Publications by authors named "Raymond Steptoe"

61 Publications

Tolerance induction by liposomes targeting a single CD8 epitope IGRP in a model of type 1 diabetes is impeded by co-targeting a CD4 islet epitope.

Immunol Cell Biol 2021 Oct 20. Epub 2021 Oct 20.

The University of Queensland Diamantina Institute, The University of Queensland, Woolloongabba, QLD, Australia.

The autoimmune disease type 1 diabetes is predominantly mediated by CD8 cytotoxic T-cell destruction of islet beta cells, of which islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is a dominant target antigen specificity. Previously, we found that a liposome-based antigen-specific immunotherapy encapsulating the CD4 T-cell islet epitope 2.5 together with the nuclear factor-κB inhibitor calcitriol induced regulatory T cells and protected from diabetes in NOD mice. Here we investigated whether the same system delivering IGRP could induce antigen-specific CD8 T-cell-targeted immune regulation and delay diabetes. Subcutaneous administration of IGRP /calcitriol liposomes transiently activated and expanded IGRP-specific T-cell receptor transgenic 8.3 CD8 T cells. Liposomal co-delivery of calcitriol was required to optimally suppress endogenous IGRP-specific CD8 T-cell interferon-γ production and cytotoxicity. Concordantly, a short course of IGRP /calcitriol liposomes delayed diabetes progression and reduced insulitis. However, when IGRP /calcitriol liposomes were delivered together with 2.5 /calcitriol liposomes, disease protection was not observed and the regulatory effect of 2.5 /calcitriol liposomes was abrogated. Thus, tolerogenic liposomes that target either a dominant CD8 or a CD4 T-cell islet epitope can delay diabetes progression but combining multiple epitopes does not enhance protection.
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http://dx.doi.org/10.1111/imcb.12506DOI Listing
October 2021

Short Duration Alagebrium Chloride Therapy Prediabetes Does Not Inhibit Progression to Autoimmune Diabetes in an Experimental Model.

Metabolites 2021 Jun 28;11(7). Epub 2021 Jun 28.

Glycation and Diabetes Complications, Mater Research Institute, The University of Queensland, Translational Research Institute, Brisbane, QLD 4102, Australia.

Mechanisms by which advanced glycation end products (AGEs) contribute to type 1 diabetes (T1D) pathogenesis are poorly understood. Since life-long pharmacotherapy with alagebrium chloride (ALT) slows progression to experimental T1D, we hypothesized that acute ALT therapy delivered prediabetes, may be effective. However, in female, non-obese diabetic (NOD) mice, ALT administered prediabetes (day 50-100) did not protect against experimental T1D. ALT did not decrease circulating AGEs or their precursors. Despite this, pancreatic β-cell function was improved, and insulitis and pancreatic CD45.1 cell infiltration was reduced. Lymphoid tissues were unaffected. ALT pre-treatment, prior to transfer of primed GC98 CD8 T cell receptor transgenic T cells, reduced blood glucose concentrations and delayed diabetes, suggesting islet effects rather than immune modulation by ALT. Indeed, ALT did not reduce interferon-γ production by leukocytes from ovalbumin-pre-immunised NOD mice and NOD recipients given diabetogenic ALT treated NOD splenocytes were not protected against T1D. To elucidate β-cell effects, NOD-derived MIN6N8 β-cell major histocompatibility complex (MHC) Class Ia surface antigens were examined using immunopeptidomics. Overall, no major changes in the immunopeptidome were observed during the various treatments with all peptides exhibiting allele specific consensus binding motifs. As expected, longer MHC Class Ia peptides were captured bound to H-2D than H-2K under all conditions. Moreover, more 10-12 mer peptides were isolated from H-2D after AGE modified bovine serum albumin (AGE-BSA) treatment, compared with bovine serum albumin (BSA) or AGE-BSA+ALT treatment. Proteomics of MIN6N8 cells showed enrichment of processes associated with catabolism, the immune system, cell cycling and presynaptic endocytosis with AGE-BSA compared with BSA treatments. These data show that short-term ALT intervention, given prediabetes, does not arrest experimental T1D but transiently impacts β-cell function.
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http://dx.doi.org/10.3390/metabo11070426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8305727PMC
June 2021

Peripheral Tolerance Checkpoints Imposed by Ubiquitous Antigen Expression Limit Antigen-Specific B Cell Responses under Strongly Immunogenic Conditions.

J Immunol 2020 09 24;205(5):1239-1247. Epub 2020 Jul 24.

University of Queensland Diamantina Institute, The University of Queensland, Woolloongabba, Queensland 4102, Australia

A series of layered peripheral checkpoints maintain self-reactive B cells in an unresponsive state. Autoantibody production occurs when these checkpoints are breached; however, when and how this occurs is largely unknown. In particular, how self-reactive B cells are restrained during bystander inflammation in otherwise healthy individuals is poorly understood. A weakness has been the unavailability of methods capable of dissecting physiologically relevant B cell responses without the use of an engineered BCR. Resolving this will provide insights that decipher how this process goes awry during autoimmunity or could be exploited for therapy. In this study, we use a strong adjuvant to provide bystander innate and adaptive signals that promote B cell responsiveness in conjunction with newly developed B cell detection tools to study in detail the ways that peripheral tolerance mechanisms limit the expansion and function of self-reactive B cells activated under these conditions. We show that although self-reactive B cells are recruited into the germinal center, their development does not proceed, possibly because of rapid counterselection. Consequently, differentiation of plasma cells is blunted, and Ab responses are transient and devoid of affinity maturation. We propose this approach, and these tools can be more widely applied to track Ag-specific B cell responses to more disease-relevant Ags, without the need for BCR transgenic mice, in settings where tolerance pathways are compromised or have been genetically manipulated to drive stronger insights into the biology underlying B cell-mediated autoimmunity.
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http://dx.doi.org/10.4049/jimmunol.2000377DOI Listing
September 2020

HMGB1 amplifies ILC2-induced type-2 inflammation and airway smooth muscle remodelling.

