Nicholas D. Leigh

A Flagellin-Derived Toll-Like Receptor 5 Agonist Stimulates Cytotoxic Lymphocyte-Mediated Tumor Immunity

Toll-like receptor (TLR) mediated recognition of pathogen associated molecular patterns allows the immune system to rapidly respond to a pathogenic insult. The “danger context” elicited by TLR agonists allows an initially non-immunogenic antigen to become immunogenic. This ability to alter environment is highly relevant in tumor immunity, since it is inherently difficult for the immune system to recognize host-derived tumors as immunogenic. However, immune cells may have encountered certain TLR ligands associated with tumor development, yet the endogenous stimulation is typically not sufficient to induce spontaneous tumor rejection. Of special interest are TLR5 agonists, because there are no endogenous ligands that bind TLR5. CBLB502 is a pharmacologically optimized TLR5 agonist derived from Salmonella enterica flagellin. We examined the effect of CBLB502 on tumor immunity using two syngeneic lymphoma models, both of which do not express TLR5, and thus do not directly respond to CBLB502. Upon challenge with the T-cell lymphoma RMAS, CBLB502 treatment after tumor inoculation protects C57BL/6 mice from death caused by tumor growth. This protective effect is both natural killer (NK) cell- and perforin-dependent. In addition, CBLB502 stimulates clearance of the B-cell lymphoma A20 in BALB/c mice in a CD8+ T cell-dependent fashion. Analysis on the cellular level via ImageStream flow cytometry reveals that CD11b+ and CD11c+ cells, but neither NK nor T cells, directly respond to CBLB502 as determined by NFκB nuclear translocation. Our findings demonstrate that CBLB502 stimulates a robust antitumor response by directly activating TLR5-expressing accessory immune cells, which in turn activate cytotoxic lymphocytes.

A TLR5 Agonist Enhances CD8+ T Cell-Mediated Graft-versus-Tumor Effect without Exacerbating Graft-versus-Host Disease

Allogeneic hematopoietic cell transplantation is an established treatment for hematologic and nonhematologic malignancies. Donor-derived immune cells can identify and attack host tumor cells, producing a graft-versus-tumor (GVT) effect that is crucial to the effectiveness of the transplantation therapy. CBLB502 is a novel agonist for TLR5 derived from Salmonella flagellin. On the basis of TLR5-mediated immunomodulatory function, we examined the effect of CBLB502 on GVT activity. Using two tumor models that do not express TLR5, and thereby do not directly respond to CBLB502, we found that CBLB502 treatment significantly enhanced allogeneic CD8+ T cell-mediated GVT activity, which was evidenced by decreased tumor burden and improved host survival. Importantly, histopathologic analyses showed that CBLB502 treatment did not exacerbate the moderate graft-versus-host disease condition caused by the allogeneic CD8+ T cells. Moreover, mechanistic analyses showed that CBLB502 stimulates CD8+ T cell proliferation and enhances their tumor killing activity mainly indirectly through a mechanism that involves the IL-12 signaling pathway and the CD11c+ and CD11b+ populations in the bone marrow cells. This study demonstrates a new beneficial effect of CBLB502, and suggests that TLR5-mediated immune modulation may be a promising approach to improve GVT immunity without exacerbating graft-versus-host disease.

Granzyme B–Mediated Damage of CD8+ T Cells Impairs Graft-versus-Tumor Effect

Allogeneic hematopoietic cell transplantation is an established treatment for hematologic and other malignancies. Donor-derived immune cells can identify and attack host tumor cells, producing a graft-versus-tumor (GVT) effect that is crucial to the treatment. Using multiple tumor models and diverse donor–host combinations, we have studied the role of granzyme B (GzmB) in GVT effect. We first confirmed previous findings that GzmB deficiency diminished the ability of a high dose of CD8+ T cells to cause lethal graft-versus-host disease. However, when GVT studies were performed using a moderate cell dose that the hosts could tolerate, GzmB−/− CD8+ T cells demonstrated a significantly enhanced GVT effect. GzmB-mediated, activation-induced cell death in wild-type CD8+ T cells was found responsible for their reduced GVT activity. Conversely, GzmB−/− CD8+ T cells exhibited enhanced expansion, skewed toward an effector or effector memory phenotype, and produced higher amounts of IFN-γ and Fas ligand that might contribute to GzmB-independent tumor control. These findings demonstrate for the first time, to our knowledge, that GzmB-mediated damage of CD8+ T cells impairs the desired GVT effect. This study suggests that inhibiting donor-derived GzmB function may represent a promising strategy to improve GVT effect without exacerbating graft-versus-host disease.

