Eliciting cytotoxic T lymphocytes against human laryngeal cancer-derived antigens: evaluation of dendritic cells pulsed with a heat-treated tumor lysate and other antigen-loading strategies for dendritic-cell-based vaccination
DC vaccines take advantage of the potent antigen-presenting capacity of DCs to stimulate
primary and secondary T and B cell immune responses. Clinical trials have been performed
for several advanced cancers such as melanoma and renal, prostate, and colon cancers,
which have shown encouraging results 5]–7]. DC vaccines targeting mutated proteins in the p53 pathway 22] and human papilloma virus peptides have been produced for the treatment of HNSCC
22], 23]. These TAA-loaded DCs stimulate immunological and clinical responses in certain patients.
However, a significant proportion of patients remain unresponsive to immunomodulatory
therapy.
Unlike specific TAAs, whole tumor cells or their derivatives may better apply to various
tumors for which few or no defined tumor-specific antigens are available, such as
for laryngeal cancer. Furthermore, whole tumor cell immunizations result in polyvalent
stimulation of both CD4+ Th cells and CD8+ CTLs against a broad range of Ags. Therefore,
DCs loaded with Ags in this manner may be used to treat laryngeal cancer.
Among the current methods for pulsing DCs with whole tumor-derived materials, tumor
cell lysates, which are prepared by repeated cycles of freezing and thawing with solid
debris removed by centrifugation, have been widely applied in early stage clinical
trials. However, an increasing number of trials have shown that the immune response
elicited by DC-TSL does not generate a sufficient antitumor potency 10], 13], 14]. Recently, several studies have demonstrated that freeze-thawed lysates suppress
DC maturation and function in vitro, and are ineffective for loading DCs for therapies
in vivo 15], 16]. Our preliminary study also suggests that DCs cultured in the presence of human laryngeal
cancer-derived lysates decreased surface expression of B7–1, B7–2, and MHC molecules,
and inhibit T cell proliferation (data not shown). Potential explanations for these
findings include the use of different tumor stains, alternate vaccination schedules,
and the use of DCs at various stages of maturity 10].
To elicit an effective immune response, we used three strategies for loading DCs with
tumor-derived Ags and compared their ability to stimulate CTL activity. The loading
strategies included using DC-TCS, DC-TSL, and DC-ITC. Our results show that human
laryngeal cancer-derived TSL was an effective TAA source for pulsing DCs. DC-TSL not
only induced the most potent CTL activity, but also induced the strongest MHC class
I TAA-specific T response (proliferation of TAA-specific CD8
+
T cells), the highest expansion of TAA-specific T cells (based on the number of TAA-specific
T cells), and the strongest Th1 cytokine response (elevated levels of IFN-? and IL-2
production). Interestingly, DC-TSL induced both class I and class II TAA-specific
responses, suggesting that DC-TSL are capable of both class I and II cross-presentation.
These results emphasize that the choice of Ag-loading strategy is critical to the
strength of the immune response.
We identified alterations in the DC phenotype after treatment with tumor-derived Ags.
DC-TSL expressed the highest level of HLA-DR and CD86, whereas DC-ITC also had elevated
HLA-DR expression but expressed the lowest levels of co-stimulatory molecules (CD80,
CD86, and CD40). HLA-DR expression on the surface of DC-TCS remained at the same level
as that in DC controls. A significant body of evidence has shown that stimulating
a stress response in tumor cells increases the production of HSPs, which may expand
the repertoire of TAAs and enhance TAA delivery to professional antigen-presenting
cells (APCs) 17], 24], 25]. Additionally, stress-induced HSPs stimulate DCs and induce APC cytokine and chemokine
secretion 26]–29]. Our findings support the idea that the stressed lysis strategy has advantages by
providing a larger TAA repertoire. Moreover, elevation of co-stimulatory molecules
such as CD86 may enhance DC capacity to prime T cell responses. In contrast, cell
culture supernatants may not contain sufficient or adequate Ags secreted by laryngeal
cancer cells. Although one study reported that DC antigen presenting function can
be improved by supernatants from retinoblastoma cells 18], it is not surprising that laryngeal cancer has a distinct tumor milieu to interact
with DCs. LSCC cells may evade the host immune system through manipulation of their
own immunogenicity, production of immunosuppressive mediators, and promotion of immunomodulatory
cell types 30]–32]. Consistent with some studies 33], there was a reduction of CD80, CD86, and CD40 in DC-ITC. Various mechanisms through
which intact tumor cells suppress DC functions have been described, including cytokines
(e.g., TGF-?, IL-10, and vascular endothelial growth factor), ceramide 34], and other tumor-derived lipids 35]. However, a complete understanding of the mechanisms by which DC suppression functions
will require additional study.
Studies have shown that CD8
+
and Th1 cells play an important role in controlling tumor growth. For example, CD8
+
T cells mediate antitumor immunity 36]. Th1 cells, a subset of CD4
+
T cells, constitutively express IFN-? and TNF-?, and play a role in priming tumor-specific
CTLs through the release of soluble IL-2 in the proximity of CTLs 37]. In addition, induction of MHC class I tumor-specific immunity requires epitope linkage
between Th1 and CTL epitopes, which is important for CTL induction 38]. In the current study, DC-TSL pulsing was the only effective method to prime CD8
+
and CD4
+
T cells. This result indicates that a TSL is not only processed for MHC class I presentation,
but also cross-presented by the MHC class II pathway, consistent with the elevation
of class II molecules (HLA-DR) on DC-TSL. Further experimentation showed that both
the percentage and number of Th1 cells (including IFN-?- and IL-2-secreting cells)
were increased in T cell and DC-TSL co-culture, which strongly supports the choice
of TSL-pulsed as a promising CTL activator against laryngeal cancer. It is worth noting
that the stronger and broader T cell response induced by DC-TSL may benefit from production
of HSPs, such as HSP70 and HSP90 39], 40], whose antitumor activity is exerted through various mechanisms. The inefficiency
of DC-ITC to induce Ag-specific T cell responses can be explained by the inefficient
cross-presentation of TAAs by DCs that express low levels of co-stimulatory molecules.
Although apoptotic tumor cells induced by irradiation effectively prime APCs in vitro,
they likely cannot stimulate DCs to generate an antitumor immune response in the absence
of additional maturation 41].
The incompetence of DC-TCS, which express low levels of MHC molecules (e.g., HLA-DR)
and maturation markers (e.g., CD83), may result from inhibition by the tumor milieu
and fewer laryngeal cancer cell-secreted Ags. Although CTL activity of DC-TCS was
lower than that of DC-TSL, which induced the highest as expected, there was no significant
difference between them. The increased CTL activity of DC-TCS-stimulated PBLs may
be due to more active natural killer and natural killer T cells, which comprise the
majority of non-CD8 T lymphocyte effector cells 42]. Additionally, to ensure that the Ag-MHC complex was recognized by CTLs, we used
Ag-pulsed DCs as the source of MHC-matched target cells, which may not accurately
reflect tumor cell susceptibility to CTL lysis 43].