Dawn Shepherd

Plasmacytoid dendritic cells prime IFN-γ-secreting melanoma-specific CD8 lymphocytes and are found in primary melanoma lesions

Plasmacytoid dendritic cells (PDC) are a small population of leukocytes specialized in the production of type I IFN. It has been shown that PDC have a potent T cell stimulatory capacity in allogeneic mixed lymphocyte reaction, However, their role in initiating primary immune responses remains elusive. We report that blood PDC efficiently prime naive CD8+ lymphocytes specific forthe melan‐A26–35 epitope to become IFN‐γ producing cells in vitro. In addition, we found that CD40L‐stimulated PDC induce expression on primed melan‐A‐specific T cells of cutaneous lymphocyte antigen and L‐selectin (CD62L), homing receptors that allow the migration of effector cells to the inflamed skin. Finally, we show that PDC can be found in the peri‐tumoralarea of most primary cutaneous melanomas in vivo and that type I IFN‐containing supernatants derived from PDC increase melanoma cell surface expression of CD95 and MHC class I and class II molecules in vitro. Our results suggest a new immunomodulatory role for tissue infiltrating PDC, which may prime tumor‐specific T cell responses and affect tumor growth via soluble factors.

Modulation of human natural killer T cell ligands on TLR-mediated antigen-presenting cell activation.

Invariant natural killer T (iNKT) cells are a subset of nonconventional T cells recognizing endogenous and/or exogenous glycolipid antigens in the context of CD1d molecules. It remains unclear whether innate stimuli can modify the profile of endogenous lipids recognized by iNKT cells on the surface of antigen-presenting cells (APCs). We report that activation of human APCs by Toll-like receptor ligands (TLR-L) modulates the lipid biosynthetic pathway, resulting in enhanced recognition of CD1d-associated lipids by iNKT cells, as defined by IFN-γ secretion. APC-derived soluble factors further increase CD1d-restricted iNKT cell activation. Finally, using soluble tetrameric iNKT T cell receptors (TCR) as a staining reagent, we demonstrate specific up-regulation of CD1d-bound ligand(s) on TLR-mediated APC maturation. The ability of innate stimuli to modulate the lipid profile of APCs resulting in iNKT cell activation and APC maturation underscores the role of iNKT cells in assisting priming of antigen-specific immune responses.

Cutting Edge: Nonglycosidic CD1d Lipid Ligands Activate Human and Murine Invariant NKT Cells

Invariant NKT cells (iNKT cells) recognize CD1d/glycolipid complexes. We demonstrate that the nonglycosidic compound threitolceramide efficiently activates iNKT cells, resulting in dendritic cell (DC) maturation and the priming of Ag-specific T and B cells. Threitolceramide-pulsed DCs are more resistant to iNKT cell-dependent lysis than α-galactosylceramide-pulsed DCs due to the weaker affinity of the human iNKT TCR for CD1d/ threitolceramide than CD1d/α-galactosylceramide complexes. iNKT cells stimulated with threitolceramide also recover more quickly from activation-induced anergy. Kinetic and functional experiments showed that shortening or lengthening the threitol moiety by one hydroxymethylene group modulates ligand recognition, as human and murine iNKT cells recognize glycerolceramide and arabinitolceramide differentially. Our data broaden the range of potential iNKT cell agonists. The ability of these compounds to assist the priming of Ag-specific immune responses while minimizing iNKT cell-dependent DC lysis makes them attractive adjuvants for vaccination strategies.

Dendritic Cell Function Can Be Modulated through Cooperative Actions of TLR Ligands and Invariant NKT Cells

The quality of signals received by dendritic cells (DC) in response to pathogens influences the nature of the adaptive response. We show that pathogen-derived signals to DC mediated via TLRs can be modulated by activated invariant NKT (iNKT) cells. DC maturation induced in vivo with any one of a variety of TLR ligands was greatly improved through simultaneous administration of the iNKT cell ligand α-galactosylceramide. DC isolated from animals treated simultaneously with TLR and iNKT cell ligands were potent stimulators of naive T cells in vitro compared with DC from animals treated with the ligands individually. Injection of protein Ags with both stimuli resulted in significantly improved T cell and Ab responses to coadministered protein Ags over TLR stimulation alone. Ag-specific CD8+ T cell responses induced in the presence of the TLR4 ligand monophosphoryl lipid A and α-galactosylceramide showed faster proliferation kinetics, and increased effector function, than those induced with either ligand alone. Human DC exposed to TLR ligands and activated iNKT cells in vitro had enhanced expression of maturation markers, suggesting that a cooperative action of TLR ligands and iNKT cells on DC function is a generalizable phenomenon across species. These studies highlight the potential for manipulating the interactions between TLR ligands and iNKT cell activation in the design of effective vaccine adjuvants.

