Kate H. Gartlan

Polyethyleneimine is a potent mucosal adjuvant for viral glycoprotein antigens

There are no mucosal adjuvant formulations licensed for human use, despite protection against many mucosally-transmitted infections probably requiring immunity at the site of pathogen entry1. Polyethyleneimines (PEI) are organic polycations used as nucleic acid transfection reagents in vitro, and gene and DNA vaccine delivery vehicles in vivo2, 3. Here we show that PEI has unexpected and unusually potent mucosal adjuvant activity in conjunction with viral subunit glycoprotein antigens. Single intranasal administration of influenza HA or HSV-2 gD with PEI elicited robust protection from otherwise lethal infection, and was superior to existing experimental mucosal adjuvants. PEI formed nanoscale complexes with antigen that were taken up by antigen presenting cells in vitro and in vivo, promoted DC trafficking to draining lymph nodes and induced non-proinflammatory cytokine responses. PEI adjuvanticity required release of host dsDNA that triggered Irf-3-dependent signaling. PEI therefore merits further investigation as a mucosal adjuvant for human use.Protection against mucosally transmitted infections probably requires immunity at the site of pathogen entry, yet there are no mucosal adjuvant formulations licensed for human use. Polyethyleneimine (PEI) represents a family of organic polycations used as nucleic acid transfection reagents in vitro and DNA vaccine delivery vehicles in vivo. Here we show that diverse PEI forms have potent mucosal adjuvant activity for viral subunit glycoprotein antigens. A single intranasal administration of influenza hemagglutinin or herpes simplex virus type-2 (HSV-2) glycoprotein D with PEI elicited robust antibody-mediated protection from an otherwise lethal infection, and was superior to existing experimental mucosal adjuvants. PEI formed nanoscale complexes with antigen, which were taken up by antigen-presenting cells in vitro and in vivo, promoted dendritic cell trafficking to draining lymph nodes and induced non-proinflammatory cytokine responses. PEI adjuvanticity required release of host double-stranded DNA that triggered Irf3-dependent signaling. PEI therefore merits further investigation as a mucosal adjuvant for human use.

Reactive Carbonyls Are a Major Th2-Inducing Damage-Associated Molecular Pattern Generated by Oxidative Stress

Oxidative stress is widespread and entwined with pathological processes, yet its linkage to adaptive immunity remains elusive. Reactive carbonyl (RC) adduction, a common feature of oxidative stress, has been shown to target proteins to the adaptive immune system. Because aldehydes are important mediators of carbonylation, we explored the immunomodulatory properties of model Ags modified by common bioactive aldehyde by-products of oxidative stress: 4-hydroxy-2-nonenal, malondialdehyde, and glycolaldehyde. Ag modification with all three aldehydes resulted in Ag-specific IgG1-dominated responses in adjuvant-free murine immunizations in an RC-dependent manner. The central role of RCs was confirmed, as their reduction into nonreactive groups abrogated all adaptive responses, despite the presence of other well-known aldehyde-driven adducts such as Nε-carboxymethyllysine and glycolaldehyde–pyridine. Moreover, Ag-specific Ab responses robustly correlated with the extent of RC adduction, regardless of the means of their generation. T cell responses mirrored the Th2-biased Ab isotypes by Ag-specific splenocyte production of IL-4, IL-5, and IL-13, but not IFN-γ. The RC-induced Th2 response was in sharp contrast to that induced by Th1/Th2 balanced or Th1-biasing adjuvants and was maintained in a range of mouse strains. In vitro studies revealed that RC adduction enhanced Ag presentation with Th2 polarization in the absence of conventional dendritic cell activation. Taken together, these data implicate commonly occurring RC as an important oxidation-derived Th2 immunomodulatory damage-associated molecular pattern with potentially important roles in health and disease.

Lung parenchyma-derived IL-6 promotes IL-17A-dependent acute lung injury after allogeneic stem cell transplantation.

Idiopathic pneumonia syndrome (IPS) is a relatively common, frequently fatal clinical entity, characterized by noninfectious acute lung inflammation following allogeneic stem cell transplantation (SCT), the mechanisms of which are unclear. In this study, we demonstrate that immune suppression with cyclosporin after SCT limits T-helper cell (Th) 1 differentiation and interferon-γ secretion by donor T cells, which is critical for inhibiting interleukin (IL)-6 generation from lung parenchyma during an alloimmune response. Thereafter, local IL-6 secretion induces donor alloantigen-specific Th17 cells to preferentially expand within the lung, and blockade of IL-17A or transplantation of grafts lacking the IL-17 receptor prevents disease. Studies using IL-6(-/-) recipients or IL-6 blockade demonstrate that IL-6 is the critical driver of donor Th17 differentiation within the lung. Importantly, IL-6 is also dysregulated in patients undergoing clinical SCT and is present at very high levels in the plasma of patients with IPS compared with SCT recipients without complications. Furthermore, at the time of diagnosis, plasma IL-6 levels were higher in a subset of IPS patients who were nonresponsive to steroids and anti-tumor necrosis factor therapy. In sum, pulmonary-derived IL-6 promotes IPS via the induction of Th17 differentiation, and strategies that target these cytokines represent logical therapeutic approaches for IPS.

