UNLEASH:
We work on cytokine control of memory lymphocytes, with a particular interest in the molecular regulators of vaccination and pathological antibodies in different disease states. Experimentally, we use i) mouse model systems of both vaccination and RBC alloimmunization as well as ii) study cytokine control of lymphocyte development in healthy humans and at-risk patients.

My research is focused on understanding how cytokine signals drive immune memory, with specific interest in the molecular mechanisms by which external signaling events become translated into the genetic programs required for the self-renewal of memory T and B cells. We employ several different mouse models of human disease including classical infectious models ( Vaccinia virus and Listeria monocytogenes infection) as well as more clinically relevant models ( Graft vs. Host Disease and Red Blood Cell Alloimmunization). The lab is currently pursuing three major areas of investigation: 1) cytokine and transcriptional control of red blood cell alloimmunization, 2) immunologic mechanisms responsible for the immune suppression induced by extracorporeal phototherapy, and 3) differential signaling and transcriptional profiles induced by IL-7 and IL-15 in memory T cells.
1) Cytokine and transcriptional control of red blood cell alloimmunization and memory cell generation. A key feature of our immune response is its ability to remember previous exposures and respond more rapidly upon re-challenge. While memory immune responses clearly serve as the basis for successful vaccination, in certain settings they can also be pathogenic. One clinically important setting is red blood cell (RBC) alloimmunization, where re-exposure to allogeneic RBCs can lead to potentially life threatening hemolytic reactions as well as hemolytic disease of the fetus and newborn (HDFN) (Luckey and Silberstein Blood 2013). Clinically significant alloimmunization requires more than just an initial response of a recipient to the transfusion. Indeed, the initial response must also generate long-lived memory B and T cells in that recipient for many years. Clinical examples include delayed hemolytic reactions and progressive worsening of HDFN with subsequent pregnancies in alloimmunized mothers. Virtually nothing is known about how memory cells are generated in the setting of the relatively weak immune stimulus of RBC alloimmunization. We have adopted two recently developed mouse models of RBC alloimmunization to study the cytokine signals and underlying genetic drivers of memory responses to RBC alloantibodies.
It has been previously shown that transfusion of mouse RBCs that have been stored under conditions similar to those employed in modern blood banks resulted in significant increases in both alloantibody production as well as a host of circulating pro-inflammatory cytokines. Given IL-6âs known role in promoting antibody responses by supporting the differentiation of both T Follicular Helper cells (TFH) as well as germinal center B cells, we initially tested whether IL-6 in general or it specific receptor (IL-6RA) expressed on either T or B cells was required for RBC alloimmunization. We have successfully demonstrated that i) IL-6 is induced by transfusion of stored RBCs, ii) alloantibody responses to stored RBCs is critically dependent on IL-6, iii) IL-6R expression on T but not B cells is required for anti-RBC antibody production, iv) transfusion of stored RBCs drives antigen specific TFH production and v) IL-6 signals are required for maximal TFH cell expansion in response to stored blood transfusions. We are now in the process of determining why storage of RBCâs leads to IL-6 production, which cells are responding to stored blood, and are further investigating whether IL-6 plays a central role in development of alloantibodies to other antigen systems and in the setting of HDFN.
We are also investigating the intrinsic transcriptional control of RBC alloimmunization, building upon my labâs recent generation of a conditional mouse model of a transcription factor (Pou6f1). Given that memory lymphocytes share functional attributes typically reserved for stem cells such as homeostatic self-renewal, we and others have hypothesized that they have reactivated a portion of the hematopoietic stem cell genetic program (Luckey and Weaver Cell Stem Cell 2012). In support of this idea, we published a common transcriptional signature shared between memory B cells, memory CD8+ T cells and hematopoietic stem cells (Luckey et al PNAS 2006). We have gone on to describe a transcription factor (Pou6f1) whose expression is selectively up-regulated in memory B and CD8+ T cells relative to shorter-lived cells. Pou6f1 is a member of the Pou-domain family of transcription factors and is a paralog of Pou5f1 (aka Oct4). Oct4 serves as one of the master regulators of embryonic stem cell self-renewal; establishing a stable, self-reinforcing genetic circuit that directs active transcription of self-renewal genes while maintaining differentiation specific transcription factors in an off, but poised state. We believe Pou6f1 functions in memory T and B cells in a manner similar to Oct4 in embryonic stem cells. The central hypothesis of this work is that Pou6f1 directs memory antibody responses to RBC alloimmunization by regulating memory T and/or memory B cell generation and homeostatic self-renewal. Preliminary data suggest Pou6f1 is required for maximal initial anti-RBC antibody production, and ongoing studies will address its role in long-lived, memory antibody responses.
