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

4.4 Monitoring the Spontaneous CTL Responses to Tumor Antigens
says most often used were chromium release to measure cytolytic activity and an
ELISA-based detection of specific release of IFN-g. It became obvious that melanoma
patients frequently responded to stimulation with melanoma differentiation antigens
such as Melan-A/MART-1or gp100 [56, 57]. In contrast, there was an apparent
paucity of responses to the two MAGE-derived peptides [58]. An in-depth search for
the CTL precursors specific for the MAGE-A3 peptide indicated that they occur at
very low frequencies in HLA-A1individuals [59].
The next wave of screening for tumor antigen-specific T cells was performed with
fluorescently labeled class I MHC/peptide complexes commonly referred to as tetramers
[48, 49]. Ex vivo, a high frequency of A2/Melan-A/MART-1tetramer + CD8 + T
lymphocytes is readily detectable in two-thirds of HLA-A2 + melanoma patients [60].
Interestingly, a comparable proportion of healthy HLA-A2 + individuals also contain a
high frequency of these Melan-A/MART-1-specific T cells in their circulating CD8
compartment. The mean frequency is around 0.07%, which implies the presence of
one specific cell in 1400 CD8 + lymphocytes. Extensive characterization of the surface
phenotype of these cells directly ex vivo indicates that in healthy donors they display
a functionally naive phenotype. This is characterized mainly by the expression of the
high molecular weight of the common lymphocyte antigen, CD45RA and the CCR7
chemokine receptor. In addition, these cells are also CD45RO ± , CD28 + , CD27 + ,
CD57 ± , CD62L high . In contrast, in about 30% of melanoma patients with circulating
Melan-A/MART-1tetramer + T cells, these express a mixed surface phenotype. Indeed
variable proportions of these T cells display characteristics of an activated/memory
phenotype (CD45RA low , CCR7 ± ) [60, 61]. The majority of these cells are also
CD45RO high , CD28 + , CD27 + .
Apart from the Melan-A/MART-1specific Tcell repertoire, few other tumor antigen
specificities are directly detectable ex vivo in the circulation with tetramers. In one report,
a high frequency of tyrosinase-specific T cells were found to make up to 2% of
the circulating CD8 + T lymphocyte pool. These cells displayed the characteristics of
activated cells but were found to be profoundly anergic in in vitro functional assays
of antigen recognition [62]. However, the latter seems to be a special case because
other studies, while confirming the existence of high levels of HLA tumor antigen
peptide-specific T cells, fail to detect major functional defects [63±65].
The apparent paucity of directly detectable HLA-tumor antigen peptide tetramer +
lymphocytes, other than Melan-A/MART-1, in the blood of the majority of cancer patients
may reflect either a specific lack of responsiveness to CTL-defined tumor antigens
or low frequencies of specific T cells below the detection limit of tetramers. In
this regard, the detection limit for tetramers is imposed largely by background in the
flow cytometers and it may be around one in 5000 lymphocytes. Purification of
CD8 + from PBMC to close to 100% purity brings the possibility to establish a power
of detection down to a frequency of 0.02% within this subpopulation. This limit may
vary for different tetramers and may depend on the relative quality of the commercially
available fluorescent streptavidin conjugates. Introduction of a single round of
in vitro stimulation with peptide allows us to expand the tumor-specific T cells to a level
that is easily detectable by tetramers. This turned out to be clearly the case for a
number of tumor antigens starting by the tyrosinase peptide 368±376 and presented
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