Donor colonic CD103+ dendritic cells determine the severity of acute graft-versus-host disease

Koyama et al. show that GVHD markedly enhances alloantigen presentation within the mesenteric lymph nodes, mediated by donor CD103+CD11b− DCs that migrate from the colon under the influence of CCR7. This antigen presentation imprints gut-homing integrin signatures on donor T cells, leading to their migration to the GI tract where they mediate fulminant disease.

Dry roasting enhances peanut-induced allergic sensitization across mucosal and cutaneous routes in mice

the HPV cohort. This could have introduced an ascertainment bias for HPV detection and may explain our lower overall rate of identified HPV. However, it is clear that post-HSCT, the cohort without HPV disease had markedly improved NK cell engraftment when compared with their counterparts with HPV disease. Investigators have proposed that in gc receptor deficiency, dendritic cells may remain dysfunctional post-HSCT and thus permissive toHPVinfection despite donorT cell engraftment.No specific data supports this theory. Our study revealed a lower incidence of HPV disease than in other cohorts, likely related to the lowermean age of ourHPV-negative cohort and excellent engraftment of T, B, and NK cells in our entire cohort. We propose that poor NK cell engraftment and function represents a more likely contributor to the development of severe HPV disease in our 4 patients. It is possible that pre-HSCT myeloablation improved NK cell engraftment and function, allowing for improved response to HPVexposure in later life and thus disease limitation. With improvements in early diagnosis and curative treatment in SCID, it is imperative to review and determine the etiology of long-term adverse events like severe HPV disease following transplant.

Polyethyleneimine is a potent systemic adjuvant for glycoprotein antigens

Polyethyleneimine (PEI) is an organic polycation used extensively as a gene and DNA vaccine delivery reagent. Although the DNA targeting activity of PEI is well documented, its immune activating activity is not. We recently reported that PEI has robust mucosal adjuvanticity when administered intranasally with glycoprotein antigens. Here, we show that PEI has strong immune activating activity after systemic delivery. PEI administered subcutaneously with viral glycoprotein (HIV-1 gp140) enhanced antigen-specific serum IgG production in the context of mixed Th1/Th2-type immunity. PEI elicited higher titers of both antigen binding and neutralizing antibodies than alum in mice and rabbits and induced an increased proportion of antibodies reactive with native antigen. In an intraperitoneal model, PEI recruited neutrophils followed by monocytes to the site of administration and enhanced antigen uptake by antigen-presenting cells. The Th bias was modulated by PEI activation of the Nlrp3 inflammasome; however its global adjuvanticity was unchanged in Nlrp3-deficient mice. When coformulated with CpG oligodeoxynucleotides, PEI adjuvant potency was synergistically increased and biased toward a Th1-type immune profile. Taken together, these data support the use of PEI as a versatile systemic adjuvant platform with particular utility for induction of secondary structure-reactive antibodies against glycoprotein antigens.

Acute graft-versus-host disease is regulated by an IL-17–sensitive microbiome

Donor T-cell-derived interleukin-17A (IL-17A) can mediate late immunopathology in graft-versus-host disease (GVHD), however protective roles remain unclear. Using multiple cytokine and cytokine receptor subunit knockout mice, we demonstrate that stem cell transplant recipients lacking the ability to generate or signal IL-17 develop intestinal hyper-acute GVHD. This protective effect is restricted to the molecular interaction of IL-17A and/or IL-17F with the IL-17 receptor A/C (IL-17RA/C). The protection from GVHD afforded by IL-17A required secretion from, and signaling in, both hematopoietic and nonhematopoietic host tissue. Given the intestinal-specificity of the disease in these animals, we cohoused wild-type (WT) with IL-17RA and IL-17RC-deficient mice, which dramatically enhanced the susceptibility of WT mice to acute GVHD. Furthermore, the gut microbiome of WT mice shifted toward that of the IL-17RA/C mice during cohousing prior to transplant, confirming that an IL-17-sensitive gut microbiota controls susceptibility to acute GVHD. Finally, induced IL-17A depletion peritransplant also enhanced acute GVHD, consistent with an additional protective role for this cytokine independent of effects on dysbiosis.

