Summary:
Prothymosin α (proTα) was initially isolated from rat thymus in 1984. It is, in
principle, a nuclear protein with a distinctive dual role. Intracellularly, it
participates in the regulation of cell proliferation and cell death, while
extracellularly proTα acts pleiotropically, inducing and enhancing immune
responses. In the frame of its immunomodulatory role, both the intact molecule
and its carboxyterminal immunoreactive peptide proTα(100-109) induce the
phenotypic maturation of dendritic cells, which comprise the professional
antigen-presenting cells of the immune system. The purpose of the present PhD
thesis was the investigation of the ability of proTα and proTα(100-109) to act
as adjuvants via activating dendritic cells, focusing on: V. The identification
of the surface binding site(s)/receptor(s) and the intracellular proteins that
are activated downstream thereof and mediate the effect of proTα and proTα(100-
109) on human dendritic cells, VI. The thorough analysis of the phenotype and
functionality of the proTα- and proTα(100-109)-matured dendritic cells and the
type of T cell responses that they induce in vitro,VII. The investigation of
the anticancer activity of proTα and proTα(100-109) in an invivo melanoma model
in mice and the ex vivo analysis of the proTα- and proTα(100-109)-induced
immune responses, and VIII. The association of the two distinct roles
(intracellular and extracellular) of proTα and the investigation of the
possibility that the intact molecule and/or its immunoreactive decapeptide
proTα(100-109) can act extracellularly as «danger signals». First, taking into
consideration recent data reporting that proTα signals through Toll-like
receptors (TLRs) on human monocytes and binds to TLR-4 on murine macrophages,
we sought to investigate whether the same receptor is also activated in human
dendritic cells. Using flow cytometry, we studied TLR-4 surface expression on
dendritic cells differentiated from human peripheral blood monocytes upon their
maturation with LPS, proTα or proTα(100-109) for specific time intervals. We
observed that all three maturation factors led to a similar transient increase
of TLR-4 expression on the dendritic cells’ surface. Using Western blot
analysis, we also observed that the increased TLR-4 expression was accompanied
by an analogous intracellular activation, since the expression levels of the
TLR-4 adaptor proteins, MyD88, TIRAP and TRIF, were found elevated upon
stimulation with LPS, proTα or proTα(100-109). These data comprise the first
evidence that the ability of proTα and proTα(100-109) to mature human dendritic
cells, similarly to LPS, is at least partially mediated by TLR-4, downstream of
which the MyD88- and TRIF-dependent signalling pathways are activated. Our next
goal was to investigate whether dendritic cells that mature in the presence of
proTα or proTα(100-109) express specific dendritic cell markers and produce T
cell stimulating cytokines. In accordance with already published data, proTα
and proTα(100-109) enhanced HLA-DR, CD11b, CD80, CD83, CD86 and CD40 surface
expression on dendritic cells, to levels comparable to those achieved by LPS.
These expression levels were also comparable to those induced in the presence
of TNF-α, a maturation factor which has been widely used in dendritic
cell-based clinical trials. Moreover, the analysis of dendritic cell culture
supernatants showed that the proTα- and proTα(100-109)-matured dendritic cells,
similarly to standard (LPS- or TNF-α-matured) dendritic cells, produced in
principle the pro-inflammatory cytokine IL-12, indicating their ability to
promote ΤH1-type of immune responses. This ability was further verified by
studying the functionality of these dendritic cells in vitro. In detail, proTα-
and proTα(100-109)-matured dendritic cells were used to stimulate autologous T
cells, both memory and naive, in the presence of viral and tumorderived
peptide-antigens, respectively. The assessment of helper T cell cytokine
production via multi-color flow cytometry in both experimental systems,
revealed enhanced production of pro-inflammatory (TNF-α, IFN-γ, IL-2) and
reduced production of anti-inflammatory (IL-4, IL-5, IL-10, IL-13) cytokines,
thus verifying the polarization toward ΤH1-type immune responses. The cytokine
profile of CD8+ T cells was analogous, indicating their differentiation to
TC1-type. Moreover, the tumor-specific cytotoxic T cells generated in the naive
T cell stimulation cultures were polyfunctional and exhibited an
antigen-specific MHC class I-restricted cytotoxicity. Similarly, the generated
tumorspecific T cell lines exhibited an antigen-specific MHC class
