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

46 4 T Cells In Tumor Immunity
to T cells by the HLA-A2 molecule. About 60 % of HLA-A2 + melanoma patients have
a vigorous response that becomes detectable with tetramers in 1-week peptide-stimulated
cultures of PBMCs [66]. Responses to several other antigens also become detectable
in this short-term assay, including gp100, NY-ESO-1 157±165 [67] and
MAGE-A10 [68]. However, as mentioned above, other responses occur at much lower
frequencies such as the MAGE-A3.A1-specific T cells. In this case, a frequency below
one in 1000 000 was estimated based on polymerase chain reaction with clonotypicspecific
primers [69].
4.4.2
Evidence of Tumor Antigen-specific T Cell Responses at the Tumor Sites
The successful use of TILs as a rich source of tumor-reactive CTL lines and clones
was an early indication of selective accumulation of tumor antigen-specific T cells at
the tumor sites, particularly in melanoma, renal cell carcinoma or ovarian carcinoma.
Several TIL lines obtained by continuous culture of melanoma TILs in highdose
IL-2 were used to clone several melanoma associated tumor antigens including
gp100, tyrosinase and Melan-A/MART-1 [70]. When it became possible to directly address
the presence of antigen-specific T cells in melanoma using fluorescent tetramers,
it was clear that Melan-A/MART-1tetramer + CD8 + lymphocytes were readily
detectable at relatively high frequencies in both TILs and tumor-infiltrated lymph
nodes (TILNs) [71]. As illustrated in Fig. 4.1, these frequencies can be very high. In a
series of HLA-A2 + TILNs analyzed ex vivo, most lesions had detectable high frequencies
of Melan-A + CD8 + lymphocytes with frequencies directly ex vivo ranging around
1±15% of the CD8 + subpopulation.
High as the frequencies of Melan-A/MART-1-specific T lymphocytes may appear,
they still constitute a numerically small contingent of specific T cells in the metastasis
cell suspension analyzed directly ex vivo. As illustrated in Fig. 4.2 in cartoon fashion,
the majority of the cells recovered from reducing the metastatic lymph nodes to
single-cell suspensions are melanoma tumor cells. Only 10 % or less of the cells may
be lymphocytes. Most of these cells are CD3 + T cells when analyzed by flow cytometry
and, in the majority of cases analyzed in our laboratory, over 80 % of these are sin-
Fig. 4.1 Melan-A/MART-1 tetramer + lymphocytes accumulate
at tumor sites. Using tetramers, we have observed
that Melan-A/MART-1-specific Tcells are frequently accumulated
at metastatic melanoma lesions. Here,TILs from
a metastasis in the back from a HLA-A2 + melanoma patient
were stained with A2/Melan-A tetramers and anti-
CD8 antibody ex vivo. Among the CD8 Tcells in the lesion,
12% were specific for the Melan-A peptide directly ex vivo,
i. e. in a cell suspension freshly prepared by dissociation
of the tumor mass. These data confirm previous findings
that patients with metastatic melanoma develop substantial
CD8 Tcell responses against their tumor [71]. Reprinted
with permission from [50]