Cross-Dressing by Donor Dendritic Cells after Allogeneic Bone Marrow Transplantation Contributes to Formation of the Immunological Synapse and Maximizes Responses to Indirectly Presented Antigen

The stimulation of naive donor T cells by recipient alloantigen is central to the pathogenesis of graft-versus-host disease after bone marrow transplantation (BMT). Using mouse models of transplantation, we have observed that donor cells become “cross-dressed” in very high levels of recipient hematopoietic cell–derived MHC class I and II molecules following BMT. Recipient-type MHC is transiently present on donor dendritic cells (DCs) after BMT in the setting of myeloablative conditioning but is persistent after nonmyeloablative conditioning, in which recipient hematopoietic cells remain in high numbers. Despite the high level of recipient-derived alloantigen present on the surface of donor DCs, donor T cell proliferative responses are generated only in response to processed recipient alloantigen presented via the indirect pathway and not in response to cross-dressed MHC. Assays in which exogenous peptide is added to cross-dressed MHC in the presence of naive TCR transgenic T cells specific to the MHC class II–peptide combination confirm that cross-dressed APC cannot induce T cell proliferation in isolation. Despite failure to induce T cell proliferation, cross-dressing by donor DCs contributes to generation of the immunological synapse between DCs and CD4 T cells, and this is required for maximal responses induced by classical indirectly presented alloantigen. We conclude that the process of cross-dressing by donor DCs serves as an efficient alternative pathway for the acquisition of recipient alloantigen and that once acquired, this cross-dressed MHC can assist in immune synapse formation prior to the induction of full T cell proliferative responses by concurrent indirect Ag presentation.

Imaging the immunological synapse between dendritic cells and T cells.

Immunological synapse formation between antigen-specific T cells and antigen presenting cells (APC) involves reorganization of the cellular cytoskeleton (polymerization of filamentous actin) and recruitment of adhesion molecules (e.g. LFA-1, ICAM-1). This engagement is critical for the generation of specific immune responses. Until recently, quantitative, high-throughput measurements of these interactions have not been possible. Instead, previous assessment was reliant on qualitative microscopy of live cells, where typically the APC is adhered to a surface and the suspended T cell is required to migrate to facilitate synapse formation. While this methodology can demonstrate the capacity for synapse formation, it cannot accommodate quantification of large numbers of interacting cell pairs, nor does it allow for statistically robust comparison between test conditions. We have developed a method for assessing immunological synapse formation between purified ex vivo dendritic cells (DCs) and responder antigen-specific CD4(+) T cells using imaging flow cytometry, allowing us to quantify LFA-1 and f-actin rearrangement at the interface between DC/T cell pairs. This novel application of imaging flow cytometry represents a major advance in dendritic cell function and immunological synapse research as it facilitates quantitative, high throughput analysis of the interaction between live, ex vivo DC and T cells.

Eomesodermin promotes the development of Type-1 Regulatory T (TR1) cells

TR1 cells are the major regulatory population generated after allogeneic bone marrow transplantation. Regulatory T cells sans FoxP3 Although expression of FoxP3 is largely synonymous with regulatory T (Treg) cell identity in mice, type 1 regulatory T (TR1) cells are an exception. TR1 cells produce interleukin-10 but are FoxP3−. In comparison with FoxP3+ Treg cells, the development and functions of TR1 cells are poorly understood. Here, Zhang et al. report that TR1 cells play a critical regulatory role after allogeneic bone marrow transplantation (BMT) in mice and use this model to delineate the molecular circuits driving commitment to the TR1 cell lineage. By documenting the presence of TR1 cells after BMT in humans, they propose that modulation of TR1 cells could be a therapeutic venue for increasing BMT success rates in the clinic. Type 1 regulatory T (TR1) cells are Foxp3− interleukin-10 (IL-10)–producing CD4+ T cells with potent immunosuppressive properties, but their requirements for lineage development have remained elusive. We show that TR1 cells constitute the most abundant regulatory population after allogeneic bone marrow transplantation (BMT), express the transcription factor Eomesodermin (Eomes), and are critical for the prevention of graft-versus-host disease. We demonstrate that Eomes is required for TR1 cell differentiation, during which it acts in concert with the transcription factor B lymphocyte–induced maturation protein-1 (Blimp-1) by transcriptionally activating IL-10 expression and repressing differentiation into other T helper cell lineages. We further show that Eomes induction in TR1 cells requires T-bet and donor macrophage–derived IL-27. Thus, we define the cellular and transcriptional control of TR1 cell differentiation during BMT, opening new avenues to therapeutic manipulation.