Engineering Pancreatic Islets to Transiently Codisplay on Their Surface Thrombomodulin and CD47 Immunomodulatory Proteins as a Means of Mitigating Instant Blood-Mediated Inflammatory Reaction following Intraportal Transplantation.

Most pancreatic islets are destroyed immediately after intraportal transplantation by an instant blood-mediated inflammatory reaction (IBMIR) generated through activation of coagulation, complement, and proinflammatory pathways. Thus, effective mitigation of IBMIR may be contingent on the combined use of agents targeting these pathways for modulation. CD47 and thrombomodulin (TM) are two molecules with distinct functions in regulating coagulation and proinflammatory responses. We previously reported that the islet surface can be modified with biotin for transient display of novel forms of these two molecules chimeric with streptavidin (SA), that is, thrombomodulin chimeric with SA (SA-TM) and CD47 chimeric with SA (SA-CD47), as single agents with improved engraftment following intraportal transplantation. This study aimed to test whether islets can be coengineered with SA-TM and SA-CD47 molecules as a combinatorial approach to improve engraftment by inhibiting IBMIR. Mouse islets were effectively coengineered with both molecules without a detectable negative impact on their viability and metabolic function. Coengineered islets were refractory to destruction by IBMIR ex vivo and showed enhanced engraftment and sustained function in a marginal mass syngeneic intraportal transplantation model. Improved engraftment correlated with a reduction in intragraft innate immune infiltrates, particularly neutrophils and M1 macrophages. Moreover, transcripts for various intragraft procoagulatory and proinflammatory agents, including tissue factor, HMGB1 (high-mobility group box-1), IL-1ß, IL-6, TNF-α, IFN-γ, and MIP-1α, were significantly reduced in coengineered islets. These data demonstrate that the transient codisplay of SA-TM and SA-CD47 proteins on the islet surface is a facile and effective platform to modulate procoagulatory and inflammatory responses with implications for both autologous and allogeneic islet transplantation.

Oncolytic Adenovirus Armed with a Novel Agonist of the CD137 Immune Checkpoint Stimulator Suppresses Tumor Growth.

Natural 4-1BBL (CD137L) is a cell membrane-bound protein critical to the expansion, effector function, and survival of CD8+ T cells. We reported the generation of an active soluble oligomeric construct, SA-4-1BBL, with demonstrated immunoprevention and immunotherapeutic efficacy in various mouse tumor models. Herein, we developed an oncolytic adenovirus (OAd) for the delivery and expression of SA-4-1BBL (OAdSA-4-1BBL) into solid tumors for immunotherapy. SA-4-1BBL protein expressed by this construct produced T-cell proliferation in vitro. OAdSA-4-1BBL decreased cell viability in two mouse lung cancer cell lines, TC-1 and CMT64, but not in the non-cancerous lung MM14.Lu cell line. OAdSA-4-1BBL induced programmed cell death types I and II (apoptosis and autophagy, respectively), and autophagy-mediated adenosine triphosphate (ATP) release was also detected. Intratumoral injection of OAdSA-4-1BBL efficiently expressed the SA-4-1BBL protein in the tumors, resulting in significant tumor suppression in a syngeneic subcutaneous TC-1 mouse lung cancer model. Tumor suppression was associated with a higher frequency of dendritic cells and an increased infiltration of cytotoxic CD8+ T and NK cells into the tumors. Our data suggest that OAdSA-4-1BBL may present an efficacious alternative therapeutic strategy against lung cancer as a standalone construct or in combination with other immunotherapeutic modalities, such as immune checkpoint inhibitors.

Bending the Rules: Amplifying PD-L1 Immunoregulatory Function Through Flexible Polyethylene Glycol Synthetic Linkers.

