Jeniffer B. Hernandez

Enhanced T Cell Apoptosis within Drak2-Deficient Mice Promotes Resistance to Autoimmunity

Clonal expansion of T cells is vital to adaptive immunity, yet this process must be tightly controlled to prevent autoimmune disease. The serine/threonine kinase death-associated protein kinase-related apoptosis-inducing kinase 2 (DRAK2) is a negative regulator of TCR signaling and sets the threshold for the activation of naive and memory T cells and selected thymocytes. Despite enhanced T cell activation, Drak2−/− mice are resistant to experimental autoimmune encephalomyelitis, an autoimmune demyelinating disease that resembles multiple sclerosis. However, the basis for this autoimmune resistance is currently unknown. In this study, we show that, in the absence of DRAK2 signaling, T cells require greater tonic signaling for maintenance during clonal expansion. Following stimulation, Drak2−/− T cells were more sensitive to an intrinsic form of apoptosis that was prevented by CD28 ligation, homeostatic cytokines, or enforced Bcl-xL expression. T cell-specific Bcl-xL expression also restored the susceptibility of Drak2−/− mice to experimental autoimmune encephalomyelitis and enhanced thymic positive selection. These findings demonstrate that DRAK2 is selectively important for T cell survival and highlight the potential that DRAK2 blockade may lead to permanent autoimmune T cell destruction via intrinsic apoptosis pathways.

Modulation of DRAK2 autophosphorylation by antigen receptor signaling in primary lymphocytes

Death-associated protein-related apoptotic kinase-2 (DRAK2), a member of the death-associated protein-like family of serine/threonine kinases, is highly expressed in lymphoid organs and is a negative regulator of T cell activation. To investigate the regulation of DRAK2 activity in primary lymphocytes, we employed mass spectrometry to identify sites of autophosphorylation on DRAK2. These studies have revealed a key site of autophosphorylation on serine 12. Using a phospho-specific antibody to detect Ser12 phosphorylation, we found that autophosphorylation is induced by antigen receptor stimulation in T and B cells. In Jurkat T cells, resting B cells and thymocytes, DRAK2 was hypophosphorylated on Ser12 but rapidly phosphorylated with antigen receptor ligation. This increase in phosphorylation was dependent on intracellular calcium mobilization, because BAPTA-AM blocked DRAK2 kinase activity, whereas the SERCA inhibitor thapsigargin promoted Ser12 phosphorylation. Our results show that DRAK2 kinase activity is regulated in a calcium-dependent manner and that Ser12 phosphorylation is necessary for optimal suppression of T cell activation by this kinase, suggesting a potential feedback loop may act to modulate the activity of this kinase following antigen receptor signaling.

Loss of DRAK2 Signaling Enhances Allogeneic Transplant Survival by Limiting Effector and Memory T Cell Responses

Here, we demonstrate that loss of DRAK2 signaling significantly promotes the acceptance of allogeneic engraftment in two separate transplant models without promoting generalized immunosuppression. Drak2−/− T cells failed to reject allogeneic tumors, and were defective in rejecting Balb/C allogeneic skin grafts on C57BL6/J recipients. A significant fraction of alloreactive Drak2−/− T cells underwent apoptosis following activation, whereas enforced expression of Bcl‐xL in Drak2−/− T cells restored allograft rejection. Formation of allogeneic memory was also greatly hampered in T cells lacking the Drak2 gene. Adoptive transfer of memory T cells from Drak2−/− mice failed to promote the rejection of allogeneic tumors, and such cells led to significantly delayed rejection of skin allografts in the Balb/C‐>C57BL/6J model. Costimulatory blockade by in vivo administration of Cytotoxic T‐Lymphocyte Antigen 4 fusion protein (CTLA4‐Ig) synergized with the DRAK2 deficiency and led to long‐term allogeneic skin graft acceptance. Overall, these results demonstrate that DRAK2 plays an important role in primary and memory T cell responsiveness to allografts. Because previous studies have demonstrated that a loss of DRAK2 does not negatively impact antiviral immunity, the studies here underscore the potential utility of pharmacological blockade of DRAK2 to achieve transplant maintenance without the imposition of generalized immunosuppression.