Studies to define the host immune response to human tumor has concentrated in part on characterizing the T cells within the tumor bed. The picture that has immerged is complex with respect to the functional responsiveness of these cells. Tumor infiltrating lymphocytes (TIL) contain responsive T cells that display normal effector functions. It is also clear that a significant portion of TIL from a variety of tumors are defective in their proliferative response. We have demonstrated that TIL derived from human RCC and B cell lymphomas have a reduced proliferative response that is reflected by a decrease in the IL2Ralpha surface expression and mRNA levels. While TIL can produce comparable levels of IFNgamma when compared to PBL they also display a diminished capacity to produce IL2. Our hypothesis is that while a segment of TIL are responsive the majority display a state of unresponsiveness that is rather limited in scope. Other functional parameters are thought to be intact even though most TIL have a diminished ability to proliferate and produce IL2.
Aim 1 will determine which subsets of TIL are most affected. Cell sorting experiments """"""""will determine if the proliferative defect is unequally distributed among T cell subsets (CD4+ and CD8+ cells) and if the defect relates to the state of activation of T cells in the tumor bed.
Aim 2 will define the extent of the unresponsiveness by determining if other functional parameters are intact in the affected populations. Purified TIL subsets will be used to document whether TIL are defective in their ability to produce IL2. Whether the defect is limited to IL2 will he determined by testing TIL for the production of other cytokines such as IL4, TNFalpha and GM-CSF. Experiments will also determine if TIL are as competent as PBL to produce mRNA for perforin and serine esterases. Additional work will define the role various stimulation routes play in the unresponsiveness. An analysis of multiple functions induced by IL2R and TCR stimulation will help define the extent to which each of these receptors are affected. Experiments will also determine if triggering TIL via CD28 is normal since this pathway appears to be distinct from that induced by TCR.
Aim 3 will determine if the reduced IL2Ralpha surface expression and IL2 production of TIL is related to alterations in transcription. RT/PCR will be used to quantitate mRNA levels of TIL and PBL. Included will be studies that determine if the reduction in mRNA levels is due to changes in the kinetics of mRNA accumulation, rate of transcription or mRNA stability. The goal of the proposed work is to begin to understand how broad the defect is, and to identify the subsets affected as well as which receptor pathways are altered in the unresponsive TIL. It is important to characterize quantitatively and qualitatively the T cell unresponsiveness within the tumor. An understanding of this anergy may provide ways to reverse or bypass this defect allowing for T cell activation and the development of a potent host immune response to tumors. Understanding T cell anergy in disease states such as cancer will be important for the development of new treatment strategies.
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