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Objective: To show the high analytical specificity of our multiplex microsphere polymerase chain reaction (mmPCR) method, which offers the simultaneous detection of both general (eg, Gram type) and specific (eg, Pseudomonas species) clinically relevant genetic targets in a single modular multiplex reaction., Materials and Methods: Isolated gDNA of 16S/rRNA Sanger-sequenced and Basic Local Alignment Tool-identified bacterial and fungal isolates were selectively amplified in a custom 10-plex Luminex MagPlex-TAG microsphere-based mmPCR assay. The signal/noise ratio for each reaction was calculated from flow cytometry standard data collected on a BD LSR Fortessa II flow cytometer. Data were normalized to the no-template negative control and the signal maximum. The analytical specificity of the assay was compared to single-plex SYBR chemistry quantitative PCR., Results: Both general and specific primer sets were functional in the 10-plex mmPCR. The general Gram typing and pan-fungal primers correctly identified all bacterial and fungal isolates, respectively. The species-specific and antibiotic resistance-specific primers correctly identified the species- and resistance-carrying isolates, respectively. Low-level cross-reactive signals were present in some reactions with high signal/noise primer ratios., Conclusion: We found that mmPCR can simultaneously detect specific and general clinically relevant genetic targets in multiplex. These results serve as a proof-of-concept advance that highlights the potential of high multiplex mmPCR diagnostics in clinical practice. Further development of specimen-specific DNA extraction techniques is required for sensitivity testing., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society for Clinical Pathology.)

Perforin is a key effector of lymphocyte-mediated cell death pathways and contributes to transplant rejection of immunologically mismatched grafts. We have developed a novel series of benzenesulfonamide (BZS) inhibitors of perforin that can mitigate graft rejection during allogeneic bone marrow/stem cell transplantation. Eight such perforin inhibitors were tested for their murine pharmacokinetics, plasma protein binding, and their ability to block perforin-mediated lysis in vitro and to block the rejection of major histocompatibility complex (MHC)-mismatched mouse bone marrow cells. All compounds showed >99% binding to plasma proteins and demonstrated perforin inhibitory activity in vitro and in vivo . A lead compound, compound 1 , that showed significant increases in allogeneic bone marrow preservation was evaluated for its plasma pharmacokinetics and in vivo efficacy at multiple dosing regimens to establish a pharmacokinetic/pharmacodynamic (PK/PD) relationship. The strongest PK/PD correlation was observed between perforin inhibition in vivo and time that total plasma concentrations remained above 900 μM, which correlates to unbound concentrations similar to 3× the unbound in vitro IC 90 of compound 1 . This PK/PD relationship will inform future dosing strategies of BZS perforin inhibitors to maintain concentrations above 3× the unbound IC 90 for as long as possible to maximize efficacy and enhance progression toward clinical evaluation., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Immunopathology and intestinal stem cell (ISC) loss in the gastrointestinal (GI) tract is the prima facie manifestation of graft-versus-host disease (GVHD) and is responsible for significant mortality after allogeneic bone marrow transplantation (BMT). Approaches to prevent GVHD to date focus on immune suppression. Here, we identify interferon-λ (IFN-λ; interleukin-28 [IL-28]/IL-29) as a key protector of GI GVHD immunopathology, notably within the ISC compartment. Ifnlr1-/- mice displayed exaggerated GI GVHD and mortality independent of Paneth cells and alterations to the microbiome. Ifnlr1-/- intestinal organoid growth was significantly impaired, and targeted Ifnlr1 deficiency exhibited effects intrinsic to recipient Lgr5+ ISCs and natural killer cells. PEGylated recombinant IL-29 (PEG-rIL-29) treatment of naive mice enhanced Lgr5+ ISC numbers and organoid growth independent of both IL-22 and type I IFN and modulated proliferative and apoptosis gene sets in Lgr5+ ISCs. PEG-rIL-29 treatment improved survival, reduced GVHD severity, and enhanced epithelial proliferation and ISC-derived organoid growth after BMT. The preservation of ISC numbers in response to PEG-rIL-29 after BMT occurred both in the presence and absence of IFN-λ-signaling in recipient natural killer cells. IFN-λ is therefore an attractive and rapidly testable approach to prevent ISC loss and immunopathology during GVHD., (© 2021 by The American Society of Hematology.)

