Abstract
Graft-versus-host-disease (GVHD) is an alloimmune response complicating allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although donor T lymphocytes and recipient antigen presenting cells (APCs) are the primarily mediators of GVHD, the molecular and cellular basis are not well understood. Recent published studies investigated T cell migration and homing in GVHD, especially effects of adhesion molecules including chemokines and integrins. Intermediate filaments (IFs) are cytoskeletal polymers encoded by a large family of differentially expressed genes that provide crucial structural support. As the most abundant IF protein and the only known IF protein in leukocytes, vimentin plays an important role in stabilizing intracellular architecture, maintaining cellular integrity and providing resistance against stress. Vimentin-deficient (Vim-/-) mice are viable with impaired wound healing, and defects in PMN and lymphocyte adhesion to endothelial cells. In addition, Vimentin has been reported to be involved in colitis, Crohn's disease and allograft rejection. Herein, we report that vimentin regulates the organization of proteins in cell adhesion, migration and signaling, all of which are important for T cell activation in GVHD.
Using homotypic aggregation assay, we found there was significantly reduced aggregation of primary T cells isolated from Vim-/- mice compared to WT control. However, the expression of both LFA-1 and ICAM-1 are similar between WT and Vim-/- T cells, thus the reduced T cell adhesion is not attributed to LFA-1 expression. We further investigated whether vimentin regulates mouse primary T cell proliferation upon activation in mixed lymphocyte reactions (MLR), and demonstrated there was a significant reduction of both CD4+ and CD8+ T cell proliferation in the absence of vimentin (Vim-/-) compared to WT control. Moreover, the frequency of IFN-γ (Th1) and IL-17 (Th17) producing CD4+ cells was significantly reduced in T cells isolated from Vim-/- mice compared to WT control. To investigate the role of vimentin in regulating TCR-induced activation, we examined the immune synapse formation in mouse CD8+ T cells, and found vimentin was enriched proximal to the membrane and associated with the prominent CD3/LFA-1 cluster upon TCR stimulation. In the control without any TCR stimulation, the predominant pattern was different with vimentin evenly distributed beneath the cell membrane. In the absence of vimentin (Vim-/-), immune synapse failed to form in mouse CD8+ T cells upon TCR stimulation. The data demonstrate that vimentin can regulate immunological synapse function and signal transduction in T cell activation and proliferation.
To determine whether vimentin expression in T cells plays a role in GVHD, the MHC class I and II disparate model, C57BL/6 (H-2b) to BALB/c (H-2d) transplantation, was used to establish GVHD. T cells from WT or Vim-/- (C57BL/6 background) mice were used as donors and Balb/c mice as recipients. Irradiated BALB/c mice received 5x106 T cell-depleted bone marrow cells (WT) and 10×106 T cells (WT or Vim-/-). In comparison to WT control, mice received Vim-/- T cells showed a lower mortality rate. Within 8 weeks post-transplantation, about 65% of mice received Vim-/- T cells survived, compared with only less than 10% of control mice received WT T cells (P=0.036; n=15 in each group). Control recipients had severe GVHD in the skin, intestine and liver. Mice received Vim-/- T cells exhibited only mild changes in these organs, reflected in their significantly lower GVHD scores. There were significantly reduced donor-derived CD4+ and CD8+ T cells in secondary lymphoid organs. Thus, the reduced homing and proliferation of Vim-/- T cells in vivo led to the reduced mortality and morbidity associated with GVHD.
In summary, we have shown that there are significantly reduced T cell adhesion, proliferation and Th1/17 polarization in Vim-/- T cells, and vimentin participates in TCR clustering and immunological synapse formation in CD8+ T cells. Furthermore, vimentin plays an important role in GVHD through regulating donor T cell adhesion, proliferation and activation. Our data will not only significantly advance our knowledge of GVHD, but also define a new function of vimentin and IF proteins in health and diseases, and thus provide a rationale for using vimentin inhibitors as potential novel therapeutic interventions for GVHD.
Disclosures
No relevant conflicts of interest to declare.
Stimulation of naive T-cell adhesion and immunological synapse formation by chemokine-dependent and -independent mechanisms