Immune checkpoint signaling, such as programmed cell death protein-1 (PD-1), is a key target for immunotherapy due to its role in dampening immune responses. PD-1 signaling in T cells is regulated by complex physicochemical and mechanical cues. However, how these mechanical forces are integrated with biochemical responses remains poorly understood. Our previous work demonstrated that the use of an immobilizing polyethylene glycol (PEG) linker on synthetic microgels for the presentation of a chimeric form of PD-L1, SA-PD-L1, lead to local regulatory responses capable of abrogating allograft rejection in a model of cell-based transplantation. We herein provide evidence that enhanced immune regulating function can be obtained when presentation of SA-PD-L1 is achieved through a longer more flexible PEG chain. Presentation of SA-PD-L1 through a linker of high molecular weight, and thus longer length (10 kDa, 60 nm in length), led to enhance conversion of naive T cells into T regulatory cells (Tregs) in vitro. In addition, using a subcutaneous implant model and protein tethered through three different linker sizes (6, 30, and 60 nm) to the surface of PEG hydrogels, we demonstrated that longer linkers promoted PD-1 immunomodulatory role in vivo through three main functions: (1) augmenting immune cell recruitment at the transplant site; (2) promoting the accumulation of naive Tregs expressing migratory markers; and (3) dampening CD8+ cytolytic molecule production while augmenting expression of exhaustion phenotypes locally. Notably, accumulation of Treg cells at the implant site persisted for over 30 days postimplantation, an effect not observed when protein was presented with the shorter version of the linkers (6 and 30 nm). Collectively, these studies reveal a facile approach by which PD-L1 function can be modulated through external tuning of synthetic presenting linkers. Impact statement Recently, there has been a growing interest in immune checkpoint molecules as potential targets for tolerance induction, including programmed cell death protein-1 (PD-1). However, how the mechanics of ligand binding to PD-1 receptor affect downstream activation signaling pathways remains unresolved. By taking advantage of the effect of polyethylene glycol chain length on molecule kinetics in an aqueous solution, we herein show that PD-L1 function can be amplified by adjusting the length of the grafting linker. Our results uncover a potential facile mechanism that can be exploited to advance the role of immune checkpoint ligands, in particular PD-L1, in tolerance induction for immunosuppression-free cell-based therapies.

Protocol for transplanting pancreatic islets into the parametrial fat pad of female mice.

Although the male epididymal fat pad is an effective site for islet transplantation, females lack this tissue. Here, we present a protocol to assess the parametrial fat pad (PFP) adjacent to the uterine horn in females as an alternative site for islet transplantation. We describe steps for islet isolation from the pancreas, counting, transplantation into PFP, and monitoring for engraftment. Transplantation into PFP is minimally invasive, time efficient, and supports long-term engraftment of syngeneic islets and rejection of allogeneic islets. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).1.

Delayed graft rejection in autoimmune islet transplantation via biomaterial immunotherapy.

The induction of operational immune tolerance is a major goal in beta-cell replacement strategies for the treatment of type 1 diabetes. Our group previously reported long-term efficacy via biomaterial-mediated programmed death ligand 1 (PD-L1) immunotherapy in islet allografts in nonautoimmune models. In this study, we evaluated autoimmune recurrence and allograft rejection during islet transplantation in spontaneous nonobese diabetic (NOD) mice. Graft survival and metabolic function were significantly prolonged over 60 days in recipients of syngeneic islets receiving the biomaterial-delivered immunotherapy, but not in control animals. The biomaterial-mediated PD-L1 immunotherapy resulted in delayed allograft rejection in diabetic NOD mice compared with controls. Discrimination between responders and nonresponders was attributed to the enriched presence of CD206+ program death 1+ macrophages and exhausted signatures in the cytotoxic T cell compartment in the local graft microenvironment. Notably, draining lymph nodes had similar remodeling in innate and adaptive immune cell populations. This work establishes that our biomaterial platform for PD-L1 delivery can modulate immune responses to transplanted islets in diabetic NOD mice and, thus, can provide a platform for the development of immunologic strategies to curb the allo- and autoimmune processes in beta-cell transplant recipients.

A novel agonist of 4-1BB costimulatory receptor shows therapeutic efficacy against a tobacco carcinogen-induced lung cancer.