Mature Dendritic Cells Prime Functionally Superior Melan-A-Specific CD8+ Lymphocytes as Compared with Nonprofessional APC

Priming of melan-A26/27–35-specific CTL occurs only in a fraction of late stage melanoma patients, whereas during the early stages of the disease and in healthy volunteers, melan-A CTL have functional and phenotypic markers consistent with a naive phenotype. To study the requirements for expansion of naive melan-A CTL from healthy donors, we set up an in vitro priming protocol and, using tetramer assays, we demonstrate that the activity and phenotype of the expanded melan-A CTL are profoundly influenced by the type of APC used. Priming by nonprofessional APC leads to expansion of melan-A CTL with reduced cytolytic activity and low level of IFN-γ secretion. In contrast, mature dendritic cells (DC) expand cytolytic and IFN-γ-producing melan-A CTL. Priming by mature DC is also efficient at low peptide concentration and requires only one round of stimulation. Finally, we observed that a significant fraction of CD45RO+ melan-A CTL primed by mature DC expresses high levels of the homing receptor CD62L, whereas CTL primed by nonprofessional APC express CD62L in lower percentages and at lower levels. These results suggest that suboptimal priming by nonprofessional APC could account for the presence in vivo of dysfunctional cells and strongly support the immunotherapeutic use of mature DC for expansion of effector and memory Ag-specific CTL.

Diverse Endogenous Antigens for Mouse NKT Cells: Self-Antigens That Are Not Glycosphingolipids

NKT cells with an invariant Ag receptor (iNKT cells) represent a highly conserved and unique subset of T lymphocytes having properties of innate and adaptive immune cells. They have been reported to regulate a variety of immune responses, including the response to cancers and the development of autoimmunity. The development and activation of iNKT cells is dependent on self-Ags presented by the CD1d Ag-presenting molecule. It is widely believed that these self-Ags are glycosphingolipids (GSLs), molecules that contain ceramide as the lipid backbone. In this study, we used a variety of methods to show that mammalian Ags for mouse iNKT cells need not be GSLs, including the use of cell lines deficient in GSL biosynthesis and an inhibitor of GSL biosynthesis. Presentation of these Ags required the expression of CD1d molecules that could traffic to late endosomes, the site where self-Ag is acquired. Extracts of APCs contain a self-Ag that could stimulate iNKT cells when added to plates coated with soluble, rCD1d molecules. The Ag(s) in these extracts are resistant to sphingolipid-specific hydrolase digestion, consistent with the results using live APCs. Lyosphosphatidylcholine, a potential self-Ag that activated human iNKT cell lines, did not activate mouse iNKT cell hybridomas. Our data indicate that there may be more than one type of self-Ag for iNKT cells, that the self-Ags comparing mouse and human may not be conserved, and that the search to identify these molecules should not be confined to GSLs.

Expression of MHC class I-related Chain B (MICB) molecules on renal transplant biopsies.