PLoS Pathog 2020 07 13;16(7):e1008651. Epub 2020 Jul 13.

School of Biomedical Sciences, The University of Queensland, Queensland, Australia.

Type-2 immunity elicits tissue repair and homeostasis, however dysregulated type-2 responses cause aberrant tissue remodelling, as observed in asthma. Severe respiratory viral infections in infancy predispose to later asthma, however, the processes that mediate tissue damage-induced type-2 inflammation and the origins of airway remodelling remain ill-defined. Here, using a preclinical mouse model of viral bronchiolitis, we find that increased epithelial and mesenchymal high-mobility group box 1 (HMGB1) expression is associated with increased numbers of IL-13-producing type-2 innate lymphoid cell (ILC2s) and the expansion of the airway smooth muscle (ASM) layer. Anti-HMGB1 ablated lung ILC2 numbers and ASM growth in vivo, and inhibited ILC2-mediated ASM cell proliferation in a co-culture model. Furthermore, we identified that HMGB1/RAGE (receptor for advanced glycation endproducts) signalling mediates an ILC2-intrinsic IL-13 auto-amplification loop. In summary, therapeutic targeting of the HMGB1/RAGE signalling axis may act as a novel asthma preventative by dampening ILC2-mediated type-2 inflammation and associated ASM remodelling.
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http://dx.doi.org/10.1371/journal.ppat.1008651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7377495PMC
July 2020

Transfer of antigen-encoding bone marrow under immune-preserving conditions deletes mature antigen-specific B cells in recipients and inhibits antigen-specific antibody production.

Cytotherapy 2020 08 13;22(8):436-444. Epub 2020 Jun 13.

University of Queensland Diamantina Institute, The University of Queensland, Woolloongabba, Australia. Electronic address:

Background Aims: Pathological activation and collaboration of T and B cells underlies pathogenic autoantibody responses. Existing treatments for autoimmune disease cause non-specific immunosuppression, and induction of antigen-specific tolerance remains an elusive goal. Many immunotherapies aim to manipulate the T-cell component of T-B interplay, but few directly target B cells. One possible means to specifically target B cells is the transfer of gene-engineered BM that, once engrafted, gives rise to widespread specific and tolerogenic antigen expression within the hematopoietic system.

Methods: Gene-engineered bone marrow encoding ubiquitous ovalbumin expression was transferred after low-dose (300-cGy) immune-preserving irradiation. B-cell responsiveness was monitored by analyzing ovalbumin-specific antibody production after immunization with ovalbumin/complete Freund's adjuvant. Ovalbumin-specific B cells and their response to immunization were analyzed using multi-tetramer staining. When antigen-encoding bone marrow was transferred under immune-preserving conditions, cognate antigen-specific B cells were purged from the recipient's preexisting B-cell repertoire and the repertoire that arose after bone marrow transfer.

Results: OVA-specific B-cell deletion was apparent within the established host B-cell repertoire as well as that developing after gene-engineered bone marrow transfer. OVA-specific antibody production was substantially inhibited by transfer of OVA-encoding BM and activation of OVA-specific B cells, germinal center formation and subsequent OVA-specific plasmablast differentiation were all inhibited. Low levels of gene-engineered bone marrow chimerism were sufficient to limit antigen-specific antibody production.

Results: These data show that antigen-specific B cells within an established B-cell repertoire are susceptible to de novo tolerance induction, and this can be achieved by transfer of gene-engineered bone marrow. This adds further dimensions to the utility of antigen-encoding bone marrow transfer as an immunotherapeutic tool.
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http://dx.doi.org/10.1016/j.jcyt.2020.04.041DOI Listing
August 2020

Long-lived regulatory T cells generated during severe bronchiolitis in infancy influence later progression to asthma.

Mucosal Immunol 2020 07 17;13(4):652-664. Epub 2020 Feb 17.

Respiratory Immunology, QIMR Berghofer Medical Research Institute, Herston, Queensland, 4006, Australia.

The type-2 inflammatory response that promotes asthma pathophysiology occurs in the absence of sufficient immunoregulation. Impaired regulatory T cell (Treg) function also predisposes to severe viral bronchiolitis in infancy, a major risk factor for asthma. Hence, we hypothesized that long-lived, aberrantly programmed Tregs causally link viral bronchiolitis with later asthma. Here we found that transient plasmacytoid dendritic cell (pDC) depletion during viral infection in early-life, which causes the expansion of aberrant Tregs, predisposes to allergen-induced or virus-induced asthma in later-life, and is associated with altered airway epithelial cell (AEC) responses and the expansion of impaired, long-lived Tregs. Critically, the adoptive transfer of aberrant Tregs (unlike healthy Tregs) to asthma-susceptible mice failed to prevent the development of viral-induced or allergen-induced asthma. Lack of protection was associated with increased airway epithelial cytoplasmic-HMGB1 (high-mobility group box 1), a pro-type-2 inflammatory alarmin, and granulocytic inflammation. Aberrant Tregs expressed lower levels of CD39, an ectonucleotidase that hydrolyzes extracellular ATP, a known inducer of alarmin release. Using cultured mouse AECs, we identify that healthy Tregs suppress allergen-induced HMGB1 translocation whereas this ability is markedly impaired in aberrant Tregs. Thus, defective Treg programming in infancy has durable consequences that underlie the association between bronchiolitis and subsequent asthma.
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http://dx.doi.org/10.1038/s41385-020-0268-8DOI Listing
July 2020

Cytokine/chemokine profiles in squamous cell carcinoma correlate with precancerous and cancerous disease stage.

Sci Rep 2019 11 28;9(1):17754. Epub 2019 Nov 28.

The University of Queensland Diamantina Institute, Faculty of Medicine, The University of Queensland, Translational Research Institute, Brisbane, QLD, Australia.

