Download File - JOHN J. HADDAD, Ph.D.

Diagnostic Approaches to Maximize Therapeutic Effect 193
Immune Response Monitoring as Potential Tool for Therapy Guidance
MKC’s cancer vaccine is an active immunotherapy aimed at inducing or augmenting
tumor-specific T cells in vivo that leads to tumor regression and survival
benefit. With the initiation of various vaccination trials, accurate and
reliable assays for testing T-cell function is crucial for the evaluation, comparison,
and further development of these approaches. The cellular immune
responses have been evaluated using methods measuring cytotoxicity, proliferation,
or release of cytokines in a bulk culture. However, these assays often
require in vitro stimulation prior to performing them. A selection bias is automatically
introduced with culturing of the effector cells, and the results subsequently
obtained from these assays may not reflect in vivo T-cell function.
The emergence of ex vivo assays represented by tetramer analysis, ELI-
SPOT assay, and intracellular cytokine staining has significantly improved our
ability to measure T-cell response to vaccine attributing to their capability of
detecting antigen-specific cell at the single cell level and therefore providing
quantitative information. The evolved multiparameter flow cytometry allows us
to characterize T-cell subpopulations and provide a better understanding of
antitumor immunity.
The immune function varies among individuals, and the variation is
amplified among cancer patients. It is common that patients respond to cancer
immunotherapy heterogeneously. Therefore, it is important to monitor each
individual’s immune response to vaccine treatment and adjust the treatment
strategy accordingly to achieve clinical benefit. It is logical to measure the
increase of tumor-reactive T cells, in vivo if any, by tetramer and ELISPOT
assays, after vaccine administration. However, recent findings indicate that
generation of a large in vivo population of tumor-reactive CD8 T cells alone is
insufficient to achieve clinically significant tumor regression. Studies applying
multiparameter analysis of T-cell phenotypes and functions demonstrate that it is
the effective memory response that has a superior antitumor activity (35–37). No
doubt, the multiparameter flow cytometry is a valuable addition to tetramer and
ELISPOT assay for monitoring immune responses to vaccines.
Tetramer Analysis
The use of MHC1/peptide tetrameric technology to directly visualize and
quantify antigen-specific CTLs was first described by Altman et al. in 1996 (38)
in which soluble, fluorescently labeled, multimeric MHC/peptide complex bind
stably, specifically, and avidly to antigen-specific T cells. This assay is easy to
perform; generally 30 minutes staining of tetramer at room temperature is sufficient.
Both fresh and cryopreserved PBMC samples have been successfully
analyzed and have achieved comparable results (39). The tetramer is able to
identify all the T cells specifically recognizing the MHC1/peptide complex
composing the tetramer regardless of their functional status. Since the tetramer
analysis is a flow cytometry–based assay, it can be used together with other cell