We determined the efficacy of tocilizumab (TCZ) in preventing grade 2-4 acute graft-versus-host disease (aGVHD) in patients with acute leukemia or myelodysplasia undergoing matched sibling donor (MSD) or volunteer unrelated donor (VUD) allogeneic stem cell transplantation after myeloablative or reduced-intensity conditioning across 5 Australian centers. A total of 145 patients (50 MSD, 95 VUD) were randomly assigned to placebo or TCZ on day -1. All patients received T-cell-replete peripheral blood stem cell grafts and graft-versus-host disease (GVHD) prophylaxis with cyclosporin/methotrexate. A planned substudy analyzed the VUD cohort. With a median follow-up of 746 days, the incidence of grade 2-4 aGVHD at day 100 for the entire cohort was 36% for placebo vs 27% for TCZ (hazard ratio [HR], 0.69; 95% confidence interval [CI], 0.38-1.26; P = .23) and 45% vs 32% (HR, 0.61; 95% CI, 0.31-1.22; P = .16) for the VUD subgroup. The incidence of grade 2-4 aGVHD at day 180 for the entire cohort was 40% for placebo vs 29% for TCZ (HR, 0.68; 95% CI, 0.38-1.22; P = .19) and 48% vs 32% (HR, 0.59; 95% CI, 0.30-1.16; P = .13) for the VUD subgroup. Reductions in aGVHD were predominantly in grade 2 disease. For the entire cohort, transplant-related mortality occurred in 8% vs 11% of placebo-treated vs TCZ-treated patients, respectively (P = .56), and overall survival was 79% vs 71% (P = .27). Median day to neutrophil and platelet engraftment was delayed by 2 to 3 days in TCZ-treated patients, whereas liver toxicity and infectious complications were similar between groups. In this phase 3 randomized double-blind trial, TCZ showed nonsignificant trends toward reduced incidence of grade 2-4 aGVHD in recipients from HLA-matched VUDs but no improvements in long term-survival., (© 2021 by The American Society of Hematology.)

Panobinostat is a pan-deacetylase inhibitor that modulates the expression of oncogenic and immune-mediating genes involved in tumour cell growth and survival. We evaluated panobinostat-induced post-transplant responses and identified correlative biomarkers in patients with multiple myeloma who had failed to achieve a complete response after autologous transplantation. Patients received panobinostat 45 mg administered three-times weekly (TIW) on alternate weeks of 28-day cycles commencing 8-12 weeks post-transplant. Twelve of 25 patients (48%) improved their depth of response after a median (range) of 4·3 (1·9-9·7) months of panobinostat. In responders, T-lymphocyte histone acetylation increased after both three cycles (P < 0·05) and six cycles (P < 0·01) of panobinostat when compared to baseline, with no differences in non-responders. The reduction in the proportion of CD127 + CD8 + T cells and CD4:CD8 ratio was significantly greater, after three and six cycles of panobinostat compared to pre-transplant, in non-responders when compared to responders. Whole marrow RNA-seq revealed widespread transcriptional changes only in responders with baseline differences in genes involved in ribosome biogenesis, oxidative phosphorylation and metabolic pathways. This study confirmed the efficacy of panobinostat as a single agent in multiple myeloma and established acetylation of lymphocyte histones, modulation of immune subsets and transcriptional changes as pharmacodynamic biomarkers of clinical benefit., (© 2020 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

The adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) is known to facilitate caspase-1 activation, which is essential for innate host immunity via the formation of the inflammasome complex, a multiprotein structure responsible for processing IL1β and IL18 into their active moieties. Here, we demonstrated that ASC-deficient CD8 + T cells failed to induce severe graft-versus-host disease (GVHD) and had impaired capacity for graft rejection and graft-versus-leukemia (GVL) activity. These effects were inflammasome independent because GVHD lethality was not altered in recipients of caspase-1/11-deficient T cells. We also demonstrated that ASC deficiency resulted in a decrease in cytolytic function, with a reduction in granzyme B secretion and CD107a expression by CD8 + T cells. Altogether, our findings highlight that ASC represents an attractive therapeutic target for improving outcomes of clinical transplantation., (©2020 American Association for Cancer Research.)

