Neuroblastoma as a target for effector mechanisms of the immune system

Neuroblastoma (NB) is the most common solid extracranial tumor in children. Despite intensive multimodal treatment, the prognosis for high-risk NB patients remains poor and therefore new treatment modalities are needed. The main aim of my studies was to assess NB as a target for cytotoxic T lymphocyte (CTL)-mediated immunotherapy. CTLs induce target cell death through two major pathways, namely the cytotoxic granule exocytosis- and the death receptor-mediated pathway. While an efficient T cell-mediated lysis through the release of perforin/granzyme-containing cytotoxic granules requires the expression of the relevant HLA class I complexes as well as adhesion molecules on the target cells, killing through the death receptor pathway involves the binding of the ligands to death receptors in the tumor cell membrane, and requires an intact intracellular molecular machinery that conveys the signal to execute tumor cell death. However, a number of features including low or absent surface HLA class I as well as a defective expression of caspase-8, may hamper the development of immunotherapeutic modalities for NB. Nevertheless, strategies to enhance the immunogenicity of NB cells could render them into good targets for CTL-mediated immunotherapy. Alternatively, NB cells can be targeted by means of bystander activated CTLs in an HLA-independent fashion. Both these approaches have been investigated in the studies presented in this thesis.

We demonstrated that retinoids, differentiating agents currently in use as a treatment modality for high-risk NB, act as enhancers of HLA class I antigen processing and presentation as well as increase the susceptibility of NB to CTL-mediated effector mechanisms. We found that retinoids induced the expression of proteolytic and regulatory subunits of the immunoproteasome, increased the half-life of HLA class I complexes, and enhanced the sensitivity of NB cells to both HLA class I-restricted peptide-specific and HLA class I non-restricted lysis by CTLs. Our data suggest that the application of retinoids and CTL-based immunotherapy may be an effective combination for the treatment of NB (Paper I). Though numerous studies have demonstrated the ability of IFNγ to induce components of the antigen processing machinery (APM) in NB, the impact on subsequent tumor recognition by CTLs has not been investigated in detail. Therefore, we investigated the IFNγ-induced effects on selected components of the APM in NB cells as well as on CTL recognition. Although IFNγ-treatment efficiently induced surface HLA class I on NB cells, this effect did not translate into an efficient increase in HLA-restricted killing by CTLs. We suggest two possible molecular explanations for this phenomenon; (i) an IFNγ-induced expression of the inhibitory HLA-E on NB cells, and/or (ii) a decreased susceptibility of IFNγ-treated tumor cells to the cytolytic granule content of CTLs (Paper III).

To investigate whether CTLs may eliminate NB cells in an HLA-independent manner without the involvement of perforin/granzyme-mediated cytotoxicity, we used NB-non-specific CTLs activated on third party targets as well as soluble factors released from these CTLs. We found that both caspasedependent and-independent cell death were induced in NB cells in a bystander manner, and, in addition, TNFα released from activated CTLs was defined as an important effector molecule (Paper II). Besides inducing NB cell death, soluble factors released by activated CTLs modulated the levels of cell surface molecules implicated in HLA-restricted and HLA-independent NB cell recognition, induced the expression of caspase-8, as well as increased the susceptibility of NB cells to death receptor-mediated killing. Importantly, soluble factors released by activated CTLs skewed the surface immune phenotype of both long term cultured and primary NB cells (Paper IV).

Altogether, the results presented in this thesis suggest that recruiting activated CTLs into the proximity of the tumor may have a therapeutic effect in patients with NB. CTLs may simultaneously activate different death pathways and override the “silent” immune phenotype of NB cells. We also suggest that CTL-mediated therapy could be combined with retinoid treatment of NB.

List of scientific papers

I. Vertuani S, De Geer A, Levitsky V, Kogner P, Kiessling R, Levitskaya J (2003). "Retinoids act as multistep modulators of the major histocompatibility class I presentation pathway and sensitize neuroblastomas to cytotoxic lymphocytes." Cancer Res 63(22): 8006-13
https://pubmed.ncbi.nlm.nih.gov/14633733

II. De Geer A, Kiessling R, Levitsky V, Levitskaya J (2006). "Cytotoxic T lymphocytes induce caspase-dependent and -independent cell death in neuroblastomas in a MHC-nonrestricted fashion." J Immunol 177(11): 7540-50
https://pubmed.ncbi.nlm.nih.gov/17114423

III. De Geer A, Rakhmanaliev E, Handke D, Carlson LM, Kiessling R, Kogner P, Seliger B, Levitskaya J (2007). "Interferon gamma-induced changes either do not promote or negatively affect MHC class I-restricted T-cell recognition of neuroblastoma cells." (Submitted)

IV. De Geer A, Carlson LM, Kogner P, Levitskaya J (2007). "Soluble factors released by activated cytotoxic T lymphocytes interfere with death receptor pathways in neuroblastoma." (Submitted)