Immunotherapy utilizing checkpoint inhibitors has shown remarkable success in the treatment of cancers. In addition to immune checkpoint inhibitors, immune co-stimulation has the potential to enhance immune activation and destabilize the immunosuppressive tumor microenvironment. CD137, also known as 4-1BB, is one of the potent immune costimulatory receptors that could be targeted for effective immune co-stimulation. The interaction of the 4-1BB receptor with its natural ligand (4-1BBL) generates a strong costimulatory signal for T cell proliferation and survival. 4-1BBL lacks costimulatory activity in soluble form. To obtain co-stimulatory activity in soluble form, a recombinant 4-1BBL protein was generated by fusing the extracellular domains of murine 4-1BBL to a modified version of streptavidin (SA-4-1BBL). Treatment with SA-4-1BBL inhibited the development of lung tumors in A/J mice induced by weekly injections of the tobacco carcinogen NNK for eight weeks. The inhibition was dependent on the presence of T cells and NK cells; depletion of these cells diminished the SA-4-1BBL antitumor protective effect. The number of lung tumor nodules was significantly reduced by the administration of SA-4-1BBL to mice during ongoing exposure to NNK. The data presented in this paper suggest that utilizing an immune checkpoint stimulator as a single agent generate a protective immune response against lung cancer in the presence of a carcinogen. More broadly, this study suggests that immune checkpoint stimulation can be extended to a number of other cancer types, including breast and prostate cancers, for which improved diagnostics can detect disease at the preneoplastic stage.

Bioengineered omental transplant site promotes pancreatic islet allografts survival in non-human primates.

The transplanting islets to the liver approach suffers from an immediate posttransplant loss of islets of more than 50%, progressive graft dysfunction over time, and precludes recovery of grafts should there be serious complications such as the development of teratomas with grafts that are stem cell-derived islets (SC-islets). The omentum features an attractive extrahepatic alternative site for clinical islet transplantation. We explore an approach in which allogeneic islets are transplanted onto the omentum, which is bioengineered with a plasma-thrombin biodegradable matrix in three diabetic non-human primates (NHPs). Within 1 week posttransplant, each transplanted NHP achieves normoglycemia and insulin independence and remains stable until termination of the experiment. Success was achieved in each case with islets recovered from a single NHP donor. Histology demonstrates robust revascularization and reinnervation of the graft. This preclinical study can inform the development of strategies for ß cell replacement including the use of SC-islets or other types of novel cells in clinical settings.

Engineering pancreatic islets with a novel form of thrombomodulin protein to overcome early graft loss triggered by instant blood-mediated inflammatory reaction.

The instant blood-mediated inflammatory reaction (IBMIR) is initiated by innate immune responses that cause substantial islet loss after intraportal transplantation. Thrombomodulin (TM) is a multifaceted innate immune modulator. In this study, we report the generation of a chimeric form of thrombomodulin with streptavidin (SA-TM) for transient display on the surface of islets modified with biotin to mitigate IBMIR. SA-TM protein expressed in insect cells showed the expected structural and functional features. SA-TM converted protein C into activated protein C, blocked phagocytosis of xenogeneic cells by mouse macrophages and inhibited neutrophil activation. SA-TM was effectively displayed on the surface of biotinylated islets without a negative effect on their viability or function. Islets engineered with SA-TM showed improved engraftment and established euglycemia in 83% of diabetic recipients when compared with 29% of recipients transplanted with SA-engineered islets as control in a syngeneic minimal mass intraportal transplantation model. Enhanced engraftment and function of SA-TM-engineered islets were associated with the inhibition of intragraft proinflammatory innate cellular and soluble mediators of IBMIR, such as macrophages, neutrophils, high-mobility group box 1, tissue factor, macrophage chemoattractant protein-1, interleukin-1ß, interleukin-6, tumor necrosis factor-α, interferon-γ. Transient display of SA-TM protein on the islet surface to modulate innate immune responses causing islet graft destruction has clinical potential for autologous and allogeneic islet transplantation.

Engineering donor lymphocytes with Fas ligand protein effectively prevents acute graft-versus-host disease.