Perforin is a key effector protein in the vertebrate immune system and is secreted by cytotoxic T lymphocytes and natural killer cells to help eliminate virus-infected and transformed target cells. The ability to modulate perforin activity in vivo could be extremely useful, especially in the context of bone marrow stem cell transplantation where early rejection of immunologically mismatched grafts is driven by the recipient's natural killer cells, which overwhelmingly use perforin to kill their targets. Bone marrow stem cell transplantation is a potentially curative treatment for both malignant and nonmalignant disorders, but when the body recognizes the graft as foreign, it is rejected by this process, often with fatal consequences. Here we report optimization of a previously identified series of benzenesulfonamide-based perforin inhibitors for their physicochemical and pharmacokinetic properties, resulting in the identification of 16 , the first reported small molecule able to prevent rejection of transplanted bone marrow stem cells in vivo by blocking perforin function.

Graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (alloSCT) is characterized by interleukin-6 (IL-6) dysregulation. IL-6 can mediate effects via various pathways, including classical, trans, and cluster signaling. Given the recent availability of agents that differentially inhibit these discrete signaling cascades, understanding the source and signaling and cellular targets of this cytokine is paramount to inform the design of clinical studies. Here we demonstrate that IL-6 secretion from recipient dendritic cells (DCs) initiates the systemic dysregulation of this cytokine. Inhibition of DC-driven classical signaling after targeted IL-6 receptor (IL-6R) deletion in T cells eliminated pathogenic donor Th17/Th22 cell differentiation and resulted in long-term survival. After engraftment, donor DCs assume the same role, maintaining classical IL-6 signaling-dependent GVHD responses. Surprisingly, cluster signaling was not active after transplantation, whereas inhibition of trans signaling with soluble gp130Fc promoted severe, chronic cutaneous GVHD. The latter was a result of exaggerated polyfunctional Th22-cell expansion that was reversed by IL-22 deletion or IL-6R inhibition. Importantly, inhibition of IL-6 classical signaling did not impair the graft-versus-leukemia effect. Together, these data highlight IL-6 classical signaling and downstream Th17/Th22 differentiation as important therapeutic targets after alloSCT., (© 2019 by The American Society of Hematology.)

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has recently emerged as an important pathogenic cytokine in acute graft-versus-host disease (GVHD), but the nature of the T-cell lineages secreting the cytokine and the mechanisms of action are less clear. Here we used interleukin 17A-fate reporter systems with transcriptional analysis and assays of alloantigen presentation to interrogate the origins of GM-CSF-secreting T cells and the effects of the cytokine on antigen-presenting cell (APC) function after experimental allogeneic stem cell transplantation (SCT). We demonstrated that although GM-CSF-secreting Th17 and non-Th17 cells expanded in the colon over time after SCT, the Th17 lineage expanded to represent 10% to 20% of the GM-CSF secreting T cells at this site by 4 weeks. Donor T-cell-derived GM-CSF expanded alloantigen-presenting donor dendritic cells (DCs) in the colon and lymph nodes. In the mesenteric lymph nodes, GM-CSF-dependent DCs primed donor T cells and amplified acute GVHD in the colon. We thus describe a feed-forward cascade whereby GM-CSF-secreting donor T cells accumulate and drive alloantigen presentation in the colon to amplify GVHD severity. GM-CSF inhibition may be a tractable clinical intervention to limit donor alloantigen presentation and GVHD in the lower gastrointestinal tract., (© 2019 by The American Society of Hematology.)

This unit describes methods for quantifying phagocytosis and imaging the immunological synapse between T cells and antigen-presenting cells (APCs), with both techniques delivering valuable information about APC function. These aspects of APC biology have traditionally been challenging to quantify, and imaging flow cytometry, which harnesses the high-throughput nature of flow cytometry combined with the capacity of microscopy to deliver spatial localization, facilitates analysis of these APC functions in a fashion that was previously not possible. Imaging flow cytometry allows large numbers of events to be captured and large amounts of fluorescence data to be quantified at the physical location of markers of interest, both on the cell surface and in intracellular compartments, combining key features of traditional flow cytometry and fluorescence microscopy. © 2019 by John Wiley & Sons, Inc., (© 2019 John Wiley & Sons, Inc.)

