Kylie J. McDonald

Human CD141+ (BDCA-3)+ dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens

The characterization of human dendritic cell (DC) subsets is essential for the design of new vaccines. We report the first detailed functional analysis of the human CD141+ DC subset. CD141+ DCs are found in human lymph nodes, bone marrow, tonsil, and blood, and the latter proved to be the best source of highly purified cells for functional analysis. They are characterized by high expression of toll-like receptor 3, production of IL-12p70 and IFN-β, and superior capacity to induce T helper 1 cell responses, when compared with the more commonly studied CD1c+ DC subset. Polyinosine-polycytidylic acid (poly I:C)–activated CD141+ DCs have a superior capacity to cross-present soluble protein antigen (Ag) to CD8+ cytotoxic T lymphocytes than poly I:C–activated CD1c+ DCs. Importantly, CD141+ DCs, but not CD1c+ DCs, were endowed with the capacity to cross-present viral Ag after their uptake of necrotic virus-infected cells. These findings establish the CD141+ DC subset as an important functionally distinct human DC subtype with characteristics similar to those of the mouse CD8α+ DC subset. The data demonstrate a role for CD141+ DCs in the induction of cytotoxic T lymphocyte responses and suggest that they may be the most relevant targets for vaccination against cancers, viruses, and other pathogens.

Human CD1c (BDCA-1)+ myeloid dendritic cells secrete IL-10 and display an immuno-regulatory phenotype and function in response to Escherichia coli

Human blood myeloid DCs can be subdivided into CD1c (BDCA‐1)+ and CD141 (BDCA‐3)+ subsets that display unique gene expression profiles, suggesting specialized functions. CD1c+ DCs express TLR4 while CD141+ DCs do not, thus predicting that these two subsets have differential capacities to respond to Escherichia coli. We isolated highly purified CD1c+ and CD141+ DCs and compared them to in vitro generated monocyte‐derived DCs (MoDCs) following stimulation with whole E. coli. As expected, MoDCs produced high levels of the proinflammatory cytokines TNF, IL‐6, and IL‐12, were potent inducers of Th1 responses, and processed E. coli‐derived Ag. In contrast, CD1c+ DCs produced only low levels of TNF, IL‐6, and IL‐12 and instead produced high levels of the anti‐inflammatory cytokine IL‐10 and regulatory molecules IDO and soluble CD25. Moreover, E. coli‐activated CD1c+ DCs suppressed T‐cell proliferation in an IL‐10‐dependent manner. Contrary to their mouse CD8+ DC counterparts, human CD141+ DCs did not phagocytose or process E. coli‐derived Ag and failed to secrete cytokines in response to E. coli. These data demonstrate substantial differences in the nature of the response of human blood DC subsets to E. coli.

Hodgkin's Lymphoma Cell Lines Express a Fusion Protein Encoded by Intergenically Spliced mRNA for the Multilectin Receptor DEC-205 (CD205) and a Novel C-type Lectin Receptor DCL-1

Classic Hodgkins lymphoma (HL) tissue contains a small population of morphologically distinct malignant cells called Hodgkin and Reed-Sternberg (HRS) cells, associated with the development of HL. Using 3′-rapid amplification of cDNA ends (RACE) we identified an alternative mRNA for the DEC-205 multilectin receptor in the HRS cell line L428. Sequence analysis revealed that the mRNA encodes a fusion protein between DEC-205 and a novel C-type lectin DCL-1. Although the 7.5-kb DEC-205 and 4.2-kb DCL-1 mRNA were expressed independently in myeloid and B lymphoid cell lines, the DEC-205/DCL-1 fusion mRNA (9.5 kb) predominated in the HRS cell lines (L428, KM-H2, and HDLM-2). The DEC-205 and DCL-1 genes comprising 35 and 6 exons, respectively, are juxtaposed on chromosome band 2q24 and separated by only 5.4 kb. We determined the DCL-1 transcription initiation site within the intervening sequence by 5′-RACE, confirming that DCL-1 is an independent gene. Two DEC-205/DCL-1 fusion mRNA variants may result from cotranscription of DEC-205 and DCL-1, followed by splicing DEC-205 exon 35 or 34–35 along with DCL-1 exon 1. The resulting reading frames encode the DEC-205 ectodomain plus the DCL-1 ectodomain, the transmembrane, and the cytoplasmic domain. Using DCL-1 cytoplasmic domain-specific polyclonal and DEC-205 monoclonal antibodies for immunoprecipitation/Western blot analysis, we showed that the fusion mRNA is translated into a DEC-205/DCL-1 fusion protein, expressed in the HRS cell lines. These results imply an unusual transcriptional control mechanism in HRS cells, which cotranscribe an mRNA containing DEC-205 and DCL-1 prior to generating the intergenically spliced mRNA to produce a DEC-205/DCL-1 fusion protein.