II-restricted proliferation ability. In continuation to our in vitro results,
we studied proTα’s and proTα(100-109)’s ability to induce tumor-reactive immune
responses in vivo in a melanoma model. For the development of mouse melanoma
tumors, we used B16.F1 cells, as these cells are known to be antigenic,
immunogenic and can form solid tumors when inoculated subcutaneously in
syngeneic C57BL/6 mice. Both proTα and its immunoreactive peptide proTα(100-
109) were administered to the animals in conjunction with B16.F1-derived tumor
peptide-antigens, eluted from the same cancer cell line and anticipated to
trigger the generation of tumor-reactive immune responses in vivo. We developed
two protocols, one of heterochronous and one of simultaneous administration of
immunoenhancing factors and cancer cells. By recording the development of the
tumors, we observed that administration of proTα and proTα(100-109) was
beneficial, as it reduced tumor growth rates and prolonged the overall survival
of the animals in both immunotherapeutic protocols. Of interest, ex vivo
analysis of splenocytes isolated from selected animals of all groups verified
that the observed inhibition of melanoma cell growth was attributed to the in
vivo induction of tumor antigen-specific, as well as of non-specific immune
responses, mediated by activated T and NK/LAK cells, respectively. According to
the literature, intracellularly, proTα regulates cell proliferation and exerts
an anti-apoptotic role. According to the findings of the present PhD thesis,
the same polypeptide and its immunoreactive decapeptide proTα(100-109),
extracellularly, enhance cell-mediated immune responses both in vitro and in
vivo. In an attempt to link these two roles, we studied the conditions under
which proTα(100-109) is produced and exocytosed in order to exert its
extracellular role. It is already known that early during apoptosis, proTα is
translocated to the cytoplasm, where it is truncated by activated caspases 3
and 7 at aminoacid (D) 99. ProTα’s fragment 1-99 has already been detected in
the cytoplasm, while to date proTα(100-109) has not been intracellularly
detected. Assuming that the decapeptide could be possibly released/excreted
extracellularly, we first showed that proTα(100-109) remains intact and is not
further cleaved by activated proteases during apoptosis. Further, the
extracellular medium of apoptotic HeLa cells was collected and analyzed using
RP-HPLC and mass spectrometry. In the mass spectras acquired, a peak
corresponding to the theoretical molecular weight of proTα(100-109) was
recorded, indicating that the decapeptide is exocytosed under apoptotic
conditions, via an, as yet, unknown mechanism. Of interest, as we additionally
showed, the extracellular release of proTα(100-109) is associated only with
apoptosis and not with other types of cell death, such as necrosis or oncosis.
In summary, in the present PhD thesis, we present significant data on the
immunoenhancing activity of proTα and proTα(100-109). Studying the expression
of TLR-4, as well as that of the intracellular proteins participating in
signalling pathways downstream thereof, we verified that this receptor is
mediating, at least partially, the maturation of dendritic cells, induced by
the two peptides. The proTα- and proTα(100-109)-matured dendritic cells express
high levels of MHC class II and costimulatory molecules and produce
pro-inflammatory cytokines. The assessment of their functionality showed that,
apart from phenotypically mature, these dendritic cells are also functionally
immunocompetent and can stimulate antigen-specific T cells in vitro, regardless
of their cell type (memory and naive T cells) and/or the nature of the antigen
(viral- and tumor-derived). The immune responses that they induce are mediated
by TH1- and TC1-type Τ cells. In parallel, both intact proTα and its
immunoreactive fragment proTα(100-109) enhanced, in principle, tumor-specific
immune responses in an in vivo mouse melanoma model, leading to the reduction
of tumor growth and the prolongation of the overall survival of the animals.
Combining the afore presented data with the first evidence for the specific
exocytosis of proTα(100-109) under apoptotic conditions, the results of the
present PhD thesis support the ability of proTα and proTα(100-109) to act as
«danger signals», inducing and promoting immune responses in vivο. If this
hypothesis holds true, new perspectives are opened as for the potential use of
the two peptides as adjuvants to improve, in principle, the efficacy of
anticancer vaccines that are based on tumor antigens administered in
conjunction with dendritic cells.