Purpose: Inducible caspase 9 ( iCasp9 ) is a cellular safety switch that can make T-cell therapy safer. The purpose of this phase I trial was to investigate the use of iCasp9 -transduced T-cell addback in adult patients undergoing haploidentical stem cell transplantation for high-risk hematologic malignancies., Patients and Methods: Patients undergoing myeloablative, CD34-selected haploidentical stem cell transplantation were treated with 0.5-1.0 × 10 6 /kg donor-derived iCasp9 -transduced T cells on day +25 or 26 post-transplant, with additional doses allowed for disease relapse, infection, or mixed chimerism., Results: Three patients were enrolled. iCasp9 -transduced T cells were readily detectable by 4 weeks post-infusion in all patients and remained at high level (114 cells/μL, 11% of T cells) in 1 patient alive at 3.6 years. One patient developed donor-derived Epstein-Barr virus-associated post-transplant lymphoproliferative disease (EBV-PTLD), which was followed by a marked expansion of iCasp9 T cells and cytokine release syndrome (CRS). These iCasp9 -transduced T cells infiltrated the affected lymph nodes and secreted IFNγ and IL-10. They peaked at 1,848 cells/μL and were found to be monoclonal by T-cell receptor (TCR) clonotype and oligoclonal by viral integrant analysis, representing a 6-log in vivo expansion of the dominant T-cell clone. These T cells were not autonomous and contracted with the resolution of EBV-PTLD, which did not recur., Conclusions: iCasp9 -transduced T cells could persist long-term. They retained very high in vivo clonotypic proliferative capacity and function, and could cause CRS in response to de novo lymphoma development., (©2019 American Association for Cancer Research.)

Transplantation with autologous hematopoietic progenitors remains an important consolidation treatment for patients with multiple myeloma (MM) and is thought to prolong the disease plateau phase by providing intensive cytoreduction. However, transplantation induces inflammation in the context of profound lymphodepletion that may cause hitherto unexpected immunological effects. We developed preclinical models of bone marrow transplantation (BMT) for MM using Vk*MYC myeloma-bearing recipient mice and donor mice that were myeloma naive or myeloma experienced to simulate autologous transplantation. Surprisingly, we demonstrated broad induction of T cell-dependent myeloma control, most efficiently from memory T cells within myeloma-experienced grafts, but also through priming of naive T cells after BMT. CD8+ T cells from mice with controlled myeloma had a distinct T cell receptor (TCR) repertoire and higher clonotype overlap relative to myeloma-free BMT recipients. Furthermore, T cell-dependent myeloma control could be adoptively transferred to secondary recipients and was myeloma cell clone specific. Interestingly, donor-derived IL-17A acted directly on myeloma cells expressing the IL-17 receptor to induce a transcriptional landscape that promoted tumor growth and immune escape. Conversely, donor IFN-γ secretion and signaling were critical to protective immunity and were profoundly augmented by CD137 agonists. These data provide new insights into the mechanisms of action of transplantation in myeloma and provide rational approaches to improving clinical outcomes.

CD8 T cells protect the liver against viral infection, but can also cause severe liver damage that may even lead to organ failure. Given the lack of mechanistic insights and specific treatment options in patients with acute fulminant hepatitis, we develop a mouse model reflecting a severe acute virus-induced CD8 T cell-mediated hepatitis. Here we show that antigen-specific CD8 T cells induce liver damage in a perforin-dependent manner, yet liver failure is not caused by effector responses targeting virus-infected hepatocytes alone. Additionally, CD8 T cell mediated elimination of cross-presenting liver sinusoidal endothelial cells causes endothelial damage that leads to a dramatically impaired sinusoidal perfusion and indirectly to hepatocyte death. With the identification of perforin-mediated killing as a critical pathophysiologic mechanism of liver failure and the protective function of a new class of perforin inhibitor, our study opens new potential therapeutic angles for fulminant viral hepatitis.