Actinic Keratosis (AK), Intraepidermal Carcinoma (IEC), and Squamous Cell Carcinoma (SCC) are generally considered to be advancing stages of the same disease spectrum. However, while AK often regress spontaneously, and IEC often regress in response to immune-activating treatments, SCC typically do not regress. Therefore, it is vital to define whether fundamental immunological changes occur during progression to SCC. Here we show that proinflammatory cytokine expression, chemokine expression, and immune cell infiltration density change during progression to SCC. Our findings suggest a switch from predominantly proinflammatory cytokine production to chemokine production is a key feature of progression from precancer to cancer. Together, these observations propose a model that can underpin current research and open new avenues of exploration into the clinical significance of these profiles with respect to immunotherapeutic or other treatment outcomes.
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http://dx.doi.org/10.1038/s41598-019-54435-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6882799PMC
November 2019

Simple, rapid and inexpensive typing of common HLA class I alleles for immunological studies.

J Immunol Methods 2019 02 8;465:72-76. Epub 2018 Dec 8.

University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Woolloongabba, Queensland, Australia. Electronic address:

Current HLA-typing methods are typically designed to provide exquisitely-detailed identification of multiple HLA-alleles to satisfy the requirements for organ and bone marrow transplantation or genetic studies. Many human immunological studies, on the other hand, focus around only a small number of HLA alleles that are abundant or of relevance to specific diseases. Consequently, for such studies, many HLA typing approaches are not cost-effective and are potentially complicated, slow and not easily performed in-house. Work-flow would be streamlined by a simple, inexpensive and rapid typing method able to be performed in-house. We outline a straightforward approach that provides appropriate data for much immunological research. In a predominantly Caucasian population, flow cytometry using anti-HLA-A2, -B8 and -B7 antibodies consistently and accurately screened for samples carrying the highly-abundant HLA class I alleles HLA-A*02:01, -B*08:01 and -B*07:02 that form the focus of immunological studies. Next, we describe a straightforward and simple strategy for design and use of allele-specific PCR primers to identify, at high-resolution, alleles of interest. When combined with a simple gDNA extraction technique this provides reliable, simple and inexpensive in-house HLA typing demonstrated here for highly-abundant HLA class I alleles.
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http://dx.doi.org/10.1016/j.jim.2018.12.002DOI Listing
February 2019

Clinically-Relevant Rapamycin Treatment Regimens Enhance CD8 Effector Memory T Cell Function In The Skin and Allow their Infiltration into Cutaneous Squamous Cell Carcinoma.

Oncoimmunology 2018;7(9):e1479627. Epub 2018 Jul 30.

The University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, QLD Australia.

Patients receiving immunosuppressive drugs to prevent organ transplant rejection exhibit a greatly increased risk of developing cutaneous squamous cell carcinoma (SCC). However, not all immunosuppressive drugs confer the same risk. Randomised, controlled trials demonstrate that switching renal transplant recipients receiving calcineurin inhibitor-based therapies to mammalian target of rapamycin (mTOR) inhibitors results in a reduced incidence of SSC formation, and can even result in the regression of pre-existing premalignant lesions. However, the contribution played by residual immune function in this setting is unclear. We examined the hypotheses that mTOR inhibitors promote the enhanced differentiation and function of CD8 memory T cells in the skin. Here, we demonstrate that the long-term oral administration of rapamycin to achieve clinically-relevant whole blood drug target thresholds, creates a "low rapamycin dose" environment in the skin. While both rapamycin and the calcineurin inhibitor tacrolimus elongated the survival of OVA-expressing skin grafts, and inhibited short-term antigen-specific CD8 T cell responses, rapamycin but not tacrolimus permitted the statistically significant infiltration of CD8 effector memory T cells into UV-induced SCC lesions. Furthermore, rapamycin uniquely enhanced the number and function of CD8 effector and central memory T cells in a model of long-term contact hypersensitivity provided that rapamycin was present during the antigen sensitization phase. Thus, our findings suggest that patients switched to mTOR inhibitor regimens likely experience enhanced CD8 memory T cell function to new antigen-challenges in their skin, which could contribute to their lower risk of SSC formation and regression of pre-existing premalignant lesions.
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http://dx.doi.org/10.1080/2162402X.2018.1479627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6140608PMC
July 2018

Temporal Regulation of Natural Killer T Cell Interferon Gamma Responses by β-Catenin-Dependent and -Independent Wnt Signaling.

Front Immunol 2018 16;9:483. Epub 2018 Mar 16.

The University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, QLD, Australia.

Natural killer T (NKT) cells are prominent innate-like lymphocytes in the liver with critical roles in immune responses during infection, cancer, and autoimmunity. Interferon gamma (IFN-γ) and IL-4 are key cytokines rapidly produced by NKT cells upon recognition of glycolipid antigens presented by antigen-presenting cells (APCs). It has previously been reported that the transcriptional coactivator β-catenin regulates NKT cell differentiation and functionally biases NKT cell responses toward IL-4, at the expense of IFN-γ production. β-Catenin is not only a central effector of Wnt signaling but also contributes to other signaling networks. It is currently unknown whether Wnt ligands regulate NKT cell functions. We thus investigated how Wnt ligands and β-catenin activity shape liver NKT cell functions in response to the glycolipid antigen, α-galactosylceramide (α-GalCer) using a mouse model. Pharmacologic targeting of β-catenin activity with ICG001, as well as myeloid-specific genetic ablation of , to specifically target Wnt protein release by APCs, enhanced early IFN-γ responses. By contrast, within several hours of α-GalCer challenge, myeloid-specific deficiency, as well as pharmacologic targeting of Wnt release using the small molecule inhibitor IWP-2 impaired α-GalCer-induced IFN-γ responses, independent of β-catenin activity. These data suggest that myeloid cell-derived Wnt ligands drive early Wnt/β-catenin signaling that curbs IFN-γ responses, but that, subsequently, Wnt ligands sustain IFN-γ expression independent of β-catenin activity. Our analyses in ICG001-treated mice confirmed a role for β-catenin activity in driving early IL-4 responses by liver NKT cells. However, neither pharmacologic nor genetic perturbation of Wnt production affected the IL-4 response, suggesting that IL-4 production by NKT cells in response to α-GalCer is not driven by released Wnt ligands. Collectively, these data reveal complex temporal roles of Wnt ligands and β-catenin signaling in the regulation of liver NKT cell activation, and highlight Wnt-dependent and -independent contributions of β-catenin to NKT cell functions.
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http://dx.doi.org/10.3389/fimmu.2018.00483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5864864PMC
April 2019

Tetramer-based identification of naïve antigen-specific B cells within a polyclonal repertoire.

