Immunotherapy for Infectious Diseases

Dendritic Cells 105
chaperones such as heat shock proteins (e.g., hsp96) to deliver antigens via the class I
pathway (96). Finally, as noted earlier, DCs express the CD1 family of antigens, which
appear to play important roles in the presentation of nonprotein antigens to T-cells.
As noted above, a number of studies indicate the existence of DC subpopulations.
Some controversy remains as to whether these subpopulations represent distinct lineages
or cells in different stages of maturation. Regardless of the answer, the possibility
that the DC subpopulations differ in their functions has significant implications for
DC-based treatment strategies. In humans, the classical “myeloid” DCs, which constitute
the majority of circulating DCs and DCs in the periphery, are derived from a committed
precursor of granulocyte/monocyte lineage. Myeloid DCs appear in the blood
as lymphoid-appearing cells that express high levels of MHC class II antigens and lack
most lineage-specific markers. Myeloid DCs can also be derived from several cell
types previously thought to be terminally committed. For example, monocytes and
committed granulocyte precursors can differentiate into DCs when exposed, in vitro,
to appropriate combinations of cytokines including granulocyte/macrophage colonystimulating
factor (GM-CSF) and TNF-�, with or without IL-4 (97,98). In addition to
myeloid DC, a human DC subpopulation (plasmacytoid DC) expressing high levels of
CD123 (IL-3 receptor) and CD4 and lacking the CD11c myeloid DC marker has been
described (99). These CD123� precursors require IL-3 for their survival and an activation
signal, such as CD40L, for maturation. They reportedly bias CD4� T-cell priming
to a Th2 response, in contrast to the classical myeloid CD11c� DC, which induce
a Thl biased response (100). However, viral infections predominantly induce a Thl
response (101). These CD123 hi DC also play an important role in innate immunity by
producing type 1 interferons upon viral exposure (33).
Recent reports have appeared describing CD8�� DCs in the lymph nodes and
spleen of mice. These DCs appear to bias CD4� T-cell priming to a Th1 response,
whereas CD8� negative sign DCs appear to bias toward a Th2 response (102,103).
Because CD8 is primarily expressed on a subset of T-cells, these CD8� DC have been
postulated to be of lymphoid origin. However, recent studies from our group show
clearly that these cells are of myeloid origin and that CD8 is induced on the cells when
they migrate from the periphery to lymphoid organs (104). No human CD8� DCs have
been described. Human DCs derived from CD10� lymphoid precursors have been
reported, although their role is not clearly understood (105,106).
METHODS OF DENDRITIC CELL ENRICHMENT
Human DC precursors in blood constitute about 1% of peripheral blood mononuclear
cells (30,107). The low frequency of such DCs and the lack of specific DCs markers
have been barriers to the isolation of sufficient numbers of DCs for routine study
let alone development of DC vaccines. However, DCs can be enriched from the blood
using density-based purification (46). Most DC precursors present in peripheral blood
are lymphoid-appearing cells that can be separated from monocytes on the basis of
their different buoyant densities. Our group currently uses centrifugation through a
solution of Percoll for this purpose. After 24–48 hours of culture, the buoyant density
of DC precursors decreases, enabling their separation from lymphocytes by centrifugation
through solutions of metrizamide, Nycodenz, or Percoll. Our group has made
extensive use of this approach to prepare DCs for clinical trials (46,108).