Autologous stem cell transplantation (SCT) remains a standard of care for multiple myeloma (MM) patients and prolongs progression-free survival. A small cohort of patients achieve long-term control of disease, but the majority of patients ultimately relapse, and the mechanisms permitting disease progression remain unclear. In this study, we used a preclinical model of autologous SCT for myeloma where the disease either progressed (MM relapsed) or was controlled. In the bone marrow (BM), inhibitory receptor expression on CD8 + T cells correlated strongly with myeloma progression after transplant. In conjunction, the costimulatory/adhesion receptor CD226 (DNAM-1) was markedly downregulated. Interestingly, DNAM-1 - CD8 + T cells in MM-relapsed mice had an exhausted phenotype, characterized by upregulation of multiple inhibitory receptors, including T-cell immunoglobulin and ITIM domains (TIGIT) and programmed cell death protein 1 (PD-1) with decreased T-bet and increased eomesodermin expression. Immune checkpoint blockade using monoclonal antibodies against PD-1 or TIGIT significantly prolonged myeloma control after SCT. Furthermore, CD8 + T cells from MM-relapsed mice exhibited high interleukin-10 (IL-10) secretion that was associated with increased TIGIT and PD-1 expression. However, while donor-derived IL-10 inhibited myeloma control post-SCT, this was independent of IL-10 secretion by or signaling to T cells. Instead, the donor myeloid compartment, including colony-stimulating factor 1 receptor-dependent macrophages and an IL-10-secreting dendritic cell population in the BM, promoted myeloma progression. Our findings highlight PD-1 or TIGIT blockade in conjunction with SCT as a potent combination therapy in the treatment of myeloma., (© 2018 by The American Society of Hematology.)

IL-6 mediates broad physiological and pathological effects through its receptor signal transducing unit gp130. Due to the reportedly wide cellular expression of gp130, IL-6 is thought to signal ubiquitously via gp130 complex formation with membrane-bound IL-6Rα or soluble IL-6Rα. gp130 signaling primarily induces p-STAT3 and p-STAT1. In contrast to the previous dogma, we show in this article that circulating mouse and human granulocytes are unable to induce p-STAT3 or p-STAT1 after stimulation with IL-6 or an IL-6/soluble IL-6R complex. Furthermore, we demonstrate that this is due to a lack of gp130 expression on mouse and human granulocytes, despite their expression of membrane-bound IL-6R. Importantly, the absence of gp130 is not only a feature of mature granulocytes in healthy individuals, it is also observed after allogeneic stem cell transplantation. Moreover, granulocyte gp130 expression is lost during maturation, because granulocyte-monocyte progenitor cells express gp130 and respond to IL-6. Given that granulocytes constitute 50-70% of circulating leukocytes, this indicates a significantly smaller scope of IL-6 signaling than previously anticipated and has important implications for therapeutic IL-6 inhibition and the mechanisms of action thereof., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Bloodstream infection is a significant clinical problem, particularly in vulnerable patient groups such as those undergoing chemotherapy and bone marrow transplantation. Clinical diagnostics for suspected bloodstream infection remain centered around blood culture (highly variable timing, in the order of hours to days to become positive), and empiric use of broad-spectrum antibiotics is therefore employed for patients presenting with febrile neutropenia. Gram-typing provides the first opportunity to target therapy (e.g., combinations containing vancomycin or teicoplanin for Gram-positives; piperacillin-tazobactam or a carbapenem for Gram-negatives); however, current approaches require blood culture. In this study, we describe a multiplexed microsphere-PCR assay with flow cytometry readout, which can distinguish Gram-positive from Gram-negative bacterial DNA in a 3.5 h time period. The combination of a simple assay design (amplicon-dependent release of Gram-type specific Cy3-labeled oligonucleotides) and the Luminex-based readout (for quantifying each specific Cy3-labeled sequence) opens opportunities for further multiplexing. We demonstrate the feasibility of detecting common Gram-positive and Gram-negative organisms after spiking whole bacteria into healthy human blood prior to DNA extraction. Further development of DNA extraction methods is required to reach detection limits comparable to blood culture.

