Cancer Immune Therapy Edited by G. Stuhler and P. Walden ...

44 4 T Cells In Tumor Immunity
cells [45, 46]. In one case, several mimotopes efficiently recognized by a CTL clone directed
against a cutaneous T cell lymphoma (CTCL) in an HLA-B8-restricted fashion
were identified. Moreover, close to 80 % of a panel of HLA-B8 CTCL patients appeared
to have detectable frequencies of mimotope-reactive CD8 T cells in the circulating
lymphocyte compartment. However, the native tumor antigenic peptide could not be
identified in the current gene databases [45]. In the other case, a validation of the
combinatorial peptide library approach was conducted using tumor-reactive CTL
clones of well-defined specificity and restricted by the commonly expressed HLA-A2
molecule [46]. To better guide the identification of the stimulating peptides present in
the highly complex peptide mixtures of the libraries, a biometric matrix was designed
[47]. This is used to search protein databases. Millions of peptides are retrieved with a
score that reflects the relative T cell-stimulating weight. Clearly, the antigenic peptide
is consistently placed among the first thirty peptides in the list of peptides ranked according
to their score. Although encouraging, these results indicate the need to
further refine this promising approach to Tcell ligand(s) identification.
4.4
Monitoring the Spontaneous CTL Responses to Tumor Antigens
Knowledge of the molecular identity of CTL-defined tumor antigens opened the possibility
to track well-defined antigen-specific CTL responses in cancer patients.
Again, technological advances in the detection of antigen-specific T lymphocytes
have greatly facilitated monitoring CTL responses directly ex vivo without the need
for repeated in vitro stimulation to obtain expansion of the specific T cells. Indeed, a
direct approach for the visual identification of antigen-specific CD8 T cells was introduced
for the first time in 1996 [48]. It is based on the use of soluble and fluorescently
labeled tetramers of MHC class I molecules complexed with the antigenic
peptide of interest [49, 50]. The question we have tried to address is whether the specific
CTL clones isolated from a limited number of cancer patients, for tumor antigen
identification, reflect frequent responses arising in all the patients with the appropriate
expression of both antigen and MHC presenting allele or whether, in contrast,
they represent only rare responses in isolated cases.
4.4.1
Monitoring Specific CTL in the PBMC Compartment
The initial screening was obviously performed using the first tumor antigenic peptides
that became available after the identification of the genes encoding tumor antigens,
i. e. MAGE-A1 161±169 presented by HLA-A1 [51], MAGE-A3 168±176 also presented
by HLA-A1[52] and several melanoma differentiation antigens presented by
the HLA-A2 molecule [53±55]. The most widely used screening procedure was applied
to the latter antigens using PBMCs. It consisted of three or more rounds of
weekly in vitro stimulations with peptide and cytokines followed by an assay to assess
functionally active antigen-specific T cells expanded in this culture system. The as-