Eur J Immunol 2018 07 17;48(7):1251-1254. Epub 2018 Apr 17.

The University of Queensland, Translational Research Institute, University of Queensland Diamantina Institute, Brisbane, Australia.

Detecting naïve antigen-specific B cells can be challenging. Use of multiple, complementary tetramers with different fluorochromes enhances sensitivity and specificity allowing naïve antigen-specific B cells to be readily distinguished within a polyclonal repertoire. Activated, affinity-matured B cells, however, can be detected effectively using a single tetramer.
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http://dx.doi.org/10.1002/eji.201747447DOI Listing
July 2018

Flt-3L Expansion of Recipient CD8α Dendritic Cells Deletes Alloreactive Donor T Cells and Represents an Alternative to Posttransplant Cyclophosphamide for the Prevention of GVHD.

Clin Cancer Res 2018 04 24;24(7):1604-1616. Epub 2018 Jan 24.

QIMR Berghofer Medical Research Institute, Brisbane, Australia.

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. 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. 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. 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. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-2148DOI Listing
April 2018

Re-educating immunity in respiratory allergies: the potential for hematopoietic stem cell-mediated gene therapy.

J Mol Med (Berl) 2018 01 17;96(1):21-30. Epub 2017 Nov 17.

The University of Queensland, Translational Research Institute, University of Queensland Diamantina Institute, Level 6, 37 Kent Street, Woolloongabba, Queensland, 4102, Australia.

Respiratory allergies represent a significant disease burden worldwide affecting up to 300 million people globally. Medication and avoidance of known triggers do not address the underlying pathology. Traditional immunotherapies for allergy aim to reinstate immune homeostasis but require years of treatment and have poor long-term efficacy. Novel approaches, such as gene-engineered hematopoietic stem cell transplantation, induce profound antigen-specific tolerance in autoimmunity. Recent evidence shows this approach may also have therapeutic utility for allergy. Here, we review the mechanisms of antigen-specific tolerance and the potential of stem cell-mediated gene therapy to induce tolerance in allergic respiratory diseases.
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http://dx.doi.org/10.1007/s00109-017-1611-8DOI Listing
January 2018

The effect of interleukin-22 treatment on autoimmune diabetes in the NOD mouse.

Diabetologia 2017 Nov 4;60(11):2256-2261. Epub 2017 Aug 4.

Inflammatory Diseases Biology and Therapeutics, Mater Research Institute - The University of Queensland, Translational Research Institute, Level 4/37 Kent Street, Woolloongabba, Brisbane, QLD, 4102, Australia.

Aims/hypothesis: The aim of this study was to determine whether therapy with the cytokine IL-22 could be used to prevent the development of, or treat, autoimmune diabetes in the NOD mouse.

Methods: Six-week-old NOD mice were administered bi-weekly either recombinant mouse IL-22 (200 ng/g) or PBS (vehicle control) intraperitoneally until overt diabetes was diagnosed as two consecutive measurements of non-fasting blood glucose ≥ 11 mmol/l. At this time, NOD mice in the control arm were treated with LinBit insulin pellets and randomised to bi-weekly therapeutic injections of either PBS or IL-22 (200 ng/g) and followed until overt diabetes was diagnosed, as defined above.

Results: IL-22 therapy did not delay the onset of diabetes in comparison with the vehicle-treated mice. We did not observe an improvement in islet area, glycaemic control, beta cell residual function, endoplasmic reticulum stress, insulitis or macrophage and neutrophil infiltration as determined by non-fasting blood glucose, C-peptide and histological scoring. Therapeutic administration of IL-22 did not reduce circulating lipopolysaccharide, a marker of impaired gut mucosal integrity.

Conclusions/interpretation: Our study suggests that, at this dosing regimen introduced either prior to overt diabetes or at diagnosis of diabetes, recombinant mouse IL-22 therapy cannot prevent autoimmune diabetes, or prolong the honeymoon period in the NOD mouse.
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http://dx.doi.org/10.1007/s00125-017-4392-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6448904PMC
November 2017

Antigen presenting cell-targeted proinsulin expression converts insulin-specific CD8 T-cell priming to tolerance in autoimmune-prone NOD mice.

Eur J Immunol 2017 09 18;47(9):1550-1561. Epub 2017 Jul 18.

The University of Queensland Diamantina Institute, University of Queensland, Brisbane, Australia.

Type 1 diabetes (T1D) results from autoimmune destruction of insulin-producing pancreatic β cells. Therapies need to incorporate strategies to overcome the genetic defects that impair induction or maintenance of peripheral T-cell tolerance and contribute to disease development. We tested whether the enforced expression of an islet autoantigen in antigen-presenting cells (APC) counteracted peripheral T-cell tolerance defects in autoimmune-prone NOD mice. We observed that insulin-specific CD8 T cells transferred to mice in which proinsulin was transgenically expressed in APCs underwent several rounds of division and the majority were deleted. Residual insulin-specific CD8 T cells were rendered unresponsive and this was associated with TCR downregulation, loss of tetramer binding and expression of a range of co-inhibitory molecules. Notably, accumulation and effector differentiation of insulin-specific CD8 T cells in pancreatic lymph nodes was prominent in non-transgenic recipients but blocked by transgenic proinsulin expression. This shift from T-cell priming to T-cell tolerance exemplifies the tolerogenic capacity of autoantigen expression by APC and the capacity to overcome genetic tolerance defects.
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http://dx.doi.org/10.1002/eji.201747089DOI Listing
September 2017

CD4CD8β double-positive T cells in skin-draining lymph nodes respond to inflammatory signals from the skin.

J Leukoc Biol 2017 09 21;102(3):837-844. Epub 2017 Jun 21.