Alloreactive T-effector cells (Teffs) are the major culprit of acute graft-versus-host disease (aGVHD) associated with hematopoietic stem cell transplantation. Ex vivo nonspecific depletion of T cells from the donor graft impedes stem cell engraftment and posttransplant immune reconstitution. Teffs upregulate Fas after activation and undergo Fas ligand (FasL)-mediated restimulation-induced cell death (RICD), an important mechanism of immune homeostasis. We targeted RICD as a means to eliminate host-reactive Teffs in vivo for the prevention of aGVHD. A novel form of FasL protein chimeric with streptavidin (SA-FasL) was transiently displayed on the surface of biotinylated lymphocytes, taking advantage of the high-affinity interaction between biotin and streptavidin. SA-FasL-engineered mouse and human T cells underwent apoptosis after activation in response to alloantigens in vitro and in vivo. SA-FasL on splenocytes was effective in preventing aGVHD in >70% of lethally irradiated haploidentical mouse recipients after cotransplantation with bone marrow cells, whereas all controls that underwent transplantation with nonengineered splenocytes developed aGVHD. Prevention of aGVHD was associated with an increased ratio of CD4+CD25+FoxP3+ T regulatory (Tregs) to Teffs and significantly reduced transcripts for proinflammatory cytokines in the lymphoid organs and target tissues. Depletion of Tregs from the donor graft abrogated the protection conferred by SA-FasL. This approach was also effective in a xenogeneic aGVHD setting where SA-FasL-engineered human PBMCs were transplanted into NSG mice. Direct display of SA-FasL protein on donor cells as an effective means of eliminating alloreactive Teffs in the host represents a practical approach with significant translation potential for the prevention of aGVHD.

Fas Ligand-Modified Scaffolds Protect Stem Cell Derived ß-Cells by Modulating Immune Cell Numbers and Polarization.

Stem cell derived ß-cells have demonstrated the potential to control blood glucose levels and represent a promising treatment for Type 1 diabetes (T1D). Early engraftment post-transplantation and subsequent maturation of these ß-cells are hypothesized to be limited by the initial inflammatory response, which impacts the ability to sustain normoglycemia for long periods. We investigated the survival and development of immature hPSC-derived ß-cells transplanted on poly(lactide-co-glycolide) (PLG) microporous scaffolds into the peritoneal fat, a site being considered for clinical translation. The scaffolds were modified with biotin for binding of a streptavidin-FasL (SA-FasL) chimeric protein to modulate the local immune cell responses. The presence of FasL impacted infiltration of monocytes and neutrophils and altered the immune cell polarization. Conditioned media generated from SA-FasL scaffolds explanted at day 4 post-transplant did not impact hPSC-derived ß-cell survival and maturation in vitro, while these responses were reduced with conditioned media from control scaffolds. Following transplantation, ß-cell viability and differentiation were improved with SA-FasL modification. A sustained increase in insulin positive cell ratio was observed with SA-FasL-modified scaffolds relative to control scaffolds. These results highlight that the initial immune response can significantly impact ß-cell engraftment, and modulation of cell infiltration and polarization may be a consideration for supporting long-term function at an extrahepatic site.

A hydrogel platform for co-delivery of immunomodulatory proteins for pancreatic islet allografts.

Type 1 diabetes (T1D), an autoimmune disorder in which the insulin-producing ß-cells in the islets of Langerhans in the pancreas are destroyed, afflicts over 1.6 million Americans. Although pancreatic islet transplantation has shown promise in treating T1D, continuous use of required immunosuppression regimens limits clinical islet transplantation as it poses significant adverse effects on graft recipients and does not achieve consistent long-term graft survival with 50%-70% of recipients maintaining insulin independence at 5 years. T cells play a key role in graft rejection, and rebalancing pathogenic T effector and protective T regulatory cells can regulate autoimmune disorders and transplant rejection. The synergy of the interleukin-2 (IL-2) and Fas immunomodulatory pathways presents an avenue for eliminating the need for systemic immune suppression by exploiting IL-2's role in expanding regulatory T cells and leveraging Fas ligand (FasL) activity on antigen-induced cell death of effector T cells. Herein, we developed a hydrogel platform for co-delivering an analog of IL-2, IL-2D, and FasL-presenting microgels to achieve localized immunotolerance to pancreatic islets by targeting the upregulation of regulatory T cells and effector T cells simultaneously. Although this hydrogel provided for sustained, local delivery of active immunomodulatory proteins, indefinite allograft survival was not achieved. Immune profiling analysis revealed upregulation of target regulatory T cells but also increases in Granzyme B-expressing CD8+ T cells at the graft site. We attribute the failed establishment of allograft survival to these Granzyme B-expressing T cells. This study underscores the delicate balance of immunomodulatory components important for allograft survival - whose outcome can be dependent on timing, duration, modality of delivery, and disease model.