Mucosal-associated invariant T (MAIT) cells are a unique innate-like T cell subset that responds to a wide array of bacteria and yeast through recognition of riboflavin metabolites presented by the MHC class I-like molecule MR1. Here, we demonstrate using MR1 tetramers that recipient MAIT cells are present in small but definable numbers in graft-versus-host disease (GVHD) target organs and protect from acute GVHD in the colon following bone marrow transplantation (BMT). Consistent with their preferential juxtaposition to microbial signals in the colon, recipient MAIT cells generate large amounts of IL-17A, promote gastrointestinal tract integrity, and limit the donor alloantigen presentation that in turn drives donor Th1 and Th17 expansion specifically in the colon after BMT. Allogeneic BMT recipients deficient in IL-17A also develop accelerated GVHD, suggesting MAIT cells likely regulate GVHD, at least in part, by the generation of this cytokine. Indeed, analysis of stool microbiota and colon tissue from IL-17A-/- and MR1-/- mice identified analogous shifts in microbiome operational taxonomic units (OTU) and mediators of barrier integrity that appear to represent pathways controlled by similar, IL-17A-dependent mechanisms. Thus, MAIT cells act to control barrier function to attenuate pathogenic T cell responses in the colon and, given their very high frequency in humans, likely represent an important population in clinical BMT.

Purpose: Allogeneic bone marrow transplantation (BMT) provides curative therapy for leukemia via immunologic graft-versus-leukemia (GVL) effects. In practice, this must be balanced against life threatening pathology induced by graft-versus-host disease (GVHD). Recipient dendritic cells (DC) are thought to be important in the induction of GVL and GVHD. Experimental Design: We have utilized preclinical models of allogeneic BMT to dissect the role and modulation of recipient DCs in controlling donor T-cell-mediated GVHD and GVL. Results: We demonstrate that recipient CD8α + DCs promote activation-induced clonal deletion of allospecific donor T cells after BMT. We compared pretransplant fms-like tyrosine kinase-3 ligand (Flt-3L) treatment to the current clinical strategy of posttransplant cyclophosphamide (PT-Cy) therapy. Our results demonstrate superior protection from GVHD with the immunomodulatory Flt-3L approach, and similar attenuation of GVL responses with both strategies. Strikingly, Flt-3L treatment permitted maintenance of the donor polyclonal T-cell pool, where PT-Cy did not. Conclusions: These data highlight pre-transplant Flt-3L therapy as a potent new therapeutic strategy to delete alloreactive T cells and prevent GVHD, which appears particularly well suited to haploidentical BMT where the control of infection and the prevention of GVHD are paramount. Clin Cancer Res; 24(7); 1604-16. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Graft-versus-host disease (GVHD) is the major cause of nonrelapse morbidity and mortality after allogeneic stem cell transplantation (allo-SCT). Prevention and treatment of GVHD remain inadequate and commonly lead to end-organ dysfunction and opportunistic infection. The role of interleukin (IL)-17 and IL-22 in GVHD remains uncertain, due to an apparent lack of lineage fidelity and variable and contextually determined protective and pathogenic effects. We demonstrate that donor T cell-derived IL-22 significantly exacerbates cutaneous chronic GVHD and that IL-22 is produced by highly inflammatory donor CD4 + T cells posttransplantation. IL-22 and IL-17A derive from both independent and overlapping lineages, defined as T helper (Th)22 and IL-22 + Th17 cells. Donor Th22 and IL-22 + Th17 cells share a similar IL-6-dependent developmental pathway, and while Th22 cells arise independently of the IL-22 + Th17 lineage, IL-17 signaling to donor Th22 directly promotes their development in allo-SCT. Importantly, while both IL-22 and IL-17 mediate skin GVHD, Th17-induced chronic GVHD can be attenuated by IL-22 inhibition in preclinical systems. In the clinic, high levels of both IL-17A and IL-22 expression are present in the skin of patients with GVHD after allo-SCT. Together, these data demonstrate a key role for donor-derived IL-22 in patients with chronic skin GVHD and confirm parallel but symbiotic developmental pathways of Th22 and Th17 differentiation., (© 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.)