Housing Temperature-Induced Stress Is Suppressing Murine Graft-versus-Host Disease through β2-Adrenergic Receptor Signaling.

Graft-versus-host disease (GVHD) is the major complication of allogeneic hematopoietic cell transplantation, a potentially curative therapy for hematologic diseases. It has long been thought that murine bone marrow–derived T cells do not mediate severe GVHD because of their quantity and/or phenotype. During the course of experiments testing the impact of housing temperatures on GVHD, we discovered that this apparent resistance is a function of the relatively cool ambient housing temperature. Murine bone marrow–derived T cells have the ability to mediate severe GVHD in mice housed at a thermoneutral temperature. Specifically, mice housed at Institutional Animal Care and Use Committee–mandated, cool standard temperatures (∼22°C) are more resistant to developing GVHD than are mice housed at thermoneutral temperatures (∼30°C). We learned that the mechanism underlying this housing-dependent immunosuppression is associated with increased norepinephrine production and excessive signaling through β-adrenergic receptor signaling, which is increased when mice are cold stressed. Treatment of mice housed at 22°C with a β2-adrenergic antagonist reverses the norepinephrine-driven suppression of GVHD and yields similar disease to mice housed at 30°C. Conversely, administering a β2-adrenergic agonist decreases GVHD in mice housed at 30°C. In further mechanistic studies using β2-adrenergic receptor–deficient (β2-AR−/−) mice, we found that it is host cell β2-AR signaling that is essential for decreasing GVHD. These data reveal how baseline levels of β-adrenergic receptor signaling can influence murine GVHD and point to the feasibility of manipulation of β2-AR signaling to ameliorate GVHD in the clinical setting.

Defining Immunological Impact and Therapeutic Benefit of Mild Heating in a Murine Model of Arthritis

Traditional treatments, including a variety of thermal therapies have been known since ancient times to provide relief from rheumatoid arthritis (RA) symptoms. However, a general absence of information on how heating affects molecular or immunological targets relevant to RA has limited heat treatment (HT) to the category of treatments known as “alternative therapies”. In this study, we evaluated the effectiveness of mild HT in a collagen-induced arthritis (CIA) model which has been used in many previous studies to evaluate newer pharmacological approaches for the treatment of RA, and tested whether inflammatory immune activity was altered. We also compared the effect of HT to methotrexate, a well characterized pharmacological treatment for RA. CIA mice were treated with either a single HT for several hours or daily 30 minute HT. Disease progression and macrophage infiltration were evaluated. We found that both HT regimens significantly reduced arthritis disease severity and macrophage infiltration into inflamed joints. Surprisingly, HT was as efficient as methotrexate in controlling disease progression. At the molecular level, HT suppressed TNF-α while increasing production of IL-10. We also observed an induction of HSP70 and a reduction in both NF-κB and HIF-1α in inflamed tissues. Additionally, using activated macrophages in vitro, we found that HT reduced production of pro-inflammatory cytokines, an effect which is correlated to induction of HSF-1 and HSP70 and inhibition of NF-κB and STAT activation. Our findings demonstrate a significant therapeutic benefit of HT in controlling arthritis progression in a clinically relevant mouse model, with an efficacy similar to methotrexate. Mechanistically, HT targets highly relevant anti-inflammatory pathways which strongly support its increased study for use in clinical trials for RA.