A modified surgical procedure using minimally invasive ileocolic vein perfusion in a mouse intrahepatic islet transplant model.

Murine intrahepatic islet transplantation is a clinically relevant but technically challenging surgical procedure because of frequent lethal postoperative bleeding. Here, we describe a protocol for mouse pancreatic islet isolation, purification, and culture. Besides, we also describe a protocol for intrahepatic islet transplantation through the ileocolic vein. Intrahepatic islet transplantation through the ileocolic vein, as opposed to traditional islet perfusion via the main portal vein, has the advantage of improving recovery after surgery and may facilitate islet survival and function in preclinical settings. For complete details on the use and execution of this protocol, please refer to Shrestha et al. (2020).

FasL microgels induce immune acceptance of islet allografts in nonhuman primates.

Islet transplantation to treat insulin-dependent diabetes is greatly limited by the need for maintenance immunosuppression. We report a strategy through which cotransplantation of allogeneic islets and streptavidin (SA)-FasL-presenting microgels to the omentum under transient rapamycin monotherapy resulted in robust glycemic control, sustained C-peptide levels, and graft survival in diabetic nonhuman primates for >6 months. Surgical extraction of the graft resulted in prompt hyperglycemia. In contrast, animals receiving microgels without SA-FasL under the same rapamycin regimen rejected islet grafts acutely. Graft survival was associated with increased number of FoxP3+ cells in the graft site with no significant changes in T cell systemic frequencies or responses to donor and third-party antigens, indicating localized tolerance. Recipients of SA-FasL microgels exhibited normal liver and kidney metabolic function, demonstrating safety. This localized immunomodulatory strategy succeeded with unmodified islets and does not require long-term immunosuppression, showing translational potential in ß cell replacement for treating type 1 diabetes.

Immunotherapy via PD-L1-presenting biomaterials leads to long-term islet graft survival.

Antibody-mediated immune checkpoint blockade is a transformative immunotherapy for cancer. These same mechanisms can be repurposed for the control of destructive alloreactive immune responses in the transplantation setting. Here, we implement a synthetic biomaterial platform for the local delivery of a chimeric streptavidin/programmed cell death-1 (SA-PD-L1) protein to direct "reprogramming" of local immune responses to transplanted pancreatic islets. Controlled presentation of SA-PD-L1 on the surface of poly(ethylene glycol) microgels improves local retention of the immunomodulatory agent over 3 weeks in vivo. Furthermore, local induction of allograft acceptance is achieved in a murine model of diabetes only when receiving the SA-PD-L1-presenting biomaterial in combination with a brief rapamycin treatment. Immune characterization revealed an increase in T regulatory and anergic cells after SA-PD-L1-microgel delivery, which was distinct from naïve and biomaterial alone microenvironments. Engineering the local microenvironment via biomaterial delivery of checkpoint proteins has the potential to advance cell-based therapies, avoiding the need for systemic chronic immunosuppression.

Localized Immunomodulation with PD-L1 Results in Sustained Survival and Function of Allogeneic Islets without Chronic Immunosuppression.

Allogeneic islet transplantation is limited by adverse effects of chronic immunosuppression used to control rejection. The programmed cell death 1 pathway as an important immune checkpoint has the potential to obviate the need for chronic immunosuppression. We generated an oligomeric form of programmed cell death 1 ligand chimeric with core streptavidin (SA-PDL1) that inhibited the T effector cell response to alloantigens and converted T conventional cells into CD4+Foxp3+ T regulatory cells. The SA-PDL1 protein was effectively displayed on the surface of biotinylated mouse islets without a negative impact islet viability and insulin secretion. Transplantation of SA-PDL1-engineered islet grafts with a short course of rapamycin regimen resulted in sustained graft survival and function in >90% of allogeneic recipients over a 100-d observation period. Long-term survival was associated with increased levels of intragraft transcripts for innate and adaptive immune regulatory factors, including IDO-1, arginase-1, Foxp3, TGF-ß, IL-10, and decreased levels of proinflammatory T-bet, IL-1ß, TNF-α, and IFN-γ as assessed on day 3 posttransplantation. T cells of long-term graft recipients generated a proliferative response to donor Ags at a similar magnitude to T cells of naive animals, suggestive of the localized nature of tolerance. Immunohistochemical analyses showed intense peri-islet infiltration of T regulatory cells in long-term grafts and systemic depletion of this cell population resulted in prompt rejection. The transient display of SA-PDL1 protein on the surface of islets serves as a practical means of localized immunomodulation that accomplishes sustained graft survival in the absence of chronic immunosuppression with potential clinical implications.

