Immunological recognition and tumor escape mechanisms of ovarian carcinoma

Ovarian carcinoma is a leading cause of cancer mortality and there is an urgent need for new and better therapeutic modalities that result in improved long-term outcome for this severe malignancy. One promising treatment modality for patients suffering from ovarian carcinoma is immunotherapy. Several different immunotherapeutic approaches targeting ovarian carcinoma, including infusion of monoclonal antibodies, adoptive transfer of T cells and administration of tumor vaccines, are currently being tested in the clinic. To date, the clinical efficacy of these treatments has been limited, partially because of the development of tumor cell escape and induction of immune suppression. In order to improve the success rate of future immunotherapy protocols it will be important to gain a further understanding of the molecular mechanisms underlying the escape of ovarian carcinoma from the immune system.

This thesis deals with several mechanisms that all lead to lack of immune recognition, and describes one possibility of exploiting this new knowledge in the design of alternative treatment strategies.

The first part of my thesis demonstrates that metastatic ovarian carcinoma often exhibit heterogeneous human leukocyte antigen -A2 (HLA-A2) expression. The down-regulation may reflect a specific and progressive loss of this HLA allele. One underlying molecular mechanism was found to be haplotype loss, associated with the presence of HLA-A2-restricted HER-2/neu specific Tcell immunity. Based on these findings, this study suggested that anti-tumoral cytotoxic T lymphocytes (CTL) favor tumor escape variants lacking HLA-A2 expression, which is required for most TceU-based anti-tumoral immunotherapeutic strategies. Moreover, our studies also showed that short-term tumor cell lines from patients with advanced ovarian carcinoma (OVACs) were protected from lysis by peptide- and allospecific CD8+ T cells upon interferon-γ IFN-γ) treatment. This paradoxical phenomenon was dependent on enhanced inhibitory signaling via CD94/NKG2A receptors expressed on the CTL. IFN-γ treatment of OVACs induced HLA-G expression, and data suggested that the underlying mechanism of protection was increased surface expression of HLA-E molecules, binding a peptide derived from the leader sequence of HLA-G. This study reveals that IFN-y modulation may shift the balance of triggering and inhibitory signals leading to tumor escape from CTL-mediated immunity.

Natural killer (NK) cell function in cancer patients is often impaired and we describe how down-modulation of the expression of multiple activating receptors on NK cells may be one underlying mechanism. We found that in particular DNAM-1, which is one of the activating receptors, was severely down-modulated in tumor associated NK cells as compared to NK cells in autologous peripheral blood and blood from healthy donors. NK cells in the tumor environment also had lower expression of CD16 and the costimulatory receptor 2B4 while exhibiting moderately increased levels of both NKG2D and the natural cytotoxicity receptor NKp46. There was also a significant overrepresentation of regulatory C1356bright NK cells in ascites. These complex perturbations resulted in an impaired functional capacity of tumor associated NK cells as demonstrated by their poor ability to recognize K562 cells. These results provide mechanistic insights into the failure of innate immunity to control progression of ovarian carcinoma.

Loss or reduction of HLA class 1 expression should render tumor cells susceptible to NK cell lysis in agreement with the missing-self hypothesis. Indeed, freshly isolated ovarian carcinoma cells triggered degranulation of resting allogeneic NK cells. This lead to detectable levels of granzyme B and caspase-6 activation in the tumor cells and induction of significant tumor cell lysis. Ovarian carcinoma cells exhibited ubiquitous expression of the poliovirus receptor (PVR, a ligand for DNAM-1) and sparse/ heterogeneous expression of NKG2D ligands. Blocking experiments suggested that DNAM-1 engagement was critical for the direct NK cell recognition of ovarian carcinoma, while NKG2D and natural cytotoxicity receptor signaling resulted in a complementary contribution to tumor cell recognition. These results demonstrate that resting NK cells readily recognize and kill freshly isolated human tumor cells, and identify ovarian carcinoma as a potential target for adoptive NK cell-based therapy.

In conclusion, this work provides new insights into the mechanisms of tumor escape of ovarian carcinoma and describes successful targeting of this tumor type by resting allogeneic NK cells.

List of scientific papers

I. Norell H, Carlsten M, Ohlum T, Malmberg KJ, Masucci G, Schedvins K, Altermann W, Handke D, Atkins D, Seliger B, Kiessling R (2006). Frequent loss of HLA-A2 expression in metastasizing ovarian carcinomas associated with genomic haplotype loss and HLA-A2-restricted HER-2/neu-specific immunity. Cancer Res. 66(12): 6387-94.
https://doi.org/10.1158/0008-5472.CAN-06-0029

II. Malmberg KJ, Levitsky V, Norell H, de Matos CT, Carlsten M, Schedvins K, Rabbani H, Moretta A, Soderstrom K, Levitskaya J, Kiessling R (2002). IFN-gamma protects short-term ovarian carcinoma cell lines from CTL lysis via a CD94/NKG2A-dependent mechanism. J Clin Invest. 110(10): 1515-23.
https://doi.org/10.1172/JC1200215564

III. Norell H, Carlsten M, Schedvins K, Ljunggren HG, Kiessling R, Malmberg KJ (2006). Altered Receptor Repertoire and Impaired Function of Tumor Associated NK Cells in Ovarian Carcinoma. [Manuscript]

IV. Carlsten M, Bjorkstrom N, Norell H, Bryceson Y, van Hall T, Bauman B, Hanson M, Schedvins K, Ljunggren HG, Malmberg KJ (2006). Direct Recognition of Ovarian Carcinoma by Resting Human Natural Killer cells. [Submitted]