Research Project On The Role Of The Innate Immunity In The Pathogenesis Of Pediatric Malignancies And Autoimmune Disease

Project location: Italy, Rome
Project start date: July 2009 - Project end date: January 2010
Project number: 2009-41
Beneficiary: Arcobaleno ONLUS


Timeline of the present activity report:1-3-2011 30-11-2011


Natutal Killer (NK) cells are professional cytotoxic cells capable of eliminating hematological and epithelial malignant cells in vitro and in vivo. The role of NK cells in human haematological and epithelial malignancies is not completely understood. We have recently found that NK cells barely infiltrate epithelial tumors including colorectal carcinoma (CRC) and renal cell carcinoma (RCC). Further studies, carried out on tumor tissue microarrays have demonstrated that NK cells do not interfere with the progression of the indicated epithelial malignancies. In contrast, NK cells seem to play an important clinicopathological role in controlling myeloid leukemias. Ruggeri et al have demonstrated that, after haploidentical hematopoietic stem cell transplantation for acute myeloid leukemia, an efficient regeneration of haploidentical donor NK cells was associated with prolong survival of myeloid leukemia patients. The beneficial effect of NK cells was also demonstrated in acute leukemia patients undergoing HLA identical allogeneic hematopietic stem cell transplantation by Bipin et al. This information suggests that NK cell could be usefully utilized for optimizing cell based cellular immunotherapy of acute myeloid leukemia. However, literature data indicate that either leukemia cells or epithelial cancer cells deploy a variety of mechanisms allowing them to escape NK cell recognition by NK cell apoptosis and FC-gamma-RIII (CD16) down-regulation. To date, the mechanism, specificity, and extent of this phenomenon remain unclear. Thus, in this project there has been interest in testing the following hypothesis
Accomplished Results
Leukemia and cancer cells avoid NK cell recognition by triggering NK cell elimination through an apoptotic/necrotic mechanism
The mechanism of NK cell elimination involves CD16 expressed on NK cells
The interaction between beta cells and NK cell results in beta cell elimination

In this research, the following results has been achieved.
1. A wide number of leukemia and epithelial cancer cell lines caused human NK cell apoptosis, NK cell elimination and CD16 down-regulation
2. Leukemia and lymphoma allogeneic cell lines induced mouse NK cell apoptosis and elimination (Figure 1)
3. Leukemia and epithelial cancer cell dependent NK cell elimination and CD16 down-regulation involved preferentially CD16+CD56+ and CD56+CD16- NK cells while CD16+ monocytes were not.
4. This phenomenon was quite specific since NK cell elimination was preferentially induced by allogeneic tumor cells but not by normal allogeneic or tumor xenogeneic cells
5. CD16 directly recognized cell surface molecules of tumor cells
Thus, in the second part of this project much emphasis has been put on understanding mechanism by which post-activated NK cells could overcome cancer cell dependent NK cell apoptosis and CD16 down-regulation
To understand which pro-apoptotic signalling pathway is activated by cancer cells after NK cell engagement, a series of metabolic inhibitors were utilized. Cancer cell independent apoptosis was capsase, metalloproteases, PI-3 kinase, tyrosine kinases, histamine and DMSO independent. However, interestingly upon conjugation with cancer cells, long term activated NK cells with interleukin-2 (IL-2) become resistant to cancer cell dependent apoptosis and CD16-downregulation without losing their cytotoxic activity against myeloid leukemia and epithelial cancer cells.
Particularly, NK cell resistance to cancer cell dependent NK cell elimination required 10 to 14 days activation with high dose of IL-2.
Considering that NK cell have an important clinicopathological role in counteracting acute myeloid leukemia in vitro and in vivo, this is an interesting finding since provides the opportunity to generate more efficient NK cells. These cells could be utilized for optimizing NK cell based immunotherapy after haploidentical or HLA-matched hematopoietic bone marrow transplantation or outside transplant setting perhaps after chemotherapy.
The future plan of this project will be to continue this research with the objective to demonstrate that (i) cancer cell dependent NK cell apoptosis and CD16-downregulation is not restricted to leukemia cell lines but is also mediated by primary leukemia cells in vitro and (ii) long-term cultured NK cells with IL-2 will protect immunodeficient mice from human myeloid leukemia more than short term activated human NK cells with IL-2 or resting NK cells.
Then the results of the indicated studies will be utilized for translational investigations whose final objective will be to perform a bench-to-bedside clinical trial were allogeneic NK cells are going to be tested for controlling acute myeloid leukemia minima residual disease in children or adults.
In this projects the investigator has also been interested in testing whether the concept of cancer cell dependent elimination of NK would be applicable to autoimmune disease such as type I diabetes. In this situation mouse cancer cells were replaced by mouse beta cells obtained from pancreatic islets of normal or diabetic mice
Given to the difficulties of this setting, to date, the study has not able to draw definitive conclusions. As reported in the project there will be an effort to continue the investigation of this issue in future studies. In particular methods of beta cell selection will be optimized and when possible human NK cells will be tested with human beta islets of type I diabetic patients.

Pubblications and Abstracts

1.Roberto Arriga, Sara Caratelli , Katia Basello, Andrea Coppola , Barbara Capuani , Davide Lauro , Giulia Donadel, Spagnoli Giulio Cesare and Giuseppe Sconocchia Leukemia and epithelial cancer cells triggered NK cell storming: The NK cell alloperturbation phenomenon (manuscript in preparation) 2012
2. Sconocchia G, Zlobec I, Lugli A et al. Tumor infiltration by FcgammaRIII (CD16)+ myeloid cells is associated with improved survival in patients with colorectal carcinoma. Int J Cancer 2011; 128:2663-2672
3. Sconocchia G, Zlobec I, Capuani B, Caratelli S, Donadel G, Arriga R, Iorio B, Terracciano L, Lauro D, Spagnoli GC. Impaired NK sell infiltration into the tumor microenvironment by cancer sell dependent NK cell elimination involves CD16. Bone marrow Transplantation 2010, 45S, 174. EBMT, Vien
4. Sconocchia G, Arriga R, Caratelli S, Basello K, Coppola A, Capuani B, Donadel G, Lauro D, Spagnoli GC. Leukaemia and epithelial cancer cells trigger NK cell storming : the NK cell alloperturbation phenomenon. Bone Marrow Transplantation 2011; 46S:325. EBMT Paris

Figure 1: Schematic representation of mouse leukemia/lymphoma dependent NK cell elimination : (1) B6 splenocytes were cultured in the presence of Balb/C derived allogeneic monocytic leukemia cells ; (2) B6 derived lymphoma cells were cultured in the presence of allogeneic Balb/C splenocytes. After 5 hours culture cells were stained with murine anti-NK cell markers monoclonal antibodies and analyzed by flow cytometry for NK cell apoptosis and elimination. Black circles and white circles indicate NK cell elimination induced by leukemia cells. 
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