The University of Queensland Diamantina Institute, Faculty of Medicine, University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia;

CD4CD8 double-positive (DP), mature, peripheral T cells are readily detectable in a variety of species and tissues. Despite a common association with autoimmune and malignant skin disorders, however, little is understood about their role or function. Herein, we show that DP T cells are readily detectable in the blood, spleen, and peripheral lymph nodes of naïve C57BL/6 mice. DP T cells were also present in Jα18 and CD1d mice, indicating that these cells are not NK-T cells. After skin administration of CASAC adjuvant, but not Quil A adjuvant, both total DP T cells and skin-infiltrating DP T cells increased in number. We explored the possibility that DP T cells could represent aggregates between CD4 and CD8 single-positive T cells and found strong evidence that a large proportion of apparent DP T cells were indeed aggregates. However, the existence of true CD4CD8 DP T cells was confirmed by Amnis ImageStream (Millipore Sigma, Billerica, MA, USA) imaging. Multiple rounds of FACS sorting separated true DP cells from aggregates and indicated that conventional analyses may lead to ∼10-fold overestimation of DP T cell numbers. The high degree of aggregate contamination and overestimation of DP abundance using conventional analysis techniques may explain discrepancies reported in the literature for DP T cell origin, phenotype, and function.
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http://dx.doi.org/10.1189/jlb.1AB0217-065RDOI Listing
September 2017

APC-targeted proinsulin expression inactivates insulin-specific memory CD8 T cells in NOD mice.

Immunol Cell Biol 2017 10 14;95(9):765-774. Epub 2017 Jun 14.

The University of Queensland Diamantina Institute, Brisbane, Queensland, Australia.

Type 1 diabetes (T1D) results from T-cell-mediated autoimmune destruction of pancreatic β cells. Effector T-cell responses emerge early in disease development and expand as disease progresses. Following β-cell destruction, a long-lived T-cell memory is generated that represents a barrier to islet transplantation and other cellular insulin-replacement therapies. Development of effective immunotherapies that control or ablate β-cell destructive effector and memory T-cell responses has the potential to prevent disease progression and recurrence. Targeting antigen expression to antigen-presenting cells inactivates cognate CD8 effector and memory T-cell responses and has therapeutic potential. Here we investigated this in the context of insulin-specific responses in the non-obese diabetic mouse where genetic immune tolerance defects could impact on therapeutic tolerance induction. Insulin-specific CD8 memory T cells transferred to mice expressing proinsulin in antigen-presenting cells proliferated in response to transgenically expressed proinsulin and the majority were rapidly deleted. A small proportion of transferred insulin-specific Tmem remained undeleted and these were antigen-unresponsive, exhibited reduced T cell receptor (TCR) expression and H-2K/insB tetramer binding and expressed co-inhibitory molecules. Expression of proinsulin in antigen-presenting cells also abolished the diabetogenic capacity of CD8 effector T cells. Therefore, destructive insulin-specific CD8 T cells are effectively inactivated by enforced proinsulin expression despite tolerance defects that exist in diabetes-prone NOD mice. These findings have important implications in developing immunotherapeutic approaches to T1D and other T-cell-mediated autoimmune diseases.
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http://dx.doi.org/10.1038/icb.2017.48DOI Listing
October 2017

Allergen-encoding bone marrow transfer inactivates allergic T cell responses, alleviating airway inflammation.

JCI Insight 2017 Jun 2;2(11). Epub 2017 Jun 2.

The University of Queensland Diamantina Institute, University of Queensland, Brisbane, Australia.

Memory Th2 cell responses underlie the development and perpetuation of allergic diseases. Because these states result from immune dysregulation, established Th2 cell responses represent a significant challenge for conventional immunotherapies. New approaches that overcome the detrimental effects of immune dysregulation are required. We tested whether memory Th2 cell responses were silenced using a therapeutic approach where allergen expression in DCs is transferred to sensitized recipients using BM cells as a vector for therapeutic gene transfer. Development of allergen-specific Th2 responses and allergen-induced airway inflammation was blocked by expression of allergen in DCs. Adoptive transfer studies showed that Th2 responses were inactivated by a combination of deletion and induction of T cell unresponsiveness. Transfer of BM encoding allergen expression targeted to DCs terminated, in an allergen-specific manner, Th2 responses in sensitized recipients. Importantly, when preexisting airway inflammation was present, there was effective silencing of Th2 cell responses, airway inflammation was alleviated, and airway hyperreactivity was reversed. The effectiveness of DC-targeted allergen expression to terminate established Th2 responses in sensitized animals indicates that exploiting cell-intrinsic T cell tolerance pathways could lead to development of highly effective immunotherapies.
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http://dx.doi.org/10.1172/jci.insight.85742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5453705PMC
June 2017

Short-course rapamycin treatment enables engraftment of immunogenic gene-engineered bone marrow under low-dose irradiation to permit long-term immunological tolerance.

Stem Cell Res Ther 2017 03 9;8(1):57. Epub 2017 Mar 9.

The University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Brisbane, QLD, Australia.

Background: Application of genetically modified hematopoietic stem cells is increasingly mooted as a clinically relevant approach to protein replacement therapy, immune tolerance induction or conditions where both outcomes may be helpful. Hematopoietic stem and progenitor cell (HSPC)-mediated gene therapy often requires highly toxic pretransfer recipient conditioning to provide a 'niche' so that transferred HSPCs can engraft effectively and to prevent immune rejection of neoantigen-expressing engineered HSPCs. For widespread clinical application, reducing conditioning toxicity is an important requirement, but reduced conditioning can render neoantigen-expressing bone marrow (BM) and HSC susceptible to immune rejection if immunity is retained.

Methods: BM or HSPC-expressing OVA ubiquitously (actin.OVA) or targeted to MHC II+ cells was transferred using low-dose (300 cGy) total body irradiation. Recipients were administered rapamycin, cyclosporine or vehicle for 3 weeks commencing at BM transfer. Engraftment was determined using CD45 congenic donors and recipients. Induction of T-cell tolerance was tested by immunising recipients and analysing in-vivo cytotoxic T-lymphocyte (CTL) activity. The effect of rapamycin on transient effector function during tolerance induction was tested using an established model of tolerance induction where antigen is targeted to dendritic cells.

