Samenvatting
Natural killer T (NKT) cells are T-lymphocytes that co-express conventional T-cell (CD3) and NK cell (NK1.1) surface receptors, while invariant NKT cells (iNKTs) also express a unique semi-invariant TCR alpha-chain encoded by Va14.Ja18 in mice and Va24.Ja18 in human. These iNKTs can recognize glycolipid antigens such as alpha-Galactosylceramide (a-GalCer) presented by the class I-like major histocompatibility complex (MHC) molecule CD1d. Activation of iNKTs can lead to an anti-tumor Th1 (IFN-gamma) response or an immunosuppressive Th2 (IL-4) response. Clinical studies in MM patients (1, 2) showed a low frequency and dysfunction of iNKTs which resulted in a low IFN-gamma secretion. This defect could be overcome in vitro by stimulating the iNKTs with a-GalCer loaded DCs. Furthermore, when MM patients were injected with loaded DCs their iNKT pool expanded 100 fold. This make MM cells an interesting target for iNKT therapy. However, the data on NKT activity in MM patients is limited and the use of a-GalCer as a drug has not been preclinically evaluated yet. Therefore, in this study, we investigated the characteristics of iNKTs in the syngeneic 5T33MM murine model, which is an immunocompetent model of myeloma which mimics the human disease closely.
We first investigated the frequency of iNKTs in the blood, BM, spleen and liver of both healthy and terminally diseased 5T33MM mice. The highest percentage of iNKTs was found in the liver (naive 7%) with a significant decrease in 5T33MM mice (2.6%). The percentage was also slightly decreased in spleen (from 1.5% in naive to 0.7% in 5T33MM mice) while no significant differences were observed in the other tissues. Next, we followed the frequency of iNKTs in the liver and spleen of MM mice during the development of the disease. We analyzed the number of iNKTs in the first, second, third and terminal week. We found that the percentage of iNKTs declined at the end stage of MM disease. To analyze the activity of iNKTs in vitro, liver iNKTs were cocultured with naive matured BM derived DCs in the presence or absence of 100 ng/ml a-GalCer. Naive iNKTs could secrete up to 2.3 ng/ml IFN-gamma when stimulated with a-GalCer, and this level increased with the progression of MM to reach 3.3 ng/ml at week 2. However, the activity of the iNKTs dropped to undetectable levels upon further progression of the disease (week 4). In contrast, very slight IL-4 production was observed indicating that liver iNKTs are skewed to a Th1 profile and can therefore be used as an immunotherapeutic tool in MM. The activity of the NKTs was also followed in vivo. The serum level of IFN-gamma peaked at 18h after a-GalCer injection in naive and non-terminal diseased mice and returned to baseline by 48h, however, the response of IFN-gamma in diseased mice was twice (6 ng/ml) that measured in naive mice, confirming the possibility of inducing Th1 responses with a-GalCer in vivo in healthy and diseased mice. No response could be detected from terminally diseased mice. It has been described previously that CD1d is significantly downregulated in patients with advanced stages of MM (3). To investigate if this is similar in the 5T33MM model, we followed the expression of CD1d on spleen and BM cells during the course of the disease. Results showed a significant downregulation of CD1d expression on spleen cells from 93% CD1d (naive) to 68% at end stage. On BM cells, CD1d was less expressed compared to spleen cells, 52% in naive mice and expression declined significantly to 35%. CD1d expression on the MM cells themselves was high (79%) and did not alter during the course of the disease.
We finally evaluated the effect of a-GalCer on the survival of MM mice. Survival was significantly increased when mice were injected with a-GalCer loaded DCs on the same day of 5T33MM inoculation (29 days survival) compared to mice injected with unloaded DCs (22 days survival).
Taken together, our data demonstrate for the first time the possibility of using a murine model as a preclinical MM model to study the effects of a-GalCer on iNKTs and shows promising results of treating MM patients with a low tumorload.
We first investigated the frequency of iNKTs in the blood, BM, spleen and liver of both healthy and terminally diseased 5T33MM mice. The highest percentage of iNKTs was found in the liver (naive 7%) with a significant decrease in 5T33MM mice (2.6%). The percentage was also slightly decreased in spleen (from 1.5% in naive to 0.7% in 5T33MM mice) while no significant differences were observed in the other tissues. Next, we followed the frequency of iNKTs in the liver and spleen of MM mice during the development of the disease. We analyzed the number of iNKTs in the first, second, third and terminal week. We found that the percentage of iNKTs declined at the end stage of MM disease. To analyze the activity of iNKTs in vitro, liver iNKTs were cocultured with naive matured BM derived DCs in the presence or absence of 100 ng/ml a-GalCer. Naive iNKTs could secrete up to 2.3 ng/ml IFN-gamma when stimulated with a-GalCer, and this level increased with the progression of MM to reach 3.3 ng/ml at week 2. However, the activity of the iNKTs dropped to undetectable levels upon further progression of the disease (week 4). In contrast, very slight IL-4 production was observed indicating that liver iNKTs are skewed to a Th1 profile and can therefore be used as an immunotherapeutic tool in MM. The activity of the NKTs was also followed in vivo. The serum level of IFN-gamma peaked at 18h after a-GalCer injection in naive and non-terminal diseased mice and returned to baseline by 48h, however, the response of IFN-gamma in diseased mice was twice (6 ng/ml) that measured in naive mice, confirming the possibility of inducing Th1 responses with a-GalCer in vivo in healthy and diseased mice. No response could be detected from terminally diseased mice. It has been described previously that CD1d is significantly downregulated in patients with advanced stages of MM (3). To investigate if this is similar in the 5T33MM model, we followed the expression of CD1d on spleen and BM cells during the course of the disease. Results showed a significant downregulation of CD1d expression on spleen cells from 93% CD1d (naive) to 68% at end stage. On BM cells, CD1d was less expressed compared to spleen cells, 52% in naive mice and expression declined significantly to 35%. CD1d expression on the MM cells themselves was high (79%) and did not alter during the course of the disease.
We finally evaluated the effect of a-GalCer on the survival of MM mice. Survival was significantly increased when mice were injected with a-GalCer loaded DCs on the same day of 5T33MM inoculation (29 days survival) compared to mice injected with unloaded DCs (22 days survival).
Taken together, our data demonstrate for the first time the possibility of using a murine model as a preclinical MM model to study the effects of a-GalCer on iNKTs and shows promising results of treating MM patients with a low tumorload.
| Originele taal-2 | English |
|---|---|
| Pagina's (van-tot) | 938-938 |
| Aantal pagina's | 1 |
| Tijdschrift | Blood |
| Volume | 120 |
| Nummer van het tijdschrift | 21 |
| Status | Published - dec 2012 |
| Evenement | 54th ASH Annual Meeting and Exposition - Atlanta, GA, United States Duur: 8 dec 2012 → 11 dec 2012 |