Polyomavirus large tumor-antigen (LTag) interacts with a number of host molecules involved with cell cycle, including the tumor-suppressor gene product p53 and has been identified as an important target of cancer immunity in murine models[38
]. Early studies in SV40 infected mice have reported on conserved CD8+ T cells immune responses against SV40 LTag[39
]. In humans, however, despite continuous efforts aimed at identifying potential HLA class I restricted LTag epitopes[40
], an epitope-specific cytotoxic immune response against this antigens or its portions is still poorly characterized [42
Upon viral entry in non-permissive cells, in the context of an abortive infection, viral DNA is fully integrated in the host genome or resides in host cells as plasmid, thereby favouring the persistence of the virus without production of progeny virions[8
]. Within these modalities of virus-cell interaction, LTag is the most highly expressed BKV antigen in non permissive infected cells and might encompass viral epitopes generated by the endogenous class I restricted antigen processing pathway and, possibly, targeted by specific CTL responses. Thus, the identification of LTag derived antigenic peptides might provide decisive advances for the analysis of BKV specific immune responses against infected non permissive cells.
Importantly, LTag regions required for viral transformation, including pRb domains aa101–118; p53 domains aa351–450 and aa533–626, accounting for about 80% of the entire sequence, are less likely to be mutated or lost, and are thus highly genetically conserved[44
HLA-class I restricted immune responses against defined viral antigens can be frequently induced by ex vivo stimulation of peptide-specific T cells from seropositive subjects[45
]. Taking advantage of well studied algorithms, in this study we comprehensively analysed CTL responses against BKV LTag-related epitopes in HLA-A*0201+ BKV LTag experienced individuals. In particular, we focused on antigen triggered cytokine gene expression and lytic capacity in BKV specific CTL.
A first important result of our study is represented by the validation of a rapid, previously characterized[31
], screening technology. Indeed, we found a highly significant correlation between IFN-γ gene expression induced by a 3-hour stimulation of CD8+ T cells and the corresponding epitope specific cytotoxic activity detectable following 2–3 week cultures in the presence of peptides and IL-2.
Most interestingly, since antigen triggered cytokine gene expression is detectable after a 3-hour culture only, it is unlikely to be related to primary "in vitro" sensitization, but rather to the stimulation of antigen experienced specific CD8+ T cells, present in the peripheral blood samples under investigation[46
]. Moreover, this pattern of epitope immunorecognition "ex vivo" and following "in vitro" culture was consistently reproducible in serial tests performed on cells from the same donors for over one year, thus suggesting that these sustained responses are not associated to specific phases of the host-virus interaction.
LTag 406, 410 and 579 emerged as the most immunogenic peptides among those studied. The latter appeared to be naturally processed in non professional HLA-A*0201 positive antigen presenting cells expressing LTag. The relatively weak response detected following co-cultures of peptide specific expanded T cells with LTag transfected targets (only 3 of 5 cultures produced detectable levels of IFN-γ following stimulation) could be ascribed either to: i) low expression of LTag protein (in spite the relevant production of LTag mRNA following transient transfection) and subsequent impaired peptide-processing; or ii) T cell anergy due to the lack of professional antigen presenting cells (i.e. mDC).
Four out of five HLA-A*0201+ BKV LTag seropositive donors responded to LTag 579 and LDA data indicate that the frequency of CTLp specific for this peptide may be comparable to that of CTLp specific for the highly immunogenic HLA-A*0201-restricted influenza matrix 58–66 epitope (Michel Adamina, manuscript in preparation), as detectable in seropositive individuals.
CTL specific for antigens from viruses responsible for persistent infections are frequently characterized by specific phenotypic profiles. For instance, EBV or CMV specific CTL responses are elicited by distinct subsets of memory T lymphocytes reported as early effectors for EBV (CD45RA-(+)/CCR7+) or late effectors for CMV (CD45RA+/CCR7-)[47
]. Our studies indicate that BKV LTag 579 specific CTL responders exquisitely belong to a CD45RA+/CCR7+(-) CD8+ T cell population. Indeed "ex vivo" studies clearly demonstrate CD8+ T cells characterized by a relatively dim positivity for LTag 579/HLA-A*0201 pentamers and displaying this phenotypic profile in the peripheral blood of BKV seropositive donors. In addition 2–3 week cultures of separated cell populations indicate that specific CTL can only be generated from CD45RA+ cells. Importantly, even after these culture times, LTag 579 specific CD8+ T cells retain a CD45RA+/CCR7- phenotype, a relatively infrequent event for CTL recognizing BKV unrelated peptides.
Our study demonstrates for the first time that virtually all HLA-A*0201+ BKV seropositive donors mount a powerful CTL response towards epitopes encompassed by a highly phylogenetically conserved region of the LTag implicated in the viral replicative activity and in the p53 mediated control of the cell cycle of host cells.
These results set the stage for further research aiming at identifying appropriate formulations of LTag immunogenic peptides that might be used to stimulate BKV specific CTL responses in patients at risk of infection reactivation and at clarifying whether this specific immunosurveillance fails in patients bearing tumors potentially associated to BKV mediated oncogenic transformation.
Interestingly, these results may possibly also be extended to JCV-seropositive individuals, as already seen for VP1[48
], due to the high homology that the two LTag-p53 binding domains share among the human polyomavirus BK and JC strains. Clearly, in this context, our data may suggest combined use of epitopes from capsid proteins and LTag leading to innovative approaches in the treatment and prevention of human polyomavirus related diseases.