2) Development of a patient guided preclinical animal model for extracorporeal photopheresis. Extracorporeal photopheresis (ECP) is a poorly understood cellular therapy wherein transfusion of a patientâs own white cells that have been induced to undergo apoptosis leads to significant immune modulation and subsequent therapeutic benefit. ECP requires harvest of autologous mononuclear cells via apheresis, treatment of these cells with 8-methoxy-soralen (8-MOP) and their exposure to UV-A light to induce cellular apoptosis. The apoptotic cells are then transfused back into patients suffering from a diversity of steroid-refractory immune-mediated diseases including chronic cutaneous graft versus host disease (GVHD). In this setting, ECP effects are partial but have been shown to facilitate decreased steroid dosing in a subset of patients. Given the relatively benign side-effect profile of ECP and the significant toxicities of high-dose glucocorticoids, ECPâs clinical use is growing. Despite the growing use of ECP, we currently do not understand ECPâs fundamental cellular and molecular mechanisms of action. Specifically, we do not definitively know (i) which cells in patients are responsible for the observed immune suppression induced by ECP, (ii) which cytokines are responsible for mediating the immune suppression or (iii) the optimal dosing and scheduling parameters required for the induction of adequate immune suppression in different organ systems. Indeed, ECPâs partial effectiveness and low side effect profile suggest its therapeutic index has yet to be determined, and its full therapeutic potential has not been explored. We are currently developing a clinically guided, experimentally tractable animal model where the MNC population treated, dosing regimen, and disease treated more accurately reflect those currently used in patients. The central hypotheses are that ECP treatment will ameliorate established chronic cutaneous GVHD in mice in a dose dependent manner, ECP effects will require regulatory T cell function, and ECP will synergize with low dose IL-2 therapy. By establishing a mouse model of ECP guided by clinically realistic therapeutic parameters in the treatment of established chronic cutaneous GVHD, this proposal will first and foremost serve as a pre-clinical model for the investigation of novel therapeutic strategies that can be directly applied to patients. Specifically, our studies investigating the dose dependence of ECP should be readily translatable into an ECP dose-escalation trial in the treatment of patients suffering from chronic cutaneous GVDH. Similarly, studies investigating synergism between ECP and low-dose IL-2 will provide important scientific support for ongoing clinical efforts investigating combination ECP and low-dose IL-2 therapy. Furthermore, by definitively identifying the fundamental cellular and cytokine mediated mechanisms by which ECP works to ameliorate tissue specific inflammation, our work should provide essential data on how best to apply ECP in the treatment of other immune mediated disease processes such as solid organ rejection and Crohnâs disease.
3) IL-7 and IL-15 control of memory T cell survival and homeostatic self-renewal. IL-7 and IL-15 govern T cell homeostasis in both mice and humans. Thus it is not surprising that they play essential roles in several important clinical situations. In patients undergoing stem cell transplant, signals via IL-7 and IL-15 are essential for the reconstitution of the mature peripheral T cell compartment. In T cell based cellular therapies, IL-15 supports the ex vivo culture and expansion of various clinically useful T cell populations such as Chimeric Antigen Receptor bearing T cells. In mice, IL-7 and IL-15 have been shown to control memory CD8+ T cell survival and homeostatic self-renewal respectively in vivo, yet the underlying molecular mechanisms for their differential effects remain unclear. Given that IL-7 and IL-15 both signal through the same common gamma chain yet induce very different functional outcomes, we applied the large-scale proteomic and genomic approaches to better understand how IL-7 and IL-15 drive different functional outcomes in human memory CD8+ T cells. By comparing equipotent doses of IL-7 and IL-15 as measured by STAT5, we have discovered that IL-15 drives much stronger PI3K/PDK1/AKT signaling than IL-7. Furthermore, IL-15 but not IL-7 leads to Notch1 signaling in memory T cells. Using both PDK1, AKT and Notch pathway loss of function assays, we have shown that each of these signals is required for the maximal cell mass increases and proliferative responses induced by IL-15. Our current work is aimed at understanding how these differential signaling pathways become translated into the unique epigenetic and transcriptional programs that drive different functional outcomes in memory T cells.