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Cd4+ Cells Are Key Regulators Of Pathologic Changes In Lymphedema
Daniel A. Cuzzone, MD, Nicholas J. Albano, BS, Swapna Ghanta, MD, Seth Z. Aschen, BS, Ira L. Savetsky, MD, Jason C. Gardenier, MD, Walter J. Joseph, BS, Jeremy S. Torrisi, BA, Gina T. Farias-Eisner, BA, Babak J. Mehrara, MD.
Memorial Sloan Kettering Cancer Setting, New York, NY, USA.
Lymphedema is a common complication of cancer treatment afflicting about 1 in 3 women who undergo lymph node dissection for breast cancer. We have previously shown that the absence of CD4+ cells either by antibody depletion or in transgenic CD4 knockout mice prevents development of lymphedema after lymphatic injury suggesting that CD4+ inflammation is necessary for this process. However, it remains unclear if CD4+ cells alone are sufficient to induce the pathologic changes that occur after lymphatic injury. This distinction is important since it implies that CD4+ cells play an active role in the pathology of this disease. The purpose of this study was therefore to determine how adoptive transferred CD4+ cells differentiate and regulate development of lymphedema in transgenic mice lacking CD4 cells (CD4KO).
Adult female CD4KO mice underwent microsurgical excision of the superficial and deep lymphatics of the mid portion of the tail. Beginning two weeks postoperatively and weekly thereafter, experimental animals (n=8) were adoptively transferred using retro-orbital injections with 10e7 CD4+ cells harvested from the spleens of syngeneic female C57B6 mice using negative magnetic selection and expanded in vitro. Control animals (n=8) received retro-orbital injections of PBS. Tail volumes were recorded weekly using caliper measurements and calculated using the truncated cone formula. Lymphatic function (uptake of Tc99) as well as histological changes in the tail were analyzed using a variety of techniques 6-weeks postoperatively based on our previous studies demonstrating onset of lymphedema changes at this time point.
In vitro expanded CD4+ cells maintained CD4+ markers but did not demonstrate evidence of T cell maturation or activation as analyzed by flow cytometry. Adoptive transfer of 10e7 cells resulted in significantly increased numbers of these cells in the spleens and lymph nodes of recipient mice (554 and 75 fold change respectively). Adoptively transferred experimental mice had significantly greater tail volume and decreased lymphatic transport capacity (Tc99) as compared with controls (both p<0.001) 6 weeks after tail surgery. In addition, consistent with histological changes noted in clinical lymphedema, we found that CD4+ cell adoptive transfer promoted significant subcutaneous adipose deposition in the regions of the tail distal to the zone injury (p<0.05). Moreover, using flow cytometry and immunohistochemistry we found that there was significant accumulation of CD4+ cells in the dermis/subcutaneous fat (p<0.001) and that this response was associated with a significant fibrotic response (type I collagen deposition; p<0.03 compared with controls). Interestingly, our preliminary studies have not demonstrated differences in Th1/ Th2 differentiation, however, these studies are ongoing.
We have shown that the adoptive transfer of CD4+ cells into CD4KO mice recapitulates the histopathologic findings associated with wild type mice following ablation of tail lymphatics. Specifically, adoptive transferred CD4+ cells home to and are sufficient to induce fibrosis, chronic inflammation, and adipose deposition in the regions of the tail distal to the zone of injury. These findings therefore suggest that CD4+ cells play an active role in the pathology of lymphedema. Future studies will determine how CD4+ cell differentiation contributes to this process.
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