Effects of intracellular and extracellular heat shock proteins on anti-tumor immune responses

The goal of this study was to investigate the function of the heat
shock protein 70 family members, expressed in tumors under
physiological and stress conditions and to dissect their role in
tumor immune recognition as a function of intra- versus
extracellular location. Another goal was to investigate whether
heat-treatment at clinically relevant thermal doses affects the
immunophenotype of a given tumor, as defined by tumor cell
sensitivity to immune effector cells. For these questions, the
human melanoma system was selected because it is well characterized
with regards to tumor-associated antigens, like tyrosinase and
Melan-A/MART-1, their epitopes and restriction elements for MHC
class I presentation. In the first part of the study the focus
specifically was on the time-temperature dependent effects of heat
exposure. Two different thermal doses (41,8°C/120 minutes and
45°C/22 minutes) were selected that mimic the heterogeneity of the
achieved temperature distribution within the tumor and the
time-temperature dependent changes were determined in: a) antigen
expression (tyrosinase and Melan-A/MART-1) at the protein and mRNA
level; b) expression of the inducible HSP70 and the constitutive
HSC70; c) processing and presentation of tyrosinase and MART-1 via
MHC class I; d) susceptibility of melanoma cell lines to cytotoxic
T lymphocytes like CD8+ T cells, LAK and NK cells. It was
demonstrated that HSP70 and antigen expression display distinct
expression and kinetics that reflect the thermohistory of the
cells, i.e. exposure to high or low thermal doses. Immunologically,
a low thermal dose did not alter immune recognition of the cells
despite the fact that intracellular HSP70 and tyrosinase protein
were upregulated. High thermal dose induced a pleiotropy of
effects, including stronger upregulation of HSP70 and tyrosinase
protein but downregulation of tyrosinase at mRNA level. Concordant
with reduced HLA-A2 surface expression and tyrosinase mRNA levels,
immune recognition of the heat-treated cells was initially reduced,
but pretreatment levels were restored after 72 hours of recovery.
The observation that tumor cells treated with temperatures below
the breakpoint temperature maintain an immunological homeostasis
during the heat shock response is of critical importance for the
clinical application of hyperthermia in the treatment of tumors. In
the second part of the study, the ability of HSP70 to cross-present
a naturally expressed human tumor antigen, tyrosinase, that is of
low immunigenicity, a situation that more closely resembles the
patient situation was investigated. It was demonstrated that
HSP70-peptide complexes (HSP70-PC) purified from
tyrosinase-positive (HSP70-PC/tyr+) but not from
tyrosinase-negative (HSP70-PC/tyr-) melanoma cells deliver the
tyrosinase antigen to immature DCs for MHC class I restricted T
cell recognition. T cell stimulation by HSP70-PC/tyr+ incubated
with immature DCs with was very efficient even without additional
DC maturation signals (e.g. exogenous TNF-?) demonstrating the
ability of tumor-derived HSP70-PC to act as a chaperone for
peptides and a signal for DC maturation. HSP70-PC in exerting both
functions on DCs, delivering antigens and maturing DCs, ensures
that the peptides that are delivered to the DCs are presented in an
immunogenic context optimal for T cell stimulation. In conlusion,
induction of intracellular heat shock proteins (HSPs) by heat does
not interfere with the tumor immune recognition and when HSPs are
expressed extracellularly they acquire immunostimulatory
properties. These observations open new perspectives for the
application of hyperthermia in combination with HSP-based vaccine
in the treatment of solid tumors.