In reference to our goals listed above, the following is a summary of our most recent work (and anticipated future) work on this project: 1. We will use a variety of in vitro and in vivo assays to determine whether activation of specific signaling pathways occur in MF and SS. One of our hypotheses is that IL-1b activation plays a key role in the inflammatory environment observed in CTCL. Cleavage of IL-1b as determined by Western blot or activation of caspases will be used to assess whether the IL-1 pathway is upregulated in patient skin. The model described in 2 below suggests that IL-1b activation may play a role in tumorigenesis. 2. We have injected MBL-2 cells (a mouse T cell lymphoma line) into the skin of mice and have found very little, if any, tendency for these T cells to form tumors. However, upon the topical application of dinitrofluorobenzene (DNFB), a commonly used contact allergen that is known to activate the caspase dependent inflammasome in keratinocytes resulting in cleavage of IL-1b to its active form, we have observed striking tumor formation in the skin of treated mice. Interestingly, we have found that a peptide antagonist of CCR10 blocks inflammation mediated by topical application of DNFB, and, if used in our tumorigenesis model, also blocks the formation of MBL-2 tumors. We are currently assessing the specificity of this striking result and the mechanism by which CCR10 inactivation leads to reduction in DNFB-mediated inflammation. 3. Human SS T cells have been isolated from the peripheral blood of a patient with Sezary syndrome. Isolation was achieved using antibodies that were specific for the T cell receptor expressed by the patient's tumor cells. The SS T cells were subsequently transduced with a marker protein (for determination of transduction efficiency) and human telomerase (in order to prevent T cell senescence). Initial results suggest that malignant T cells have a growth disadvantage in culture and that cultures may become """"""""overrun"""""""" with T cells bearing the correct T cell gene rearrangement, but otherwise do not have the same phenotype as the malignant T cells in vivo. Attempts will be made to find growth factors that promote the growth of malignant T cells. Following that achievement, the development of in vivo xenograft systems employing human SS T cells in immunodeficient mice will be attempted. 4. Validation of any in vitro or in vivo result will be necessary in human patient tissues. Thus, we have written and implemented several protocols that have collected CTCL patient samples. One protocol was a proteomic study (recently published in the British J. Dermatology) that sought to distiguish differences between low molecular weight serum proteins from MF patients, patients with psoriasis, and healthy patients of similar age and sex. Interestingly, we found that distinct peaks as determined by mass spectroscopy techniques were associated with MF patients. 11 of these peaks showed high correlation with patient survival, suggesting that this technique could be used to determine prognosis in CTCL patient populations. The other protocol, a natural history protocol, offered second opinions to patients that were suspected or confirmed to have a diagnosis of CTCL. As part of the protocol, patient skin and serum samples were systematically collected and archived for future study. These samples will be valuable to both generate hypotheses as well as to validate data and conclusions obtained from our animal studies.
