In vitro-differentiated T/natural killer-cell progenitors derived from human CD34+ cells mature in the thymus.

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a treatment option for patients with hematopoietic malignancies that is hampered by treatment-related morbidity and mortality, in part the result of opportunistic infections, a direct consequence of delayed T-cell recovery. Thymic output can be improved by facilitation of thymic immigration, known to require precommitment of CD34(+) cells. We demonstrate that Delta-like ligand-mediated predifferentiation of mobilized CD34(+) cells in vitro results in a population of thymocyte-like cells arrested at a T/natural killer (NK)-cell progenitor stage. On intrahepatic transfer to Rag2(-/-)gamma(c)(-/-) mice, these cells selectively home to the thymus and differentiate toward surface T-cell receptor-alphabeta(+) mature T cells considerably faster than animals transplanted with noncultured CD34(+) cells. This finding creates the opportunity to develop an early T-cell reconstitution therapy to combine with HSCT.

Defucosylation of Tumor-Specific Humanized Anti-MUC1 Monoclonal Antibody Enhances NK Cell-Mediated Anti-Tumor Cell Cytotoxicity.

Antibodies are commonly used in cancer immunotherapy because of their high specificity for tumor-associated antigens. The binding of antibodies can have direct effects on tumor cells but also engages natural killer (NK) cells via their Fc receptor. Mucin 1 (MUC1) is a highly glycosylated protein expressed in normal epithelial cells, while the under-glycosylated MUC1 epitope (MUC1-Tn/STn) is only expressed on malignant cells, making it an interesting diagnostic and therapeutic target. Several anti-MUC1 antibodies have been tested for therapeutic applications in solid tumors thus far without clinical success. Herein, we describe the generation of fully humanized antibodies based on the murine 5E5 antibody, targeting the tumor-specific MUC1-Tn/STn epitope. We confirmed that these antibodies specifically recognize tumor-associated MUC1 epitopes and can activate human NK cells in vitro. Defucosylation of these newly developed anti-MUC1 antibodies further enhanced antigen-dependent cellular cytotoxicity (ADCC) mediated by NK cells. We show that endocytosis inhibitors augment the availability of MUC1-Tn/STn epitopes on tumor cells but do not further enhance ADCC in NK cells. Collectively, this study describes novel fully humanized anti-MUC1 antibodies that, especially after defucosylation, are promising therapeutic candidates for cellular immunotherapy.

Surface Mucin-1 does not play a role in dendritic cell migration.

Mucin-1 (MUC1) is a transmembrane glycoprotein that is upregulated upon maturation of dendritic cells (DC) in vitro or in vivo. One of the proposed functions of surface expressed MUC1 is its involvement in migration of cells. We hypothesized that MUC1 is involved in DC migration since mature DC (mDC) are highly migratory cells and MUC1 is upregulated on the surface of DC upon maturation. In this study we cultured DC using two maturation cocktails, one cocktail containing IL-4, GM-CSF, TNFalpha, PGE2, IL-1 beta and IL-6 (TP1,6-DC) and the other IL-13, GM-CSF, Ribomunyl and IFN-gamma (RI-DC). Both maturation cocktails render DC with a similar surface phenotype including CCR7 expression, but only the former induces a migratory capacity of DC to a CCL19 gradient. To analyze the role of surface-expression of MUC1 on TP1,6-DC, that are capable of migration, expression of MUC1 was prevented by adding an anti-MUC1 antibody (Ab) during the maturation process. Compared with matured DC in the absence of the Ab, no difference was observed in chemokine-induced migratory behaviour between the MUC1+ and MUC1- DC populations in a standard Transwell chemotaxis assay, nor in organotypic cultures. Our data clearly demonstrate that surface MUC1 on DC does not influence intrinsic cell-motility, nor is it involved in cell-cell and cell-matrix dependent migration.

Expression of tumor-associated differentiation antigens, MUC1 glycoforms and CEA, in human thymic epithelial cells: implications for self-tolerance and tumor therapy.

Expression of tissue-restricted self-antigens in the thymus, termed promiscuous gene expression, imposes T cell tolerance and protects from autoimmune diseases. This antigen pool also includes various types of tumor-associated antigens (TAA) previously thought to be secluded from the immune system. The scope of promiscuous gene expression has been defined by mRNA analysis at the global level of isolated medullary thymic epithelial cells (mTECs). Information at the protein level on the frequency of mTECs expressing a given antigen, on coexpression patterns, and post-translational modifications is largely missing. We report here promiscuous expression at the protein level of two TAA, MUC1 and CEA, in situ and in purified human mTECs. Both antigens are expressed in 1% to 3% of mTECs, either individually or coexpressed in the same cell. Using a panel of anti-MUC1 monoclonal antibodies recognizing different post-translational modifications, i.e., glycoforms of MUC1, we show that only fully glycosylated forms of MUC1 and the differentiation-dependent glycoforms were detected on mTECs, but not the cancer-associated glycoforms. Our findings imply that MUC1 and CEA are amenable to central tolerance induction, which might, however, be incomplete in case of tumor cell-restricted MUC1 glycoforms. Knowledge of these subtleties in promiscuous gene expression may, in the future, assist the selection of T cell tumor vaccines for clinical trials.

Cancer specific Mucin-1 glycoforms are expressed on multiple myeloma.

Present therapies cannot cure the large majority of patients with multiple myeloma (MM) and therefore new treatment strategies are imperative. This study analysed the different glycosylation profiles of Mucin-1 (MUC1) on MM and acute myeloid leukaemia (AML) cells using a series of anti-MUC1 antibodies. Seventy-three per cent of the MM patients had plasma cells that expressed the fully glycosylated forms of MUC1. In contrast to controls, normal bone marrow cells and AML cells, the differentiation-dependent and cancer-associated glycoforms of MUC1 were present on 59% and 36% MM tumour cells respectively. This indicated that aberrantly glycosylated MUC1 is a potential immunotherapeutic target in MM patients.

Mucin-1 is expressed on dendritic cells, both in vitro and in vivo.

Dendritic cells (DCs) are the best professional antigen-presenting cells to stimulate cytotoxic as well as T helper cells and are therefore appropriate candidates for establishing immunotherapy. The concept of our vaccination program is to introduce the tumor-associated antigen mucin-1 (MUC1) into DCs. Analysis of immature and mature DCs--before transducing the antigen MUC1--already demonstrated expression of MUC1 on in vitro monocyte-derived DCs upon maturation. Different culture methods as well as maturation cocktails showed similar results concerning the upregulation of MUC1 expression. Furthermore, we studied the expression of MUC1 on DCs in vivo. No MUC1 expression was found on blood DCs, or on thymic or tonsil DCs. On the other hand, synovial fluid from patients with arthritis contained DCs that were found to express MUC1. This study shows for the first time that the tumor-associated antigen MUC1 is expressed on in vivo DCs. We further show that MUC1 is also expressed on in vitro cultured bone marrow-derived DCs of human MUC1 transgenic mice, supporting the relevance of this mouse model to the human situation. The observation that MUC1 is present on in vivo DCs suggests a functional role, but this physiological function remains to be elucidated.