During HLA class II synthesis in antigen-presenting cells, the invariant chain (Ii) not only stabilizes HLA class II complexes in the endoplasmic reticulum, but also mediates their transport to... Show moreDuring HLA class II synthesis in antigen-presenting cells, the invariant chain (Ii) not only stabilizes HLA class II complexes in the endoplasmic reticulum, but also mediates their transport to specialized lysosomal antigen-loading compartments termed MIICs. This study explores an alternative HLA class II presentation pathway in leukemic blasts that involves proteasome and transporter associated with antigen processing (TAP)-dependent peptide loading. Although HLA-DR did associate with Ii, Ii silencing in the human class II-associated invariant chain peptide (CLIP)-negative KG-1 myeloid leukemic cell line did not affect total and plasma membrane expression levels of HLA-DR, as determined by western blotting and flow cytometry. Since HLA-DR expression does require peptide binding, we examined the role of endogenous antigen-processing machinery in HLA-DR presentation by CLIP- leukemic blasts. The suppression of proteasome and TAP function using various inhibitors resulted in decreased HLA-DR levels in both CLIP- KG-1 and ME-1 blasts. Simultaneous inhibition of TAP and Ii completely down-modulated the expression of HLA-DR, demonstrating that together these molecules form the key mediators of HLA class II antigen presentation in leukemic blasts. By the use of a proteasome- and TAP-dependent pathway for HLA class II antigen presentation, CLIP- leukemic blasts might be able to present a broad range of endogenous leukemia-associated peptides via HLA class II to activate leukemia-specific CD4(+) T cells. Show less
Brinke, A. ten; Schijndel, G. van; Visser, R.; Gruijl, T.D. de; Zwaginga, J.J.; Ham, S.M. van 2010
Dendritic cells (DCs) are promising antigen presenting cells for cancer treatment. Previously, we showed that the combination of monophosphoryl lipid A (MPLA) with IFN gamma generates mature DCs... Show moreDendritic cells (DCs) are promising antigen presenting cells for cancer treatment. Previously, we showed that the combination of monophosphoryl lipid A (MPLA) with IFN gamma generates mature DCs that produce IL-12 and polarize CD4(+) T cells towards a Th1 phenotype. Here, we extended these observations by showing that the DCs generated with the clinical grade maturation cocktail of MPLA/IFN gamma induce superior tumour antigen-specific CD8(+) CTL responses compared to the cytokine cocktail matured DCs that are currently used in the clinic. MPLA/IFN gamma DCs can induce CTL responses in healthy individuals as well as in melanoma patients. The CTL induction was mainly dependent on the IL-12 produced by the MPLA/IFN gamma DCs. The high amounts of induced CTLs are functional as they produce IFN gamma and lyse target cells and this cytolytic activity is antigen specific and HLA restricted. Furthermore, the CTLs proved to kill tumour cells expressing endogenous tumour antigen in vitro. Therefore, MPLA/IFN gamma DCs are very promising for the use in future cancer immunotherapy. Show less
Boks, M.A.; Zwaginga, J.J.; Ham, S.M. van; Brinke, A. ten 2010
In autoimmune diseases or transplant graft rejection, a therapy that will prevent or reduce the present immune activation is highly desired. Ex vivo generated tolerogenic dendritic cells (DC) are... Show moreIn autoimmune diseases or transplant graft rejection, a therapy that will prevent or reduce the present immune activation is highly desired. Ex vivo generated tolerogenic dendritic cells (DC) are considered to have a strong potential as cellular therapy for these diseases. One of the mechanisms of immune suppression mediated by tolerogenic DC is the induction of regulatory T-cells (Treg). Consequently, the efficacy of such DC to induce Treg will reflect their tolerogenic capacity. Because no specific markers have been described for human induced (i)Treg yet, the Treg can only be appreciated by functionality. Therefore, we have optimized an in vitro suppression assay to screen for human DC-induced-Treg activity. IL-10-generated tolerogenic DC were used to induce Treg that were previously shown to effectively suppress the proliferation of responder T-cells stimulated with allogeneic mature DC (mDC). Our results show that the suppressive capacity of IL-10 DC-induced Treg measured in the suppression assay increases with the iTreg dose and decreases with higher numbers of antigen-presenting cells (APC) as T-cell stimulation. Lowering the ratio between responder T-cells and stimulator mDC present in the coculture clearly improved the read-out of the suppression assay. Furthermore, mDC-primed T-cells in the suppression assay were shown to be an essential control condition. In conclusion, we recommend titrations of both APC and iTreg in the suppression assay and to include a negative control condition with T-cells primed by mDC, to distinguish specific and functional suppression by iTreg from possible generalized suppressive activity. Show less
Wauben, M.H.M.; Toes, R.E.M.; Bos, N.A.; Damoiseaux, J.; Ham, S.M. van; Loosdrecht, A.A. van de; Samsom, J.N. 2010