Granzyme B–Mediated Activation-Induced Death of CD4 + T Cells Inhibits Murine Acute Graft-versus-Host Disease

Granzyme B (GzmB) has previously been shown to be critical for CD8+ T cell–mediated graft-versus-host disease (GVHD) but dispensable for GVHD mediated by CD4+ T cells. However, previous studies used high doses of CD4+ T cells in MHC-mismatched models that caused rapid and lethal GVHD. Because of the hyperacute lethality, it is possible that the role of GzmB was concealed by the system. Therefore, in this study, we have titrated down the T cell dose to precisely determine the contribution of GzmB in GVHD mediated by CD4+CD25– T cells. Surprisingly, we have found that GzmB−/−CD4+CD25– T cells cause more severe GVHD compared with wild-type CD4+CD25– T cells in both MHC-matched and mismatched models. Mechanistic analyses reveal that although GzmB does not affect donor T cell engraftment, proliferation or tissue-specific migration, GzmB−/− CD4+CD25– T cells exhibit significantly enhanced expansion because of GzmB-mediated activation-induced cell death of wild-type CD4+CD25– T cells. As a result of enhanced expansion, GzmB−/− T cells produced higher amounts of proinflammatory cytokines (e.g., TNF-α and IFN-γ) that may contribute to the exacerbated GVHD. These results reveal that GzmB diminishes the ability of CD4+ T cells to cause acute GVHD, which contradicts its established role in CD8+ T cells. The differential roles suggest that targeting GzmB in selected T cell subsets may provide a strategy to control GVHD.

Host-Derived CD70 Suppresses Murine Graft-versus-Host Disease by Limiting Donor T Cell Expansion and Effector Function

Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative treatment for hematologic and immunologic diseases. However, graft-versus-host disease (GVHD) may develop when donor-derived T cells recognize and damage genetically distinct normal host tissues. In addition to TCR signaling, costimulatory pathways are involved in T cell activation. CD27 is a TNFR family member expressed on T cells, and its ligand, CD70, is expressed on APCs. The CD27/CD70 costimulatory pathway was shown to be critical for T cell function and survival in viral infection models. However, the role of this pathway in allo-HCT is previously unknown. In this study, we have examined its contribution in GVHD pathogenesis. Surprisingly, Ab blockade of CD70 after allo-HCT significantly increases GVHD. Interestingly, whereas donor T cell– or bone marrow–derived CD70 plays no role in GVHD, host-derived CD70 inhibits GVHD as CD70−/− hosts show significantly increased GVHD. This is evidenced by reduced survival, more severe weight loss, and increased histopathologic damage compared with wild-type hosts. In addition, CD70−/− hosts have higher levels of proinflammatory cytokines TNF-α, IFN-γ, IL-2, and IL-17. Moreover, accumulation of donor CD4+ and CD8+ effector T cells is increased in CD70−/− versus wild-type hosts. Mechanistic analyses suggest that CD70 expressed by host hematopoietic cells is involved in the control of alloreactive T cell apoptosis and expansion. Together, our findings demonstrate that host CD70 serves as a unique negative regulator of allogeneic T cell response by contributing to donor T cell apoptosis and inhibiting expansion of donor effector T cells.

Identification of regenerative roadblocks via repeat deployment of limb regeneration in axolotls