Immune checkpoint CD47 molecule engineered islets mitigate instant blood-mediated inflammatory reaction and show improved engraftment following intraportal transplantation.

Instant blood-mediated inflammatory reaction (IBMIR) causes significant destruction of islets transplanted intraportally. Myeloid cells are a major culprit of IBMIR. Given the critical role of CD47 as a negative checkpoint for myeloid cells, we hypothesized that the presence of CD47 on islets will minimize graft loss by mitigating IBMIR. We herein report the generation of a chimeric construct, SA-CD47, encompassing the extracellular domain of CD47 modified to include core streptavidin (SA). SA-CD47 protein was expressed in insect cells and efficiently displayed on biotin-modified mouse islet surface without a negative impact on their viability and function. Rat cells engineered with SA-CD47 were refractory to phagocytosis by mouse macrophages. SA-CD47-engineered islets showed intact structure and minimal infiltration by CD11b+ granulocytes/macrophages as compared with SA-engineered controls in an in vitro loop assay mitigating IBMIR. In a syngeneic marginal mass model of intraportal transplantation, SA-CD47-engineered islets showed better engraftment and function as compared with the SA-control group (87.5% vs 14.3%). Engraftment was associated with low levels of intrahepatic inflammatory cells and mediators of islet destruction, including high-mobility group box-1, tissue factor, and IL-1ß. These findings support the use of CD47 as an innate immune checkpoint to mitigate IBMIR for enhanced islet engraftment with translational potential.

Pancreatic islets engineered with a FasL protein induce systemic tolerance at the induction phase that evolves into long-term graft-localized immune privilege.

We have previously shown that pancreatic islets engineered to transiently display a modified form of FasL protein (SA-FasL) on their surface survive indefinitely in allogeneic recipients without a need for chronic immunosuppression. Mechanisms that confer long-term protection to allograft are yet to be elucidated. We herein demonstrated that immune protection evolves in two distinct phases; induction and maintenance. SA-FasL-engineered allogeneic islets survived indefinitely and conferred protection to a second set of donor-matched, but not third-party, unmanipulated islet grafts simultaneously transplanted under the contralateral kidney capsule. Protection at the induction phase involved a reduction in the frequency of proliferating alloreactive T cells in the graft-draining lymph nodes, and required phagocytes and TGF-ß. At the maintenance phase, immune protection evolved into graft site-restricted immune privilege as the destruction of long-surviving SA-FasL-islet grafts by streptozotocin followed by the transplantation of a second set of unmanipulated islet grafts into the same site from the donor, but not third party, resulted in indefinite survival. The induced immune privilege required both CD4+ CD25+ Foxp3+ Treg cells and persistent presence of donor antigens. Engineering cell and tissue surfaces with SA-FasL protein provides a practical, efficient, and safe means of localized immunomodulation with important implications for autoimmunity and transplantation.

Rapid On-Demand Extracellular Vesicle Augmentation with Versatile Oligonucleotide Tethers.

