proliferation and provide signals that are crucial for the generation of high-affinity antibodies against specific antigens12. Tfh cells are notably characterized by the expression of the cell surface markers CXCR5 and ICOS, the cytokine IL-21 and the transcription factors Bcl-6 and STAT312,13. In addition to playing a role in certain autoimmune diseases, such as systemic lupus erythematosus14 and juvenile dermatomyositis15, emerging data suggest a role for Tfh cells in mediating allograft rejection16,17. In a recent publication, we studied the dendritic cells (DCs) infiltrating human kidney allografts18. In biopsies with a high DC density, immunofluorescence and electron microscopy studies showed direct physical contact between DCs and T cells, and the DC density correlated with higher Ki-67-positive labeling indices in infiltrating T cells. These observations suggest that the crosstalk between DCs and T cells PFE-360 (PF-06685360) may be driving an inflammatory response within the graft. Allograft transplantation is a human model of exposure to a persistent, large load of alloantigens from the donor. However, the interaction between DCs and T cells in this context PFE-360 (PF-06685360) remains poorly understood. Based on these observations, we hypothesized that one of the mechanisms by which antibodies against HLA class II lead to increased graft loss is by preferentially instructing naive T cells to differentiate into Tfh cells through their interaction with DCs. We show, in a human allogeneic model, that HLA class II-stimulated DCs polarize naive CD4+ T cells into a Tfh phenotype. We further demonstrate in a cohort of kidney transplant recipients that patients with DSAs against HLA class II have higher frequencies of circulating Tfh cells and a higher number of lymphoid aggregates containing Tfh cells in their allograft biopsies than those with antibodies against HLA class I. Results Antibodies against HLA class II stimulate monocyte-derived DCs to mature into a CD80+CD86hiHLA-DR+BAFF+CCR7+ phenotype To investigate the effect of HLA I and HLA II on the DC phenotype, CD14+ monocytes from healthy volunteers were isolated and differentiated into immature DCs using GM-CSF and IL-4. The cells were then matured under the following conditions: unstimulated, stimulated with a pan-antibody against HLA class I, a pan-antibody against HLA class II, a corresponding IgG2a PFE-360 (PF-06685360) isotype or TLR4 (LPS). Generation of monocyte-derived DCs (moDCs) was confirmed by CD11c expression (95% of cells). The differentiation of moDCs into mature DCs with an APC phenotype predominantly occurred in the PFE-360 (PF-06685360) presence of.