Axolotl salamanders are powerful models for understanding how regeneration of complex body parts can be achieved, whereas mammals are severely limited in this ability. Factors that promote normal axolotl regeneration can be examined in mammals to determine if they exhibit altered activity in this context. Furthermore, factors prohibiting axolotl regeneration can offer key insight into the mechanisms present in regeneration-incompetent species. We sought to determine if we could experimentally compromise the axolotl’s ability to regenerate limbs and, if so, discover the molecular changes that might underlie their inability to regenerate. We found that repeated limb amputation severely compromised axolotls’ ability to initiate limb regeneration. Using RNA-seq, we observed that a majority of differentially expressed transcripts were hyperactivated in limbs compromised by repeated amputation, suggesting that mis-regulation of these genes antagonizes regeneration. To confirm our findings, we additionally assayed the role of amphiregulin, an EGF-like ligand, which is aberrantly upregulated in compromised animals. During normal limb regeneration, amphiregulin is expressed by the early wound epidermis, and mis-expressing this factor lead to thickened wound epithelium, delayed initiation of regeneration, and severe regenerative defects. Collectively, our results suggest that repeatedly amputated limbs may undergo a persistent wound healing response, which interferes with their ability to initiate the regenerative program. These findings have important implications for human regenerative medicine.Persistent healing blocks salamander limb regenerationUnderstanding a roadblock to limb regeneration in salamanders could help develop suitable regenerative therapies for humans. Jessica Whited of the Harvard Stem Cell Institute and colleagues in the US found that repeatedly amputating a limb in Mexican salamanders compromised their normal ability to regenerate it. The team found high levels of a gene encoding a damage-protecting protein, called amphiregulin, at the wound site. Artificially inducing the expression of this gene in salamanders whose limbs were amputated just once also led to slow and poor regeneration. The findings suggest that high levels of amphiregulin induce a persistent wound-healing response that interferes with the salamanders’ ability to initiate regeneration. Pushing organisms with strong regenerative abilities into a non-regenerative state can help researchers understand the factors that limit human regenerative abilities and thus develop suitable therapies.

Abstract B43: The degree of adrenergic stress signaling regulates the severity of graft versus host disease following allogeneic hematopoietic cell transplantation

Adrenergic stress is recognized to influence multiple arms of the immune system. Recent work in our lab supports the observation that conditions which cause physiologic stress (i.e., increase adrenergic stress) reduce T cell function. T cells are crucial mediators of graft versus host disease (GVHD), a common and often lethal side effect of allogeneic hematopoietic cell transplantation (allo-HCT). As many patients succumb to GVHD, being able to understand underlying factors that regulate its severity is paramount. Our recent work led us to hypothesize that stress induced β-adrenergic receptor (AR) agonists such as norepinephrine may dampen GVHD following allo-HCT. To investigate the role of β-AR signaling on GVHD we performed MHC-mismatched allo-HCT in which recipient mice were treated with PBS (β-AR signaling intact) or a β-AR antagonist, propranolol, (i.e., a β-blocker) to inhibit β-AR signaling. Following allo-HCT, GVHD was significantly increased in mice treated with propranolol as evidenced by more weight loss (P To confirm that the weight loss and decreased survival were indicative of T cell-mediated GVHD, we performed allo-HCT with T cell depleted bone marrow. No GVHD was observed in PBS- or propranolol-treated groups when the graft did not contain allogeneic T cells. Furthermore, syngeneic transplants also yielded no GVHD in PBS- or propranolol-treated groups. We next evaluated pathology in the liver, small and large intestine. We found that liver pathology was significantly increased (P We next asked whether we could regulate GVHD by manipulating endogenous levels of the β-AR agonist, norepinephrine, by exposing mice to physiological stress. To test this we used mildly cool housing temperature to activate the adrenergic stress response as temperature manipulation is a classic means to study norepinephrine-driven stress signaling. We housed mice at 22°C, a temperature in which mice are cold stressed, and 30°C, a temperature in which the adrenergic stress response is significantly reduced. Therefore in this model, we predicted that GVHD would be reduced in severity in mice housed at 22°C compared to those housed at 30°C. We first confirmed that mice housed at 22°C were more stressed by measuring norepinephrine levels, finding that norepinephrine was significantly elevated both pre- and 13 days post-allo-HCT (P Clinically, using β-blockers to achieve pharmacologic inhibition of β-AR signaling is commonly used to alleviate symptoms of hypertension or other age-related diseases in elderly patients. Of note, β-blockers are also routinely given post allo-HCT to ameliorate symptoms such as hypertension and tachycardia. Thus, following allo-HCT, it may be important to reconsider the use of β-blockers as our findings suggest that the degree of β-AR signaling regulates the severity of GVHD. Citation Format: Nicholas D. Leigh, Kathleen M. Kokolus, Jason W-L Eng, Jingxin Qiu, George L. Chen, Philip L. McCarthy, Xuefang Cao, Elizabeth A. Repasky. The degree of adrenergic stress signaling regulates the severity of graft versus host disease following allogeneic hematopoietic cell transplantation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B43.