The artemisinins are the first-line therapy for severe and uncomplicated malaria, since they cause rapid declines in parasitemia after treatment. Despite this, in vivo mechanisms underlying this rapid decline remain poorly characterised. The overall decline in parasitemia is the net effect of drug inhibition of parasites and host clearance, which competes against any ongoing parasite proliferation. Separating these mechanisms in vivo was not possible through measurements of total parasitemia alone. Therefore, we employed an adoptive transfer approach in which C57BL/6J mice were transfused with Plasmodium berghei ANKA strain-infected, fluorescent red blood cells, and subsequently drug-treated. This approach allowed us to distinguish between the initial drug-treated generation of parasites (Gen 0 ), and their progeny (Gen 1 ). Artesunate efficiently impaired maturation of Gen 0 parasites, such that a sufficiently high dose completely arrested maturation after 6h of in vivo exposure. In addition, artesunate-affected parasites were cleared from circulation with a half-life of 6.7h. In vivo cell depletion studies using clodronate liposomes revealed an important role for host phagocytes in the removal of artesunate-affected parasites, particularly ring and trophozoite stages. Finally, we found that a second antimalarial drug, mefloquine, was less effective than artesunate at suppressing parasite maturation and driving host-mediated parasite clearance. Thus, we propose that in vivo artesunate treatment causes rapid decline in parasitemia by arresting parasite maturation and encouraging phagocyte-mediated clearance of parasitised RBCs., (Copyright © 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

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., (© 2017 by The American Society of Hematology.)

Type 1 regulatory T (T R 1) 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 T R 1 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 T R 1 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 T R 1 cells requires T-bet and donor macrophage-derived IL-27. Thus, we define the cellular and transcriptional control of T R 1 cell differentiation during BMT, opening new avenues to therapeutic manipulation., (Copyright © 2017, American Association for the Advancement of Science.)

T-helper 17 (Th17) cells have been widely implicated as drivers of autoimmune disease. In particular, Th17 cytokine plasticity and acquisition of an interleukin-17A + (IL-17A + )interferon γ(IFNγ) + cytokine profile is associated with increased pathogenic capacity. Donor Th17 polarization is known to exacerbate graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (allo-SCT); however, donor Th17 cytokine coexpression and plasticity have not been fully characterized. Using IL-17 "fate-mapping" mice, we identified IL-6-dependent Th17 cells early after allo-SCT, characterized by elevated expression of proinflammatory cytokines, IL-17A, IL-22, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor. This population did not maintain lineage fidelity, with a marked loss of IL-17A and IL-22 expression late posttransplant. Th17 cells were further segregated based on IFNγ coexpression, and IL-17A + IFNγ + Th17 displayed an enhanced proinflammatory phenotype. Th17 cytokine plasticity and IFNγ production were critically dependent upon donor-derived IL-12p40, and cyclosporine (CsA) treatment regulated this differentiation pathway. This observation was highly concordant with clinical samples from allo-SCT recipients receiving CsA-based immune suppression where although the IFNγ-negative-Th17 subset predominated, IFNγ + -Th17 cells were also present. In sum, Th17 polarization and ensuing differentiation are mediated by sequential inflammatory signals, which are modulated by immunosuppressive therapy, leading to distinct phenotypes within this lineage., Competing Interests: Conflict-of-interest disclosure: G.R.H. has received funding from Roche Ltd for clinical trial studies and has served as a paid consult for Amgen Inc. The remaining authors declare no competing financial interests.

Carbopol is a polyanionic carbomer used in man for topical application and drug delivery purposes. However parenteral administration of Carbopol in animal models results in systemic adjuvant activity including strong pro-inflammatory type-1 T-cell (Th1) polarization. Here we investigated potential pathways of immune activation by Carbopol by comparison with other well-characterized adjuvants. Carbopol administration triggered rapid and robust leukocyte recruitment, pro-inflammatory cytokine secretion and antigen capture largely by inflammatory monocytes. The induction of antigen specific Th1 cells by Carbopol was found to occur via a non-canonical pathway, independent of MyD88/TRIF signaling and in the absence of pattern-recognition-receptor (PRR) activation typically associated with Th1/Ig2a induction. Using multispectral fluorescence imaging (Imagestream) and electron microscopy we demonstrated that phagocytic uptake of Carbopol particles followed by entry into the phagosomal/lysosomal pathway elicited conformational changes to the polymer and reactive oxygen species (ROS) production. We therefore conclude that Carbopol may mediate its adjuvant activity via novel mechanisms of antigen presenting cell activation and Th1 induction, leading to enhanced IgG2a responses independent of microbial pattern recognition., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