Exosomes show potential as ideal vehicles for drug delivery because of their natural role in transferring biological cargo between cells. However, current methods to engineer exosomes without negatively impacting their function remain challenging. Manipulating exosome-secreting cells is complex and time-consuming, while direct functionalization of exosome surface proteins suffers from low specificity and low efficiency. We demonstrate a rapid, versatile, and scalable method with oligonucleotide tethers to enable diverse surface functionalization on both human and murine exosomes. These exosome surface modifiers, which range from reactive functional groups and small molecules to aptamers and large proteins, can readily and efficiently enhance native exosome properties. We show that cellular uptake of exosomes can be specifically altered with a tethered AS1411 aptamer, and targeting specificity can be altered with a tethered protein. We functionalize exosomes with an immunomodulatory protein, FasL, and demonstrate their biological activity both in vitro and in vivo. FasL-functionalized exosomes, when bioprinted on a collagen matrix, allows spatial induction of apoptosis in tumor cells and, when injected in mice, suppresses proliferation of alloreactive T cells. This oligonucleotide tethering strategy is independent of the exosome source and further circumvents the need to genetically modify exosome-secreting cells.

Robust Th1 cellular and humoral responses generated by the Yersinia pestis rF1-V subunit vaccine formulated to contain an agonist of the CD137 pathway do not translate into increased protection against pneumonic plague.

Yersinia pestis is the causative agent of plague and is a re-emerging pathogen that also has the potential as a biological weapon, necessitating the development of a preventive vaccine. Despite intense efforts for the last several decades, there is currently not a vaccine approved by the FDA. The rF1-V vaccine adjuvanted with Alhydrogel is a lead candidate subunit vaccine for plague and generates a strong Th2-mediate humoral response with a modest Th1 cellular response. As immune protection against Y. pestis requires both humoral and Th1 cellular responses, modifying the rF1-V subunit vaccine formulation to include a robust inducer of Th1 responses may improve efficacy. Thus, we reformulated the subunit vaccine to include SA-4-1BBL, an agonist of the CD137 costimulatory pathway and a potent inducer of Th1 response, and assessed its protective efficacy against pneumonic plague. We herein show for the first time a sex bias in the prophylactic efficacy of the Alhydrogel adjuvanted rF1-V vaccine, with female mice showing better protection against pneumonic plague than male. The sex bias for protection was irrespective of the generation of comparable levels of rF1-V-specific antibody titers and Th1 cellular responses in both sexes. The subunit vaccine reformulated with SA-4-1BBL generated robust Th1 cellular and humoral responses. A prime-boost vaccination scheme involving prime with rF1-V + Alhydrogel and boost with the rF1-V + SA-4-1BBL provided protection in male mice against pneumonic plague. In marked contrast, prime and boost with rF1-V reformulated with both adjuvants resulted in the loss of protection against pneumonic plague, despite generating high levels of humoral and Th1 cellular responses. While unexpected, these findings demonstrate the complexity of immune mechanisms required for protection. Elucidating mechanisms responsible for these differences in protection will help to guide the development of better prophylactic subunit vaccines effective against pneumonic plague.

A Novel Form of 4-1BBL Prevents Cancer Development via Nonspecific Activation of CD4+ T and Natural Killer Cells.

Costimulation through 4-1BB (CD137) receptor generates robust CD8+ T-effector and memory responses. The only known ligand, 4-1BBL, is a trimeric transmembrane protein that has no costimulatory activity as a soluble molecule. Thus, agonistic antibodies to the receptor have been used for cancer immunotherapy in preclinical models and are currently being evaluated in the clinic. Here, we report that treatment with an oligomeric form of the ligand, SA-4-1BBL, as a single agent is able to protect mice against subsequent tumor challenge irrespective of the tumor type. Protection was long-lasting (>8 weeks) and a bona fide property of SA-4-1BBL, as treatment with an agonistic antibody to the 4-1BB receptor was ineffective in generating immune protection against tumor challenge. Mechanistically, SA-4-1BBL significantly expanded IFNγ-expressing, preexisting memory-like CD44+CD4+ T cells and NK cells in naïve mice as compared with the agonistic antibody. In vivo blockade of IFNγ or depletion of CD4+ T or NK cells, but not CD8+ T or B cells, abrogated the immunopreventive effects of SA-4-1BBL against cancer. SA-4-1BBL as a single agent also exhibited robust efficacy in controlling postsurgical recurrences. This work highlights unexpected features of SA-4-1BBL as a novel immunomodulator with implications for cancer immunoprevention and therapy. SIGNIFICANCE: This study demonstrates the unique and unexpected immunomodulatory features of SA-4-1BBL that bridge innate and adaptive immune responses with both preventive and therapeutic efficacy against cancer.