Results: Immune rejection of neoantigen-expressing BM and HSPCs after low-dose irradiation was prevented by a short course of rapamycin, but not cyclosporine, treatment. Whereas transient T-cell tolerance developed in recipients of OVA-expressing BM administered vehicle, only when engraftment of neoantigen-expressing BM was facilitated with rapamycin treatment did stable, long-lasting T-cell tolerance develop. Rapamycin inhibited transient effector function development during tolerance induction and inhibited development of CTL activity in recipients of OVA-expressing BM.

Conclusions: Rapamycin acts to suppress acquisition of transient T-cell effector function during peripheral tolerance induction elicited by HSPC-encoded antigen. By facilitating engraftment, short-course rapamycin permits development of long-term stable T-cell tolerance.
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http://dx.doi.org/10.1186/s13287-017-0508-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5345164PMC
March 2017

CRISPR-targeted genome editing of mesenchymal stem cell-derived therapies for type 1 diabetes: a path to clinical success?

Stem Cell Res Ther 2017 03 9;8(1):62. Epub 2017 Mar 9.

The School of Life Sciences, Chronic Disease Solutions Team and the Centre for Health Technologies, University of Technology Sydney, PO Box 123, Broadway, NSW, 2007, Australia.

Due to their ease of isolation, differentiation capabilities, and immunomodulatory properties, the therapeutic potential of mesenchymal stem cells (MSCs) has been assessed in numerous pre-clinical and clinical settings. Currently, whole pancreas or islet transplantation is the only cure for people with type 1 diabetes (T1D) and, due to the autoimmune nature of the disease, MSCs have been utilised either natively or transdifferentiated into insulin-producing cells (IPCs) as an alternative treatment. However, the initial success in pre-clinical animal models has not translated into successful clinical outcomes. Thus, this review will summarise the current state of MSC-derived therapies for the treatment of T1D in both the pre-clinical and clinical setting, in particular their use as an immunomodulatory therapy and targets for the generation of IPCs via gene modification. In this review, we highlight the limitations of current clinical trials of MSCs for the treatment of T1D, and suggest the novel clustered regularly interspaced short palindromic repeat (CRISPR) gene-editing technology and improved clinical trial design as strategies to translate pre-clinical success to the clinical setting.
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http://dx.doi.org/10.1186/s13287-017-0511-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5345178PMC
March 2017

The T-cell Receptor Repertoire Influences the Tumor Microenvironment and Is Associated with Survival in Aggressive B-cell Lymphoma.

Clin Cancer Res 2017 Apr 20;23(7):1820-1828. Epub 2016 Sep 20.

University of Queensland Diamantina Institute, Translational Research Institute, University of Queensland, Australia.

To investigate the relationship between the intra-tumoral T-cell receptor (TCR) repertoire and the tumor microenvironment (TME) in diffuse large B-cell lymphoma (DLBCL) and the impact of TCR on survival. We performed high-throughput unbiased TCRβ sequencing on a population-based cohort of 92 patients with DLBCL treated with conventional (i.e., non-checkpoint blockade) frontline "R-CHOP" therapy. Key immune checkpoint genes within the TME were digitally quantified by nanoString. The primary endpoints were 4-year overall survival (OS) and progression-free survival (PFS). The TCR repertoire within DLBCL nodes was abnormally narrow relative to non-diseased nodal tissues ( < 0.0001). In DLBCL, a highly dominant single T-cell clone was associated with inferior 4-year OS rate of 60.0% [95% confidence interval (CI), 31.7%-79.6%], compared with 79.8% in patients with a low dominant clone (95% CI, 66.7%-88.5%; = 0.005). A highly dominant clone also predicted inferior 4-year PFS rate of 46.6% (95% CI, 22.5%-76.6%) versus 72.6% (95% CI, 58.8%-82.4%, = 0.008) for a low dominant clone. In keeping, clonal expansions were most pronounced in the EBV DLBCL subtype that is known to express immunogenic viral antigens and is associated with particularly poor outcome. Increased T-cell diversity was associated with significantly elevated , and immune checkpoint molecules. Put together, these findings suggest that the TCR repertoire is a key determinant of the TME. Highly dominant T-cell clonal expansions within the TME are associated with poor outcome in DLBCL treated with conventional frontline therapy. .
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http://dx.doi.org/10.1158/1078-0432.CCR-16-1576DOI Listing
April 2017

Maintenance of peripheral tolerance to islet antigens.

J Autoimmun 2016 08 30;72:118-25. Epub 2016 May 30.

The University of Queensland Diamantina Institute, University of Queensland, Translational Research Institute, Brisbane, QLD, Australia.

Reestablishment of immune tolerance to the insulin-producing beta cells is the desired goal for type 1 diabetes (T1D) treatment and prevention. Immune tolerance to multiple islet antigens is defective in individuals with T1D, but the mechanisms involved are multifaceted and may involve loss of thymic and peripheral tolerance. In this review we discuss our current understanding of the varied mechanisms by which peripheral tolerance to islet antigens is maintained in healthy individuals where genetic protection from T1D is present and how this fails in those with genetic susceptibility to disease. Novel findings in regards to expression of neo-islet antigens, non-classical regulatory cell subsets and the impact of specific genetic variants on tolerance induction are discussed.
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http://dx.doi.org/10.1016/j.jaut.2016.05.009DOI Listing
August 2016

Antigen-encoding bone marrow terminates islet-directed memory CD8+ T-cell responses to alleviate islet transplant rejection.

Diabetes 2016 05 9;65(5):1328-1340. Epub 2016 Mar 9.

The University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Brisbane, QLD, AUSTRALIA.