IL-17-producing cells are important mediators of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (SCT). Here we demonstrate that a distinct CD8(+) Tc17 population develops rapidly after SCT but fails to maintain lineage fidelity such that they are unrecognizable in the absence of a fate reporter. Tc17 differentiation is dependent on alloantigen presentation by host dendritic cells (DCs) together with IL-6. Tc17 cells express high levels of multiple prototypic lineage-defining transcription factors (eg, RORγt, T-bet) and cytokines (eg, IL-17A, IL-22, interferon-γ, granulocyte macrophage colony-stimulating factor, IL-13). Targeted depletion of Tc17 early after transplant protects from lethal acute GVHD; however, Tc17 cells are noncytolytic and fail to mediate graft-versus-leukemia (GVL) effects. Thus, the Tc17 differentiation program during GVHD culminates in a highly plastic, hyperinflammatory, poorly cytolytic effector population, which we term "inflammatory iTc17" (iTc17). Because iTc17 cells mediate GVHD without contributing to GVL, therapeutic inhibition of iTc17 development in a clinical setting represents an attractive approach for separating GVHD and GVL., (© 2015 by The American Society of Hematology.)

The continued discovery and development of adjuvants for vaccine formulation are important to safely increase potency and/or reduce the antigen doses of existing vaccines and tailor the adaptive immune response to newly developed vaccines. Adjuplex is a novel adjuvant platform based on a purified lecithin and carbomer homopolymer. Here, we analyzed the adjuvant activity of Adjuplex in mice for the soluble hemagglutinin (HA) glycoprotein of influenza A virus. The titration of Adjuplex revealed an optimal dose of 1% for immunogenicity, eliciting high titers of HA-specific IgG but inducing no significant weight loss. At this dose, Adjuplex completely protected mice from an otherwise lethal influenza virus challenge and was at least as effective as the adjuvants monophosphoryl lipid A (MPL) and alum in preventing disease. Adjuplex elicited balanced Th1-/Th2-type immune responses with accompanying cytokines and triggered antigen-specific CD8(+) T-cell proliferation. The use of the peritoneal inflammation model revealed that Adjuplex recruited dendritic cells (DCs), monocytes, and neutrophils in the context of innate cytokine and chemokine secretion. Adjuplex neither triggered classical maturation of DCs nor activated a pathogen recognition receptor (PRR)-expressing NF-κB reporter cell line, suggesting a mechanism of action different from that reported for classical pathogen-associated molecular pattern (PAMP)-activated innate immunity. Taken together, these data reveal Adjuplex to be a potent and well-tolerated adjuvant with application for subunit vaccines., (Copyright © 2015, Wegmann et al.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The primacy of the gastrointestinal (GI) tract in dictating the outcome of graft-versus-host disease (GVHD) is broadly accepted; however, the mechanisms controlling this effect are poorly understood. Here, we demonstrate that GVHD markedly enhances alloantigen presentation within the mesenteric lymph nodes (mLNs), mediated by donor CD103(+)CD11b(-) dendritic cells (DCs) that migrate from the colon under the influence of CCR7. Expansion and differentiation of donor T cells specifically within the mLNs is driven by profound levels of alloantigen, IL-12, and IL-6 promoted by Toll-like receptor (TLR) and receptor for advanced glycation end products (RAGE) signals. Critically, alloantigen presentation in the mLNs imprints gut-homing integrin signatures on donor T cells, leading to their emigration into the GI tract where they mediate fulminant disease. These data identify a critical, anatomically distinct, donor DC subset that amplifies GVHD. We thus highlight multiple therapeutic targets and the ability of GVHD, once initiated by recipient antigen-presenting cells, to generate a profound, localized, and lethal feed-forward cascade of donor DC-mediated indirect alloantigen presentation and cytokine secretion within the GI tract., (© 2015 Koyama et al.)

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., (© 2015 by The American Society of Hematology.)