The Novel Endocytic and Phagocytic C-Type Lectin Receptor DCL-1/CD302 on Macrophages Is Colocalized with F-Actin, Suggesting a Role in Cell Adhesion and Migration

C-type lectin receptors play important roles in mononuclear phagocytes, which link innate and adaptive immunity. In this study we describe characterization of the novel type I transmembrane C-type lectin DCL-1/CD302 at the molecular and cellular levels. DCL-1 protein was highly conserved among the human, mouse, and rat orthologs. The human DCL-1 (hDCl-1) gene, composed of six exons, was located in a cluster of type I transmembrane C-type lectin genes on chromosomal band 2q24. Multiple tissue expression array, RT-PCR, and FACS analysis using new anti-hDCL-1 mAbs established that DCL-1 expression in leukocytes was restricted to monocytes, macrophages, granulocytes, and dendritic cells, although DCL-1 mRNA was present in many tissues. Stable hDCL-1 Chinese hamster ovary cell transfectants endocytosed FITC-conjugated anti-hDCL-1 mAb rapidly (t1/2 = 20 min) and phagocytosed anti-hDCL-1 mAb-coated microbeads, indicating that DCL-1 may act as an Ag uptake receptor. However, anti-DCL-1 mAb-coated microbead binding and subsequent phagocytic uptake by macrophages was ∼8-fold less efficient than that of anti-macrophage mannose receptor (MMR/CD206) or anti-DEC-205/CD205 mAb-coated microbeads. Confocal studies showed that DCL-1 colocalized with F-actin in filopodia, lamellipodia, and podosomes in macrophages and that this was unaffected by cytochalasin D, whereas the MMR/CD206 and DEC-205/CD205 did not colocalize with F-actin. Furthermore, when transiently expressed in COS-1 cells, DCL-1-EGFP colocalized with F-actin at the cellular cortex and microvilli. These data suggest that hDCL-1 is an unconventional lectin receptor that plays roles not only in endocytosis/phagocytosis but also in cell adhesion and migration and thus may become a target for therapeutic manipulation.

Differential uptake and cross‐presentation of soluble and necrotic cell antigen by human DC subsets

Cross‐presentation is the mechanism by which exogenous Ag is processed for recognition by CD8+ T cells. Murine CD8α+ DCs are specialized at cross‐presenting soluble and cellular Ag, but in humans this process is poorly characterized. In this study, we examined uptake and cross‐presentation of soluble and cellular Ag by human blood CD141+ DCs, the human equivalent of mouse CD8α+ DCs, and compared them with human monocyte‐derived DCs (MoDCs) and blood CD1c+ DC subsets. MoDCs were superior in their capacity to internalize and cross‐present soluble protein whereas CD141+ DCs were more efficient at ingesting and cross‐presenting cellular Ag. Whilst cross‐presentation by CD1c+ DCs and CD141+ DCs was dependent on the proteasome, and hence cytosolic translocation, cross‐presentation by MoDCs was not. Inhibition of endosomal acidification enhanced cross‐presentation by CD1c+ DCs and MoDCs but not by CD141+ DCs. These data demonstrate that CD1c+ DCs, CD141+ DCs, and MoDCs are capable of cross‐presentation; however, they do so via different mechanisms. Moreover, they demonstrate that human CD141+ DCs, like their murine CD8α+ DC counterparts, are specialized at cross‐presenting cellular Ag, most likely mediated by an enhanced capacity to ingest cellular Ag combined with subtle changes in lysosomal pH during Ag processing and use of the cytosolic pathway.

CD34+ cord blood DC-induced antitumor lymphoid cells have efficacy in a murine xenograft model of human ALL

Acute lymphocytic leukemia (ALL) patients who relapse after transplantation have few therapeutic options. An immunotherapeutic approach that enhances the graft versus leukemia effect may improve their survival. We postulate that cytotoxic T lymphocytes (CTLs) generated from total RNA loaded cord blood CD34+-derived dendritic cells can control the kinetics of leukemic growth in a nonobese diabetic/severe combined immunodeficient (NOD-SCID) mouse model of human ALL. CD34+-derived dendritic cells electroporated with total RNA from an ALL xenograft generate antileukemic CTL with specificity for the ALL xenograft while sparing autologous cord blood mononuclear cells. The CD3+ T-cell compartment of the CTL was dominated by CD4+ T cells, although CD8+ T cells accounted for an average of 30% of the CD3+ T cells present. Expansion of both CD4+ and CD8+ memory and terminal effector memory subsets from predominantly naive cells was evident. Natural killer (NK) cells accounted for an average of 13% of the final antitumor lymphoid cells produced. Blocking experiments confirmed that the CD8+ T-cell compartment was responsible for the antileukemic activity of the polyclonal CTL pool. Administration of antileukemic CTL to NOD-SCID mice bearing ALL xenograft cells was able to delay, but not prevent the growth of ALL in vivo. Coadministration of antigen-loaded antigen-presenting cells did not further improve upon the delay in ALL engraftment kinetics observed with CTL alone. The efficacy of adoptively transferred polyclonal CTL can be improved with coadministration of recombinant human interleukin-2. However, in NOD-SCID mice, the efficacy of these adoptively transferred cells is masked by interleukin-2 stimulation of murine NK cells, which facilitate killing of ALL cells. Our data highlights the role for NK cells in antileukemic responses posttransplant. Collectively, our results support the notion that ALL-specific adoptive immunotherapy could be used clinically and provide an alternative strategy for preventing and treating disease relapse posttransplant and that the success of this therapy is likely to be maximized if given in the setting of minimal residual disease.