Islet-specific memory T cells arise early in type 1 diabetes (T1D), persist for long periods, perpetuate disease and are rapidly reactivated by islet transplantation. As memory T cells are poorly controlled by 'conventional' therapies, memory T-cell mediated attack is a substantial challenge in islet transplantation and this will extend to application of personalized approaches using stem-cell derived replacement β cells. New approaches are required to limit memory autoimmune attack of transplanted islets or replacement β cells. Here we show that transfer of bone marrow encoding cognate antigen directed to dendritic cells, under mild, immune-preserving conditions inactivates established memory CD8 T-cell populations and generates a long-lived, antigen-specific tolerogenic environment. Consequently, CD8 memory T cell-mediated targeting of islet-expressed antigens is prevented and islet graft rejection alleviated. The immunological mechanisms of protection are mediated through deletion and induction of unresponsiveness in targeted memory T-cell populations. The data demonstrate that hematopoietic stem cell-mediated gene therapy effectively terminates antigen-specific memory T-cell responses and this can alleviate destruction of antigen-expressing islets. This addresses a key challenge facing islet transplantation and importantly, the clinical application of personalized β-cell replacement therapies using patient-derived stem cells.
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http://dx.doi.org/10.2337/db15-1418DOI Listing
May 2016

Reduced interleukin-2 responsiveness impairs the ability of Treg cells to compete for IL-2 in nonobese diabetic mice.

Immunol Cell Biol 2016 05 14;94(5):509-19. Epub 2016 Jan 14.

The University of Queensland Diamantina Institute, University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia.

Enhancement of regulatory T cell (Treg cell) frequency and function is the goal of many therapeutic strategies aimed at treating type 1 diabetes (T1D). The interleukin-2 (IL-2) pathway, which has been strongly implicated in T1D susceptibility in both humans and mice, is a master regulator of Treg cell homeostasis and function. We investigated how IL-2 pathway defects impact Treg cells in T1D-susceptible nonobese diabetic (NOD) mice in comparison with protected C57BL/6 and NOD congenic mice. NOD Treg cells were reduced in frequency specifically in the lymph nodes and expressed lower levels of CD25 and CD39/CD73 immunosuppressive molecules. In the spleen and blood, Treg cell frequency was preserved through expansion of CD25(low), effector phenotype Treg cells. Reduced CD25 expression led to decreased IL-2 signaling in NOD Treg cells. In vivo, treatment with IL-2-anti-IL-2 antibody complexes led to effective upregulation of suppressive molecules on NOD Treg cells in the spleen and blood, but had reduced efficacy on lymph node Treg cells. In contrast, NOD CD8(+) and CD4(+) effector T cells were not impaired in their response to IL-2 therapy. We conclude that NOD Treg cells have an impaired responsiveness to IL-2 that reduces their ability to compete for a limited supply of IL-2.
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http://dx.doi.org/10.1038/icb.2016.7DOI Listing
May 2016

Impaired T-Cell Function in B-Cell Lymphoma: A Direct Consequence of Events at the Immunological Synapse?

Front Immunol 2015 2;6:258. Epub 2015 Jun 2.

The University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute , Brisbane, QLD , Australia.

Tumors can escape immune destruction through the development of antigen loss variants and loss of antigen processing/presentation pathways, thereby rendering them invisible to T cells. Alternatively, mechanisms of peripheral T-cell tolerance that would normally be important for protection from the development of autoimmunity may also be co-opted to (i) generate an immuno-inhibitory tumor environment, (ii) promote development of regulatory cell populations, or (iii) cell-intrinsically inactivate tumor-specific T cells. Emerging evidence suggests that T-cell function is impaired in hematological malignancies, which may manifest from cognate interactions between T cells and the tumor. The immunological synapse forms the cognate T-cell and antigen-presenting cell interaction and is the site where key signalling events, including those delivered by co-inhibitory receptors, that determine the fate of T cells occur. Here, we review evidence that events at the immune synapse between T cells and malignant B cells and alterations in immune synapse function may contribute to loss of T-cell function in B-cell malignancies.
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http://dx.doi.org/10.3389/fimmu.2015.00258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4451642PMC
June 2015

Regulatory T cells prevent inducible BALT formation by dampening neutrophilic inflammation.

J Immunol 2015 May 25;194(9):4567-76. Epub 2015 Mar 25.

School of Biomedical Sciences, University of Queensland, St. Lucia, Queensland 4072, Australia; Australian Infectious Diseases Research Centre, University of Queensland, St. Lucia, Queensland 4006, Australia

Inducible BALT (iBALT) can amplify pulmonary or systemic inflammatory responses to the benefit or detriment of the host. We took advantage of the age-dependent formation of iBALT to interrogate the underlying mechanisms that give rise to this ectopic, tertiary lymphoid organ. In this study, we show that the reduced propensity for weanling as compared with neonatal mice to form iBALT in response to acute LPS exposure is associated with greater regulatory T cell expansion in the mediastinal lymph nodes. Ab- or transgene-mediated depletion of regulatory T cells in weanling mice upregulated the expression of IL-17A and CXCL9 in the lungs, induced a tissue neutrophilia, and increased the frequency of iBALT to that observed in neonatal mice. Remarkably, neutrophil depletion in neonatal mice decreased the expression of the B cell active cytokines, a proliferation-inducing ligand and IL-21, and attenuated LPS-induced iBALT formation. Taken together, our data implicate a role for neutrophils in lymphoid neogenesis. Neutrophilic inflammation is a common feature of many autoimmune diseases in which iBALT are present and pathogenic, and hence the targeting of neutrophils or their byproducts may serve to ameliorate detrimental lymphoid neogenesis in a variety of disease contexts.
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http://dx.doi.org/10.4049/jimmunol.1400909DOI Listing
May 2015

Blockade of PD-1/PD-L1 promotes adoptive T-cell immunotherapy in a tolerogenic environment.

PLoS One 2015 5;10(3):e0119483. Epub 2015 Mar 5.

UQ Diamantina Institute, University of Queensland, Brisbane, Australia.

