Whole Body Irradiation Alone or in Combination with Fever Range Hyperthermia Generates Tumor Specific Immune Response: Its Implication in Developing Dendritic Cell
based Immunotherapy for Leukemia
Prabir K Chakravarty , Zoya Niazova
Background : Though whole body irradiation is extensively used prior to bone marrow transplantation in hematological malignancies, the extent of its use alone or in combination with other regimens for immunological intervention has not been adequately explored.
We hypothesize that low dose irradiation (LIR) alone or in combination with fever range hyperthermia (FR-HT) could cause tumor cell death and dying cells when taken up by na´ve dendritic cells (DCs), should facilitate immune activation that could result in eradication of residual tumor cells.
Materials and Methods : Exponentially growing leukemic EL-4 cells(1 x 10 6) was subjected to a) LIR in a single (4 Gy) or in multiple fractions (2 Gy x 2), b) FR-HT at a constant temperature of 39.5 0C [plusminus]0.1 for 6 hours and c) a combination of both regimens (the cells were first given FR-HT followed by irradiation (2Gyx2) at 16 hours interval).
Following completion of treatment with the above regimens, the cells were assessed for 1) Growth 2) apoptosis and 3) their capacity to stimulate a T cell mediated immune response. Growth was determined by counting the viable cells in a neuber chamber 24 hours following treatment.
Apoptosis was determined by visualizing DNA fragmentation pattern using a DNA ladder kit. The immuno-stimulatory capacity was determined as follows:a) Proliferation assay: The EL-4 cells (2x10 5 cells/ml) subjected to the different regimens (as described above) were mixed with a immature DC cell line, JAWS-II,(2x10 5 cells/ml), serially diluted into 96 well microtitre plates and incubated for 4 hours.
Subsequently, autologous lymphocytes from na´ve C57BL/6 mice were added at a concentration of 1x10 5 cells/ml in a final volume of 200 [mu]l. After 5 days, cell proliferation was determined by WST-1 assay. b) Cytotoxicity assay: Na´ve splenocytes were incubated for five days with the DC+ treated EL-4 cells.
The stimulated splenocytes were tested for their cytotoxicity against fresh tumor cells by a standard LDH assay. c) RT-PCR: Total RNA was isolated from the treated EL-4 cells and the stimulated splenocytes. The expression of HSP-70 in EL-4 cells and expression of CD4 in the stimulated splenocytes were determined by RT-PCR using their specific primers.
Results : 1. LIR, FR-HT and LIR+ FR-HT caused significant growth arrest and apoptosis in EL-4 cells: 2. Lymphocyte proliferation assay revealed that at a dilution of 1: 512, the stimulation was higher in both the treated groups and the combined treatment group had a higher rate of proliferation. 3. The stimulated splenocytes from all the three groups had significantly higher cytotoxicity against tumor cells (p[lte].05). 4. HSP-70 expression was enhanced in treated EL-4 cells. 5. The CD4 expression was enhanced in stimulated splenocytes.
Conclusion : Our results indicate for the first time that the regimen of LIR and FR-HT alone or in combination is capable of stimulating apropriate immune response and could be used to develop DC based strategies for treating chronic leukemia.
AACR Abstract Number: LB-108, 2003
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