Background. MICA and MICB (MHC class I-related chain A and B) are polymorphic genes that encode molecules related to MHC class I and are expressed on epithelial cells in response to stress. Incompatible donor MIC antigens can stimulate antibody production in transplant recipients. This study was designed to determine MICB expression in kidney pretransplant and any subsequent changes in expression following transplantation and to correlate changes with inflammatory markers and clinical events. Methods. Paired renal biopsies obtained from living donor (n=10) and cadaveric allografts (n=50) before and 7 days posttransplant were stained for MICB, leukocytic infiltration, and HLA class II antigens. Results. Variable tubular MICB expression was evident in donor biopsies [high 6/60 (10%), low/negative 13/60 (22%), intermediate 41/60 (68%)]. Following transplantation, MICB was up-regulated on renal tubules of 17/60 (28%) biopsies and was associated with MHC class II antigen induction (P=0.02) and leukocyte infiltration (P=0.01). Acute tubular necrosis leading to delayed graft function (DGF) and acute rejection (AR) cause cellular stress within the transplanted kidney. We found a strong association between up-regulation of MICB and cellular stress, 15/17 biopsies with up-regulated MICB expression had AR and/or DGF (P=0.003). Conclusions. This is the first study demonstrating variable levels of MICB expression in kidneys before transplantation and induction of MICB expression following renal transplantation. MICB expression is associated with HLA class II antigen induction, leukocytic infiltration of the graft and cellular stress in the transplanted kidney. Expression of MICB could contribute significantly to the alloimmune response in mismatched donors and recipients.

Nonglycosidic Agonists of Invariant NKT Cells for Use as Vaccine Adjuvants

Based on the crystal structures of human α‐GalCer–CD1d and iNKT–α‐GalCer–CD1d complexes, nonglycosidic analogues of α‐GalCer were synthesized. They activate iNKT cells resulting in dendritic cell maturation and the priming of antigen‐specific T and B cells. Therefore, they are attractive adjuvants in vaccination strategies for cancer and infectious diseases.

Phage display-derived recombinant antibodies with TCR-like specificity against α-galactosylceramide and its analogues in complex with human CD1d molecules

The glycolipid α‐galactosylceramide (α‐GalCer) is a potent activator of invariant natural killer T (iNKT) cells and has been shown to be an effective agent against cancer, infections and autoimmune diseases. The effectiveness of α‐GalCer and its alkyl chain analogues depends on efficient loading and presentation by the antigen‐presenting molecule CD1d. To monitor the ability of CD1d to present the glycolipids, we have used a phage display strategy to generate recombinant antibodies with T cell receptor‐like (TCRL) specificity against the human CD1d (hCD1d)‐α‐GalCer complex. These Fab fragments were able to detect specifically hCD1d‐α‐GalCer complexes in cell‐free systems such as surface plasmon resonance and ELISA, as well as on the surface of hCD1d+ antigen‐presenting cells (APC) by flow cytometry and immunofluorescence microscopy, the latter of which could also detect intracellular complexes. We show that our TCRL antibodies can stain dendritic cells from CD11c‐hCD1d‐transgenic mice administered in vivo with α‐GalCer and its analogues. Furthermore, the antibody was also able to detect the presentation by hCD1d molecules of analogues of α‐GalCer with the same polar head structure. Using this reagent, we were able to confirm directly that the α‐GalCer analogue C20:2 preferentially loads onto cell surface CD1d rapidly without the need for internalization, while the loading of α‐GalCer is improved with longer incubation times on professional APC. This reagent will be essential for assessing the loading and presenting capabilities of hCD1d of α‐GalCer and its analogues.

Invariant natural killer T cells are not affected by lysosomal storage in patients with Niemann-Pick disease type C.

Invariant natural killer T (iNKT) cells are a specialised subset of T cells that are restricted to the MHC class I like molecule, CD1d. The ligands for iNKT cells are lipids, with the canonical superagonist being α‐galactosylceramide, a non‐mammalian glycosphingolipid. Trafficking of CD1d through the lysosome is required for the development of murine iNKT cells. Niemann‐Pick type C (NPC) disease is a lysosomal storage disorder caused by dysfunction in either of two lysosomal proteins, NPC1 or NPC2, resulting in the storage of multiple lipids, including glycosphingolipids. In the NPC1 mouse model, iNKT cells are virtually undetectable, which is likely due to the inability of CD1d to be loaded with the selecting ligand due to defective lysosomal function and/or CD1d trafficking. However, in this study we have found that in NPC1 patients iNKT cells are present at normal frequencies, with no phenotypic or functional differences. In addi‐tion, antigen‐presenting cells derived from NPC1 patients are functionally competent to present several different CD1d/iNKT‐cell ligands. This further supports the hypothesis that there are different trafficking requirements for the development of murine and human iNKT cells, and a functional lysosomal/late‐endosomal compartment is not required for human iNKT‐cell development.