Adoptive cellular immunotherapy using in vitro expanded CD8+ T cells shows promise for tumour immunotherapy but is limited by eventual loss of function of the transferred T cells through factors that likely include inactivation by tolerogenic dendritic cells (DC). The co-inhibitory receptor programmed death-1 (PD-1), in addition to controlling T-cell responsiveness at effector sites in malignancies and chronic viral diseases is an important modulator of dendritic cell-induced tolerance in naive T cell populations. The most potent therapeutic capacity amongst CD8+ T cells appears to lie within Tcm or Tcm-like cells but memory T cells express elevated levels of PD-1. Based on established trafficking patterns for Tcm it is likely Tcm-like cells interact with lymphoid-tissue DC that present tumour-derived antigens and may be inherently tolerogenic to develop therapeutic effector function. As little is understood of the effect of PD-1/PD-L1 blockade on Tcm-like CD8+ T cells, particularly in relation to inactivation by DC, we explored the effects of PD-1/PD-L1 blockade in a mouse model where resting DC tolerise effector and memory CD8+ T cells. Blockade of PD-1/PD-L1 promoted effector differentiation of adoptively-transferred Tcm-phenotype cells interacting with tolerising DC. In tumour-bearing mice with tolerising DC, effector activity was increased in both lymphoid tissues and the tumour-site and anti-tumour activity was promoted. Our findings suggest PD-1/PD-L1 blockade may be a useful adjunct for adoptive immunotherapy by promoting effector differentiation in the host of transferred Tcm-like cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0119483PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4351071PMC
January 2016

HPV16 E7 expression in skin induces TSLP secretion, type 2 ILC infiltration and atopic dermatitis-like lesions.

Immunol Cell Biol 2015 Jul 20;93(6):540-7. Epub 2015 Jan 20.

The University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Brisbane, QLD, Australia.

Atopic dermatitis is a common pruritic and inflammatory skin disorder with unknown etiology. Most commonly occurring during early childhood, atopic dermatitis is associated with eczematous lesions and lichenification, in which the epidermis becomes hypertrophied resulting in thickening of the skin. In this study, we report an atopic dermatitis-like pathophysiology results in a murine model following the expression of the high-risk human papillomavirus (HPV) 16 oncoprotein E7 in keratinocytes under the keratin 14 promoter. We show that HPV16 E7 expression in the skin is associated with skin thickening, acanthosis and light spongiosis. Locally, HPV16 E7-expressing skin secreted high levels of thymic stromal lymphopoietin (TSLP) and contained increased numbers of innate lymphoid cells (ILCs). High levels of circulating immunoglobulin E were associated with increased susceptibility to skin allergy in a model of cutaneous challenge, and to airway bronchiolar inflammation, enhanced airway goblet cell metaplasia and mucus production in a model of atopic march. Surprisingly, skin pathology occurred independently of T cells and mast cells. Thus, our findings suggest that the expression of a single HPV oncogene in the skin can drive the onset of atopic dermatitis-like pathology through the induction of TSLP and type 2 ILC infiltration.
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http://dx.doi.org/10.1038/icb.2014.123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4496302PMC
July 2015

CD4+/CD8+ double-positive T cells: more than just a developmental stage?

J Leukoc Biol 2015 Jan 30;97(1):31-8. Epub 2014 Oct 30.

The University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia; and

CD4(+)/CD8(+) DP thymocytes are a well-described T cell developmental stage within the thymus. However, once differentiated, the CD4(+) lineage or the CD8(+) lineage is generally considered to be fixed. Nevertheless, mature CD4(+)/CD8(+) DP T cells have been described in the blood and peripheral lymphoid tissues of numerous species, as well as in numerous disease settings, including cancer. The expression of CD4 and CD8 is regulated by a very strict transcriptional program involving the transcription factors Runx3 and ThPOK. Initially thought to be mutually exclusive within CD4(+) and CD8(+) T cells, CD4(+)/CD8(+) T cell populations, outside of the thymus, have recently been described to express concurrently ThPOK and Runx3. Considerable heterogeneity exists within the CD4(+)/CD8(+) DP T cell pool, and the function of CD4(+)/CD8(+) T cell populations remains controversial, with conflicting reports describing cytotoxic or suppressive roles for these cells. In this review, we describe how transcriptional regulation, lineage of origin, heterogeneity of CD4 and CD8 expression, age, species, and specific disease settings influence the functionality of this rarely studied T cell population.
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http://dx.doi.org/10.1189/jlb.1RU0814-382DOI Listing
January 2015

Asymmetric peptide dendrimers are effective linkers for antibody-mediated delivery of diverse payloads to b cells in vitro and in vivo.

Pharm Res 2014 Nov 22;31(11):3150-60. Epub 2014 May 22.

UQ Diamantina Institute, The University of Queensland, TRI, 37 Kent St,Woolloongabba, 4102, Queensland, Australia.

Purpose: Safe, targeted delivery of therapeutics remains a focus of drug/gene delivery, the aim being to achieve optimal efficacy while minimising off-target delivery. Dendrimers have a vast array of potential applications and have great potential as gene and drug delivery tools. We previously reported the development of peptide dendrimers that effectively complexed DNA and that have distinct advantages over conventional spherical dendrimers. Here, to expand the application of peptide-based low generation dendrimers we tested their capacity to be transformed into linkers for antibody-based targeting of diverse payloads.

Methods: Peptide-based low-generation asymmetric dendrimers were generated and conjugated to partially-reduced antibodies specific for B cell surface antigens or an irrelevant antigen. Preservation of antigen binding by the antibodies and targeting of the conjugated dendrimers carrying a small molecule (biotin) or plasmid DNA payloads was tested.

Results: Peptide-based low generation dendrimers were efficiently and site-specifically conjugated to antibodies with retention of antigen-binding capacity. Altering the branching termini of dendrimers facilitated delivery of diverse payloads in vitro and in vivo.

Conclusions: We propose that safe, non-toxic peptide dendrimers, which are readily synthesised and modifiable for a variety of applications, form the basis of a new family of biocompatible "linkers" with substantial potential for targeted delivery applications.
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http://dx.doi.org/10.1007/s11095-014-1408-1DOI Listing
November 2014
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